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Studies on Lumpy Skin Disease Virus

Thesis · October 2016


DOI: 10.13140/RG.2.2.21593.21607

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''Studies on Lumpy Skin Disease Virus''
Thesis submitted to Faculty of Veterinary Medicine

Benha University

By

Gehad Hossam ELdein Mohamed Elkady


B. V. Sc Benha University (2012)

For
The degree of M. V. Sc (Virology)

Under supervision of

Prof. Dr. Gabr Fikry El-Bagoury

Professor of Virology

Department of Virology

Faculty of Veterinary Medicine

Benha University

Dr. Ehab Mostafa El-Nahas Mohamed

Ass. Professor of Virology

Department of Virology

Faculty of Veterinary Medicine

Benha University

Dr. Ayman Saeed Emam El-habbak

Ass. Professor of Virology

Department of Virology

Faculty of Veterinary Medicine

Benha University

1
ACKNOWLEDGEMENT

First of all and for most I’m indebted to Allah; the most kind and the most merciful,
for all gifts which given to me.

I would like to take this opportunity to express my deepest appreciation and sincere
gratitude to Prof. Dr. Gabr F. El-Bagoury, professor of virology. Dept, Faculty of
Vet. Med. Benha University, for his supervision of this work.

My great appreciation to Dr. Ayman S. El-Habbaa, Assistant Prof. of Virology.


Dept. Faculty of Vet. Med. Benha University, for his supervision of this work.

I would like to express my gratitude to Dr. Ehab M. El-Nhas, Assistant Prof. of


Virology department, Faculty of Vet. Med. Benha University for his supervision of
this work.

I would like to extend my best regards to Dr- Mohamed gouda abd -elwahab who
is the professor of infectious department, faculty of veterinary medicine, Benha
University, Dr-Wessel Dirksen who is the Laboratory manager at Ohio university,
and Dr- Saeed Elshafae who is lecturer at pathology department, faculty of
veterinary medicine, Benha University for their help to design the primers.

Finally, my great gratitude to My family who adapted the optimum environment to


complete this work.

2
LIST OF CONTENTS

FRONTCOVER…………………………………………………………………………………………………………………….1

ACKNOWLEDGMENT…………………………………………………………………………………………………………………….2

LIST OF CONTENTS…………………………………………………………………………………………………………….3
LIST OF ABBREVIATIONS……………………………………………………………………………………..4

LIST OF TABLES…………………………………………………………………………………………………..10

LIST OF FIGURES…………………………………………………………………………………………………11

LIST OF PHOTOS…………………………………………………………………………………………………12

ABSTRACT………………………………………………………………………………………………………….13

1. INTRODUCTION…………………………………………………………………………………………………………….15
2. AIM OF THE STUDY………………………………………………………………………………………………………..18

3. LITERATURE REVIEW………………………………………………………………………………………………………20

3.1. HISTORY OF LSDV……………………………………………………………………………………………………..21

3.2. TAXONOMY OF LSDV…………………………………………………………………………………………………22

3.3. STRUCTURE OF LSDV…………………………………………………………………………………………………22

3.4. ANTIGENIC CHARACTERS AND RELATIONSHIPS OF LSDV……………………………………………51

3.5. RESISTANCE OF LSDV TO PHYSICAL AND CHEMICAL AGENTS…………………………………….51

3.6. BIOLOGICAL PROPERTIES OF LSDV…………………………………………………………………………….52

3.6.1. REPLICATION IN THE HOST CELL………………………………………………………………………52

3.6.2. PROPAGATION IN VITRO…………………………………………………………………………………55

3.7. PATHOGENESIS AND HISTOPATHOLOGY OF LSDV…………………………………………………….56

3.8. LABORATORY DIAGNOSIS…………………………………………………………………………………………58

3
3.8.1. SAMPLING OF LSDV…………………………………………………………………………………………58

3.8.2. ISOLATION OF LSDV………………………………………………………………………………………...58

3.8.2.1. ISOLATION OF LSDV ON EMBRYONATED CHICKEN EGGS (SPF-ECES)………….58

3.8.2.2. ISOLATION OF LSDV ON CELL CULTURE………………………………………………………………….59

3.8.3. IDENTIFICATION OF LSDV …………………………………………………………………………………………………………………………….60

3.8.3.1. NON-SEROLOGICAL TECHNIQUES……………………………………………………………….60

3.8.3.1.1. TRANSMISSION ELECTRON MICROSCOPY………………………………………….60

3.8.3.1.2. HISTOPATHOLOGICAL EXAMINATION………………………………………………..61

3.8.3.2. SEROLOGICAL TECHNIQUES…………………………………………………………………………..63

3.8.3.2.1. INDIRECT FLUORESCENCE ANTIBODY TECHNIQUE (IFAT)……………………63

3.8.3.3. MOLECULAR IDENTIFICATION…………………………………………………………………………63

3.8.3.3.1. POLYMERASE CHAIN REACTION (PCR)……………………………………………….63

3.8.3.3.2. SEQUENCING OF THE VIRAL GENOME………………………………………………69

3.9. VIRAL IMMUNE EVASION…………………………………………………………………………………………75

4. MATERIAL AND METHODS STRATEGY…………………………………………………………………………78

4.1. MATERIAL…………………………………………………………………………………………………………………79

4.2. METHODS………………………………………………………………………………………………………………..102

5. RESULTS…………………………………………………………………………………………………………………………118

6. DISCUSSION, CONCLUSION AND RECOMMENDATION…………………………………………………145

7. SUMMARY ……………………………………………………………………………………………………………………153

8. REFERENCES …………………………………………………………………………………………………………………156

9. ARABIC SUMMARY………………………………………………………………………………………………………..2

4
LIST OF ABBREVIATIONS

LSDV Lumpy Skin Disease Virus


LSD Lumpy Skin Disease
ds Double Stranded deoxyribonucleic
DNA acid
SPPV Sheep Pox Virus
GTPV Goat Pox Virus
SPF Specific Pathogen Free
ECE Embryonated Chichen Egg
MDBK Madin Darby Bovine Kidney Cell
CPE Cytopathic effect
TEM Transmission Electron Microscope
TEM Transmission Electron Microscopy
PCR Polymerase Chain Reaction
ORFS Open Reading Frames
ITRS Inverted Terminal Repeats
bp base pair
GI Gene Bank Identification
IMV Intracellular Mature Virus
IEV Intracellular Enveloped Virus
CEV Cell- Associated Extracellular
Virus
EMV Extracellular Mature Virus
IV Immature Virion
MV Mature Virus

EV Enveloped Virus

5
EEV Extracellular Enveloped Virus

NP Nucleoprotein

VTM Viral transport media


PM Post mortem
S1 Sample 1
S2 Sample 2
PTFEL Polytetrafluoroethylene laminated
I.V Intravenous
W/V Weight per volume
M.O.H Manufacturer On Hold
Na+ Sodium
CL- Chloride
W Weight
L Liter
EXP Expired
Cat. No. Catalogue Number
WFI Water for Injection
ml Milliliter
µm Micrometer

⁰c Degree Celsius

µl Microliter
g Gram
mg Milligram
USA United states of America
EMS Electron Microscopy Sciences
µg Microgram
Ca+2 Calsium
mg+2 Magnesium

6
min Minute
Sec Second
Temp Temperature
N Normality
M Molarity
NaCL Sodium chloride
NaOH Sodium hydroxide
HCL Hydrochloric acid
MEM Minimum essential medium
NaHCO3 Sodium bicarbonate
PBS Phosphate buffered saline
KCL Pottasium hydroxide
Na2 HPO4 Di-sodium hydrogen phosphate
KH2PO4 Pottasium di-hydrogen phosphate
DH2O Distilled Water
EDTA Ethylene diamine tetra acetic acid
MW Molecular weight
M Mole or mol
mM Millimole
Co2 Carbon dioxide
NSA non enyl succinic anhydride
ERL-4206 (VCD) (Vinyl Cyclohexene Dioxide)
(Vinyl-4 Cyclohexene Diepoxide),
DER 736 EPOXY RESIN diglycidyl ether of polypropylene
glycol
DMAE Dimethylaminoethanol
DW Distilled water
FITC Fluorescein isothiocyanate
CAM Chorioallantoic membrane
CAMs Chorioallantoic membranes
HA Haemagglutination

7
IFAT Indirect fluorescence antibody
technique
qRT-PCR Quantitative real-time PCR
mgCl2 Magnesium chloride
Taq Thermus aquaticus
RNase Ribonuclease
dNTP Deoxy nucleotide tri-phosphate
Tm Melting temperature
nm Nanomole
OD Optical density
cm Centimetre
Kcal Kilocalories
Ext. Coefficient Extinction coefficient
L Length
µm Micromole
TAE Tris acetic acid EDTA
FW Formula weight
POP Performance Optimized Polymers
ABC Anode Buffer Container
CBC Cathode Buffer Container
P/N Part number
rpm Round per minute
H&E Hematoxylin and eosin
RBCS Red blood cells
Pmol Picomol
ng Nanogram
Acc.no Accession number
BLAST Basic local alignment search tool
Ncbi National center for biotechnology
information
Hr Hour

8
Max Maximum
No Number
cm3 Cubic centimetre
Abs Antibodies
UV Ultraviolet
Nm Nanometer
FBT foetal bovine testes
LK Londiani Kenya
LT lamb testes
ELISA Enzyme linked immunosorbent
assay
VNT Viral neutralization test
BVD Bovine viral diarrhea
SA South Africa
EBL Embryonic bovine lung
CER Chicken embryo rough
MAFFT Multiple Alignment using Fast
Fourier Transform

9
LIST OF TABLES

TABLE.NO TITLE
TABLE_1 DNA replication and nucleotide
metabolism genes of LSDV
TABLE_2 RNA transcription and modification
genes of LSDV
TABLE_3 Structure and assembly genes of LSDV
TABLE_4 Viral virulence and host range genes of
LSDV

TABLE_5 Major gene families of LSDV

TABLE_6 Alterations on CAM and Embryo of SPF


ECE inoculated with two suspected skin
samples for LSD virus.

TABLE_7 Characteristics of suspected LSD virus


isolate strain on SPF ECE and MDBK cell
line.

10
LIST OF FIGURES

FIGURE.NO TITLE
FIGURE_1 Morphological structure LSDV

FIGURE_2 Diagram shows the surface structure of


an un enveloped virion, whereas the
other part shows the cross- section
through the center of an enveloped
virion
FIGURE_3 Linear map of the LSDV genome.
FIGURE_4 The poxvirus lifecycle

FIGURE_5 Material and method of TEM.


FIGURE_6 The partial sequence profile of LSDV
envelope protein-like gene
FIGURE_7 The partial sequence profile of LSDV
envelope protein-like gene
FIGURE_8 Alignment report of partial nucleotide
sequences for the envelope protein-like
gene
FIGURE_9 Pair wise sequence distance for the
envelope protein-like gene
FIGURE_10 Phylogenetic tree of local LSDV strain
FIGURE_11 Alignment report of partial nucleotide
sequences for the envelope protein-like
gene of local LSDV strain
FIGURE_12 Pair wise sequence distance for the
envelope protein-like gene of local LSDV
strain
FIGURE_13 Phylogenetic tree of local LSDV strain

11
LIST OF PHOTOS

PHOTO.NO TITLE
Photo_1 Suspected cattle for LSD
Photo_2 Signs of LSDV multiplication on ECE-SPF
CAM
Photo_3 Signs of LSDV multiplication on ECE-SPF
embryo
Photo_4 Control non- infected complete sheet of
MDBK cells
Photo_5 Characteristic CPE of suspected LSDV on
the sheet of MDBK cells
Photo_6 Histopathological examination of
intracytoplasmic inclusions of LSDV
Photo_7 Transmission Electron Microscopy of
LSDV
Photo_8 HA property of LSD virus isolate
Photo_9&10 Serological identification of suspected
LSD virus isolate using IFAT

Photo_11 Electrophoresis of the amplified products

12
13
ABSTRACT

Between 2013 -2014, outbreaks of LSDV were reported in Qaliubiya province,


where animals suffered from severe signs differ from the previous outbreaks such
as suffocation and deaths.
This thesis presents the first investigation that the LSDV changes its biological
properties and also its molecular properties.
The general objective of the pilot study was to study the biological and molecular
characteristic of recent isolate of LSDV.
Trial for isolation and identification of LSDV was carried out using SPF-fertile
ECE and MDBK cell line, which show new signs on inoculated egg, such as
edema and hemorrhage, with hepatomegaly and bloody liver of embryo,
hemorrhagic and slight heart hypertrophy in addition to pock lesion on CAM in the
form of white line. Moreover cell rounding and clustering on MDBK cells. The
presence of virus confirmed by non-serological techniques, such as TEM, where
the virus appeared as rounded shape with inclusion bodies, histopathological
examination, where intracytoplasmic inclusion bodies appeared, and
hemagglutination technique where button shape appeared, serological technique,
such as IFAT, where intracytoplasmic apple green fluorescence emission appered
and molecular identification, such as conventional PCR, cycle sequencing, gene
alignment and phylogenetic analysis of target gene confirmed the success isolation
of LSDV.

14
15
1- INTRODUCTION

Lumpy skin disease virus (LSDV), a DNA virus of the genus Capripoxvirus,
chordopoxvirinae subfamily and Poxviridae family. The prototype strain is
Neethling virus (Gari, G.et al,2010).
It is closely related to sheep pox (SPPV) and Goat pox virus (GTPV) (Babiuk,
S et al,2009). However, although all three viruses are considered distinct species,
they can't be differentiated serologically (Magori-Cohen at al, 2012). Therefore,
the only molecular techniques to distinguish LSDV from SPPV and GTPV. It exists
epidemically or sporadically in southern and eastern Africa and more recently in
northern Africa and the Middle East (Fenner et al,1996), It was reported in
unvaccinated cattle in Bulgaria, the Former Yugoslavian Republic of Macedonia,
Turkey, Greece (OIE a and b, 2016) and Israel (Brenner et al., 2006), and in the
Sultanate of Oman (Kumar, 2011; Tageldin et al., 2014), UAE (Abutarbush,
2015), in Jordan (Abutarbush etal, 2015) and Iraq (Al-Salihi and Hassan, 2015).
It affects cattle and occasionally buffaloes, it causes LSD which characterized by
lachrymation and nasal discharge. Subscapular and precrural lymph nodes become
markedly enlarged. High fever accompanies the appearance of highly characteristic
skin lesions of 10-50 mm in diameter. The number of the lesions may vary from a
few in mild cases, to multiple lesions, covering the entire body in severely infected
individuals. Necrotic plaques may appear in the mucous membranes of the oral and
nasal cavities, causing purulent or mucopurulent nasal discharge and excessive
salivation. Painful ulcerative lesions may appear in the cornea of one or both eyes,
leading to blindness in some cases. Pox lesions are found throughout the entire
digestive and respiratory tracts and on the surface of almost any internal organ.
Necrotic skin lesions in the legs and on top of the joints may lead to deep
subcutaneous infections complicated with secondary bacterial infections and
lameness. Pneumonia caused by the virus itself or secondary bacterial infection, is a
common complication. Silent subclinical infections are common in the field. In
experimentally infected animals approximately one third of the cattle did not show
any clinical signs, although all of the infected animals became viraemic. It is
transmitted by biting flies, especially during very high rainfall where its high
activity. Only mechanical transmission in insects has been reported, such as in Aedes
aegypti mosquitoes. The outbreak in Israel (1989) was attributed to Stomoxys
calcitrans carried by wind from Ismailiya to Egypt. Recent transmission studies with
ticks on animals demonstrated mechanical/intrastadial and transstadial transmission
by Amblyomma hebraeum and Rhipicephalus appendiculatus adult ticks.
Transovarial transmission of the virus was demonstrated in Rhipicephalus

16
decoloratus. Both intrastadial and transstadial passage of LSDV has been
demonstrated in R. appendiculatus and A. hebraeum through detection of LSDV in
saliva of adult ticks fed is either adult or nymphs respectively. However,
transmission may also occur by direct or indirect contact, via contaminated food or
water, or via artificial insemination or natural mating. The incubation period in
naturally infected animals may be up to 5 weeks. but in experimentally infected
animal is 6-9 days until the onset of fever (Eeva S. M. Tuppurainen et al, 2015).
Subcutaneous inoculation or intradermal inoculation of cattle with LSDV results in
the development of a localized swelling at the size of inoculation after 4-7 days and
the enlargement of the regional lymph nodes, while generalized eruption of skin
nodules usually occurs 7 to 19 days after inoculation. In experimentally infected
cattle LSDV was demonstrated in saliva at least for 11 days after the development
of fever, in semen for 42 days and in skin nodules for 39 days. Viraemia occurred
after the initial febrile reaction and persisted for 2 weeks. Viral replication in
pericytes, endothelial cells and probably other cells in blood vessel and lymph vessel
walls causes vasculitis and lymphagitis in some vessels in affected areas. In severe
cases infarction may result. (Coetzer et al ,2004). My recent LSDV strain (2014),
mortality occurs due to the nodules developed in the mucosa of RT particulary
trachea and lungs leading to suffocation. Investigated cutaneous and testicular
lesions with diffuse degenerative and inflamatory changes in seminiferous tubules
and blood vessels, the seminiferous tubules were devoid of primary, secondary
spermatocytes and spermatids, although spermatogonia and sertoli cells are resistant,
so transient infertility in bulls as the regeneration of the germinal epithelium depends
mainly on the persistence of the spermatogonia and sertoli cells, although extensive
fibrosis may preculde the return to fertility (Cornelius Henry Annandale et
al,2006). Immunity after recovery from natural infection is life-long in most cattle;
calves of immune cows acquire maternal antibody and are resistant to clinical
disease for about 6 months. Microscopically, the lesions vary considerably
depending on the stage of development. In the acute stage vasculitis is sometimes
with concomitant thrombosis, and infarction, as well as perivascular fibroplasia and
infiltration of macrophages and some lymphocytes and eosinophils in particulary the
dermis and subcutis. During the acute and subacute stages of the disease eosinophilic
intracytoplasmic inclusion bodies (Coetzer et al,2004).

17
18
2- AIM OF THE STUDY

Between 2013-2014, outbreaks of LSDV were reported in Qaliubiya province,


where animals suffered from severe signs differ from the previous outbreaks, such
as suffocation and deaths, so The general objective of the pilot study was to obtain
a proof of concept that there is may be a new strain of LSDV differ from the
neethling strain.

The objectives set for the thesis could be obtained through, For the first time,
isolation and identification of LSDV was demonstrated by isolation on SPF-fertile
ECE and MDBK cell line, which show new signs on inoculated egg. In addition,
the presence of virus confirmed by non-serological techniques, such as TEM,
haemagglutination technique, and histopathological examination, serological
techniques, such as indirect fluorescence antibody technique and molecular
identification by using conventional PCR, cycle sequencing, gene alignment and
phylogenetic analysis.

19
20
3- LITERATURE REVIEW

3.1. History of Lumpy Skin Disease (LSD):


3.1.1. History in Egypt:
Several outbreaks of LSD were reported among cattle from different
governorates in Egypt. In 1988, it was appeared in Suez in June, and Ismailia in
October (House et al.,1990). It was recorded also in 2006 at Beni-Suef (Tamam et
al., 2006), and in early 2006 in Fayoum, Menofia and Sharquia (El-Kholy et al.,
2008), Qaliubiya and Alexandria (Sohair et al., 2008), Damietta (Awadin et al.,
2011). At 2008, it remerged again in different localities in Egypt (Ahmed and
Kawther, 2008), (Amal et al., 2008), (Omyma, 2008). At 2010, LSDV infection
was recognized and diagnosed in Egypt (Awad et al., 2010) and diagnosed among
buffaloes from Qaliubiya governorate (EL Nahas et al., 2011) and at 2011 LSDV
was diagnosed among cattle and buffaloes in Qaliubiya (Sharawi and Abd El-
Rahim, 2011), among cattle in Ismailia (EL-Kenawy and EL-Tholoth, 2011), in
Monoufia and Fayoum (Abou Elyazeed, et al, 2012) and among cattle at Ismailia
(Ahmed and Amina, 2013). At 2013, LSDV was identified among cattle at Ismailia
(El-Haig et al., 2014) and among cattle at Qaliubiya (Aziza et al., 2015) and at July
to November 2014 in Sharquia governorate (Neamat-Allah et al., 2015).

3.1.2. History in the World:


LSD was recorded in Russia and countries of the former Soveit Union (Orlova
et al., 2006). It was recorded also in Europe, Asia and it is endemic in most African
countries and Middle East (Tuppurainen and Oura, 2012). LSD was reported
affecting cattle in Europe (Kreindel et al., 2015), the Eastern Mediterranean Basin
countries (Wainwright et al., 2013), and Greece, (Dilaveris, 2015). It was reported
in unvaccinated cattle in Bulgaria, the Former Yugoslavian Republic of Macedonia,
Turkey, Greece (OIE a and b, 2016) and Israel (Brenner et al., 2006; Stram et al.,
2008; Brenner et al., 2009; Reuma Magori-Cohen et al., 2012). An outbreak of
LSD among cattle in the Sultanate of Oman (Kumar, 2011; Tageldin et al., 2014),
UAE (Abutarbush et al., 2015), in Jordan (Abutarbush, 2015) and Iraq (Al-Salihi
and Hassan, 2015). LSD was reported in Kenya (Binepal et al., 2001) and Ethiopia
(Gari et al., 2008; Gezahegn et al., 2013; Zelalem et al., 2015). LSD was also

21
reported in South Africa in recurrent outbreaks (Hunter and Wallace, 2001;
Tulman et al., 2001; Chihota et al., 2001; Wallace and Viljoen, 2002; Kara et
al., 2003; Chihota et al., 2003; Coetzer et al., 2004; Tuppurainen et al., 2005;
Irons et al., 2005; Annandale et al., 2005; Annandale et al., 2006; Osuagwuh et
al., 2006 and 2007; Annandale et al., 2010; Tuppurainen et al., 2011; Lubinga
et al., 2013 and 2014; Tuppurainena et al., 2015).

3.2. Taxonomy of Lumpy Skin Disease Virus (LSDV):

Poxviridae is divided into two subfamilies: poxviruses affecting insects


(Entomopoxvirinae) and vertebrates (Chordopoxvirinae) and several genera. Within
the Chordopoxvirinae the genus Capripoxvirus, comprises LSDV, sheep pox virus
(SPPV) and goat pox virus (GTPV). The prototype of LSDV is Neethling strain
which was first isolated in South Africa (Alexander et al., 1957). LSDV belongs to
the family Poxviridae, subfamily Chordopoxvirinae, genus Capripoxvirus
(International Committee on Taxonomy of Viruses, ICTV, 2013; Tuppurainen
et al, 2015).

3.3. Structure of LSDV:

Figure (1): Morphological structure of LSDV (Haftu et al., 2012).

22
Figure (2): Diagram shows the surface structure of an
unenveloped virion, whereas the other part shows the cross-
section through the center of an enveloped virion. (Fenner's
Veterinary virology, fourth edition, 2011).

Pox virions are brick or oval shaped with the average size of LSDV is length
294±20 nm and width 262±22 nm (Kitching and Smale, 1986). Within the virion,
there are over 100 polypeptides, which are arranged in a core, two lateral bodies,
an outer membrane and an envelope. The core of the virus is dumbbell-shaped and
the nature of lateral bodies is unknown. The core of the viruses contains proteins
that include a transcriptase and several other enzymes (Fenner et al., 2011).
Poxviruses exist in the intracellular space, with or without an envelope and are

23
enveloped in the extracellular space (Fenner et al., 2011). Both forms are
infectious and have the same core and genetic material. “Mature virions” (MV)
(Moss, 2006) also called “intracellular mature virions” (Fenner et al., 2011) are
surrounded by a single lipid membrane with irregular arrangements of tubular
proteins on the surface. These are the most abundant form of the virus and are
believed to be responsible of host to-host spread, Intracellular enveloped virions
(IEV) (Fenner et al., 2011), more recently referred as “wrapped virions” (Moss,
2006) develop from MV, surrounded by two additional layers of membrane,
originating from the trans-Golgi apparatus or endoplasmic network. While budding
out, the outmost layer of wrapped virions fuses with the plasma membrane,
releasing extracellular enveloped viruses (EV) (Fenner et al., 2011). All vertebrate
poxviruses share a group-specific antigen (NP antigen) (Woodroofe and Fenner,
1962; Tuppurainen et al, 2015). Poxviridae comprise a diverse family of large
double-stranded DNA viruses that undergo replication exclusively in the host–cell
cytoplasm. Each virion contains a single linear genome that varies in length (130–
360 Kb) depending on the virus strain. The genomes are compact, with open
reading frames (ORFs) being closely spaced and non-overlapping with no evidence
of mRNA splicing. Although individual strains may contain more than 200 ORFs,
only 50 are thought to encode proteins essential for viral transcription, DNA
replication, or the formation of new virions. These ORFs cluster in the central
region of the genome and are well conserved in sequence and position across
different species. The remaining ORFs are more variable and tend to be distributed
more towards the terminal ends of each genome that encode factors confer
virulence, tissue tropism, or serve to expand host range. Poxviruses have captured
host genes during their evolution in order to evade immune detection and
elimination. Also, poxviruses also adapt to changes in host defense by altering
their existing repertoire of factors through accumulation of point mutations,
occurrence of unequal crossovers giving rise to chimeric factors, or transient
genomic expansions that increase the number of targets available for mutation.
Poxvirus genomes are modified in response to evolutionary pressure, several
poxvirus families show signs of ORF duplication and divergence. These include:
the ankyrin-repeat proteins, the serpin family, the C7L family, the kelch-like

24
proteins, and the Bcl-2-like proteins (Nelson et al., 2015). The 151-kbp
LSDV genome consists of a central coding region bounded by identical 2.4 kbp-
inverted terminal repeats and contains 156 putative genes. Comparison of LSDV
with chordopoxviruses of other genera reveals 146 conserved genes which encode
proteins involved in transcription and mRNA biogenesis, nucleotide metabolism,
DNA replication, protein processing, virion structure and assembly, and viral
virulence and host range. In the central genomic region, LSDV genes share a high
degree of collinearity and amino acid identity (average of 65%) with genes of other
known mammalian poxviruses, particularly suipoxvirus, yatapoxvirus, and
leporipoxviruses. In the terminal regions, collinearity is disrupted and poxvirus
homologues are either absent or share a lower percentage of amino acid identity
(average of 43%). Most of these differences involve genes and gene families with
likely functions involving viral virulence and host range (Tulman et al., 2001).
The genomes of SPPV and GTPV are very similar to that of LSDV, sharing 96%
nucleotide identity within the genus Capripoxvirus (Tulman et al., 2002).
However, molecular studies have demonstrated that LSDV, SPPV and GTPV are
phylogenetically distinct (Tulman et al., 2001 and 2002; Tuppurainen et al.,
2015).

25
Figure (3): Linear map of the LSDV genome. ORFs are numbered from
left to right based on the position of the methionine initiation codon.
ORFs transcribed to the right are located above the horizontal line; ORFs
transcribed to the left are below. Genes with similar functions and
members of gene families are colored according to the figure key. ITRs
are represented as black bars below the ORF map (Tulman et al., 2001 at
J. Virol. 2001; 75:7122-7130).

LSDV NW-LW isolate Neethling Warmbaths LW, complete genome sequence


is 150793 bp DNA that was published on the gene bank data base
(https://blast.ncbi.nlm.nih.gov/Blast.cgi) in accession number AF409137, version
AF409137.1, GI:22595533 with following organized protein coding genes in the
following tables.

26
Table (1): DNA replication and nucleotide metabolism genes.

ORF CDS Codon Product protein_id db_xref Note


complement start

1 LD018_R-L 11586..1202 =1 dUTPase AAN02584 GI:225955


6 .1 51

2 LD020_R-L 13849..1481 =1 ribonucleotide AAN02587 GI:225955


4 reductase small .1 54
subunit

3 LD039_R-L 32313..3534 =1 DNA polymerase AAN02607 GI:225955


5 .1 74

4 LD066_L-R 56798..5733 =1 thymidine kinase AAN02634 GI:225956


1 .1 01

5 LD077_L-R 69235..7018 =1 DNA topoisomerase AAN02645 GI:225956


8 .1 12

27
6 LD082_L-R 74375..7503 =1 uracil DNA AAN02650 GI:225956
1 glycosylase .1 17

7 LD083_L-R 75074..7743 =1 putative NTPase AAN02651 GI:225956 similar to vaccinia


4 .1 18 virus strain
Copenhagen D5R

8 LD112_L-R 103931..105 =1 putative DNA AAN02681 GI:225956 similar to vaccinia


223 polymerase .1 48 virus strain
processivity factor Copenhagen A20R

9 LD133_L-R 120352..122 =1 DNA ligase-like AAN02701 GI:225956


031 protein .1 68

10 LD139_L-R 131627..132 =1 putative Ser/Thr AAN02707 GI:225956 similar to vaccinia


544 protein kinase .1 74 virus strain
Copenhagen B1R

28
Table (2): RNA transcription and modification genes.

ORF CDS Codon Product protein_id db_xref note


complement start

1 LD032_R 23753..25177 =1 poly(A) polymerase AAN02600 GI:225955 similar to vaccinia virus


-L large subunit .1 67 strain Copenhagen E1L,
PAPL

2 LD036_R 27988..28593 =1 RNA polymerase AAN02603 GI:225955 similar to vaccinia virus


-L subunit .1 70 strain Copenhagen E4L,
RPO30

3 LD049_L- 42985..45015 =1 putative RNA helicase AAN02617 GI:225955 similar to vaccinia virus
R .1 84 strain Copenhagen I8R,
NPH-II

4 LD051_L- 47125..47793 =1 putative AAN02620 GI:225955 similar to vaccinia virus


R transcriptional .1 87 strain Copenhagen G2R
elongation factor

29
5 LD055_L- 49454..49645 =1 RNA polymerase AAN02623 GI:225955 similar to vaccinia virus
R subunit .1 90 strain Copenhagen
G5.5R, RPO7

6 LD058_L- 51334..52116 =1 putative late AAN02626 GI:225955 similar to vaccinia virus


R transcription factor .1 93 strain Copenhagen G8R ,
VLTF-1

7 LD068_L- 58056..59057 =1 poly(A) polymerase AAN02636 GI:225956 similar to vaccinia virus


R small subunit .1 03 strain Copenhagen J3R,
PAPS

8 LD069_L- 58972..59529 =1 RNA polymerase AAN02637 GI:225956 similar to vaccinia virus


R subunit .1 04 strain Copenhagen J4R ,
RPO22

9 LD071_L- 60022..63879 =1 RNA polymerase AAN02639 GI:225956 similar to vaccinia virus


R subunit .1 06 strain Copenhagen J6R ,
RPO147

30
10 LD075_R 65982..68378 =1 RNA polymerase- AAN02643 GI:225956 similar to vaccinia virus
-L associated protein .1 10 strain Copenhagen H4L ,
RAP94

11 LD076_L- 68522..69193 =1 putative late AAN02644 GI:225956 similar to vaccinia virus


R transcription factor .1 11 strain Copenhagen H5R ,
VLTF-4

12 LD079_L- 70682..73210 =1 mRNA capping AAN02647 GI:225956 similar to vaccinia virus


R enzyme large subunit .1 14 strain Copenhagen D1R

ORF CDS Codon Product protein_id db_xref note


complement start

31
13 LD084_L- 77431..79338 =1 putative early AAN02652 GI:225956 similar to vaccinia virus
R transcription factor .1 19 strain Copenhagen D6R ,
small subunit VETFS

14 LD085_L- 79363..79854 =1 RNA polymerase AAN02653 GI:225956 similar to vaccinia virus


R subunit .1 20 strain Copenhagen D7R ,
RPO18 "

15 LD087_L- 80536..81297 =1 multi motif protein AAN02655 GI:225956 putative gene expression
R .1 22 regulator; similar to
vaccinia virus strain
CopenhagenD10R

16 LD088_R 81303..83210 =1 putative transcription AAN02656 GI:225956 similar to vaccinia virus


-L termination factor .1 23 strain Copenhagen D11L
, NPH-I

17 LD089_R 83237..84100 =1 mRNA capping AAN02657 GI:225956 similar to vaccinia virus


-L enzyme small subunit .1 24 strain Copenhagen D12L

32
18 LD091_R 85816..86268 =1 putative late AAN02659 GI:225956 similar to vaccinia virus
-L transcription factor .1 26 strain Copenhagen A1L ,
VLTF-2

19 LD092_R 86298..86996 =1 putative late AAN02660 GI:225956 similar to vaccinia virus


-L transcription factor .1 27 strain Copenhagen A2L ,
VLTF-3

20 LD096_L- 89865..90377 =1 RNA polymerase AAN02664 GI:225956 similar to vaccinia virus


R subunit .1 31 strain Copenhagen A5R ,
RPO19

21 LD098_R 91522..93666 =1 putative early AAN02666 GI:225956 similar to vaccinia virus


-L transcription factor .1 33 strain Copenhagen A7L ,
large subunit VETFL

22 LD099_L- 93723..94595 =1 putative intermediate AAN02667 GI:225956 similar to vaccinia virus


R transcription factor .1 34 strain Copenhagen A8R ,
subunit VITF-3

33
23 LD110_L- 101937..1033 =1 putative DNA helicase AAN02678 GI:225956 similar to vaccinia virus
R 79 transcriptional .1 45 strain Copenhagen A18R
elongation factor

24 LD115_L- 105723..1068 =1 putative intermediate AAN02683 GI:225956 similar to vaccinia virus


R 80 transcription factor .1 50 strain Copenhagen A23R
subunit

25 LD116_L- 106911..1103 =1 RNA polymerase AAN02684 GI:225956 similar to vaccinia virus


R 81 subunit .1 51 strain Copenhagen A24R ,
RPO132

26 LD119_R 111265..1121 =1 RNA polymerase AAN02687 GI:225956 similar to vaccinia virus


-L 76 subunit .1 54 strain Copenhagen A29L ,
RPO35

34
Table (3): structure and assembly genes

ORF CDS Codo Product protein_id db_xref note


complement n

Start

1 LD025_R- 16655..17998 =1 putative Ser/Thr AAN02592 GI:225955 similar to vaccinia virus


L protein kinase .1 59 strain Copenhagen F10L

2 LD027_R- 18951..20867 =1 putative EEV AAN02595 GI:225955 similar to vaccinia virus


L maturation protein .1 62 strain Copenhagen F12L

3 LD028_R- 20874..21986 =1 putative palmitylated AAN02596 GI:225955 similar to vaccinia virus


L virion envelope .1 63 strain Copenhagen F13L
protein

4 LD031_L- 23435..23749 =1 putative DNA-binding AAN02599 GI:225955 similar to vaccinia virus


R virion core .1 66 strain Copenhagen F17L
phosphoprotein

35
5 LD040_L- 35379..35666 =1 putative redox AAN02608 GI:225955 similar to vaccinia virus
R protein .1 75 strain Copenhagen E10R

6 LD041_R- 35663..36055 =1 putative virion core AAN02609 GI:225955 similar to vaccinia virus
L protein .1 76 strain Copenhagen E11L

7 LD043_R- 38202..39146 =1 putative DNA- AAN02611 GI:225955 similar to vaccinia virus


L binding virion core .1 78 strain Copenhagen I1L
protein

8 LD046_R- 40247..40483 =1 hypothetical protein AAN02614 GI:225955


L .1 81

9 LD048_R- 41678..42979 =1 putative virion core AAN02616 GI:225955 similar to vaccinia virus
L protein .1 83 strain Copenhagen I7L

36
10 LD050_R- 45012..46802 =1 putative AAN02618 GI:225955 similar to vaccinia virus
L metalloprotease .1 85 strain Copenhagen G1L

11 LD053_R- 47757..48137 =1 putative glutaredoxin AAN02621 GI:225955 similar to vaccinia virus


L .1 88 strain Copenhagen G4L

12 LD057_R- 50183..51304 =1 putative virion core AAN02625 GI:225955 similar to vaccinia virus
L protein .1 92 strain Copenhagen G7L

13 LD060_L- 53154..53891 =1 putative myristylated AAN02628 GI:225955 similar to vaccinia virus


R IMV envelope .1 95 strain Copenhagen L1R
protein

14 LD063_L- 55198..55959 =1 putative DNA- AAN02631 GI:225955 similar to vaccinia virus


R binding virion core .1 98 strain Copenhagen L4R
protein ,VP8

37
ORF CDS Codo Product protein_id db_xref note
complement n
start

15 LD072_R- 63884..64399 =1 putative protein- AAN02640 GI:225956 similar to vaccinia virus


L tyrosine phosphatase .1 07 strain Copenhagen H1L

16 LD074_R- 64984..65952 =1 putative IMV AAN02642 GI:225956 similar to vaccinia virus


L envelope protein .1 09 strain Copenhagen H3L,
p35

17 LD080_R- 73172..73639 =1 putative virion AAN02648 GI:225956 similar to vaccinia virus


L protein .1 15 strain Copenhagen D2L

18 LD081_L- 73641..74378 =1 putative virion AAN02649 GI:225956 similar to vaccinia virus


R protein .1 16 strain Copenhagen D3R

19 LD090_R- 84140..85789 =1 putative rifampicin AAN02658 GI:225956 similar to vaccinia virus


L resistance protein .1 25 strain Copenhagen D13L

38
20 LD094_R- 87229..89214 =1 putative virion core AAN02662 GI:225956 similar to vaccinia virus
L protein .1 29 strain Copenhagen A3L ,
P4b

21 LD095_R- 89339..89824 =1 putative virion core AAN02663 GI:225956 similar to vaccinia virus
L protein .1 30 strain Copenhagen A4L

22 LD100_R- 94619..94855 =1 putative IMV AAN02668 GI:225956 similar to vaccinia virus


L membrane protein .1 35 strain Copenhagen A9L

23 LD101_R- 94856..97570 =1 putative virion core AAN02669 GI:225956 similar to vaccinia virus
L protein .1 36 strain Copenhagen A 10L,
P4a

24 LD103_R- 98535..99107 =1 putative virion core AAN02671 GI:225956 similar to vaccinia virus
L protein .1 38 strain Copenhagen A12L

39
25 LD104_R- 99172..99375 =1 putative IMV AAN02672 GI:225956 similar to vaccinia virus
L membrane protein .1 39 strain Copenhagen A13L

26 LD105_R- 99457..99744 =1 putative IMV AAN02673 GI:225956 similar to vaccinia virus


L membrane protein .1 40 strain Copenhagen A14L

27 LD109_R- 101332..1019 =1 Putative AAN02677 GI:225956 similar to vaccinia virus


L 22 phosphorylated IMV .1 44 strain Copenhagen A17L
membrane protein

28 LD117_R- 110395..1108 =1 putative fusion AAN02685 GI:225956 similar to vaccinia virus


L 41 protein .1 52 strain Copenhagen A27L

40
ORF CDS Codo protein_id db_xref note
complement n
Product
start

29 LD121_R- 112548..1133 =1 putative DNA AAN02689 GI:225956 similar to vaccinia virus


L 12 .1 56 strain Copenhagen A32L
packaging protein

30 LD122_L- 113444..1140 =1 putative EEV AAN02690 GI:225956 similar to vaccinia virus


R 34 glycoprotein .1 57 strain Copenhagen A33R

31 LD123_L- 114067..1145 =1 putative EEV protein AAN02691 GI:225956 similar to vaccinia virus
R 82 .1 58 strain Copenhagen A34R

32 LD126_L- 116147..1166 =1 putative EEV AAN02694 GI:225956 similar to vaccinia virus


R 92 glycoprotein .1 61 strain Copenhagen A36R

41
33 LD141_L- 133347..1340 =1 putative EEV AAN02709 GI:225956 similar to vaccinia virus
R 21 .1 76 strain Copenhagen B5R
host range protein

Table (4): Viral virulence and host range genes

ORF CDS Codon Product protein_id db_xref NOTE


complement start

1 LD003_R- 1433..2155 =1 putative ER- AAN02569 GI:225955 similar to myxoma virus


L localized apoptosis .1 36 M004; similar to ORF
regulator LD154

2 LD005_L- 2450..2962 =1 interleukin-10- AAN02571 GI:225955


R like protein .1 38

3 LD006_R- 2973..3668 =1 interleukin-1 AAN02572 GI:225955


L receptor-like .1 39
protein

42
4 LD008_R- 4841..5668 =1 putative soluble AAN02574 GI:225955 similar to myxoma virus
L interferon gamma .1 41
M007
receptor

5 LD010_R- 6445..6933 =1 LAP/PHD-finger AAN02576 GI:225955


L protein .1 43

6 LD011_R- 6978..8111 =1 CC chemokine AAN02577 GI:225955 similar to GenBank


L receptor-like .1 44 Accession Number
protein S78201

7 LD013a_R 8932..9918 =1 interleukin-1 AAN02579 GI:225955 LD013a differs from


-L receptor-like .1 46 LSDV013 due to a
protein frameshift at the C-
terminus end producing a
stop

8 LD014_R- 9978..10247 =1 putative eIF2 AAN02580 GI:225955 similar to vaccinia virus


L alpha-like PKR .1 47 strain Copenhagen K3L
inhibitor

43
9 LD015_R- 10234..10719 =1 putative AAN02581 GI:225955
L interleukin-18 .1 48
binding protein

10 LD016_R- 10757..11026 =1 EGF-like growth AAN02582 GI:225955


L factor .1 49

11 LD017_R- 11017..11547 =1 putative integral AAN02583 GI:225955 apoptosis regulator;


L membrane protein .1 50 similar to myxoma virus
M011L

12 LD034_R- 27393..27926 =1 putative PKR AAN02602 GI:225955 similar to vaccinia virus


L inhibitor .1 69 strain Copenhagen E3L

13 LD067_L- 57404..57997 =1 putative host AAN02635 GI:225956 similar to vaccinia virus


R range protein .1 02 strain Copenhagen C7L

44
ORF CDS Codon Product protein_id db_xref NOTE
complement start

14 LD128_R- 117528..1184 =1 CD47-like protein AAN02696 GI:225956


L 33 .1 63

LD131_L- 119272..1197 =1 superoxide AAN02699 GI:225956


R 57 dismutase -like .1 66
15
protein

16 LD135_L- 128334..1294 =1 putative IFN-alpha AAN02703 GI:225956 similar to vaccinia virus


R 16 /beta binding .1 70 strain Copenhagen B19R
protein

17 LD138_L- 131028..1315 =1 Ig domain OX-2- AAN02706 GI:225956


R 88 like protein .1 73

45
18 LD140_L- 132576..1332 =1 putative RING AAN02708 GI:225956 similar to rabbit fibroma
R 98 finger host range .1 75 virus N1R
protein

19 LD142_L- 134023..1344 =1 putative secreted AAN02710 GI:225956 similar to vaccinia virus


R 27 virulence factor .1 77 strain Copenhagen N1L

20 LD143_L- 134464..1353 =1 tyrosine protein AAN02711 GI:225956


R 72 kinase-like protein .1 78

21 LD146_L- 139264..1405 =1 phospholipase D- AAN02714 GI:225956 similar to vaccinia virus


R 05 like protein .1 81 strain Copenhagen K4L

22 LD149_L- 143480..1444 =1 serpin-like protein AAN02717 GI:225956


R 93 .1 84

46
23 LD154_L- 148639..1493 =1 putative ER- AAN02722 GI:225956 similar to myxoma virus
R 61 localized apoptosis .1 89 M004; similar to LD003
regulator

Table (5): Major gene families

1-ANKYRIN REPEAT

ORF CDS complement Codon Product protein_id db_xref note

Start

1 LD012_R- 8218..8853 =1 ankyrin repeat AAN02578.1 GI:22595545 similar to GenBank Acc.


L protein No. S78201

2 LD145_L- 137231..139135 =1 ankyrin repeat AAN02713.1 GI:22595680


R protein

3 LD147_L- 140572..142068 =1 ankyrin repeat AAN02715.1 GI:22595682


R protein

47
4 LD148_L- 142116..143459 =1 ankyrin repeat AAN02716.1 GI:22595683
R protein

5 LD152_L- 146780..148249 =1 ankyrin repeat AAN02720.1 GI:22595687


R protein

2-KELCH LIKE

ORF CDS complement Codon Product protein_id db_xref note

start

1 LD019a_R- 12073..12945 =1 kelch-like AAN02585.1 GI:22595552 LD019a and LD019b


L protein appear to be a
frameshifted homolog of
LSDV019

2 LD019b 12972..13784 =1 kelch-like AAN02586.1 GI:22595553 LD019a and LD019b


protein appear to be a

48
frameshifted homolog of
LSDV019

3 LD144_L-R 135541..137193 =1 kelch-like AAN02712.1 GI:22595679


protein

4 LD151_L-R 145059..146714 =1 kelch-like AAN02719.1 GI:22595686


protein

3-A52R LIKE

ORF CDS complement Codon Product protein_id db_xref note

Start

1 LD001_R- 238..717 =1 hypothetical AAN02567.1 GI:22595534 similar to ORF LD156


L protein

2 LD009_R- 5701..6393 =1 putative alpha AAN02575.1 GI:22595542 similar to vaccinia virus


L amanitin-sensitive strain Copenhagen N2L
protein

49
3 LD136_L- 129464..129925 =1 hypothetical AAN02704.1 GI:22595671
R protein

4 LD150_L- 144532..145017 =1 hypothetical AAN02718.1 GI:22595685


R protein

5 LD156_L- 150077..150556 =1 hypothetical AAN02724.1 GI:22595691 similar to LD001


R protein

50
3.4. Antigenic characters and relationships of LSDV:

All isolates of LSDV from collected a large number of samples from active
cases in South Africa, Kenya and Malawi were belonged to Poxviridae (Prydie
and Coackley, 1959; Weiss 1962 and 1966). They were antigenically similar and
showed complete reciprocal cross-neutralization with the "Neethling" prototype
strain. Complete cross-immunity between the South African "Neethling" virus and
the Londiani strain, isolated in Kenya, has also been demonstrated (Prydie and
Coackley, 1959). There is only one immunological type of LSDV responsible for
true LSD. Antigenic relationship of LSDV to sheep pox was investigated. Cattle
inoculated intradermally with sheep pox virus (Isiolo strain) isolated from sheep
showing lesions of sheep pox, developed lesions indistinguishable from those of
LSD and acquired immunity to challenge with LSDV (Capstick, 1959). It was
thought that true sheep pox virus did not protect cattle as well against LSD because
sheep pox virus (Isiolo strain) showed a closer relationship to goat pox virus
(Andrewes, 1964). Later on, the immunological relationship of LSDV to sheep
pox virus was further substantiated and it could be concluded that cattle could be
protected against LSD by vaccination with a strain of sheep pox virus grown in
tissue culture (Weiss et al,1968).

3.5. Resistance of LSDV to physical and chemical agents:

LSDV was remarkably stable between pH 6.6 and 8.6 and showed no
significant reduction in titer after exposure for 5 days at 37°C within the pH range
mentioned above. The virus was readily inactivated by the detergent sodium-
dodecyl-sulphate, chloroform and ether suggesting that lipid is incorporated in the
structure of the virus (Plowright and Ferris, 1959; Weiss, 1959).

In the skin lesions of infected animals, the virus can persist for at least 33
days even though the necrotic portions of skin have become completely dried out
prior to sequestration. It has also been shown that the virus remained viable for 18
days in the lesions and superficial epidermal scrapings from such lesions in air-
dried portions of hide kept at room temperature (Alexander and Weiss, 1959). The
virus was also recovered from intact skin nodules kept at -80°C for 10 years (Weiss,
1967), and from infected tissue culture fluid kept at 4°C for 6 months (Alexander
and Weiss, 1959). Virus in tissue culture fluid has remained viable for at least 10
years under dry-ice refrigeration (Weiss, 1967). This is contrary to the report by

51
HAIG (1957) that prolonged storage of preparations of lumpy skin disease virus
under dry-ice refrigeration reduced the infectivity (Weiss et al, 1968).

3.6. Biological properties of LSDV:

3.6.1. Replication in the host cell:

There are three main stages in the poxvirus multiplication cycle (Roberts
and Smith, 2008) including:

1) Virus attachment to host cell membrane and release of the poxvirus core.

2) Viral DNA replication and transcription of viral genes.

3) Assembly of infectious viral particles then release via budding or cell


lysing.
Entry of mature non-enveloped LSDV into the host cell occurs by
micropinocytosis triggered by the viral surface protein phosphatidylserine and the
receptors for the poxvirus thought to be Glycosaminoglycans (GAGs), (Haftu,
2012). Initiation of the process requires activation of the cellular p21-activated
kinase1 (PAK-1) by the virus (Mercer and Helenius, 2008).
In contrast, enveloped virus enters the host cell by endocytosis. Inside the
endocytic vesicle the envelope is lysed, releasing mature virion (MV). The core of
the virus is then released into the cytoplasm of the host cell by the fusion of the
outer membrane of MV with the vesicle membrane (Fenner et al., 1987; Moss,
2006; Tuppurainen et al., 2015).
After entry of the poxvirus core to the host cell, the infection process is
initiated, poxvirus core (contains structural proteins, a linear double-stranded DNA
genome and the enzymes required for early gene regulation) is transported along
microtubules deeper into the cytoplasm (Gemma et al., 2003).

Cores accumulate of the poxvirus cores in the perinuclear regions of the cell,
forming viral factories, where transcription of viral early mRNAs occurs using the
virus associated DNA-dependent RNA polymerase. The stages of the initiated
replication process are determined by the three groups of poxvirus genes; early,
intermediate and late. Expression of early gene occurs within 2hour post-infection
where early gene products function mainly to modify the host cell environment and
aid in viral escape from host immune responses (Carroll and Moss, 1997).

Early gene expression is followed by poxvirus DNA replication. During


DNA replication, intermediate gene expression occurs. Intermediate genes are
fewer in number compared to early genes and their main function is to regulate the

52
expression of late genes. Late genes, in turn, generally encode final stage structural
proteins responsible for virion formation, enzymes and transcription factors
required for the next round of replication. Following late gene expression, a
crescent shaped structure is formed (crescent structure consists of a host derived
single layer lipid membrane and viral proteins) which marks the early stages of
virion formation (Rosel et al., 1986).

The infectious poxvirus occurs in four distinct forms (Richard et al., 2006):

1- Intracellular mature virus (IMV), non-enveloped and makes up the


majority of virus particles.
2- Intracellular enveloped virus (IEV).
3- Cell-associated extracellular virus (CEV), enveloped.
4- Extracellular mature virus (EMV), non-enveloped.
During early morphogenesis viral crescents grow to form spherical shapes
that encapsulate the virus core components to form immature virions (IV). These
IVs then mature into infectious brick shaped IMV through the proteolytic cleavage
of core proteins. IMV is the first infectious form of the virus to occur and is
encapsulated in a single lipid membrane. This form of the virus remains within the
host cell until the cell lyses. Small subsets of IMV particles are enveloped by host
cellular membranes (derived from endosomes or the Golgi network) to form IEVs.
IEVs are transported along microtubules from the viral factories toward the
periphery of cells. Here at the outer membrane of the host cell the outer membrane
of IEVs fuse with the plasma host membrane. The virus may remain associated
with the extracellular cell surface forming CEV which makes use of cellular actin
filaments to aid in cell to cell viral transmission. Particles are also released from
the cell surface forming EMVs which are thought to aid in long range transmission
of the virus. The wrapping of viral particles in host membrane aid in viral evasion
of the host immune response. The IMV and EMV disseminate through the host
(Smith et al., 2002).

53
Figure (4): An overview of poxvirus multiplication including entry of EEV into
host cell, membrane attachment and fusion of the IMV membrane with the host
membrane, formation of viral factories, synthesis of crescent membranes are
synthesized enclosing viral DNA and proteins to form circular immature virion
(IV), formation of brick shaped intracellular mature virus (IMV), formation of the
intracellular enveloped virus (IEV) by wrapping of IMV in Golgi membrane,
finally IEV released as extracellular enveloped virus (EEV) through exocytosis but
cell-associated enveloped virus (CEV) forms actin tails aid in cell to cell
transmission of the virus (Smith et al., 2002).

A hallmark of the acute to subacute stages of the lesions was the presence of
intracytoplasmic eosinophilic inclusions in various cell types. The inclusions
consisted of the viroplasm which was identified as aggregates of electron-dense,
finely granular to fibrillar deposits in which membrane enclosed virions and
occasional groups of tubular structures were observed. (Prozesky L, Barnard BJ.,
1982).

54
3.6.2. Propagation in vitro:

LSDV could be inoculated into rabbits causing generalized skin lesions


(Alexander et al., 1957).

The virus can also be propagated in the chorioallantoic membranes (CAM)


of embryonated chicken eggs aged 9–12 days, causing macroscopic pock lesions
(Alexander et al., 1957; Van Rooyen et al., 1969; House et al., 1990; Omyma
et al., 2008; Sohair et al., 2008; Sharawi and Abd El-Rahim, 2011; EL-Kenawy
et al., 2011; El-Nahas et al., 2011; Abou Elyazeed, et al., 2012).

Capripoxviruses grow slowly in cell cultures and may require several


passages. They grow on a wide variety of bovine and ovine cells, causing easily
recognizable cytopathic effects (CPE) on cell monolayers (Alexander et al., 1957,
Munz and Owen, 1966, Prydie and Coackley, 1959).

Microscopic examination of LSDV infected monolayers, stained with


haematoxylin eosin or haematoxylin-phloxin, showed that cytopathic changes are
accompanied by the development of intracytoplasmic inclusion (Thomas and
Mare, 1945). In tissue cultures, these inclusions at first appear as small round
basophilic bodies surrounded by a halo. As they increase in size, they become more
acidophilic and some inclusions appear to have basophilic "inner bodies", which
have been shown to consist of cytoplasmic RNA by histochemical staining methods
(Weiss and Broekman, 1965). Some inclusion bodies are round and others have
an irregular outline and show small protuberances at their margins.

LSDV could be cultured in a variety of cell cultures including lamb and calf
kidney cells, calf testis cells, sheep kidney cells, lamb and or calf adrenal or thyroid
cultures, foetal lamb and calf muscle cells, sheep embryonic kidney or lung cells,
rabbit foetal kidney or skin cells, chicken embryo fibroblasts, adult vervet monkey
kidney cell line (AVK 58), equine lung and baby hamster kidney cells (BHK/21)
(Alexander et al., 1957; Prydie and Coackley, 1959; Weiss, 1968).

Primary lamb testis (LT), bovine dermis cells and commercially available
LT cell line were the most commonly used cells for the propagation of LSDV
(Babiuk et al., 2007).
55
LSDV was adapted to multiply on VERO cells with CPE appeared after the
third passage characterized by granulation of cells followed by cell rounding and
aggregated together in a separate manner 4- 5days post inoculation (Rizkallah,
1994; Mangana- Vougiouka et al., 2000; Amal, et al., 2008).

Cultivation of the virus was tried on MDBK cell line cultivated in Minimal
Essential Media (MEM) supplemented with fetal calf serum and gentamycin (El-
Nahas et al,2011; Abou Elyazeed, et al., 2012).

3.7. Pathogenesis and Histopathology of LSDV:

Subcutaneous inoculation or intradermal inoculation of cattle with LSDV


results in the development of a localized swelling at the size of inoculation after
four to seven days and the enlargement of the regional lymph nodes, while
generalized eruption of skin nodules usually occurs seven to 19 days after
inoculation. In experimentally infected cattle LSDV was demonstrated in saliva at
least for 11 days after the development of fever, in semen for 42 or 22 days and in
skin nodules for 39 or 33 days, but not in urine or faeces. Viraemia occurred after
the initial febrile reaction and persisted for two weeks. A variety of cell types,
including epithelial and endothelial cells, pericytes and fibroblasts are infected by
the virus. Viral replication in pericytes, endothelial cells and probably other cells
in blood vessel and lymph vessel walls causes vasculitis and lymphangitis in some
vessels in affected areas. In severe cases infarction may result. Lumpy skin disease
virus is present in skin nodules, normal skin, lymph nodes, liver, kidneys, skeletal
muscle, saliva and semen of infected animals. Viral concentrations at the above
sites, however, have not been determined (Coetzer et al,2004). After skin
inoculation, virus replicates in epidermis and dermis, Infecting macrophages in
regional lymph nodes for further replication, Resulting in enlargement of the
regional lymph nodes, e.g. prescapular and prefemural lymph nodes. Macrophage
associated viraemia , then further viral multiplication in many different organs
including the liver, spleen, lung then secondary viraemia ,disseminating the virus
to various tissues, skin and endothelium, Damaged endothelium results in
vasculitis, thrombosis, marked dermal edema, infarction, Nodules are
circumscribed, round, slightly raised, firm and painful and involve the entire skin
and the mucosa of the GIT, RT, genital tract, developing inverted conical necrosis
called the “sit fast”. Secondary bacterial infections develop in the necrotic cores of
the nodules. Metastatic abscesses in the regional lymph nodes, lungs and other
organs. Mortality is due to secondary infection. Poxviruses are generally

56
epitheliotrophic and can cause localized or systemic disease. Initial multiplication
of the virus occurs at the entry site of the virus into the body. In systemic infections,
further viral replication takes place in the draining lymph nodes, followed by
viraemia and further viral multiplication in many different organs including the
liver, spleen and lungs (Fenner et al. 1987). The later multiplication leads to
establishment of secondary viraemia and subsequent infection and development of
disseminated focal lesions in the skin. Viral replication takes place in the cytoplasm
of cells. Viral particles are enveloped when mature virus particles move to the
Golgi complex; most particles are however non- enveloped and are released by cell
disruption. Both enveloped and non- enveloped particles are infectious (Fenner et
al.1987). The earliest description Thomas and Mare (1945) highlighted the
histological changes. Their findings were confirmed to a large extent by Prozesky
and Barnard (1982) who found that LSDV exerts its pathogenic effects by
infiltrating a variety of cell types, including epithelial and endothelial cells,
pericytes and fibroblasts, resulting in lymphangitis and vasculitis. The later report
highlighted the difference in histological changes asssociated with acute or chronic
lesions. During the acute stage vasculitis and lymphangitis with concomitant
thrombosis and infarction resulted in oedema and necrosis (Prozesky and
Barnard 1982). The lesions were initially infiltrated by neutrophils and
macrophages, and later on these cells were replaced by lymphocytes, plasma cells
and macrophages as well as fibroblasts (Prozesky and Barnard 1982).
Coagulation necrosis is the result of thrombi in the blood vessels. It seems not to
be determined whether a single cell type is responsible for the spread of LSDV
around the body and its localization in various organs. Nagi (1990) investigated
cutanaeous and testicular lesions in an outbreak of LSD in Egypt 1989. It was noted
that diffuse degenerative and inflamatory changes could be observed in the
seminiferous tubules and blood vessels. The seminiferous tubules were devoid of
primary and secondary spermatocytes and spermatids, although the spermatogonia
and Sertoli cells appeared resistant. The author speculated that the possible
infertility due to LSDV infection may be transient as the regeneration of the
germinal epithelium depends mainly upon the persistence of the spermatogonia and
Sertoli cells, although extensive fibrosis may preclude the return of fertility.
(Cornelius Henry Annandale,2006). Microscopic examination of histological
sections of the skin lesions of animals suffering from LSDV infection showed the
development of intracytoplasmic inclusion (De Lange, 1959; Prydie and
Coackley, 1959; Burdin, 1959).

57
Cells may contain one to several inclusion bodies of varying sizes. Affected
cells become rounded and shrunken, the cytoplasm becomes intensely eosinophilic
and the nuclei show degenerative changes consisting of margination of chromatin,
juxtaposition of the nucleoli to the nuclear membrane, and eventual pyknosis and
distortion (De Lange, 1959; Prydie and Coackley, 1959; Weiss et al, 1968).

3.8. Laboratory Diagnosis of LSDV:

3.8.1. Sampling of LSDV:

The most suitable specimens for LSDV isolation collected as biopsy or at


post-mortem include skin nodules, lung lesions, lymph nodes, buffy coat, semen
from suspected animals (Davies, 1971; Tuppurainen, 2005; OIE, 2010), and
milk, (S.S.A. Sharawi & I.H.A. Abd El-Rahim, 2011).

For transportation of the viral samples over long distances, 50% glycerol
saline as a viral transport medium was added to tissue samples kept on ice tanks.
For storage of these tissue samples used for LSDV isolation, they should be kept
at – 20°C (OIE, 2010).

3.8.2. Isolation of LSDV:

3.8.2.1. Isolation of LSDV on Specific Pathogen Free- Embryonated Chicken


Eggs (SPF-ECEs):

LSDV could be propagated in the chorioallantoic membranes (CAM) of 9 -


11 day old embryonated chicken eggs (ECEs) (Alexander et al., 1957).

The supernatant fluid from the ground skin lesions of LSDV infected cattle was
inoculated as 0.1 - 0.2 ml from the prepared samples via CAM, by drop membrane
method (Van Rooyen et al., 1969; and House et al., 1990).

Successful isolation of LSDV from prepared samples on CAM showed


specific typical pock lesion on CAMs harvested 4days post inoculation (Omyma
et al., 2008; Sohair et al., 2008), thickening and congestion (Tuppurainen et al.,
2015).

Isolation of LSDV from buffaloes revealed the characteristic pock lesion on


CAM of ECE as numerous, small, scattered white foci (El-Nahas et al., 2011).

Examination of CAMs from ECEs inoculated with LSDV from cattle


showed small hemorrhagic areas at site of inoculation at day one post inoculation

58
(PI) considered as nonspecific, thickening, congestion and hemorrhage at day 3 PI,
white and opaque area around site of inoculation at day 4 PI, this white and opaque
area increase in size at day 5 PI and opaque pin point pock lesions arranged in
streaks at day 6 PI but that of control non inoculated eggs showed no gross lesions
(El-Kenawy et al., 2011).

LSDV could be grown on embryonated chicken eggs of 7 – 9 days old


embryos with maximum yield of the virus was obtained from CAM of infected
eggs incubated at 33.5°C and 35°C for 5 – 6 days (Sharawi and Abd El-Rahim,
2011).

Isolation of LSDV via CAM of ECES aged 9 days was tried and eggs
incubated at 37ºC were examined by trans-illumination once or twice a day to
determine the time of death. In the same time, sample eggs were opened at different
times after inoculation, and the membranes, as well as the embryos, carefully
examined for macroscopic pock lesions (Abou Elyazeed et al., 2012).

3.8.2.2. Isolation of LSDV on cell culture:

Capripoxviruses grow slowly in cell cultures and may require several


passages. They grow on a wide variety of bovine and ovine cells, causing easily

recognizable cytopathic effects (CPE) on cell monolayers (Alexander et al., 1957;


Prydie and Coackley, 1959; Munz and Owen, 1966).

In addition, LSDV could be cultured in lamb and calf kidney cells, calf testis cells,
sheep kidney cells, lamb and or calf adrenal or thyroid cultures, foetal lamb and
calf muscle cells, sheep embryonic kidney or lung cells, rabbit foetal kidney or skin
cells, chicken embryo fibroblasts, adult vervet monkey kidney cell line (AVK 58),
equine lung and baby hamster kidney cells (BHK/21) (Alexander et al., 1957,
Prydie and Coackley, 1959, Weiss, 1968).

Primary lamb testis (LT) and bovine dermis cells, or a commercially


available LT cell line were the most commonly used cells for the propagation of
LSDV (Babiuk et al., 2007).

Madin Darby bovine kidney (MDBK) cell line was used for LSDV isolation.
Infected cells develop a characteristic CPE consisting of retraction of the cell
membrane from surrounding cells, and eventually rounding of cells and
margination of the nuclear chromatin (OIE, 2010; Abou Elyazeed et al., 2012).

59
prominent CPE on MDBK cells started from third day post inoculation until
complete destruction of cell sheet. Characteristic CPE of LSDV in the form of
clusters of cell rounding, cell aggregations and vacuoles then cell beginning of
detachment. (El-Nahas et al,2011).

LSDV could be isolated on VERO cell culture (Mangana- Vougiouka et


al., 2000). LSDV was adapted on VERO cells and gave CPE after 3 successive
passages with the CPE appeared as granulation of cells, cell rounding and
aggregation at 4 – 5 days post inoculation (Rizkallah, 1994; Amal et al., 2008).

3.8.3. Identification of LSDV:

3.8.3.1. Non-Serological Techniques:

3.8.3.1.1. Transmission electron microscopy:

For electron microscopic diagnosis of LSDV, preparation and negative


staining of skin specimens were made (Tuppurainen et al., 2005). Semithin
sections were carried out by fixing in 5%, glutaraldehyde then possessed for
sectioning by ultra-microtome in thickness of 1micron. The sections were stained
by Toluidine blue (Bancroft et al., 1990; Salib and Osman, 2011). Ultra-thin
sections from skin nodules and CAM of ECE was stained with uranyl acetate and
lead citrate then examined using electron microscope (Bozzala and Russell, 1992;
Sohair et al., 2008). Electron microscopic examination determined LSDV size to
range from 300 - 350 nm with crescent or ovoid shape (Madbouly et al., 2005;
Ahmed and Kawther, 2008). Transmission electron microscope showed that
capripox virion was brick shaped, covered in short tubular elements and measures
approximately 290 × 270 nm. A host-cell-derived membrane may surround some
of the virions, and as many as possible should be examined to confirm their
appearance (Kitching and Smale, 1986; OIE, 2010; Haftu, 2012).

Ultrastructurally, LSDV appeared as large cuboidal virus particles were


found in both skin lesion and inoculated CAM. (Abou Elyazeed et al., 2012).

Prepared suspensions of skin specimens from infected animals were stained


with 3% phosphotungstic acid for electron microscopic examination. The viral
particles appeared by negative staining roughly brick shaped particles with ridges
covering them. (OIE, 2004; Tuppurainen, 2004; Omyma, 2008; El-Nahas et al.,

60
2011; Tageldin et al., 2014), ovoid in shape, with rounded ends and characteristic
ball of wool appearance (Aziza et al., 2015).

3.8.3.1.2. Histopathological Examination:

Microscopic examination of stained sections of excised skin lesions and


infected monolayers (Thomas and Mare, 1945) showed the characteristic
histopathological changes and intracytoplasmic inclusion bodies associated with
infection by LSDV (De Lange, 1959; Prydie and Coackley, 1959; Burdin, 1959).
Lymphocytes, macrophages, plasma cells and fibroblast proliferation appear in the
later stages, and if secondary infection occurs, polymorphonuclear leukocytes and
red cells are seen (Thomas and Mare, 1945; Burdin, 1959).

In tissue cultures, these inclusions at first appear as small round basophilic


bodies surrounded by a halo. As they increase in size, they become more
acidophilic and some inclusions appear to have basophilic "inner bodies", which
have been shown to consist of cytoplasmic RNA by histochemical staining methods
(Weiss and Broekman, 1965).

Some inclusion bodies are round and others have an irregular outline and
show small protuberances at their margins. Cells may contain one to several
inclusion bodies of varying sizes. Affected cells become rounded and shrunken, the
cytoplasm becomes intensely eosinophilic and the nuclei show degenerative
changes consisting of margination of chromatin, juxtaposition of the nucleoli to the
nuclear membrane, and eventual pyknosis and distortion (De Lange, 1959; Prydie
and Coackley, 1959; Weiss et al, 1968). Intracytoplasmic inclusions were seen in
cells stained with haematoxylin eosin (Nawathae et al., 1978).

Histologically, skin lesions in the acute stage characterized by vasculitis,


perivasculitis, lymphangitis, thrombosis, oedema, necrosis and infarction.
(Thomas and Mare, 1945; Prozesky and Barnard, 1982).

There was cuffing of blood vessels by leukocytes, and eosinophilic,


intracytoplasmic pox inclusion bodies may be seen in the epithelioid cells, and cells
of hair follicles, smooth muscle, and skin gland (Thomas and Mare, 1945;
Burdin, 1959, Prozesky and Barnard 1982). Biopsies and tissues obtained at
necropsy obtained from skin of the lower lip and chin showed acanthosis, including
elongation of rete-ridges. The acanthotic skin and hair follicles had locally
extensive epidermal necrosis with ballooning degeneration coalescing into multiple

61
intraepithelial or subcorneal microvesicles with acantholysis. The microvesicles
coalesced to form larger vesicles. Individual cells in the epithelium were necrotic,
and some epithelial cells had vacuolated nuclei and a round to oval eosinophilic
intracytoplasmic inclusion body. These latter cells, known as “pox cells,” were
found in most affected tissues, including keratinocytes, dermis, respiratory
epithelial cells, and sebaceous gland epithelium. There was both superficial and
deep dermatitis. Necrosis and mononuclear inflammation were also present in the
sebaceous glands. There was extensive edema of the dermal papillae with some
hemorrhage. A vasculitis with a mononuclear cell perivascular reaction with
thrombosis leading to necrosis was evident in the dermis. There was lymphoid
hyperplasia and reticuloendothelial hyperplasia (House et al., 1990). The
histopathological examination of skin nodules of different cases (39) revealed
hyperkeratosis, parakeratosis and acanthosis within the epidermis. The prickle cell
layer was swollen and vacuolated. Coagulative necrosis and ulceration within
epidermal layer was seen associated with presence of numerous homogenous
eosinophilic intracytoplasmic inclusion bodies that were confirmed by phloxin
tartrazin stain (Sohair et al., 2008).

Histopathological examinations of lumpy skin disease revealed ballooning


degeneration of stratum spinosum with microvesicles formation. Eosinophilic
intracytoplasmic inclusion bodies were also noticed. (Salib and Osman, 2011).

Histopathological examination of skin biopsy from living animals and skin


samples of died animals showed round to oval eosinophilic intracellular inclusion
bodies (Abou Elyazeed, 2012).

Histopathological skin lesion of LSD on cattle as the epidermis was


extensively necrotic, while in the intact areas, some ballooning degeneration of
squamous epithelial cells with occasional intra-cytoplasmic inclusions were seen
(Ahmed and Amina, 2013) that could be numerous intracytoplasmic eosinophilic
inclusion bodies in some cases (Aziza et al., 2015). These characteristic
pathognomonic eosinophilic intracytoplasmic inclusion bodies were present in the
prickle cell layer (Neamat-Allah, 2015).

62
3.8.3.2. Serological Techniques:

3.8.3.2.1. Indirect Fluorescence Antibody Technique (IFAT):

Indirect FAT was successfully carried out on the suspected LSDV samples
isolated on CAM. Sixteen identified positive samples out of 23 showing bright
greenish yellow fluorescence in CAM (Sohair et al., 2008). Capripoxvirus antigen
can also be identified on the infected cover-slips or tissue culture slides using FAT.
Indirect FAT carried out using immune cattle sera was subject to high background
colour and nonspecific reactions, so, uninfected tissue culture should be included
as a negative control as cross-reactions can cause problems due to antibodies to
cellular components. However, a direct conjugate can be prepared from sera from
convalescent cattle (or from sheep or goats convalescing from capripox) or from
rabbits hyperimmunised with purified capripoxvirus (OIE, 2010).

Indirect FAT was used to LSDV in ultrathin sections of the collected tissues
with aid of using prepared rabbit hyperimmune serum and anti-rabbit FITC
conjugate (EL-Kenawy and EL-Tholoth, 2011)
Impression smear from CAMs that showing pock lesion or from skin lesions
were stained with anti-capripoxvirus FITC conjugate and read with a fluorescence
microscope. The conjugated anti-LSDV hyperimmune serum demonstrated typical
apple green positive intra-cytoplasmic fluorescent reaction in the cells of all CAMs
showing pock lesions (Abou Elyazeed, 2012, Aziza et al., 2015)

3.8.3.3. Molecular Identification:

3.8.3.3.1. Polymerase Chain Reaction (PCR):

The polymerase chain reaction (PCR) is a scientific technique in molecular


biology to amplify a single or a few copies of a piece of DNA across several orders
of magnitude, generating thousands to millions of copies of a particular DNA
sequence. The conventional gel based PCR method is a simple, fast and sensitive
method for the detection of Capripoxvirus genome. In EDTA blood, biopsy, semen
or tissue culture samples. However, it does not allow differentiation between LSD
and sheep and goat pox viruses. Primers for the viral attachment protein gene and
the viral fusion protein gene are specific for all the strains within the genus
Capripoxvirus (Ireland and Binepal, 1998).

63
PCR was performed for diagnosis of LSDV in semen samples from affected
bulls using commercially available primers for LSDV. The forward and reverse
primers had the sequences 5’-TTTCCTGATTTTTCTTACTAT-3’ and 5’-
AAATTATATACGTAAATAAC-3’ respectively, rendering an amplicon of 192
bp. A positive control consisting of bovine semen spiked with LSDV and a negative
bovine semen control as well as a water control were included in the PCR.
Amplified products were analysed using a 100 bp DNA ladder as a molecular
marker on 1.5% agarose gels. Amplicons were visualized using an UV
transilluminator at a wavelength of 590 nm and positive reactions were confirmed
according to size (Annandale et al., 2005). Also, semen was tested by PCR using
primers developed from the gene for the viral attachment protein. The forward and
reverse primers had the sequences 5’TTTCCTGATTTTTCTTACTAT3’ and
5’AAATTATATACGTAAATAAC3’ respectively, rendering an amplicon of
192bp (Irons et al., 2005).

PCR was successfully performed for diagnosis of LSDV in skin biopsies


(Tuppurainen et al., 2005). The PCR primers were developed from the viral
attachment protein encoding gene and have the following sequences (Ireland &
Binepal 1998): Forward primer 5’-d TTTCCTGATTTTTCTTACTAT-3’Reverse
primer 5’-d AAATTATATACGTAAATAAC-3’. The size of the amplicon was
192 bp. PCR started with one cycle of 42 °C for 2 min and 94 °C for 10 min. The
initial cycle was 94 °C for 1 min, 50 °C for 30 s and 72 °C for 1 min. This was
followed by 40 cycles of 94°C for 1 min, 50°C for 30 s, and 72°C for 1 min, and a
final elongation step of 72 °C for 1 min to complete the extension of the primers.

PCR was successfully performed for diagnosis of LSDV in extracted DNA.


The PCR primers were developed from the viral attachment protein encoding gene
and have the following sequences: forward primer 5'-d TTTCCTGATTTTTC
TTACTAT-3' and reverse primer 5'-d AAATTATATACGTAAATAAC-3’
(Ireland and Binepal 1998). DNA amplification was carried out in a final volume

of 25 μl containing 12.5 μl Platinium® Quantitative PCR SuperMix-UDG, 1 μl


0.20 mM each primer, 9.5 μl distilled water and 1 μl DNA sample. The PCR started
with one cycle of 42°C for 2 min and 94°C for 10 min. The initial cycle was 94°C
for 1 min, 50°C for 30 s and 72°C for 1 min. This was followed by 40 cycles of
94°C for 1 min, 50°C for 30 s, and 72°C for 1 min, and a final elongation step of

64
72°C for 1 min to complete the extension of the primers. PCR assay indicated that
there is a band at 192 bp which belonged to viral attachment protein encoding gene
(Ahmed and Kawther, 2008).

PCR was used for diagnosis of LSDV in extracted DNA from culture
supernatant from the LSDV infected MDBK cells and skin biopsy specimens. PCR
primers were chosen from unique LSDV sequences within the gene for viral
attachment protein (Ireland and Binepal,1998). PCR reaction was applied in a
total volume of 50 ml containing: 1X PCR buffer (20 mM Tris HCl pH 8.4 and 50
mM KCl); 1.5 mM MgCl2; 0.2 mM deoxynucleosides triphosphates mixture
(dATP, dCTP, dGTP and dTTP); 20 pmol of each primer; 2.5 units (U) Thermus
aquaticus Taq polymerase 0.1mg of extracted viral DNA and nuclease-free sterile
double distilled water up to 50.0 ml. Then, the resulting mixture was subjected to
precise thermal profile in a programmable thermocycler as follows: One cycle of:
94 oC for 2 min; 40 cycles of: 94oC for 50 sec, 50oC for 50 sec and 72oC for 1 min;
followed by one final cycle of 72oC for 10 min. Analysis of PCR amplification
products (amplicons) showed that the PCR amplicons of proper predicted size
(about 192 bp) that were gel purified using DNA gel purification kit then
quantitated and subjected for direct sequencing of PCR amplicons ( El-Kholy et
al., 2008).

PCR was used for diagnosis of LSDV in extracted viral DNA from skin
biopsy and blood in EDTA (Awad et al., 2010). The PCR primers were developed
from the gene for viral attachment protein with the following sequences; forward
primer: 5′-TTTCCTGATTTTTCTTACTAT-3′ and reverse primer: 5′-AAATTAT
ATACGTAAATAAC-3′. PCR was performed according to the procedures of
Ireland and Binepal (1998) and the amplicon size of the PCR product is 192 bp.

PCR was used for diagnosis of LSDV in extracted viral DNA from blood in
EDTA, semen or tissue culture supernatant, skin and other tissue samples. The
primers were developed from the viral attachment protein encoding gene. The size
of the expected amplicon is 192 bp (Ireland & Binepal, 1998). The primers have
the following gene sequences:

Forward primer 5’-TCC-GAG-CTC-TTT-CCT-GAT-TTT-TCT-TAC-TAT-3’

Reverse primer 5’- TAT- GGT-ACC-TAA-ATT-ATA-TAC-GTA-AAT-AAC-3’.

65
DNA amplification is carried out in a final volume of 50 μl containing: 5 μl of 10
× PCR buffer, 1.5 μl of MgCl2 (50 mM), 1 μl of dNTP (10 mM), 1 μl of forward
primer, 1 μl of reverse primer, 1 μl of DNA template (~10 ng), 0.5 μl of Taq DNA
polymerase and 39 μl of nuclease-free water. The volume of DNA template
required may vary and the volume of nuclease-free water must be adjusted to the
final volume of 50 μl. Run the samples in a thermal cycler: first cycle: 2 minutes at
95°C, second cycle: 45 seconds at 95°C, 50 seconds at 50°C and 1 minute at 72°C.
Repeat the second cycle 34 times. Last cycle: 2 minutes at 72°C and hold at 4°C
until analysis (OIE, 2010).

PCR was used for diagnosis of LSDV in extracted viral DNA from infected
CAM and MDBK cells. It was performed according to the procedures of Ireland
and Binepal, (1998) using primers developed from the gene for viral attachment
protein with the following sequences: forward primer 5'-TTTCCTGA
TTTTTCTTACTAT-3'and reverse primer 5'-AAATTATATACGTAAATAAC-3'.
The specific primers set amplified a DNA fragment of 192 bp equivalent to the
expected amplification product (amplicon) size from LSDV without significant
differences between the LSDV reference strain and the local isolate from skin
nodules, infected CAM and MDBK cells (El-Nahas et al., 2011).

PCR was used for diagnosis of LSDV in extracted viral DNA from tissue
samples. The DNA extracted from each sample was amplified using the protocol
published by Ireland and Binepal, (1998). Briefly, each reaction mixture (50 μl)
contained 250 ng of total DNA, 2 mM MgCl2, 50 pmol of each primer (forward
primer 5´-TTTCCTGATTTTTCTTACTAT-3´ and reverse primer 5´-
AAATTATATACGTAAATAAC-3´), 200 μM of each dNTP and 2 U of DNA
polymerase (Biotool, USA) in a reaction buffer (10×) containing 75 mM Tris-HCl
(pH 9), 2 mM MgCl2, 50 mM KCl, 20 mM (NH4)2SO4 and 0.001% bovine serum
albumin. Amplification was carried out in an MJ Thermal Cycler (MJ Corporation,
USA), programmed to perform a denaturation step of 95°C for 5 min, followed by
40 cycles consisting of 1 min at 94°C for denaturation, 1 min at 50°C for primer
annealing and 1 min at 72°C for extension. The last extension step was 10 min
longer. A10 μl volume of PCR products was mixed with 2 μl gel loading buffer
(Sigma-Aldrich) and electrophoresed in 1% agarose gel containing 1 μg/ml
ethidium bromide in Tris-acetate buffer (0.04 M Tris-acetate and 0.001 M EDTA,
pH 8). The resulting DNA fragments were visible at band of the appropriate size
192 bp, (Sharawi and Abd El-Rahim, 2011).

66
PCR was used for diagnosis of LSDV in extracted viral DNA from 22 blood
samples. PCR was carried out for the confirmation of the disease by using
commercial capripoxviru PCR kit‟ with the sequence of forward primer
(SpGpRNAPol F) 5‟-TCTATGTCTTGATATGTGGTGGTAG-3‟ and reverse
primer (SpGpRNAPol R) 5‟-AGTGATTAGGTGGTGTATTATTTTCC-3‟,
amplifies CaPV homologues of the vaccinia virus E4L gen which encodes the 30
KDa DNA-dependent RNA polymerase subunit (Charles et al., 2011). DNA
amplification was carried out in a final volume of 50 μl containing the following:
5 μl of (10Mm) PCR buffer, 1.5 μl of MgCl2 (25Mm), 1 μl of dNTP mixture (10
Mm), 1 μl of (50 Mm) forward primer, 1 μl of (50 Mm) reverse primer, 5 μl of
DNA template, 0.5 μl of Taq DNA polymerase and 35 μl of RNAas free water. All
PCR experiments performed using the following amplification program: initial
denaturation at 95oC for 1 min; 40 cycles of denaturation at 95oC for 30s, annealing
at 55oC for 30s and elongation at 72oC for 1 min. An additional elongation step was
performed at 72oC for 5 min and the PCR products were stored at 4°C until
analysis. Amplified products were analyzed and positive results were confirmed as
172bp product with positive samples (Haftu,2012).

DNA was extracted from whole blood samples of infected cattle and was
used as templates for PCR diagnosis of LSDV (El-Haig et al., 2013). The primers
used were directed to the viral attachment protein encoding gene (forward primer
5′-TTTCCTGATTTTTCTTACTAT-3′, reverse primer 5′-AAATTATATACGT
AAATAAC-3′, the amplicon size of PCR product is 192 bp (Ireland & Binepal,
1998) and LSD-specific primers (lsd43U 5′-GTGGAAGCCAATTAAGTAGA-3′,
lsd1262L 5′-TAAGAGGGACATTAGTTCT-3′), the amplicon size of PCR
product is 1237 bp, (Stram et al., 2008). Then, PCR was carried out in a
programmable thermocycler as follow: one cycle of 95°C for 1 min., this was
followed by 35 cycles of 94°C for 30s, 58°C for 30s and 72°C for 70s and a final
extension step of 72°C for 5min.

PCR was carried out for diagnosis of LSDV in thin tissue section removed
from each sample. The primers were designed from sequence data derived from the
South African Onderstepoort vaccine strain and Warm baths field isolate of LSDV
(Kara et al. 2003). Primer pair 1, consisting of primer DW-TK (5′-GCCGAT
AACATATATAGACCC-3′) and primer OP49 (5′-GTGCTATCTAGTGCAGCT
AT-3′), is used to amplify a 434-bp LSDV genomic fragment between positions
56698–57132, and primer pair 2, consisting of primer L132F (5′- CACTTCCCT
TTTAAGC-3′) and primer L132R (5′- CATTCTACAATCTCCATGCG-3′),

67
amplifies a 492-bp fragment between genomic positions 119801–120292.
Template DNA was denatured initially for 90 s at 95 °C, followed by 35 cycles of
denaturation (45 s at 95 °C), primer annealing (45 s at 56 °C) and strand extension
(60 s at 72 °C), ending with a final strand extension step for 7 min at 72 °C. These
conditions were used for both primer pairs. The two primer pairs used for virus
identification are homologous to regions of the LSDV thymidine kinase (TK) and
ORF132 genes, respectively. The TK gene is highly conserved among the
capripoxviruses and thus primer pair 1 also binds to the TK genes of sheep pox and
goat pox viruses. However, LSDV ORF132 is unique to LSDV and thus primer
pair 2 only binds to LSDV DNA. Amplification products of the expected sizes for
LSDV were obtained for all the samples, including the positive LSDV controls
(Tageldin et al., 2014).

PCR was carried out for diagnosis of LSDV using extracted DNA from
tissue samples taken from skin nodules, lymph nodes, lung and liver (Aziza et al.,
2015). It was performed using with commercially available primers for LSDV
developed from the gene for viral attachment (Ireland and Binepal, 1998). The
forward and reverse primers had the sequences 5'-TTCCTGATTTTTCTTACTAT-
3' and 5'-AAATTATATACGTAAATAAC-3', respectively. The precise thermal

profile was as follows: an initial denaturation cycle of 94 °C for 2 min; followed


by 40 cycles at 94°C for 50 seconds, 50 seconds at 50°C and 1 minute at 72°C for;
followed by one final extension cycle of 72° C for 10 minutes, rendering an
amplicon of 192bp.

PCR was carried out for diagnosis of LSDV using extracted DNA from tick
species associated in LSD infection in comparison with extracted DNA from scabs
and skin lesions collected from experimentally infected donor animals
(Tuppurainen, 2015). Primers were designed from the viral attachment gene
(Ireland and Binepal, 1998) with the following sequences: Forward primer 5'-
TCCGAGCTCTTTCCTGATTTTTCTTACTAT-3', Reverse primer 5'-
TATGGTACCTAAATTATATACGTAAATAAC-3’. The thermal profile was 1 x
42 °C for 2 min and 94 °C for 10 min, 1x 94 °C for 1 min, 50 °C for 30 sec and 72
°C for 1 min, followed by 40 x 94 °C for 1 min, 50 °C for 30 sec, and 72 °C for 1
min and 1 x 72 °C for 1 min. Positive samples gave products of the expected size
of 192 bp.

68
3.8.3.3.2. Sequencing of the viral genome:

Multiple sequence alignments showed high homology percentage (≥ 99 %) of the


nucleotide sequences among local isolates of LSDV. Nevertheless, blast searches
over the Genebank database together with the phylogenetic analyses and
sequence alignments revealed that local isolates of LSDV are highly related (≥ 95
%) to not only other LSDV strains but also other Capripoxviruses (goat and sheep
pox). These results coincide with the theory of that all capripoxviruses are
genetically related and originated from one ancestor lineage (Black etal., 1986,
Fenner et al., 1987 and Tulman et al., 2001). (Alaa. A. El-Kholy et al.,2008).

1->gi|194031729|gb|EU807974.1|:1-172 Lumpy skin disease virus isolate


Egy/2006 nonfunctional attachment protein (P32) gene, partial sequence
AAATTATATACGTAAATAACATACCTGCTTAAAACCATAGTAATTTAGAATTCAAATCCAAAA
TTATCATTATTATAATAAATAAAATAATAAGTGCTCCTATTATACTAATATCAAATATACCAA
AAATTGAAACCAATGGATGGGATACATAGTAAGAAAAATCAGGAAA

>gi|14994025|gb|AF325528.1|:64986-65157 Lumpy skin disease virus NI-2490


isolate Neethling 2490, complete genome
AAATTATATACGTAAATAACATACCTGCTAAAAACCATAGTAATTTAGAATTCAAATCAAAAA
TTATCATTATTATAATAAATAAAATAATAAGTGCTCCTATTATACTAATATCAAATATACCAA
AAAATGAAACCAATGGATGGGATACATAGTAAGAAAAATCAGGAAA

Sequence ID: gb|AF325528.1|Length: 150773, Number of Matches: 1

Related Information, Range 1: 64986 to 65157,

Alignment statistics for match #1


Score Expect Identities Gaps Strand

302 bits (163) 1e-83 169/172(98%) 0/172(0%) Plus/Plus

69
Query 1 AAATTATATACGTAAATAACATACCTGCTTAAAACCATAGTAATTTAGAATTCAAATCCa 60

||||||||||||||||||||||||||||| |||||||||||||||||||||||||||| |

Sbjct 64986 AAATTATATACGTAAATAACATACCTGCTAAAAACCATAGTAATTTAGAATTCAAATCAA 65045

Query 61 aaattatcattattataataaataaaataataagtgctcctattatactaatatcaaata 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 65046 AAATTATCATTATTATAATAAATAAAATAATAAGTGCTCCTATTATACTAATATCAAATA 65105

Query 121 taCCAAAAATTGAAACCAATGGATGGGATACATAGTAAGAAAAATCAGGAAA 172

||||||||| ||||||||||||||||||||||||||||||||||||||||||

Sbjct 65106 TACCAAAAAATGAAACCAATGGATGGGATACATAGTAAGAAAAATCAGGAAA 65157

/ gene="LD074 "

CDS complement (64984..65952 )

/ gene="LD074 "

/ note="similar to vaccinia virus strain Copenhagen H3L ,

p35; similar to GenBank Accession Number AF124516 "

/ codon_start=1

/ product="putative IMV envelope protein "

/ protein_id="AAN02642.1 "

/ db_xref="GI:22595609 "

2- >gb|GQ202146.1| Lumpy skin disease virus isolate Egypt/Mansoura08


nonfunctional attachment protein gene, partial sequence, Length=192

>gb|AF325528.1| Lumpy skin disease virus NI-2490 isolate Neethling 2490,


complete genome, Length=150773

70
Score = 235 bits (127), Expect = 2e-58, Identities = 159/174 (91%), Gaps =
4/174 (2%), Strand=Plus/Plus

Query 1 AAATTATATACGTAAATAACATACCTGCTTAAAACCATAGTAATTTAGAATTCAAATCCA 60

||||||||||||||||||||||||||||| |||||||||||||||||||||||||||| |

Sbjct 64986 AAATTATATACGTAAATAACATACCTGCTAAAAACCATAGTAATTTAGAATTCAAATCAA 65045

Query 61 AAATTATCAT--TCCTAATAAATAAAATGGGAAGTGCTCCTATTATACTAATATCAAACT 118

|||||||||| | ||||||||||||| ||||||||||||||||||||||||||| |

Sbjct 65046 AAATTATCATTATTATAATAAATAAAATAATAAGTGCTCCTATTATACTAATATCAAA-T 65104

Query 119 ATACCATTTGATTGAAACCAATGGATGGGATACATAGTAAGAAAAATCAGGAAA 172

|||||| | ||||||||||||||||||||||||||||||||||||||||||

Sbjct 65105 ATACCA-AAAAATGAAACCAATGGATGGGATACATAGTAAGAAAAATCAGGAAA 65157

/ gene="LD074 "

CDS complement (64984..65952 )

/ gene="LD074 "

/ note="similar to vaccinia virus strain Copenhagen H3L ,

p35; similar to GenBank Accession Number AF124516 "

/ codon_start=1

/ product="putative IMV envelope protein "

/ protein_id="AAN02642.1 "

/ db_xref="GI:22595609 "

3- Query= KU298637.1 Lumpy skin disease virus isolate 956/Egy/2015 envelope


protein gene, partial cds, Length=331

>AF325528.1 Lumpy skin disease virus NI-2490 isolate Neethling 2490,


complete genome, Length=150773

71
Score = 612 bits (331), Expect = 1e-176, Identities = 331/331 (100%), Gaps =
0/331 (0%), Strand=Plus/Plus

Query 1 AAGAGCATTACATAATCCAGAAAAATATTCTGTAAAATTTTCAACACCTCCTGATTTTTC 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 65692 AAGAGCATTACATAATCCAGAAAAATATTCTGTAAAATTTTCAACACCTCCTGATTTTTC 65751

Query 61 TACCTTTTCCCATATAAGGAACTTATATGATAAACTGATATCTTTTTTATCTTTaaaaaa 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 65752 TACCTTTTCCCATATAAGGAACTTATATGATAAACTGATATCTTTTTTATCTTTAAAAAA 65811

Query 121 aaaaTTTACATCTGAATTTTTAAAATCTTTTACTGTGTCAACTTTTTTATAAAATATATC 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 65812 AAAATTTACATCTGAATTTTTAAAATCTTTTACTGTGTCAACTTTTTTATAAAATATATC 65871

Query 181 ATTGTCACTTTTTAATTCTGGAACTACATCTGAAATTTCGCGACCAACGATTGGTATAAC 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 65872 ATTGTCACTTTTTAATTCTGGAACTACATCTGAAATTTCGCGACCAACGATTGGTATAAC 65931

Query 241 ATATAATGGGATATCTGCCATTTTTGATAATTAGTTATCTAAAGCACTATTTAGTTATTA 300

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 65932 ATATAATGGGATATCTGCCATTTTTGATAATTAGTTATCTAAAGCACTATTTAGTTATTA 65991

Query 301 AAATTAAAGAAGTATAGCTCTCTAATTTTAG 331

|||||||||||||||||||||||||||||||

Sbjct 65992 AAATTAAAGAAGTATAGCTCTCTAATTTTAG 66022

gene complement (64984..65952 )

/ gene="LD074 " CDS complement(64984..65952 )

/ gene="LD074 " / note="similar to vaccinia virus strain Copenhagen H3L ,

p35; similar to GenBank Accession Number AF124516 " / codon_start=1

/ product="putative IMV envelope protein " / protein_id="AAN02642.1 "

/ db_xref="GI:22595609, " gene complement(65982..68378 )

72
/ gene="LD075 ", CDS complement(65982..68378 )

/ gene="LD075 ", / note="similar to vaccinia virus strain Copenhagen H4L ,

RAP94 " ,/ codon_start=1 ,/ product="RNA polymerase-associated protein "

/ protein_id="AAN02643.1 ", / db_xref="GI:2259561

4- Query= KJ561442.1 Lumpy skin disease virus strain Egypt/BSU-1, G-protein-


coupled chemokine receptor (GCPR) gene, partial cds, Length=557

>AF325528.1 Lumpy skin disease virus NI-2490 isolate Neethling 2490,


complete genome, Length=150773, Score = 968 bits (524), Expect = 0.0,

Identities = 556/569 (98%), Gaps = 12/569 (2%), Strand=Plus/Minus

Query 1 CTTAGTACAGTTAGTAGCGCAACCATGTATAATAGTAGCAGTAATATTACCACTATAGCT 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 8106 CTTAGTACAGTTAGTAGCGCAACCATGTATAATAGTAGCAGTAATATTACCACTATAGCT 8047

Query 61 ACTACAATTATTA-----------A-TACAATTTCAACTAATCAAAATAATGTTACAACG 108

||||||||||||| | |||||||||||| |||||||||||||||||||||

Sbjct 8046 ACTACAATTATTAGTACAATTCTCAGTACAATTTCAACAAATCAAAATAATGTTACAACG 7987

Query 109 CCTTCAACTTATGAAAATACAACAACGATATCTAATTATACAACCGCATATAATACAACT 168

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 7986 CCTTCAACTTATGAAAATACAACAACGATATCTAATTATACAACCGCATATAATACAACT 7927

Query 169 TATTATAGCGATGATTATGATGATTATGAAGTGAGCATAGTCGATATCCCACATTGTGAT 228

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 7926 TATTATAGCGATGATTATGATGATTATGAAGTGAGCATAGTCGATATCCCACATTGTGAT 7867

Query 229 GATGGTGTGGATACTACAAGTTTTGGACTGATTACTTTATATTCGACTATATTCTTTCTT 288

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 7866 GATGGTGTGGATACTACAAGTTTTGGACTGATTACTTTATATTCGACTATATTCTTTCTT 7807

73
Query 289 GGATTATTTGGAAATATAATTGTGTTAACTGTTCTTCGTAAATATAAGATAAAAACAATA 348

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 7806 GGATTATTTGGAAATATAATTGTGTTAACTGTTCTTCGTAAATATAAGATAAAAACAATA 7747

Query 349 CAGGATATGTTTTTGCTTAATTTGACACTGTCTGATTTAATTTTCGTGTTGGTGTTTCCT 408

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 7746 CAGGATATGTTTTTGCTTAATTTGACACTGTCTGATTTAATTTTCGTGTTGGTGTTTCCT 7687

Query 409 TTTAATTTATACGATAGTATCGCTAAACAATGGAGTTTAGGAGATTGTTTGTGTAAATTT 468

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 7686 TTTAATTTATACGATAGTATCGCTAAACAATGGAGTTTAGGAGATTGTTTGTGTAAATTT 7627

Query 469 AAAGCTATGTTTTACTTTGTTGGTTTTTACAATAGCATGTCATTTATAACATTGATGAGT 528

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 7626 AAAGCTATGTTTTACTTTGTTGGTTTTTACAATAGCATGTCATTTATAACATTGATGAGT 7567

Query 529 ATTGATAGATACCTAGCTGTAGTTCACCC 557

|||||||||||||||||||||||||||||

Sbjct 7566 ATTGATAGATACCTAGCTGTAGTTCACCC 7538

gene complement(6978..8111 )
/ gene="LD011 "
CDS complement(6978..8111 )
/ gene="LD011 "
/ note="similar to GenBank Accession Number S78201 "
/ codon_start=1
/ product="CC chemokine receptor-like protein "
/ protein_id="AAN02577.1 "
/ db_xref="GI:22595544

74
3.9. Viral immune evasion:

Successful transmission by viruses in the face of vigorous innate and acquired


host immunity requires the ability to evade, obstruct, or subvert critical elements
that mediate host antiviral responses. To that end, viruses with larger genomes,
such as poxviruses, encode multiple classes of immunomodulatory proteins that
have evolved specifically to inhibit such diverse processes as apoptosis, the
production of interferons, chemokines, and inflammatory cytokines, and the
activity of cytotoxic T lymphocytes (CTLs), natural killer (NK) cells,
complement, and antibodies. Often, the evolutionary origins of these virus-
encoded immunomodulatory proteins are difficult to trace. The obvious sequence
similarity between some immunomodulatory poxvirus genes and the cDNA
versions of related cellular counterparts suggests that they were once captured by
ancestral retrotranscription and/or recombination events and then reassorted into
individual virus isolates during coevolution with vertebrate hosts. However, other
poxviral immunomodulators have no known cellular counterpart or have putative
functions that cannot be predicted based on similarity to known cellular proteins.
The origins of these orphan regulators may be obscure, but their potential for
immune subversion can be profound ( J. B. Johnston et al,2003).

ADVANCES IN VIRAL GENOMICS: GENES APLENTY

Poxvirus immunomodulatory proteins can be operationally divided by function


into a trinity of distinct strategic classes, which we will refer to here as
virostealth, virotransduction, and viromimicry. Virostealth is characterized by
masking of the visible signals associated with virus infection, for example, by
reducing the capacity of effector leukocytes to recognize and eliminate infected
cells. Virotransducers are intracellular viral proteins that inhibit innate antiviral
pathways, such as apoptosis, proinflammatory cascades, or the induction of the
antiviral state. Virotransducers can also target host signal transduction pathways
that influence host range. Viromimicry is exemplified by virokines and
viroreceptors, which are virus-encoded proteins that mimic host cytokines or their
receptors, respectively. These proteins block extracellular communication signals
and promote a protected microenvironment for the virus within normally
immuno-exposed tissues.This commentary focuses on a few select examples
within each strategy and the reader is referred to other recent reviews for more
comprehensive exegeses on this expanding subject. ( J. B. Johnston et al,2003).

75
1- VIROSTEALTH: SLEEPING WITH THE ENEMY

The participation of innate and educated cytolytic immune cells, especially NK


cells and CTLs, is critical for the rapid identification and clearance of virus-
infected cells. Thus, both small and large viruses attempt to subvert the non self
discrimination pathway, most commonly by down regulating recognition
receptors and/or blocking the presentation of viral antigens to immune cells. The
capacity to decrease expression of the class I MHC receptors that normally
present endogenous viral antigens to circulating CD8_ CTLs correlates with the
extent of systemic spread and replication within diverse tissues ( J. B. Johnston et
al,2003). Poxviridae comprise a diverse family of large double-stranded DNA
viruses that undergo replication exclusively in the host–cell cytoplasm. Poxvirus
virions are easily identified by their characteristic brick-shaped appearance in
electron micrographs. Each virion contains a single linear genome that varies in
length (130–360 Kb) depending on the virus strain. The genomes are compact,
with open reading frames (ORFs) being closely spaced and non-overlapping with
no evidence of mRNA splicing. Although individual strains may contain more
than 200 ORFs, only _50 are thought to encode proteins essential for viral
transcription, DNA replication, or the formation of new virions . These ORFs
cluster in the central region of the genome and are well conserved in sequence
and position across different species. The remaining ORFs are more variable and
tend to be distributed more towards the terminal ends of each genome. These
ORFs likely encode factors that confer virulence, tissue tropism, or serve to
expand host range. Ample evidence suggests that poxviruses have captured host
genes during their evolution in order to evade immune detection and elimination.
Yet it is clear from evolutionary studies comparing the sequences of ORFs across
different genomes that poxviruses also adapt to changes in host defense by
altering their existing repertoire of factors. Possible mechanisms to explain the
observed alterations include accumulation of point mutations, the occurrence of
unequal crossovers giving rise to chimeric factors, or transient genomic
expansions that increase the number of targets available for mutation. In support
of the idea that poxvirus genomes are modified in response to evolutionary
pressure, several poxvirus families show signs of ORF duplication and
divergence. These include: the ankyrin-repeat proteins, the serpin family, the C7L
family, the kelch-like proteins, and the Bcl-2-like proteins. From a structural
point of view, each of these families can be thought of as sharing an easily

76
identified fold. Within each family, individual members likely derive from an
ancestral factor that was used as a common structural scaffold and modified
repeatedly to create different binding specificities for host molecules. These
modifications were presumably driven by host-mediated selective pressure. (A.
Nelson et al, 2015).

77
78
4. MATERIAL AND METHODS STRATEGY

4.1. Material:

4.1.1. History of the outbreak:

Lumpy skin disease (LSD) was suspected among cattle at different localities in
Qaliubiya province, Egypt during summer 2013-2014. Suspected cattle used as
source for viral samples collection demonstrate skin nodules and scabs scattered
all over the body parts (Photo_1&2). The animals showed 20 days duration of
illness, and history of vaccination with local modified live sheep pox vaccine
before 7 months and 1.5 months of the occurrence of infection.

4.1.2. Viral Samples:

Two skin biopsies from cutaneous scabs (S1 from a 2 year old Frisian bull and S2
from 2 years old Frisian cow) were collected on 50% glycerol saline. These
samples were used for isolation of Lumpy Skin Disease Virus (LSDV) on
Specific Pathogen Free-Embryonated Chicken Egg (SPF-ECE) and MDBK cell
line.

79
A

80
C

D
81
E

82
H

(Photo_ 1): Suspected cattle for LSD showing skin lesions as skin nodules
scattered all over the body (A and B (S2)), including vulva (C) and udder
(D). Suspected cattle for LSD showing skin lesions as skin scabs (E_S1),
ulcerative lesions (F) complicated to necrotic skin lesions on legs, especially
on top of the joints (G) and accompanied with limb edema (H).

83
4.1.3. Reagents for sample preparation:

1- Physiological saline 0.9%, pH 7-ready to use.

Sterile I.V infusion 0.9% W/V & pyrogen free, 9 gram in 500 ml solution
with Na+ 154 m. Mol/L and Cl– 154 m. Mol/L, M.O.H
Reg.No:27829/2012.

Single dose container (500ml), Isotonic, Batch No: 109141632,


Manufactured by ATECO PHARMA EGYPT, EL Obour City- industrial
Area (B) No:122.

2- antibiotic.

Garamycin®, Gentamicim (as sulphate), 80mg/2ml, Memphis, B.N. ET-


13-AMKB-46, an antimicrobial spectrum for Gram negative and Gram
positive bacteria.

3- antifungal .

Mycostatin™, Nystatin,100,000 units/ml, Manufactured by


GlaxoSmithKline Egypt, Elsalam City, Cairo, Batch No: A507926.

4- Working solution of saline: (Payment and Trudel,1993, modified).

1. physiological saline (0.9%) 500 ml

2. gentamycin 1 ml

3. mycostatin 100-200µl

4. mixing well.

5. Filter sterilized and pH was adjusted to 7.5 by N/10 HCl or 1N NaOH


then stored at 2-8°C.

4.1.4. Specific Pathogen Free-Embryonated Chicken Eggs (SPF-ECE):

SPF-ECE days old were obtained from the SPF production Farm,
Koum Oshiem, Fayoum, Egypt. The fertilized eggs were incubated at the
37°C and 80% humidity till reach suitable age (10-12 days old). It was used
for isolation of LSDV from the suspected samples and IFAT.
84
4.1.5. Madin – Darby Bovine Kidney (MDBK) cell line:

MDBK cell line was produced by Ames, Iowa Laboratory, USA


and it was obtained from Cell Culture Department, VACSERA, Agouza,
Giza., Egypt and maintained at Virology Department, Faculty of
Veterinary Medicine, Benha University. It was used for virus isolation and
IFAT.

4.1.6. Tissue culture media and Solutions:

4.1.6.1. Minimum Essential Medium (MEM):

It was purchased from Biochrom, Leonorenstr: 2-6. D-12247


Berlin, Germany. Minimum Essential Medium (MEM) with Earle's salts
and stable L-glutamine and 2.2 g/l NaHCo3, Cat. No. FG 0325, Lot. No.
1298A, was used for the maintenance and growth of cell cultures. The
maintenance medium was supplemented with 2% new born calf serum,
while the growth medium was supplemented with 10% new born calf
serum. The final pH of the growth and maintenance media were
approximately adjusted to 7.2.

4.1.6.2. New born calf serum:

Virus and mycoplasma screened sterile new born calf serum


™.
PAN , Cat. No. P30-0402, Lot. No. P122207N was purchased from
Biotech, Australia. It was used to supplement cell culture media.

4.1.6.3. Cell culture growth media: (Payment and Trudel, 1993,


modified).

MEM (minimum essential media) Earle's supplemented with 10% new


calf serum+ Antibiotic and antifungal: such as previously used in sample
preparation, but with different concentrations, Gentamycin: 50µg/ml
media, mycostatin:100 ug/ml, then adjust pH with NaHCO3 buffer (0.1M)
with variable amount till pH reach 7.4,

85
4.1.6.4. Phosphate buffered saline (PBS), 1X: (Sambrook et al., 1989)

NaCl 8g
KCl 0.2 g
Na2HPO4 1.44 g
KH2PO4 0.24 g
Distilled water up to(DW) 1000 ml.

pH was adjusted 7.4 with N/10HCl or 1N NaOH and the solution


was dispensed into aliquots then sterilized by autoclaving 121ºC/20
minutes and stored at 4ºC, for washing of cell sheet.

4.1.6.5. Phosphate buffered saline (PBS) - without Ca+2 and mg+2:

(Payment and Trudel, 1993)

NaCl 8.00 g
KCl 0.20 g
Na2 HPO4 1.15 g
KH2PO4 0.20 g
Glucose 0.20 g
Double distilled H2O up to 1000 ml
Adjust pH to 7.4 by NaHCO3 buffer (0.1M) and sterilized by
autoclaving 121ºC/ 20 min. This solution could be used for up to 2 years if
kept at -20oC for three months at 4oC, for subculture of cells.

4.1.6.6. Trypsin-versene solution: (Payment and Trudel, 1993)

- Trypsin 0.05 g
- EDTA-disodium salt 0.02g
- PBS without Ca+2 and mg+2 100 ml
The total volume was sterilized by filtration (0.22µm diameter
PTFEL plastic syringe filter), and stored in screw capped bottles. Adjust
pH to 7.5 by NaHCO3 buffer (0.1M) and measured by pH meter. This
solution could be used for up to 2 years if kept at -20oC, for three months
at 4oC, for subculture of cells.

86
4.1.6.7. Cell culture buffers: (Hell Creek Life © 1997-2010
Phillip Bigelow Revised 1/24/2010)
4.1.6.7.1. Sodium bicarbonate solution (NaHCO3) 0.1 mol:

It is consisted of NaHCO3 (MW = 84.007 g/ mol). NaHCO3 (0.1M)


is prepared by dissolving 8.4007 g of NaHCO3 in 1000 ml double distilled
water and sterilized by autoclaving. It was used to adjust the required pH
of the cell culture media and solutions.

4.1.6.7.2. Sodium hydroxide solution (NaOH) 1 M:

It is consisted of NaOH (MW = 40 g/ mol). NaOH (1 M) is prepared


by dissolving 40 g of NaOH in 1000 ml double distilled water and sterilized
by autoclaving. It was used to adjust the required pH of the cell culture
solutions.

4.1.6.7.3. Hydrochloric acid (HCl) 100% as N/10:

Equivalent mass of HCl = molar mass/ No. of hydrogen ions


100% = 36.5/ 1= 36.5 g
N/10 HCl mass in grams = N required x mass equivalent x amount in
liters
= 0.1x 36.5 x 1 = 3.65 g
Volume of N/10 HCl 100% =Mass in grams/(HCl conc. x sp. gravity of
HCl)
= 3.65/ (1x 1.189)= 4.34 ml HCl

It is consisted of 4.34 ml of HCl 100% dissolved in 1000 ml double distilled


water and sterilized by autoclaving. It was used to adjust the required pH
of the cell culture solutions.

4.1.8. Material for Transmission Electron Microscopy:


4.1.8.1. Sodium cacodylate Buffer (0.2 M): (Mercer and birbeck, 1966)
-Sodium cacodylate(Na(CH3)2AsO2· 3H2O, MW= 21.4 g
214.02)
- distilled water 500 ml

87
It was adjusted to pH 7.3 by adding N/10 hydrochloric acid. (was supplied
by EMS, Washington, USA)

4.1.8.2. Glutaraldehyde 25%:

Glutaraldehyde 25% (OCHCH2CH2CH2CHO, F.W. 100.12 CAS


#111-30-8) was supplied by EMS, Washington, USA), and was used for
preparation of the fixation buffer of the samples.

4.1.8.3. Fixation Buffers: (Mercer and birbeck, 1966)

4.1.8.3.1. Glutaraldehyde 1-5% in Sodium cacodylate buffer 0.1 M:

It was prepared as 1-5% glutaraldehyde in 0.1 M sodium


cacodylate buffer as follow:

- 25% glutaraldehyde 20 ml
- distilled water 30 ml
- 0.2 M sodium cacodylate buffer, PH 7.3 50 ml
(was supplied by EMS, Washington, USA
)
4.1.8.3.2. Osmium tetroxide 1% (OsO4, F.W. 254.20) in 0.1 M Sodium
cacodylate buffer:

Add 2% aqueous solution of osmium tetroxide to an equal volume


of 0.2 M sodium cacodylate buffer.

(was supplied by EMS, Washington, USA)

4.1.8.4. Ehyl alcohol 100%:

Ethyl alcohol 100% (CH3CH2OH, 46.07 g/ mol,) Cat. No. 15055,


was supplied by ADWIC company, Egypt. Ethyl alcohol was prepared in
different concentrations including 30%, 50%, 70%, 80%, 90% and 100%
and were used for gradual dehydration of tissue samples at 4 ºC.

4.1.8.5. Infiltration and embedding media:

All reagents were obtained from Electron Microscopy Sciences -


EMS,231MorrisRoad,Washington,https://www.emsdiasum.com/microsco
py/

88
4.1.8.5.1. Acetone 100% (C3H6O, 58.08 g/mol, CAS #: 67-64-1):

It is miscible to alcohol (some types of resin kits is not miscible to


alcohol) and used to ensure dehyderation of sample after alcohol.

4.1.8.5.2. Epoxy Resin (Low Viscosity Embedding Media Spurr's Kit):


Low viscosity embedding media was supplied by Spurr kit (EMS,
Hatfield, PA, USA) with the following components:

1- NSA (non enyl succinic anhydride), MW: 227, Cat. # 19050.

2- ERL-4206 (VCD) (Vinyl Cyclohexene Dioxide=Vinyl-4


Cyclohexene Diepoxide, MW: 140.18, Cat. #15000).

3- DER 736 (Epoxy Resin, MW: 380, Cat. #13000).

4- DMAE (Dimethylaminoethanol accelerator, MW:89.14, Cat.


#13300).

It was used for hardening of the samples in the following formula:


NSA: ERL: DER (26: 10: 6) + DMAE (0.3 ml). DMAE was used as an
accelerator (hardening and polymerization of tissue resin mixture at oven
to form the tissue blocks).

4.1.8.6. Stains of the electron microscope: (Mercer and birbeck, 1966)

All reagents were obtained from Electron Microscopy Sciences -


EMS,231MorrisRoad,Washington,
https://www.emsdiasum.com/microscopy/

4.1.8.6.1. Stain for semithin section:

Methylene blue (0.1%)_ MW: 319.85. 0.1 g


Distilled water. 100 ml

4.1.8.6.2. Stain for ultrathin section:

4.1.8.6.2.1. Uranyl acetate:

Uranyl acetate 0.2 g

89
Distilled water 10 ml

Shake for 15 min till all crystals are dissolved then filter and store
the solution in a dark bottle in refrigerator, used at room temperature).

4.1.8.6.2.2. Lead citrate: (Raylonds, 1963)

Lead nitrate 1.55 g


Sodium citrate 1.76 g
Shake well for 5 min then leave to stand for 30 min and add 8 ml
N/10 sodium hydroxide aqueous solution, finally add distilled water up to
50 ml then adjusted to pH12 by N/10 aqueous solution of sodium
hydroxide and filter.

4.1.8.7. Equipments: (was supplied by EMS, Washington, USA)

1-Ultrasonicator (CREST, Germany).

2- Tissue cutter.

3-Reichert-Jung Ultra-cut 701701 Ultra Microtome (Stock:


22479, Dimensions: 20LX 14WX 18H).

4- Grid.

5- Grid holder.

6- Holder of grid holder.

7- Grid forceps.

8- Forceps of grid holder.

9- Grid fixer.

10- Grid rack.

11- Filter paper.

12- Transmission electron microscope JEM2100-Joel-Japan.

90
4.1.9. Material for histopathological examination: (Bancroft and
Gamble, 2002)

4.1.9.1. Buffered neutral formalin 10%:

A 10% formalin solution was used as fixative as follow:

Formalin (37%) BP 93, ELnaser. 270 ml


Normal saline to 1000 ml
It is used for preparation of the fixative solution.

4.1.9.2. Fixative solution:

10% buffered neutral formalin 100 ml


Anhydrous disodium phosphate 6.5 g
Acid sodium phosphate monohydrate 4.0 g
Distilled water 900 ml
4.1.9.3. Xylene: (Technical grade, ADWIC, X0018131)

4.1.9.4. Ethyl alcohol 100%: (MW: 46.07, Elsalam for chemical


industries)

It was used for preparation of grades of ethyl alcohol from 50%-


100%.

4.1.9.5. Paraffin wax: (Diapath. SPA diawax, melting point: 56-58).

4.1.9.6. Harris alum haematoxylin stain: Bancroft, J. D. and Gamble,


M. (2002)

Haemmatoxylin crystals 1g
Absolute alcohol 10 ml
Ammonium or Potassium alum sulphate 20 g
Mercuric oxide 0.5 g
Distilled water 200 ml

- Dissolve hematoxylin in alcohol then added


to alum which previously dissolved in hot
water, bring quickly to the boil and add
mercuric oxide when the solution turn dark
purple, cool rapidly under tape, filter before
use.
91
4.1.9.7. Stock aqueous eosin stains 1%:

Eosin soluble 10 g
Distilled water 1000 ml

4.1.9.8. Equipments:
1- Ultramicrotome (Microtec®, CUT 44055, CUT 2020A, low profile I,
No. 10216380, Germany).

2- Glass slide.

3- Cover slip.

4- incubator.

5- deep freeze -20.

4.1.10. Material used in Haemagglutination test (HA): (Payment and


Trudel, 1993).

4.1.10.1. Washed Red blood cells (RBCs):

RBCs were obtained from healthy cattle, sheep, goat and chicken
on EDTA as anticoagulant. The cells were washed three times with
physiological saline 0.9% and packed cells were diluted as 1% for
Haemagglutination. The diluent

used in the HA test, for both the antigen and the erythrocytes, was the
physiological saline 0.9%, pH 7.

-preparation of red blood cells (RBCs):

1- Obtain blood by venipuncture or cardiac puncture and mix with 4 times


the volume of NaCL 0.9%, pH 7.

2- Centrifuge at 1000xg for 15 min.

3- Discard the supernatant and suspend the pellet in the initial volume of
buffer saline NaCL 0.9%.
92
4- Repeat steps 2 and 3 three times.

4.1.10.2. Equipments:

1- U-shape bottom haemagglutination plate Nunc ®.

2- Uni-channel Epindorf ® automatic pipette (10-100 ul) with tips.

4.1.11. Material for Indirect Fluorescent Antibody Technique (IFAT):


4.1.11.1. Hyperimmuneserum specific for LSDV:

LSD virus hyperimmuneserum was supplied by Department of pox


virus vaccine research and production, Veterinary Serum and Vaccine
Research Institute, Abbasia, Cairo. It was used for serological
identification of LSDV isolates using indirect FAT.

4.1.11.2. Goat anti-Bovine IgG conjugated with fluorescein


Isothiocyanate:

It is Goat anti-Bovine IgG (H+L) as an affinity purified antibody fluorescen


labelled (Cat. No. 02-12-06, Lot No. 040659- - LB 261-06) and rehydrate
and store as instructed by manufacturer, Kirkegaard and Perry
Laboratories, 2 cessana court, Gaithersburg, Maryland 20879, USA,
diluted 1:200 by isotonic NaCl 0.9%, pH7.

4.1.11.3. Acetone:

Cold acetone(-20ºC) was used for fixation of infected cells on cover


slips; it was obtained from BDH Chemicals LTD, England.

4.1.11.4. Mounting buffer (buffered glycerin): (Payment and Trudel,


1993).

1 PBS: 9 glycerin was used for mounting the infected cells fixed on
the cover slips, it was obtained from elnasser for chemical industries.

4.1.11.5. conjugate diluent (isotonic NaCl 0.9%).

4.1.11.6. PBS 1X (washing solution).

93
4.1.11.7. Equipments:
1- Incubator.

2- Fluorescence microscope (Olympus_ Tokyo_ Japan, and Leica-


Germany)

3- Automatic pipette with tips.

4.1.12. Material used for extraction of genomic DNA of LSDV from


samples:
4.1.12.1. DNeasy® Tissue Kit (QIAGEN, USA):
The kit contains DNeasy Mini Spin Columns (colorless) in 2 ml
Collection Tubes, Collection Tubes (2 ml), Buffer ATL, Buffer AL, Buffer
AW1 (concentrate), Buffer AW2 (concentrate), Buffer AE and Proteinase
K. It was used for extraction of genomic DNA of LSDV from tissue
samples. https://windward.hawaii.edu/paces/summerfiles/publications/qiagen.pdf_march,
2004.

4.1.12.2. QIAamp® DNA Mini and Blood Mini kits-Suspension


(QIAGEN, USA):
The kit contains QIAamp Mini Spin Columns, Collection Tubes (2
ml), Buffer AL, Buffer ATL, Buffer AW1, Buffer AW2, Buffer AE,
QIAGEN® Protease, Protease Solvent and Proteinase K. It was used for
extraction of genomic DNA of LSDV from prepared tissue samples
suspensions. 3rdedition, June 2012_
https://moodle.ufsc.br/mod/resource/view.php?id=805619

4.1.12.3. Equipment and Reagents to be Supplied by User:

1. Pipets and pipet tips with aerosol barrier

2. Vortexer.

3. Microcentrifuge tubes (1.5 ml).

4. Microcentrifuge with rotor for 2 ml tubes.

5. Water bath, or heating block at 56ºC.

6. PBS, pH 7.2.
94
7. Ethanol (96–100%).

4.1.13. Material for conventional polymerase chain reaction


(PCR):

4.1.13.1. QIAGEN® PCR Master mix.

Taq PCR Master Mix 2x concentrated, (Catalog no. 201443) supplied by


Qiagen, USA with the following composition: Taq PCR Master Mix (3 x
1.7 ml), RNase-Free water (3 x 1.7 ml), MgCl2 (3 mM), Taq DNA
Polymerase (250 units), Qiagen PCR Buffer (2x) and each dNTP (400 μM).

4.1.13.2. Nuclease free water.

Highly pure, nuclease-free water for use in all molecular biology


applications, 500 ml, Catalog no. 129114, qiagen USA.

4.1.13.3. Primers:

Two primer pairs were designed based on the sequence of LSDV


attachment protein gene and were used in this study as following:

4.1.13.3.1. Primer pair one:

The primer sequences were based on the sequence of LSDV attachment


protein gene as described by OIE (2008). The specific primers were
manufactured in the laboratories of the Midland Certified Reagent
company Inc. of Midland, Texas. The sequence of oligonucleotides is:

LSD1 5' -TTTCCTGATTTTTCTTACTAT- 3' (forward)


Tm (50mM NaCl) *:44.5 ᵒc
GC Content : 23.8%
AMOUNT OF OLIGO
6.1= 33.1= 0.21
OD260 nmoles mg
For 100 µM: add 331 µl
LSD1 5'-AAATTATATACGTAAATAAC- 3' (reverse)
Tm (50mM NaCl) *:37.6 ᵒc
GC Content : 15.0%

95
AMOUNT OF OLIGO

5.7= 26.3= 0.16


OD260 nmoles mg
For 100 µM: add 263 µl
The primer sequence of LSDV attachment protein gene 172 bp fragment
was selected for amplification of DNA, From positions: 64986- 65157.

4.1.13.3.2. Primer pair two:

The specific primers were obtained kindly from Dr. Wessel Dirksen, Ohio
State University Research Foundation, 925 Coffey RD, 345 Goss Lab/
College of Vet Med, Columbus, OH 43210, USA (P.O # RF01404362).
The primer sequences were selected based on the sequence of LSDV
attachment protein gene and was designedusingPrimer-BLASTsoftware
(http://www.ncbi.nlm.nih.gov/tools/primer-blast). The specific primers
(Package # 34851877) were manufactured in Integrated DNA
Technologies (IDT), 1710 Commertial Park, Coralville, Iowa 52241.

The sequence of oligonucleotides is:

LSD2 5' - GGGAAAAGGTAGAAAAATCAGGAGG- 3' (forward)


Tm (50mM NaCl) :55.6 ᵒc
*

GC Content : 44.0%
AMOUNT OF OLIGO
5.3= 19.1= 0.15
OD260 nmoles mg
For 100 µM: add 191 µl
LSD2 5' - CGCATCGGCATACGATTTCC- 3' (REVERSE)
Tm (50mM NaCl) *:56.6 ᵒc
GC Content : 55.0%

AMOUNT OF OLIGO
5.6= 30.7= 0.18
OD260 nmoles mg
For 100 µM: add 307 µl

The primer sequence of LSDV attachment protein gene 137 bp fragment


was selected for amplification of DNA, From positions: 65626- 65763.

96
4.1.13.4. Equipments

1- Thermal cycler (applied biosystem, Amp 9600 PCR system,


ramp speed 9600).

2- Laminar air flow.

3- Micro centrifuge.

4- Sterile thin wall micro centrifuge tubes 0.2 ml capacity.

5- Ice buckets.

6- Latex gloves.

7- Spectrophotometer.

4.1.14. Material used for analysis of PCR product using Agarose Gel
Electrophoresis

4.1.14.1. Agarose powder, molecular biology grade: (SPI, India).

Agarose gel was prepared at concentration of 1.5 %

4.1.14.2. Tris Acetic acid EDTA (TAE) buffer 1X:

1. 40 mM Tris base (pH 7.6), Hydroxymethyle aminomethane,


tromethamine, C3H11NO3.FW 121.14, Ultra pure, AMERICAN
BIOANALYTICAL.

2. 20 mM acetic acid, HPLC GRADE REAGENT, INDIA,

CH3COOH, M=60.05 g/mol

3. 1 mM EDTA, pH 8, Ethylenediaminetetra acetic acid disodium salt

(Disodium ethylenediaminetetraacetate), C10 H14N2Na2O8 .2H2O,


FW.292.24

97
- Preparation of EDTA 1 mM, pH 8.

a.1 M= 292.24 g / 1 liter DW.

b.1 mM = 0.29224 g / 1 liter DW.

c. dissolve EDTA by magnetic stirrer and by gradual adding 10N NaOH


till adjust to pH 8 by pH meter.

- Preparation of 20 mM acetic acid.

1M= 60.05 g/ l liter.

1 mM =0.06005 g/ 1 liter.

20 mM =1.201 ml / 1 liter.

- Preparation of 40 mM Tris.

1 M =121.14 g/ 1 liter.

1 mM=0.12114 g/ 1 liter.

40 mM=4.8456 g/ 1 liter.

- For preparation of 1 liter TAE buffer 1X:

1. Distilled water 600 ml

2. 40 mM Tris (pH 7.6) 4.8456 g

3. Add slowly acetic acid 20 mM 1.201 ml


4. Add EDTA 1 mM, pH 8 2 ml

Bulletin 6205 Rev A US/EG, 11-0864 1211 Sig 1211, Bio-Rad

Laboratories, Inc.

98
4.1.14.3. Ethidium bromide: (Qiagen)

It was prepared as stock of 10 mg/ml by dissolving 150 mg ethidium


bromide in 15 ml dd. H2O. It was used as final concentration of 1μg/ml on
stirrer for several hours to ensure complete dissolving. The solution was
stored at room temperature wrapped in aluminum foil.

4.1.14.4. Gel Pilot DNA Loading Dye, 5x: (Cat. No. 239901, Qiagen,
USA)

It was mixed as 2 ml mixed with 10 ul of the PCR product to obtain


1X then the mixture was loaded into wells of the gel matrix using mono-
channel micro-titer pipette.

4.1.14.5. Gel Pilot 100 bp plus DNA ladder: (Cat. No. 239045, Qiagen,
USA), 2% agarose. It was suitable for sizing linear double-stranded
fragments from 1500 bp to 100 bp on gel electrophoresis supplied as sachet
with data sheet.

4.1.14.6. Equipments:

1- Microwave (Panasonic).

2- Electrophoretic machine (Biometra)®.

3- UV transilluminator (Biometra)®.

4.1.15. Material used for purification of PCR product and sequencing:

4.1.15.1. QIAquick® PCR Purification Kit: septemper 2011, Link:

2012.igem.org/wiki/images/a/a3/QIAquick_PCR-purification.pdf.

It is an extraction Kit for PCR product from the gel used as recommended
by the manufacturer in the protocol of QIAquick® PCR purification kit Cat.
Nos. 28104 and 28106 (Qiagen Inc., Valencia CA) with spin columns,
buffers, and collection tubes for silica-membrane-based purification of
DNA 70 bp to 10 kb fragments from gels (up to 400 mg slices).

99
4.1.15.2. Material used for gel electrophoresis of purified PCR
product:

As Gel electrophoresis (PCR product).

4.1.15.3. Cycle sequencing:

BigDye® Terminator v3.1 Cycle Sequencing Kit. P/N 4337456, Link:


https://www3.appliedbiosystems.com/cms/groups/mcb_marketing/.../cms
_081527.pdf.

4.1.15.4. Purification after cycle sequence before injection:

Using BigDye® X Terminator™ Purification Kit.

Kit Contents: BigDye® X Terminator™ Solution and SAM™ Solution.

For details P/N 4374408, Product P/N 4376487, Insert P/N 4376151,
REV

Printed in, California, USA, Product insert 27Dec2006


https://www3.appliedbiosystems.com/cms/groups/mcb_support/documen
ts/generaldocuments/cms_042772.pdf.

4.1.15.5. Injection of sample into 3500 genetic analyzer (sequencer):

4.1.15.5.1. System Reagents:

1. Reagents Preserved at 4ºC:

1- POP-7™ 3500 Polymer (384 samples), (P/N 4393708): Performance


Optimized Polymers.

2- Big Dye terminator (buffer).

3- Conditioning Reagent 3500 series, P/N 4393718.

4- Anode Buffer Container (ABC) 3500 Series, P/N 4393927.

5- Cathode Buffer Container (CBC) 3500 Series, P/N 4408256.

6- nuclease free water.

100
2. Reagents Preserved at -20 ºC:

1- Sequencing standard V3.1.

2-Hi- Di™ formamide, (P/N 4401457).

3- Big Dye® Terminator V3.1- Cycle Sequencing Kit (1,000 rxns), P/N
4337456.

4.1.15.5.2. Equipments:

1- Capillary Array 3500 (50 cm), P/N 4404685.

2- Septa Cathode Buffer Container 3500 Series, P/N 4410715.

3- 96 well- plate with septa.

4- Genetic analyzer 3500 (Sequencer) applied biosystem.

4.1.16. Material used for phylogenetic analysis of the sequence data:


4.1.16.1. Reference sequences of LSDV strains published on Gene
Bank:
Accession number of other LSDV strains were listed in table (3). Sequence
data of these LSDV strains were published on gene bank and were used for
multiple sequence alignments and phylogenetic analysis with the local
LSDV isolated in this study.

4.1.16.2. Bioinformatics Programs:


4.1.16.2.1. BLAST and PSI-BLAST search programs:
They were used for search of LSDV reference sequences in Gene Bank
data base at National Center for Biotechnology Information (NCBI) on
line.https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSear
ch.

4.1.16.2.2. Clustal W and DNA star Mega:


It is program for sequence alignment (Clustal W) and phylogenetic
analysis (DNA star mega) of the LSDV nucleotide sequence.

101
4.2 Methods:

4.2.1. Preparation of suspected LSDV Samples: (OIE, 2010)

It was carried out on skin scabs collected from suspected cattle


using sterile scissor in sterile bottles containing 50% glycerol buffer saline
pH 7.2 as following:

1- Scabs were transferred into sterile petri dish, cut into small pieces
by sterile small scissor.

2- Transfer those pieces (about 200 mg) into 1.8 ml cryotube, with
adding 1.5 ml physiological saline with antibiotic and antifungal
solution.
3- Homogenization by homogenizer till formation of homogenized
suspension.
4- Application of 3 cycles of freezing (-20) and thawing (room
temperature) of homogenized suspension.
5- Centrifugation of thawed homogenized suspension at
3000rpm/15min.
6- Filtration of supernatant by plastic syringe filter (0.22µm-0.45µm).
7- Secondary filtration of supernatant was applied due to high
contamination at collection), before inoculation into SPF-ECE, or
stored at – 20 °C till used for isolation or PCR technique.
4.2.2. Isolation of suspected LSD virus in SPF-ECE:

This method was carried out according to Van Rooyen et al.1969, in which
SPF-ECE (4 for each sample) were examined (external and internal) and
incubated in egg incubator at 37 Cº for 10, 11, or 12 days with daily
examination by egg candler, regular shaking, ventilation and controlled
humidity (70%). They inoculated via chorio-allantoic membrane (CAM)
with dropped CAM method as following:
1- Candling of SPF-ECE for fertility, viability, then at embryo age of
10,11, or12 days with marking at sites where well developed CAM:
a. The base of air cell, triangular area at the base of air cell beside large
blood vessels, or

b. base of air cell, triangular area at large blood vessels, or

c. base of air cell, line at large blood vessels.


102
and marking of the embryo site.

2- Transfer the marked SPF-ECE eggs into sterile rack in vertical position
under laminar air flow hood.
3- Disinfection of eggs by alcohol 70%.
4- Poring of the eggs at the top of the air cell by sterile egg porer.
5- Change the position of eggs into horizontal position.
6- Making small opening at the shell at site of triangular area or on the
large blood vessels.
7- Apply the rubber teat at the top of the air cell for Suction of the air.
8- Inoculate 0.2 ml of prepared suspected viral sample by acute angle with
slight penetration of the shell.
9- Sealing of triangular area and top of the air cell with paraffin wax then
rotation of the inoculated egg around its longitudinal axis.
10- Incubate inoculated eggs in horizontal position at 37⁰C for 6- 8 days.
11- Daily examination of non-specific and specific deaths, during
daily examination, chilling of dead eggs at 4⁰c/1hr, and after the end
of incubation period, chilling of all inoculated eggs (dead or not), for
harvestation.
12- Washing of CAM 3-5 times: inside the egg then by transfer it
into petri dish containing physiological saline+ antibiotic+
antifungal solution.
13- Spread CAM well, and examined for detection of signs.
14-When lesion was not observed, two further passages were done
and samples considered negative only if no signs observed after 3
passages. With each passage non inoculated SPF-ECEs were used
as control.

4.2.3. Preparation of harvested CAM for serial passage of suspected


LSD virus on SPF-ECE, and MDBK cells:

1- Harvested CAM was placed in sterile mortar and 2 ml of saline with


antibiotic and antifungal solution was added.
2- Grinding of CAM by using a sterile pestle till homogenized suspension
was formed then transfer the suspension into 2 ml epindorf tube and
subjected for 2 cycles of freezing (-20 °C) and thawing (room
temperature) of the suspension.
3- Centrifugation of the suspension at 3000 rpm for 5min then filtration of
supernatant using syringe filter (0.22µm-0.45µm diameter), secondary
filtration of supernatant was applied, then used for inoculation into SPF-
103
ECE, or MDBK cells, or stored at – 20 °C till used for isolation and PCR
technique.
4.2.4. Propagation of suspected LSDV in MDBK cell line:

4.2.4.1. Subculture of MDBK cell line:

This method was carried out according to Payment and Trudel


(1993) as follows:
1- Examination of the sheet of cells in the vessel using inverted microscope.

2- Discard the spent cell culture media (yellow color).

3- Good washing for 3 times of the cell sheet using maintenance media
without serum.

4- Place 3 ml trypsin-versene solution (roux bottle), 0.5 ml (10 ml


prescription) and 1 ml (20 ml prescription).

5- Swirling the roux bottle or prescription at 37ºC or at room temperature


till complete sheet detachment and dissociation of cells. (take long time
at winter due to low temperature).

6- Shaking of flask or by pumping with automatic pipette for cell dispersion


into solitary cells.

7- Examination under inverted microscope for sheet detachment and


dissociation.

8- Place 200 ml growth media (Roux bottle), 20 ml (10 ml max.


prescription) and 50 ml (20 ml max. prescription).

9- Cell counting and adjustment.

10- Distribute the cell suspension (100 ml / roux bottle, 10 ml / 20 ml


prescriptions, 3 ml / tube, 200µl /well for plate).

11- Tightly closing the prescriptions, roux bottle, tubes and plates.

12- Examination under inverted microscope.

104
13- Incubation at 37ºCat dry incubator and daily examination till sheet
formation.

Cell counting using trypan blue exclusion method:

1- Mix 100µl trypan blue dye+ 400µl cell suspension by pipetting.

2- Place 10µl of mixture into haemocytometer chamber.

3- Cover the haemocytometer chamber with cover silde.

4- Counting under light microscope (adjust fine, filter).

Counting method- in 5 secondary squares:


𝑁𝑜.𝑜𝑓 𝑙𝑖𝑣𝑒 𝑐𝑒𝑙𝑙𝑠 𝑖𝑛 𝑠𝑚𝑎𝑙𝑙 𝑠𝑞𝑢𝑎𝑟𝑒
Average live cells/ small square= =
5

𝑁𝑜.𝑜𝑓 𝑑𝑒𝑎𝑑 𝑐𝑒𝑙𝑙𝑠 𝑖𝑛 𝑠𝑚𝑎𝑙𝑙 𝑠𝑞𝑢𝑎𝑟𝑒


Average dead cells/ small square =
5

5: No of secondary squares.
𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑙𝑖𝑣𝑒 𝑐𝑒𝑙𝑙𝑠
Cell viability = x100
𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑑𝑒𝑎𝑑 𝑐𝑒𝑙𝑙𝑠

𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑙𝑖𝑣𝑒 𝑐𝑒𝑙𝑙𝑠 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟


Cell density =
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑎 𝑠𝑞𝑢𝑎𝑟𝑒 (𝑚𝑙 ) (0.004µ𝑙)

Total number of cells= cell density x volume (ml)

4.2.4.2. Inoculation of suspected LSDV in MDBK cell line:

This method was carried out according to El-Nahas et al., (2011) as


follows

1-Harvested CAMs and embryo heart and liver of inoculated SPF-ECE


showing signs at the 3rd passage were prepared as previously prescribed
and filtrated using syringe filter (0.22µm-0.45µm diameter).

105
2- Examination of the cell sheet under inverted microscope for 60–70 %
confluency.

3- Under complete aseptic condition, remove the exhausted growth media


then wash the cell sheet using 1X PBS for 3 times, 3ml each with discard
of fluids completely.

4- Inoculate 0.15 ml of each prepared samples in 3 cell culture prescription


(CAM, embryo heart and liver suspensions).

5- Swirl the prescriptions, tubes and plates, and incubate at 37⁰C for 30 min
for adsorption time.

6- Place maintenance media (100ml / roux bottle, 10-20 ml / prescriptions,


3ml /tube, 200µl /plate.) then incubate at 37⁰C for 5-7 days till CPE
formation.

7- Normal non-infected cells served as control.

8- Cells were examined for CPE then the infected cells were frozen and
thawed three times, when the inoculated cell cultures show absence of
CPE, two further passages were adapted.

4.2.5. Identification of suspected LSD virus isolate:

4.2.5.1. Transmission electron microscopy of suspected LSD virus


isolate:

It was applied in Transmission Electron Microscopy Unit, National


Research Center, Dokki, Egypt. Inoculated CAM was prepared and
examined under the transmission electron microscope using positive
staining technique according to Bozzala and Russell (1992), modified as
follows:

4.2.5.1.1. Sample Fixation:

1- The samples were embedded in 5% buffered glutaraldehyde for 4 – 12


hours.

106
2- The samples washed in sodium cacodylate buffer for 45 minutes with 3
changes, each one for 15 minutes.

3- The sample was fixed by embedding in 1% osmium tetraoxide in sodium


cacodylate buffer for 2 hours.

4- Wash the sample in sodium cacodylate buffer for 45 minutes with 3


changes, each one for 15 minutes.

4.2.5.1.2. Sample Dehydration:

The tissue pieces were dehydrated in ascending grades of ethanol; from


10% to 100%, 10 minutes in each one except the finally one 100% for 30
minutes for three changes (each one for 10 minutes).

4.2.5.1.3. Infiltration and embedding:

1- Treat the tissue pieces with 3 changes of acetone (100%) for 45 min each
one for 15 minutes.

2- Tissue pieces impregnation with the epoxy resin as Epon (812) mixture,
is done by immersion in a mixture of equal parts of resin mixture and
acetone for 8 hours, then in mixture of 75/25 epoxy resin and acetone
for 8 hours, then in two changes of pure resin for each 8 hour.

3- Finally, embed the tissue pieces in pure resin and leave for 48 hours in
oven at 60ºC for resin polymerization for formation of tissue blocks.

4- Semi-thin sections were cut from these blocks by Ultracut


Ultramicrotome, stained with methylene blue and examined by the light
microscope.

5- Ultrathin sections obtained from selected blocks (by tissue cutter) by


Ultracut Ultramicrotome are mounted on copper grids, and double-
stained with uranyl acetate and lead citrate.

6- Then examined with Transmission Electron Microscope.

107
Tissue Blocks
Semithin Section Under Ultracut for selection
The semi thin section
of tissue by tissue cutter for ultracut section
2 µm

The grid on filter paper


during staining
Semithin section of CAM
under light microscope
The Ultrathin
section_1.3µm

Fig_5. material and method of TEM.

108
4.2.5.2. Histopathological examination:

The specimens were fixed in 10% buffered neutral formalin and


processed till paraffin sections were prepared for the light microscopy
study.
4.2.5.2.1. Preparation of specimens for light microscopy:

Specimens 1 cm3 were used in light microscope study according to


the method and technique quoted from Bancroft and Gamble (2002). The
specimens were washed in distilled water for removal of blood clots and
other debris, then fixed in 10% buffered neutral formalin, dehydrated in
ascending grades of ethyl alcohol, cleared in xylene and embedded in
paraffin wax. 5µm thick sections were cut, place onto glass slides, and
stained with Harris's hematoxylin and eosin stain for general histology of
the specimens.
4.2.5.2.2. Method of staining, the following steps were applied:

1- Place the slide in xylene to remove paraffin.

2- Place the slide in alcohol to remove xylene.

3- rinse with distilled water.

4- Place the slide in hematoxylin stain for 1-2 minute.

5- Rinse with distilled water.

6- Place the slide in eosin for 2-3 minute.

7- Rinse in distilled water.

8- Dehydrate in ethyl alcohol 99%.

9- Place the slide in xylene.

10- Cover the slide by cover slide using Canada balsam then representative
fields were photographed for morphology.

109
4.2.5.3. Haemagglutination test (HA):

HA test was applied according to Payment and Trudel (1993):


1- The samples of examined LSDV were prepared in sterile NaCL 0.9%
then mixed with equal amount of 1% cattle, sheep, goat, and chicken
RBCs suspension in a plastic U shape bottom micro-titer plate as 50µl
from each reactant.

2- Control negative sample was put in another well together with 50µl of
RBCS and a control RBCs was placed as 50µl of RBCs in saline into
one well of the plate.

3- Slight shaking of the plate is performed then the plate was incubated at
4ᵒC for 30 – 45 minutes until complete sedimentation of RBCs control.

4- Positive result was recorded as the occurrence of haemagglutination


which was represented by lattice formation, while negative result was
recorded as the absence of haemagglutination which was represented
by button formation.

4.2.5.4. Serological identification of suspected LSDV isolate using


Indirect Fluorescent Antibody Technique (IFAT): According to
Davies et al. (1971) and Mishra and Mallick (1997))

A- On CAM of inoculated SPF-ECE:

It was carried out as follows:

1- CAMs (which revealed the lesions) was spread on glass slide, followed
by air dryness, and then fixed in cold acetone (-20°C), then incubated at
4 °C for 30 minutes.
2- The CAM was covered by 25µl standard LSDV antiserum then
incubated at 37°C for 30 minutes (to allow antigen antibody reaction).
3- The CAM was washed for 3 times by 1 X PBS (to remove the un-
conjugated antibodies), then dried.
4- The CAM was finally stained with anti-bovine immunoglobulin
conjugated with fluorescein Isothiocyanate (diluted to 1:200).

110
5- Incubation at 37°C for 30 minutes (to allow the conjugation of labblled
antispecies antibody) with exposure to U.V light (for activation of
fluorescence dye).
6- The CAM was washed for 3 times by 1 X PBS.
7- Mounting by fine drop of buffered glycerin, then examined by
fluorescence microscope.

B- On inoculated MDBK cell

It was carried out on infected MDBK cells for detection of specific


fluorescence of LSDV as following:

1- Permanent cell line of MDBK cells were grown on glass cover slips in
Leighton tubes.
2- The culture tubes from which the growth medium had been discarded
were inoculated with 0.2 ml of each of two suspected isolates from the
third passage and incubated at 37ºC for one hour for virus adsorption.
3- After adsorption, 2 ml of medium supplied with 3% new born calf serum
was added to each tube and incubated at 37ºC.
4- After 18-24-hour post inoculation cell sheet showing 50% CPE were
used for IFAT.
5- The maintenance media was discarded and the cells attached to the cover
slips were washed three times with PBS and fixed in cold acetone ( -
200C), then acetone was discarded, air dryness, incubation at 4ºC/15 –
20 minutes.
6- The fixed cells were covered with 25µl of specific reference LSDV
antiserum, incubated at 370C for one hour.
7- The cover slips were washed 3 times in PBS (PH 7.4).
8- 25µl of anti-bovine immunoglobulin conjugated with fluorescein
Isothiocyanate stain diluted 1/200 were added to cover slips, incubated
for one hour at 37ºC.
9- The excess of the conjugate was discarded and cells were washed 3 times
with PBS, mounting, and examined under the fluorescence microscope
(Olympus- Tokyo- Japan) U.V microscope with 10X ocular and 25X
fluorite
and 40X achromatic under dark field). Negative control cover slips
consisted of non-infected MDBK cells were stained in the same way.

111
4.2.6. Molecular detection and characterization of Suspected LSDV
Isolate:

Molecular characterization of suspected isolated LSDV particles


was done using PCR according to Tuppurainen et al., (2005), using a
modified method.

4.2.6.1. Extraction of DNA:

Extraction of DNA of LSDV from tissue samples (S1 scab, S2 scab).


using DNeasy® Tissue Handbook kit, (Qiagen, USA) according the
instructions of the kit manufacturer, while extraction of DNA of LSDV
from prepared tissue samples suspensions (S1, CAM, embryo liver, cell
culture supernatant).

using QIAamp® DNA Mini and Blood Mini Handbook kit. (Qiagen,
USA) according the instructions of the kit manufacturer.

4.2.6.2. PCR:

DNA amplification was performed in 25 μl volume reaction mix as


follow:

Volume / Final
PCR reaction
reaction concentration
Qiagen® PCR Master mix. 12.5 μl 1X
Upstream primer 1 μl 10 pmol
Downstream primer 1 μl 10 pmol
DNA sample 5 μl 20-30 ng
DNA purity:1.4, 1.5, 1.6.

Nuclease free water Up to 25 μl

The thermal profile for primer 1 (LSDV1) was as following:

Stage Description Temp. Time


1 Denaturation 94 ᵒC 5 min
94 ᵒC 30 sec
Amplification
2 52 ᵒC 30 sec
40 cycles
72 ᵒC 30 min
112
3 Final extension 72 ᵒC 7 min

The thermal profile for primer 2 (LSDV2) was as follow:

Stage Description Temp. Time


1 Denaturation 94 ᵒC 5 min
94 ᵒC 30 sec
Amplification
2 52 ᵒC 30 sec
40 cycles
72 ᵒC 30 min
3 Final extension 72 ᵒC 7 min

The reaction was carried out in applied biosystem, Amp 9700 PCR
system, ramp speed 9700.

4.2.6.3. Analysis of a Polymerase Chain Reaction (PCR) product using

Agarose gel electrophoresis: E. Crandall/P.Barber, ProtocolGel - Agarose

Electrophoresis https://www.coursehero.com/file/7572635/ProtocolGel, IRELAND,

D.C. & BINEPAL, Y.S. 1998.

After the end of PCR run, the product was analyzed in agarose gel
electrophoresis using 1.5 % agarose gel in TAE buffer stained with
ethidium bromide as following:

1. Agarose powder 1.5 g

2. Tris acetic acid EDTA buffer 1X 100 ml

3. For a 1.5 % agarose gel, 1.5 gram of agarose (molecular biology grade)
was weighed in a flask with addition of 100 ml of TAE buffer.

4. Melting of the mixture at microwave, repeat heating and swirling


procedure every 10 seconds until mixture become clear with continuous
shaking and avoid gel boiling.

113
5. After complete melting (gel become clear), place 2µl ethidium bromide
(1µg/ml).

6. A slot former (comb) was placed in the gel box adjusted such that bottom
of the slots was approximately 1 millimeter above horizontal surface of
the gel box.
7. Pouring the melted gel in electrophoretic tray. (the amount of poured
gel differs according to tray size.

8. With care, remove the comb after the solution become gelled to obtain
the suitable wells.

9. the gel box filled with 1X TAE electrophoresis buffer such that the gel
was completely submerged.

10. The DNA samples were prepared 5 μl of DNA as PCR product and 3
μl of 5 X gel loading buffer (Ambion)® to obtain 1X loading dye.

11. DNA ladder 5 μl (Gel pilot 100 bp plus, ladder 100) as a molecular
marker was mixed with 3 μl of 5X gel loading buffer co-electrophoresed
with the DNA samples of PCR product.

12.Electrophoresis was performed using a submarine-type apparatus


(Biometra)® by running the gel at a constant voltage of approximately 106
V/cm, 155 amper for about 1 hour or until the bromophenol blue band has
migrated half way down the gel.

13. The gel was then examined using ultraviolet trans-illuminator at a wave
length of 590 nm and photographed.

14. Positive reactions were confirmed according to size of the amplification


product in comparison to the base pair marker.

4.2.7. Purification of PCR product and sequencing:

4.2.7.1. Purification of PCR product:

Purification of the PCR product of LSDV antigen attachment


protein gene was performed following the procedure described by the

114
manufacturer in QIAquick® PCR Purification Kit (Cat. No. 28104 and
28106),http://2012.igem.org/wiki/images/a/a3/QIAquick_PCR-
purification.pdf

Gel slices are dissolved in the buffer containing a pH indicator, allowing


easy determination of the optimal pH for DNA binding, and the mixture is
applied to the QIAquick spin column (see figure "pH Indicator Dye").
Nucleic acids adsorb to the silica membrane in the high-salt conditions
provided by the buffer. Impurities are washed away and pure DNA is eluted
with a small volume of low-salt buffer provided or water, ready to use in
all subsequent applications.

4.2.7.2. Gel electrophoresis of purified PCR product:

Gel electrophoresis of the purified PCR product was carried out on


1.5% agarose gel as mentioned before except volume of sample in the gel
well is 2µl.

4.2.7.3. Cycle Sequencing:

Sequencing of the purified product of the PCR reaction of the


amplified gene of LSDV (attachment protein gene) was performed
following the procedure described by the manufacturer in Big Dye®
Terminator v3.1 Cycle Sequencing Kit.

DNA sequencing was performed in 10 μl volume reaction mix as


following:

Component Volume
Big Dye Terminator 2 µl
5x Sequencing Buffer 2µl
Forward primer (3.2 pmol) 1µl
Template (20 ng) 1µl
Nuclease Free Water Up to 10 µl

115
Control pGEM protocol in 20 μl volume reaction mix as follow:

Component Volume
Big Dye Terminator 4 µl
5x Sequencing Buffer 4 µl
Control Primer (3.2 pmol) 4 µl
Control Template (20 ng) 1 µl
Nuclease Free Water Up to 20 µl
Total 20 µl

Thermal Profile:

Stage Description Temp. Time


1 Denaturation 96ºC 1 min
96ºC 10 sec
Amplification
2 52ºC 5 sec
25 cycles
60ºC 4 min
3 Holding 4ºC Pause
was performed using applied biosystem, Amp 9600 PCR system, Ramp
Speed must be slow (1ºC /sec.).

4.2.7.4. Purification after cycle sequence before injection:

Purification of the product of PCR amplified gene of LSDV antigen


binding protein was performed after cycle sequencing before injection
following the

procedure described by the manufacturer in BigDye® X Terminator™


Purification Kit. Kit Contents: BigDye® X Terminator™ Solution and
SAM™ Solution.

4.2.7.5. Injection of the sample into the sequencer:

Loading the sample into 96 well- plate with septa was done as
following:

1- In one well, place 10 µl Hi- Di™ formamide + 20µl sample.

116
2- In another well, place 10 µl Hi- Di™ formamide + 20µl standard
sequencing as positive control.

3-Place the plate in PCR device (applied biosystem, Amp 9600 PCR
system) for denaturation step 96ºC for 5 min, then placed on
crushed ice to avoid renaturation.

4- Place the plate in the sequencer.

5- Run the device program software.

4.2.8. Computer-assisted sequence and phylogenetic analyses (


according to Thompson et al., 1994),
4.2.8.1. BLAST and PSI-BLAST search programs: Alignment and
calculation of homology between sequences of the isolates of LSDV and
other published LSDV sequences in Gene Bank data base at National
Center for Biotechnology Information(NCBI)on
line.https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch.

4.2.8.2. Clustal W and DNA star mega: It is program for sequence


alignment and phylogenetic analysis of the nucleotide and amino acid
sequences of the isolates of LSDV and other published LSDV sequences
in Gene Bank data base at National Center for Biotechnology Information
(NCBI).

117
118
5-RESULTS

5.1. Isolation of LSD virus from Skin Nodule (scab) Samples on


Specific Pathogen Free Embryonated Chicken Eggs (SPF-ECE):

Two skin lesion samples; S1 and S2 from clinically suspected Cattle


showing signs of disease at Qaliubiya province, Egypt were inoculated in
SPF ECE via CAM route. It was found that 2 samples induced lesion on
CAMs by the 1st passage and signs on the embryos by the 2nd passage and
these changes continued till the 3rd passage. BY examination of the
collected chorio-allantoic membranes a hemorrhagic membrane with
congestion and clotted blood in blood vessels appeared by the 1st passage
then pock lesions were detected in the form of stretched white line, which
became more pronounced after 6 days of inoculation at 2nd and 3rd passages.
The dead embryos were hemorrhagic, edematous, with enlarged and
bloody liver and clotted blood inside the heart as shown in

(table_ 6) and (photo_2 & 3 ).

119
(Table _ 6 ): Alterations on CAM and Embryo of SPF-ECE inoculated

with two suspected skin samples for LSD virus.


Passage No. S1 S2
CAM Embryo CAM Embryo
Hemorrhagic Normal Hemorrhagic Normal
and and
congested
First congested
Second Congested Death, Pock lesions Normal
with clotted hemorrhage, in the form of
blood inside edema with stretched
blood vessels enlarged white line
bloody liver
and clotted
blood inside
the heart with
slight
hypertrophy
Third Congested Death, Pock lesions Normal
with clotted hemorrhage, in the form of
blood inside edema with stretched
blood vessels
enlarged white line
bloody liver
and clotted
blood inside
the heart with
slight
hypertrophy

The signs were observed 6days post-inoculation, S1: first skin suspected viral sample,
S2: second skin suspected viral sample.
120
A

B
C

(Photo_2): Characteristic lesions of two suspected skin samples (S1&S2)


st nd rd
for LSD virus on CAM of SPF-ECE after 1 (A), 2 (B) and 3 (C) passages
where the left (control), the middle (S1) and the right (S2). The black
arrows refer to lesion.

121
A

(Photo_3): Characteristic signs of two suspected skin samples (S1&S2) for


nd
LSD virus on embryo of SPF ECE where the (A) and (B) were the 2 and
rd
3 passages of S1 respectively, while S2 resembling the negative control
(C).
122
5.2. Propagation of suspected LSD virus isolate on MDBK cells:

nd
The positive CAM of the 2 passage of suspected samples was

prepared and inoculated on MDBK cell line. It was observed that infected

cell culture was characterized by cell rounding, cell aggregation, coalesce

together to form clusters that scattered all over the monolayer within

72hr post inoculation and gradually increased till 70-80 % of sheet was

completely detached. These findings were demonstrated in (photo_5) as

compared with normal control monolayer demonstrated in (photo _4).

(Photo_ 4): unstained (left, 20X) and stained (H&E, right, 40x) Control
non- infected complete sheet of MDBK cells.

123
(Photo_5): characteristic CPE of suspected LSD virus isolate 72hrs post
inoculation on MDBK cells in the form of rounding, coalescing and sheet
detachment. The left (low power 10X), and the right (high power 20X).

124
(Table_ 7): Characteristics of suspected LSD virus isolate strain on SPF-
ECE and MDBK cell line.

Suspected LSD SPF-ECE MDBK


virus isolate
strain CAM Embryo
Congested with Death, Rounding,
clotted blood haemorrhage, coalescing and
inside blood edema with sheet detachment
vessels and Pock enlarged and within 72hr post
lesions in the bloody liver and inoculation
form of stretched clotted blood
white line inside the heart
with slight
hypertrophy

125
5. 3. Non Serological identification of suspected LSD virus

isolates:

5.3.1. Histopathological examination of intracytoplasmic inclusions:

Histopathological examination of inoculated CAM with suspected

LSD viral samples showed slight proliferation in the ectodermal and

mesodermal cell layers with large eosinophilic intracytoplasmic inclusion

bodies characteristic for poxviridae

(photo _6).

(Photo_6): stained inoculated CAM with suspected LSD viral samples. The
arrows refer to eosinophilic intracytoplasmic inclusions in ectodermal
cell and mesodermal cell layers (H&E_ 100X, oil emersion lens).

126
5.3.2. Transmission Electron Microscopy (TEM):
The electron microscopic examination of inoculated CAM with
suspected LSD viral samples revealed, that the infected cells contained

few virus particles that appeared ovoid in shape, with rounded ends.

high aggregates of viral proteins were present as inclusions in the cell

cytoplasm. The characteristic virus particle was released from the cellular

membrane by budding as shown in (photo_7).

127
1
2

3
4

(Photo_7): Electron micrographs of inoculated CAM with suspected LSD


viral sample Stained with uranyl acetate and lead citrate. The arrows refer
to budding of virus particle from the membrane (plasma and Golgi
membranes 1,2). Note the aggregation of viral inclusion bodies (3). The
virus particles appeared as ovoid in shape, with rounded ends (4).
128
5.3.3. Haemagglutination (HA) property:

Examination of HA property of suspected LSD viral isolate for cattle,

sheep, goat and chicken RBCs cleared that the isolate was non HA produce

button shape resembling to the negative control sample as shown in

(photo_8).

RBCS Control - Viral


sample
contr ve

(Photo_8): HA micro-well plate with negative results of HA property


(button shapes) of LSD virus isolate compared with negative control.

129
5. 4. Serological identification of suspected LSD virus isolate

using IFAT:

Indirect FAT was adapted for identification of LSD virus protein in

infected CAM and infected MDBK monolayer with CAM and embryo liver

and heart suspensions using specific hyper immune serum against LSD

virus. It was observed, that specific intracytoplasmic yellowish green

fluorescent granules emitted from the infected CAM (S1) (photo_9) and

infected MDBK cells with CAM suspension (S1), from the infected MDBK

cells with embryo liver and heart suspensions (S1) and infected MDBK

cells with CAM suspension (S2). (photo_10).

130
(Photo_9): Stained infected S1 CAM (the upper, 4X) with FITC. Notice
intracytoplasmic apple green fluorescence emission as a positive result
for local LSD virus isolate.

131
(Photo_10): Stained infected MDBK cells with FITC after 72 hours post
inoculation (S2 CAM suspension). Notice intracytoplasmic apple green
fluorescence emission as positive result for local LSD virus isolate (20X).

132
5.5. Molecular identification of the recent local LSD virus isolate from
cattle at Qaliubiya province, Egypt:

Using Conventional Polymerase Chain Reaction (PCR):

Two types of primer pairs were used for detection of the local LSDV

strain in original skin samples (tissue & suspensions), infected CAM and

embryo liver suspensions. The first primer (primer pair 1) targeted the

LSDV attachment protein encoding gene was failed to amplify the specific

products 172bp from the extracted DNA products using PCR and dimers

appeared. The second primer (primer pair 2) targeted the LSDV envelope

protein-like gene was succeeded to amplify the specific products 137bp

from the extracted DNA products using PCR and the bands were

ambigious.

The amplified products 137bp with the second primer pair were

clear and sharp by increasing the DNA concentration from target (photo_

11).

The resulted fragments were further purified and analyzed by gel


documentation system for sequencing process and showed that three
sharp bands had the sizes 137bp and two faint bands 137bp.

133
1 2 3 4 5
L

100bp

Faint
bands

(Photo_11): Electrophoresis of the amplified products 137bp of the


envelope protein-like gene for local LSDV strain from different sources
using the second primer after target concentration. Lane L: High
molecular weight nucleic acid marker (100bp), Lane 1: S1 scab
suspension, Lane 2: S1 CAM suspension, Lane 3: embryo liver suspension,
Lane 4: S1 scab tissue, Lane 5: S2 scab tissue.

134
5.6. Partial Sequencing and Genbank submission of the envelope
protein-like gene for the recent local LSD virus strain from different
sources:

The purified PCR products for the local LSDV strain from different
sources were sequenced using automatic DNA sequencer. Analyzed
sample data is displayed as an electropherogram, a sequence of peaks in
four colors. Each color represents the base called for that peak. The fasta
format for sequence were prepared and published in Genbank with their
name and accession numbers; LSDV-Elkady-2014 (KU760905) from
original skin suspension (S1), LSDV- Elkady-3-2014 (KX236312) from
skin scab tissue (S1), LSDV-Elkady-4-2014(KX236313) from skin scab
tissue(S2), LSDV-Elkady-2-2014 (KX236311) from CAM
suspension(S1), LSDV-Elkady-5-2014 (KX250367) from CAM
suspension(S1)(repeated), LSDV-Elkady-1-2014 (KX236310) from
embryo liver of SPF-ECE (fig 6& 7). The electropherogram data looks
mostly blank for CAM (Dirty sequence) so repeated and both were
analyzed.

135
LSDV-Elkady-2014 (Acc.no .KU760905) from original skin suspension (S1)

GGTGTAAATTTTTCAGAATTATTTTTCTGGATTATGTAATGCTCTTTGTACAAAAGAGGCA
AAAAGTTCTATTGCGAAACACTTTAGTTTATGGAAATCGTATGCCGATGCGA

LSDV- Elkady-3-2014 (Acc.no.KX236312) from skin scab tissue (S1)

TTTCCAGGAAATATTTTTTTCTGGATTATGTAATGCTCTTTGTACAAAAGAGGCAAAAAGT
TCTATTGCGAAACACTTTAGTTTATGGAAATCGTATGCCGATGCGAA

LSDV-Elkady-4-2014(Acc.no.KX236313) from skin scab tissue (S2)

AAATTTTTTCTGGATTATGTAATGCTCTTTGTACAAAAGAGGCAAAAAGTTCTATTGCGAA
ACACTTTAGTTTATGGAAATCGTATGCCGATGCGAA
(Figure_ 6): The partial sequence profile of LSDV envelope protein-like
gene detected in the skin suspension, S1 and S2 samples. Note the name
and accession no. for the strain. The electropherogram showing data
analyzed with the KB™ basecaller and the fasta format for each
sequence.

136
LSDV-Elkady-2-2014 (Acc.no.KX236311) from S1 CAM suspension

GGGGGCCCGAAACGATTTTCAGGATATGTAATGCTCTTTGTACAAAAGAGGCAAAAAGTTCT
ATTGCGAAACACTTTAGTTTATGGAAATCGTATGCCGATGCG

LSDV-Elkady-5-2014 (Acc. no. KX250367) from S1 CAM suspension (repeated)

GCTGGTTAGAAGGTAAATTTCTGGATTATGTAATGCTCTTTGTACAAAAGAGGCAAAAAGTT
CTATTGCGAAACACTTTAGTTTATGGAAATCGTATGCCGATGCGA

LSDV-Elkady-1-2014 (Acc. no. KX236310) from embryo liver suspension

GCCGGCCCAAAAGGGTAAATTATTCTGGATTATGTAATGCTCTTTGTACAAAAGAGGCAAAA
AGTTCTATTGCGAAACACTTTAGTTTATGGAAATCGTATGCCGATGCGA
(Figure_ 7): The partial sequence profile of LSDV envelope protein-like
gene detected in CAM and embryo liver for our strain. The
electropherogram showing data analyzed with the KB™ basecaller and
data looks mostly blank for CAM (Dirty sequence) so repeated. Note the
name, accession no. and the sequence fasta format for the strain.

137
5.7. Partial Sequence analysis and alignment of the envelope
protein-like gene for the recent local LSD virus strain from different
sources:

The partial sequence analysis and alignment report of the envelope


protein-like gene for the recent local LSD virus strain from different
sources using Clustal W showed high nucleotide similarity of the strain in
their different sources with identity percent from 98 to 100%. The isolate
sources (CAM and the embryo source) show 100% and 98.9% homology
with their skin original samples; respectively. The embryo source shows
98.9% homology with CAM source (fig 8& 9)

The phylogenetic tree was constructed to calculate and examine the


evolutionary relationships of the sequences, in which the length of the
horizontal line connecting one sequence to another is proportional to the
estimated genetic distance between the sequences (fig 10).

138
Figure (8): alignment report of partial nucleotide sequences for the envelope
protein-like gene of local LSDV strain from different sources.

Figure (9): pair wise sequence distance for the envelope protein-like gene of local
LSDV strain from different sources.

Figure (10): phylogenetic tree of local LSDV strain from different sources based
on partial nucleotide sequence of the envelope protein-like gene.

139
5.8. Partial Sequence analysis and alignment of the envelope protein-
like gene for the recent local LSD virus strain from different sources
with the reference LSDV, Sheeppox virus and goatpox virus published
in Genbank :

The partial sequence of the envelope protein-like gene for the recent local
LSD virus strain from different sources with the reference LSDV,
Sheeppox virus and goatpox virus published in Genbank were analyzed
using Clustal W. The alignment report and the pair-wise table were showed
high nucleotide similarity of the strain in their different sources with
LSDV- NI-2490 isolate Neethling 2490 (AF325528), LSDV/ NW/LW
isolate Neethling Warmbaths LW (AF409137),
LSDV/2/Slemani/Kurdistan/2014 envelope (KM047051) and LSDV-B4
envelope protein (P32) gene (KT253438), then other LSDV strains and
followed by Sheeppox and goatpox viruses.

Both LSDV- NI-2490 isolate Neethling 2490 (AF325528) and LSDV/


NW/LW isolate Neethling Warmbaths LW (AF409137) revealed percent
homology of 100% with LSDV-Elkady-5-2014 (KX250367) , 94.3% with
LSDV-Elkady-2014 (KU760905) and 98.9% with other strain sources
include, LSDV-Elkady-1-2014(KX236310), Elkady-2-2014 (KX236311),
LSDV-Elkady-3-2014(KX236312)and LSDV-Elkady-4-
2014(KX236313).TheLSDV/2/Slemani/Kurdistan/2014 (KM047051)
revealed percent homology of 100% with LSDV-Elkady-5-2014
(KX250367), 93.7% with LSDV-Elkady-2014 (KU760905) and 98.9%
with other strain sources, while LSDV-B4 (KT253438) showed percent
homology of 100% with LSDV-Elkady-5-2014 (KX250367), 93.3% with
LSDV-Elkady-2014 (KU760905) and 98.7% with other strain sources (fig
11& 12).

140
Figure (11): Alignment report of partial nucleotide sequences for the envelope protein-
like gene of local LSDV strain from different sources with the reference LSDV,
Sheeppox virus and goatpox virus published in Genbank.

141
Figure (12): pair wise sequence distance for the envelope protein-like gene of local
LSDV strain from different sources with the reference LSDV, Sheeppox virus and
goatpox virus published in Genbank.

142
5.9. Phylogenetic analysis of the recent local LSD virus strain
from different sources with the reference LSDV, Sheeppox virus and
goatpox virus published in Genbank:

clustal W analysis of the recent local LSD virus strain from different
sources and other published LSDV, Sheeppox virus and goatpox virus
strains in Genbank was performed based on the partial nucleotide sequence
of the envelope protein-like gene as shown in figure (13).
The phylogenetic tree was constructed to calculate and examine the
evolutionary relationships of the sequences, in which the length of the
horizontal line connecting one sequence to another is proportional to the
estimated genetic distance between the sequences. The analysis can
distinguish between viruses of LSD, sheep pox and goat pox viruses are
grouped separately, even though our strain resources were related to each
other and to the other LSDV include, LSDV- NI-2490
(AF325528),LSDV/
NW/LW(AF409137),LSDV/2/Slemani/Kurdistan/
2014(KM047051) and LSDV-B4 (KT253438). Our strain seems to be
related more closely to LSD virus than to sheep pox virus and goatpox
viruses.

143
Figure (13): phylogenetic tree of local LSDV strain from different sources with
the reference LSDV, Sheep pox virus and goat pox virus published in Genbank
based on partial nucleotide sequence of the envelope protein-like gene.

144
145
6- DISCUSSION, CONCLUSION AND
RECOMMENDATION

Lumpy skin disease virus (LSDV), a member of the genus


Capripoxvirus, belongs to family poxviridae with typical poxvirus
geomorphology and closely related to the viruses of sheep and goat pox
(MacLachlan and Dubovi, 2011).

The genome is a150,000bp long double-stranded DNA, covalently


cross-linked at the ends, similarly to other pox viruses. Capripox virus
genomes sequences are highly conserved and there is more than 95%
homology amongst LSD, sheep pox and goat pox viruses (Kara et al.,
2003).

It causes an acute, subacute or chronic disease of cattle,


characterized by fever and the formation of multiple firm, circumscribed
nodules in the skin of affected animals and necrotic plaques in the mucous
membranes, as well as generalized lymphadenopathy (Buller et al., 2006).
It was transmitted by biting insects, especially blood-feeding insects, such
as the mosquito (Chihota et al., 2001), and there is a real danger that LSDV
could spread from Africa (Egypt) (House et al., 1990) into Asia and
Europe through the Middle East and vice versa. The Control of LSDV is
achieved in endemic countries through the use of attenuated live virus
vaccines (Brenner et al., 1992).

In Egypt LSD was appeared first among imported and native breed
of cattle in Suez at 1989, the disease was spread rapidly and diagnosed after
El-tall El-Kbeer (Ismailia) out break (House et al, 1990).

Reappearance of LSD was observed by EL-Bagoury et al., 1995 during


Minia outbreak. Then during late summer and autumn of 2006 in different
Egyptian governorates (Stram et al,2008), and during 2010 in cattle and
buffaloes at Qaliubiya province (El-Nahas et al,2011).

In the present study a massive outbreak of suspected LSD in cattle


population were observed during 2013 and 2014 at Qaliubiya province.

146
Because, it is clear that there are many diseases causing similar
signs like pseudo lumpy skin disease, dermodicosis, oncocercasiosis,
insect bite allergies, BVD, bovine malignant catarrhal fever, Rinderpest
(Alexander et al,1957 and weiss1968), it is important to obtain a definite
diagnosis to ensure the best preventive and control measures for the herd.
The present study concerned with the trial for isolation of LSD virus from
skin lesions of cattle from Qaliubiya province, Egypt on CAM of SPF-
ECE and MDBK cell line with further identification by non -serological
and serological means as well as advanced molecular characterization of
virus isolate using PCR and sequencing with establishment of a
phylogenetic tree between the local LSDV strain from different sources,
sheep pox and goat pox viruses.

Knowledge of capripoxvirus pathogenesis demonstrates that skin lesions


are the most useful samples for virus isolation (Bowden et al., 2008).

Our field isolate was able to replicate in CAMs and Embryos of SPF-
ECE and MDBK cell line. The CAMs of S1 were hemorrhagic with clotted
blood in blood vessels appeared by the 1st passage at the last day of
incubation (the 7th day of inoculation), and become more pronounced after
the 6th day of inoculation within the 2nd and 3rd passage, moreover the
embryo died at the 2nd and 3rd passage at the 6th day of inoculation, the dead
embryos were hemorrhagic, edematous, with enlarged and bloody liver
(hepatomegaly) and clotted blood inside the heart, with slight hypertrophy
( this may be due to the virus affects the endothelial cells), whereas the
CAMs of S2 were hemorrhagic at the 1st passage at the 9th day of
inoculation, while at the 2nd passage pock lesion were detected in the form
of stretched white line (this line may be due to the cell to cell transfer of
the virus) at the 8th day of inoculation, and become more pronounced at the
3rd passage (8th day of inoculation), while the embryos of S2 not died at
any of the three passages.

The development of lesion on CAM was firstly observed by Alexander


et al., 1957 and Van Rooyen et al., 1969 and varied from thickening and
congestion (Badr et al,2008; El-Kenawy and El- Tholoth,2011) to
clearly visible pock lesion (Woods, 1988; House et al., 1990) that
appeared like numerous, small, scattered white foci (El-Nahas et al,2011).
Characteristic pock lesions were observed after 1st passage and become
clear after 3rd passage (Tamam,2006) and become clearly observed 4days
post inoculation (Abdel-Rahim et al. 2002; El-Desawy et al, 2008).

147
Sequential detection of lesion on CAM by El-Kenawy and El-
Tholoth,2011, at day 1, small hemorrhagic areas are seen at site of
inoculation on CAM (non-specific), at day 2, thickening of the membrane
and become congested and hemorrhagic, at day 3, white and opaque area
around site of inoculation, at day 4, this white and opaque area increase in
size, at day 5, opaque, pin point pock lesions arranged in streaks, at day 6,
opaque pin point pock lesions arranged in streaks. The maximum yields of
LSDV in CAM were 5 to 6 days (Van Rooyen et al., 1969, El-Nahas et
al, 2011).

The cell rounding, aggregation, coalescing and detachment within


72hr post inoculation of our LSDV isolate on MDBK cell line confirm
the susceptibility of our isolate to this cell line. For Capripoxviruses
isolation, the recommended cells are primary or secondary culture lamb
testis or kidney cells. As an alternative, bovine cells can also be used. The
cytopathic effect (CPE) of the virus on the cells can be seen in 1 week,
although this can be up to 2–3 weeks, with several blind passages needed
(Binepal et al., 2001). Vero cells are less sensitive to capripox virus
(Bhanuprakash et al., 2006). The South African LSDV (type SA-
Neethling) vaccine strain could passaged on MDBK cells and foetal bovine
testes (FBT) cells (VanRooyen et al., 1969). The LSDV Neethling strain
2490 (LK) was originally isolated in lamb testes (LT) cells (Tulman et al.,
2001). In study by Wallace and Viljoen 2002 they demonstrated that both
wild type and lacZ recombinant of LSD virus grew to high levels in normal
MDBK cells. The most susceptible cell culture to the local Egyptian
isolates of LSDV were MDBK cells compared to EBL and CER cells as
sequentially detected by CPE and infectivity titration (El-Bagoury et al.,
1995). Also most Egyptian LSDV strains were successfully isolated on
MDBK cells (Ahmed et al., 2005; El-Nahas et al, 2011).

The surprise by our isolate was its signs on the Embryo of SPF-ECE in
addition to its lesion on CAM and CPE on the MDBK cells indicating a
new biological characteristic not observed by other Egyptian LSDV
strains. This characteristic could be expected as LSDV encodes five
proteins containing ankyrin repeat motifs that have been associated with
host range functions and inhibit virally induced apoptosis (Mossman et
al., 1996; Shchelkunov et al., 1998; Tulman et al., 2001).

Characteristic eosinophilic intracytoplasmic inclusion bodies were


detected in the ectodermal and mesodermal cell layers of stained CAM by
hematoxylin & eosin that infected with suspected LSD viral sample. These
inclusions were indicative for the presence of LSDV as a member of
148
capripoxviruses replicating in the cytoplasm and could differentiate from
pseudo lumpy skin disease, caused by bovine herpesvirus 2 causing
intranuclear inclusion bodies (MacLachlan and Dubovi, 2011). The
intracytoplasmic inclusion bodies characteristic for LSDV was described
in infected monolayers stained with hematoxylin & eosin or hematoxylin-
phloxin (Thomas and Mare, 1945) in histological sections of the skin
lesions of animals suffering from LSD (Burdin, 1959) and in trypsenized
cell of infected CAM with LSDV (Tamam 2006).

Our isolate appeared ovoid in shape, with rounded ends by transmission


electron microscope with electron dense inclusion in the cell cytoplasm
and released particles through budding indicate Capripoxviruses. All
virions of Capripoxviruses have an ovoid shape with an average size of
294×273 nm (Kitching and Smale, 1986, Woods 1988). This structure
was observed in negatively stained skin biopsies for 33day post infection
(Tuppurainen et al. (2005), in inoculated cell cultures with LSDV and the
virus size was found to range from 300 - 350 nm with crescent or ovoid
shape (Ahmed and Kawther 2008) and in inoculated CAM (El-Nahas et
al, 2011). The histopathology and transmission electron microscopy
confirm LSDV detection (Tageldin et al., 2014) and help in the
differentiation between LSD from pseudo-LSD (MacLachlan and
Dubovi, 2011).

The non haemagglutinating property of our isolate emphasized the result


of Matthews, 1982, who mentioned that Capripoxviruses are non-
haemagglutinating. Though all the haemagglutination tests consist
basically of an indirect haemagglutination test (Tiwari et al., 1996; Rao et
al., 1997).

Capripoxvirus isolation can be confirmed by immunostaining using


anti-capripoxvirus serum (Gulbahar et al., 2006; Babiuk et al., 2007).
The OIE (2004) recommended serological tests used for LSD diagnosis
are IFAT, ELISA and VNT. Gari et al. (2008) demonstrated that IFAT has
high accuracy to be used for the diagnosis and sero-surveillance analysis
of LSD in the target population. The IFAT detect the LSDV antigen in the
skin and internal organs of naturally infected cattle (El-Kenawy and El-
Tholoth, 2011). Examinations using FAT may indicate the presence of
LSD virus antigens especially in the early stages of the
disease (Davies et al., 1971). However, it is not possible to differentiate
the different members of the capripoxvirus group with the direct or indirect
FAT (Kitching et al., 1986), show brilliant, stippled cytoplasmic staining
of virus-infected cells 24hr and 72hr post infection. This bright greenish
149
yellow fluorescence was observed on CAM (House et al., 1990) and in
lamb testis cell cultures after 48 hours of inoculation (Davies et al., 1971).
A sterile fluorescein conjugated anti-LSDV IgG with dilution 1/20 was
prepared to detect LSDV in the MDBK cells using IFAT ( Razek, et al.,
2009). Two local isolates of LSDV were successfully identified by IFAT
using reference anti-LSDV hyper immune serum. Sequential detection of
a specific intracytoplasmic and membranous fluorescence was developed
in infected cells as early as 12hrs PI, become more intense and diffused at
48hr and 72hr PI (El-Bagoury et al., 1995). Several passages of LSDV on
SPF-ECE and MDBK cells were identified by IFAT (Ahmed et al., 2005;
Tuppurainen et al.,2005; El-Nahas et al, 2011).

LSD, goat pox and sheep pox viruses are serologically identical, and
so their specific identification relies exclusively on the use of molecular
tools (Le Goff et al 2009). In addition, SPPV and GTPV are extremely
host specific (Rao and Bandyopadhyay, 2000), but in some countries
both viruses can cross infect sheep and goats, which poses a problem in
diagnosis. Although recent molecular studies suggest that the
Capripoxvirus genus including SPPV, GTPV and LSDV are very similar
in terms of antigenic characteristics, these viruses are phylogenetically
similar and can be differentiated by accurate molecular techniques
(Bhanuprakash et al., 2006).

In our study, two types of primer pairs, the first targeted the LSDV
attachment protein gene was failed to amplify the specific products (172bp)
from the extracted DNA products using PCR and primer dimers appeared.
The second primer pair targeted the LSDV envelope protein-
like gene was succeeded to amplify the specific products (113bp-104(107)-
111-108-97 from the extracted DNA products and the bands were clear and
sharp by increasing the DNA concentration from target. The first primer
pair was used by Ahmed and Zaher, (2008) and El-Kholy et al. (2008)
who succeed to obtain the specific products from the original skin sample
and other isolate resources. The failed primer pair in our study may be
related to badly defined primers, incorrect primer specificity due to our
strain differs from the existing strain or needs for optimal reaction
conditions rather than mentioned by the author. The second primer pair was
sensitive to detect LSDV strain in its original skin samples and their
resources.

150
The PCR was a specific assay for specific detection of LSD virus in skin
lesion (Stram et al. 2008, El-Nahas et al, 2011), CAMs and cell culture
(El-Kholy et al. 2008, El-Kenawy and El-Tholoth, 2011), semen (Bagla
et al. 2006) and blood and skin samples (Tuppurainen et al., 2005).

The local LSDV strain from different sources were sequenced and
published in Genbank with their designations as; LSDV-Elkady-2014
(KU760905) from original skin scab suspension (S1), LSDV- Elkady-3-
2014 (KX236312)from skin scab tissue1(S1),LSDV-Elkady-4-
2014(KX236313) from skin scab tissue2(S2),LSDV-Elkady-2-2014
(KX236311) from CAM suspension (S1),LSDV-Elkady-5-2014
(KX250367) from CAM suspension (S1) (repeated), LSDV-Elkady-1-
2014 (KX236310) from embryo liver suspension of SPF-ECE. The
electropherogram data looks mostly blank for CAM suspension (Dirty
sequence), so repeated and both were analyzed.

Clustal W analysis for the recent local LSD virus strain from
different sources based on the envelope protein-like gene showed 98 to
100% identity. The isolate sources (CAM and the embryo sources) show
100% and 98.9% homology with their skin original samples respectively.
The embryo source shows 98.9% homology with CAM source. This
homology emphasis the theory of Kara et al., (2003) mentioned that
Capripoxvirus genomes sequences are highly conserved and there is more
than 95% homology amongst LSD, sheep pox and goat pox viruses and
Tulman et al. (2001) that recorded extremely conserved Capripoxvirus
isolates genome with identities of at least 96%. The high similarities
between resources indicate the low effect of passage in the mutational
process.

High nucleotide similarity of the strain in their different sources were


showed with LSDV- NI-2490 isolate Neethling 2490 (AF325528), LSDV/
NW/LW isolate Neethling Warmbaths LW (AF409137),
LSDV/2/Slemani/Kurdistan/2014 envelope (KM047051) and LSDV-B4
envelope protein (P32) gene (KT253438). Both LSDV- NI-2490 isolate
Neethling 2490 (AF325528) and LSDV/ NW/LW isolate Neethling
Warmbaths LW (AF409137) revealed percent homology of 100% with
LSDV-Elkady-5-2014 (KX250367) , 94.3% with LSDV-Elkady-2014
(KU760905) and 98.9% with other strain sources include, LSDV-Elkady-

151
1-2014 (KX236310), Elkady-2-2014 (KX236311), LSDV-Elkady-3-2014
(KX236312) and LSDV-Elkady-4-2014(KX236313). The
LSDV/2/Slemani/Kurdistan/2014 (KM047051) revealed percent
homology of 100% with LSDV-Elkady-5-2014 (KX250367), 93.7% with
LSDV-Elkady-2014 (KU760905) and 98.9% with other strain sources,
while LSDV-B4 (KT253438) showed percent homology of 100% with
LSDV-Elkady-5-2014 (KX250367), 93.3% with LSDV-Elkady-2014
(KU760905) and 98.7% with other strain sources. This report was
indicative by House et al., (1990); Al-Salihi and Hassan;
(2015); Rashid et al, (2016) who cleared that LSD of cattle was caused
by a capripox virus and was closely related to the viruses which caused
sheep and goat pox. Their isolate was related to The Neethling strain which
is the original prototype for the disease.

Our phylogenetic tree grouped viruses of LSD, sheep pox and goat pox
viruses separately, even though our strain resources were related to each
other and to the other LSDV include, LSDV- NI-2490 (AF325528),
LSDV/ NW/LW (AF409137), LSDV/2/Slemani/Kurdistan/2014
(KM047051) and LSDV-B4 (KT253438). Our strain seems to be related
more closely to LSD virus than to sheep pox virus and goatpox viruses.
The phylogenetic plot constructed by MAFFT version 7 revealed and
emphasis the high similarities in sequence between our LSDV strain
resources and reverse strand for both LSDV- NI-2490 (AF325528) and
LSDV/ NW/LW (AF409137). Hosamani et al., (2004) clustered SPPV,
GTPV and LSDV into host species-specific groups. Phylogenetic analysis
of a 466-bp fragment next to the genome ends showed that this system can
distinguish between: sheep pox, goat pox and LSD viruses and the Israeli
isolates from 2006 and 2007 were in the same clad and essentially identical
the Kenya NI-2490 isolate, and the South African LD virulent isolate
(Stram et al., 2008). All Kurdistan LSDV strains in clustered in one
lineage and releated to The Neethling strain. LSDV/2/Slemani/Kurdistan/
2014 was separated from the other LSDV isolates and it formed a different
subcluster Rashid et al, (2016).

In conclusion, there may be a new LSDV isolate in Egypt, which uniquely


shared the same characteristic sequence (partial sequence about 113bp)
with LSDV- NI-2490 Neethling isolate and
LSDV/2/Slemani/Kurdistan/2014 and differ in biological properties in
embryonated eggs. So further molecular sequencing for more specific
sufficient target were needed and cross neutralization using monoclonal
antibodies between our strain and the neethling type is recommended.

152
153
7- SUMMARY

During 2014, suspected outbreaks of LSDV were reported in Qaliubia


province, where animals suffered from severe signs differ from the
previous outbreaks, such as suffocation and deaths, so the present study
aimed to make a trial for isolation of LSD virus from skin nodule samples
with further identification using serological, non serological and molecular
techniques.

The practical study revealed that:


1. Trials for isolation of LSD virus from skin scabs samples of clinically
suspected cattle by three blind passages through CAM showed
characteristic pock lesion on CAM and dead embryos, which were
hemorrhagic, edematous, with hepatomegaly and clotted blood inside the
heart, which became more pronounced after 6 days of inoculation at 2nd
and 3rd passages.

2. Trials for isolation of LSD virus from infected CAM by three blind
passages through on MDBK cell culture showed rounding, cell
aggregation, coalesce together to form clusters that scattered all over the
monolayer within 72hr post inoculation and gradually increased till 70-80
% of sheet was completely detached.

3- Histopathological examination of inoculated CAM with suspected LSD


viral samples showed slight proliferation in the ectodermal and
mesodermal cell layers with large eosinophilic intracytoplasmic inclusion
bodies characteristic for poxviridae.

4-The electron microscopic examination of inoculated CAM with


suspected LSD viral samples revealed that the infected cells contained few
virus particles that appeared ovoid in shape, with rounded ends.

5- Examination of HA property of suspected LSD viral isolate for cattle,


sheep, goat and chicken RBCs cleared that the isolate was non HA.

154
6-Serological identification of suspected LSDV isolate on CAM and
infected MDBK monolayer revealed specific intracytoplasmic yellowish
green fluorescence emission.

7- Molecular identification of LSDV isolate from original skin samples,


infected CAM and embryo liver suspensions using PCR, an amplified
specific size products of 137 were obtained using envelope protein-like
gene targeting primer.

8-Sequencing of purified PCR products for LSDV isolate were published


in GenBank with their names and accession numbers; LSDV-Elkady-2014
(KU760905), LSDV- Elkady-3-2014 (KX236312), LSDV-Elkady-4-
2014(KX236313), LSDV-Elkady-2-2014 (KX236311), LSDV-Elkady-5-
2014 (KX250367), LSDV-Elkady-1-2014 (KX236310).

9- Phylogenetic analysis of our published sequence with other published


LSDV, sheep pox and goat pox on GenBank revealed that the recent local
LSD virus strain from different sources showed high nucleotide similarities
from 98 to 100%. Our strain was closely related to LSD virus than to sheep
pox and goat pox viruses.

155
156
8- REFERENCES

1- 2012.igem.org/wiki/images/a/a3/QIAquick_PCR-purification.pdf.

2- A. A. El-Kenawy, M. S. El-Tholoth, 2010. Sequence analysis of


attachment gene of lumpy skin disease and sheep poxviruses, Virologica
Sinica, Volume 25, pp 409-416.

3- A.A.EL-Kenawy and M.S. EL-Tholoth, Department of Virology,


Faculty of Veterinary Medicine, Mansoura University, Egypt, 2011.
Lumpy Skin Disease Virus Identification in Different Tissues of
Naturally Infected Cattle and Chorioallantoic Membrane of Embryonated
Chicken Eggs Using Immunofluorescence, Immunoperoxidase
Techniques and Polymerase Chain Reaction. International Journal of
Virology 7 (4): 158-166.
4- Abd El-Rahim, I. H. A.; El-Ballal, S. And Hussein, M., 2002. An out
break of lumphy skin disease among cattle in Upper Egypt (El-Menia
governorate). Monofia Vet. J., 2(1); 185-200.

5- Abou Elyazeed, Ebtsam a. " Samya s. Abd-el Nabi' and e.m.


Ebrahem',2012. detection of lumpy skin disease (LSD) virus in cattle in
some Egyptian governorates using different diagnostic techniques, egypt.
1. agric. res., 90 (1).
6- Abutarbush SM1, Ababneh MM2, Al Zoubi IG1, Al Sheyab OM1, Al
Zoubi MG3, Alekish MO1, Al Gharabat RJ4, 2015. Lumpy Skin Disease
in Jordan: Disease Emergence, Clinical Signs, Complications and
Preliminary-associated. Transbound Emerg Dis. 62(5):549-54.

7- Ahmed N. F. Neamat-Allah, 2015. Immunological, hematological,


biochemical, and histopathological studies on cows naturally infected
with lumpy skin disease, Veterinary World, EISSN: 2231-0916.

157
8- Ahmed, L. A.; El-Desawy, O. M. and Mohamed, A. T., 2005. Studies
on bovine field skin lesions in Fayoum Governorate. Veterinary Medical
Journal Giza; 53: 1, 73-81.

9- Alaa. A. El-Kholy*; Hatem M. T. Soliman* and Khaled A.


Abdelrahman, 2008. Polymerase chain reaction for rapid diagnosis of a
recent lumpy skin disease virus incursion to Egypt, Arab J. Biotech., Vol.
11, No. (2): 293-302.
10- Alexander, R. A., and K. E. Weiss, 1959. Unpublished observations.
11- Alexander, R.A.,; Plowright, W. and Haig,D.A.,1957.
"Cytopathogenic agents associated with LSD of cattle".
Bull.Epiz.Dis.Afr., 5: 489-492.

12- Ali Meawad Ahmed and Amina A. Dessouki, 2013. Abattoir-Based


Survey and Histopathological Findings of Lumpy Skin Disease in Cattle
at Ismailia Abattoir, International Journal of Bioscience, Biochemistry
and Bioinformatics, Vol. 3, No. 4.

13- Amal, M. A. Raof,* Shahein, M. A.* and Hoda, Abd El


Monem**,2008. Electrophoretic characterization of sheep pox virus, Goat
pox virus and lumpy skin disease virus, Egypt. J. Comp. Path. & Clinic.
Path. Vol. 21 No. 1; 15- 30.

14- Andrew1es, C., 1964. Viruses of Vertebrates. Bailliere, Tindall and


Cox, London.

15- Aziza Amin1*, Ehab El-Nahas2, Abd-Elbaset El-Mashed, 2015.


Pathological and Virological Studies on an Outbreak of Lumpy Skin
Disease among Cattle in Kalubia Governorate-Egypt. Journal of
Advanced Veterinary Research Volume 5, Issue 4, 165-175.

158
16- Babiuk, S., D.B. Wallace, S.J. Smith, T.R. Bowden, B. Dalman, G.
Parkyn, J. Copps and D.B. Boyle, 2009. Detection of Antibodies against
Capri poxviruses using an Inactivated Sheep pox Virus ELISA. Trans.
and Emer. Dis., 10: 865-1682.
17- Babiuk, S., T.R. Bowden, B. Dalman, G. Parkyn and J. Copps, 2008.
Quantification of lumpy skin disease virus following experimental
infection in cattle. Transboundary and Emerging Diseases, 55: 299-307.
18- Babiuk, S.; Parkyn, G.; Copps, J.; Larence, J. E.; Sabara, M. I.;
Bowden, T. R.; Boyle, D. B. and Kitching, R. P., 2007. Evaluation of an
ovine testis cell line (OA3.Ts) for propagation of capripoxvirus isolates
and development of an immunostaining tech1nique for viral plaque
visualization. Journal of veterinary diagnostic; Vol. 19, No. (5): pp. 486-
91.

19- Bagla, V. P.; Osuagwuh, U. I.; Annandale, C. H.; Irons, P. C.; Venter,
E. H, 2006. Elimination of toxicity and enhanced detection of lumpy skin
disease virus on cell culture from experimentally infected bovine semen
samples. Onderstepoort J. Vet. Res. Vol. 73, No. (4), pp.:263-268.

20- Bancroft, J. D. and Gamble, M., 2002. Theory and practice of


Histological Techniques. 5th Ed., Churchill Livingstone, Medical Division
of Pearson Professional Ltd.

21- Bancroft, J. D.; Stevens, A. and Tumer, D.R.,1990. "Theory and


practice of histological techniques." Third Ed.

22- Bhanuprakash, V., Indrani, B.K., Hosamani, M., Singh, R.K., 2006.
The current status of sheep pox disease. Comp. Immunol. Microbiol.
Infect. Dis. 29, 27–60.

23- Bowden, T. R., S. L. Babiuk, G. R. Parkyn, J. S. Copps, and D. B.


Boyle, 2008. Capripoxvirus tissue tropism and shedding: A quantitative
study in experimentally infected sheep and goats. Virol. 371, 380–393.

159
24- Bozzalo, J. and Russell, D.,1992. "Electron microscopy. Principals
and techniques for biologists." Jones and Bartle-tte Publishers. Boston.
U.S.A.

25- Brenner, J., 2Haimovitz, M., 3Oron E., 1Stram, Y., 1Fridgut, O.,
1
Bumbarov, V. 1Kuznetzova, L., 2Oved, Z., 2Waserman, A., 2Garazzi, S.,
4
Perl, S., 4Lahav, D., 4Edery, N. and 1Yadin, H, 2006. Lumpy Skin
Disease (LSD) IN A Large Dairy Herd in Israel, June, Israel Journal of
Veterinary Medicine.

26- Brenner, J., D. David, A. Avraham, U. Klopfer-Orgad,I. Samina, and


B. A. Peleg, 1992. Experimental infection with local lumpy skin disease
virus in cattle vaccinated with sheep pox vaccine. Isr. J. Vet. Med. 47,
17–21.

27- Buller, R. M.; Arif, B. M., Black, D. N., Dumbell, K. R.; Esposito, J.
J., Lefkourtz, E. J.; McFadden, G., Moss, V., Mercer, A. A., Moyer, R.
W., Skinner, M. A.; Tripathy, D. N, 2006. Poxviridae In Virus
Taxonomy. VII the report of the International Committee on Taxonomy
of viruses. Academic Press, PP. 117-133.

28- Bulletin 6205 Rev A US/EG, 11-0864 1211 Sig 1211, Bio-Rad.
Burdin, M. L., 1959. The use of histopathological examinations of skin
material for the diagnosis of lumpy skin disease in Kenya. Bull. epizoot.
Dis. Afr. 7, 27.

29- Burdin, M. L., and J. PRYDIE, 1959. Observations on the first outbreak
of lumpy skin disease in Kenya. Bull epizoot. Dis. Afr. 7, 21.
30- C. M. Chihota1,*, L. F. Rennie2, R. P. Kitching3, P. S. Mellor2, 2003.
Attempted mechanical transmission of lumpy skin disease virus by biting
insects. Medical and Veterinary Entomology Volume 17, pages 294–300.
31- C. M. Chihota a1, L. F. Rennie a1, R. P. Kitching a1 c1 and P.
S. Mellor a1 , 2001. Mechanical transmission of lumpy skin disease virus
by Aedes aegypti (Diptera:Culicidae). Epidemiology and
Infection ,Volume 126, pp 317-321.

160
a a b a
32- C.H. Annandale , P.C. Irons , V.P. Bagla , U.I. Osuagwuh , E.H.
b,
Venter 2005. Sites of persistence of lumpy skin disease virus in the
genital tract of experimentally infected bulls.

33- Capstic, P. B.; Prydie,J.; Coakley,W. and Burdin, N.L.,1959.


"Protection of cattle against the Neethling type virus of LSD". Vet. Rec.,
71: 122-123.
34- Carroll MW, Moss B.,1997. Poxviruses as expression vectors. Curr
Opin Biotechnol (5):573-7. Review.

35- CH Annandale1, PC Irons1, VP Bagla2, UI Osuagwuh1, EH Venter2.,


2010. Sites of Persistence of Lumpy Skin Disease Virus in the Genital
Tract of Experimentally Infected Bull. Reproduction in Domestic
Animals. Volume 45, pages 250–255.

36- Charles. L., Le Goff. B., Roland. C., David. B., Wallace. D., Vely´
Gulyaz. F., Tuppurainen. G., Madani. H., Caufour. B., Adami., El Harrak.
j., George. A., Emmanuel B., Diallo A., 2011. Use of the Capripoxvirus
homologue of Vaccinia virus 30 kDa RNA polymerase subunit (RPO30)
gene as a novel diagnostic and genotyping target: Development of a
classical PCR method to differentiate Goat poxvirus from Sheep
poxvirus. Vet Microbiology 149: 30–39.

37- Christopher A. Nelson 1,*, Megan L. Epperson 1, Sukrit Singh 2,


Jabari I. Elliott 1 and Daved H. Fremont 1,2,3, 2015. Structural
Conservation and Functional Diversity of the Poxvirus Immune Evasion
(PIE) Domain Superfamily, Viruses, 7, 4878–4898.

38- Cornelius Henry Annandale,2006. Mechanisms by which lumpy skin


disease virus is shed in semen of artificially infected bulls. University of
pertoria, Onderstepoort.
39- D. B. Wallace and G. J. Viljoen., 2002. Importance of thymidine
kinase activity for normal growth of lumpy skin disease virus (SA-
Neethling). Arch Virol 147: 659–663.

161
40- Davies F.G., Krauss H., Lund L.J. & Taylor M.,1971. The laboratory
diagnosis of lumpy skin disease. Res. Vet. Sci., 12, 123–127.
41- De lange, M., 1959. The histopathology of the cytopathogenic
changes produced in monolayer epithelial cultures by viruses associated
with lumpy skin disease. Onderstepoort J. vet. Res. 28, 245.

42- Dimitrios Dilaveris,2015. Lumpy Skin disease in Greece (Evros),


EFSA Journal. 13(1):3986.

43- E. Crandall/P.Barber,
https://www.coursehero.com/file/7572635/ProtocolGel.

44-E. R. Tulman, C. L. Afonso, Z. LU, L. Zsak, G. F. Kutish, And D. L.


Rock*, 2001. Genome of Lumpy Skin Disease Virus, Journal of
Virology, Vol. 75, p. 7122–7130.
45- E. R. Tulman,1 C. L. Afonso,1 Z. Lu,1 L. Zsak,1 J.-H. Sur,1 N. T.
Sandybaev,2 U. Z. Kerembekova,2 V. L. Zaitsev,2 G. F. Kutish,1 and D.
L. Rock, 2002. The Genomes of Sheeppox and Goatpox Viruses.
JOURNAL OF VIROLOGY, p. 6054–6061, Vol. 76.

46- E. S. M. Tuppurainen1, W. H. Stoltsz2, M. Troskie2, D. B. Wallace2,3,


C. A. L. Oura1, P. S. Mellor1, J. A. W. Coetzer2, E. H. Venter, 2011. A
Potential Role for Ixodid (Hard) Tick Vectors in the Transmission of
Lumpy Skin Disease Virus in Cattle. Transboundary and Emerging
Diseases. Volume 58, pages 93–104.

47- E.S.M. Tuppurainena,∗, E.H. Venterb, J.A.W. Coetzerb, L. Bell-


Sakyia,2015. Lumpy skin disease: Attempted propagation in tick cell
lines and presence of viral DNA in field ticks collected from naturally-
infected cattle, Ticks and Tick-borne Diseases 6,134–140.

48- E.S.Orlova,A.V. Shcherbacov, V.I. Deiv, and V.M.Zakharov, 2006.


Differetiation Of Capripoxvirus Species and Strains by Polymerase Chain
Reaction. MOLECULAR BIOLOGY, Vol 40,pp139-145.
49- Edgar Howard Mercer, M. S. C. Birbeck, 1966. Electron
Microscopy: A Handbook for Biologists.

162
50- Edward S. Reynolds, 1963. The use of lead citrate at high pH as an
electron-opaque stain in electron microscopy.

51- Eeva S. M. Tuppurainen, Evaluation of Vector Potential of


Rhipicephalus Appendiculatus, Amblyomma Hebraeum and
Rhipicephalus Decoloratus ticks for lumpy skin disease virus, 2015.phD,
Department of Veterinary Biosciences, Faculty of Veterinary Medicine,
University of Helsinki, Helsinki, Finland.

52- El-Bagoury, G. F.; Madbouly, H. M.; Iman, A. A. Farrag and Saber,


M. S.,1995. Isolation and characterization of Lumpy skin disease (LSD)
virus from cattle during natural outbreak in Minia Governorate. Alex. J.
Vet. Sci., Volume 11, 167-174.

53- El-Nahas, A.S. El-Habbaa, G.F. El-bagoury and Mervat E.I. Radwan,
2011. Isolation and Identification of Lumpy Skin Disease Virus from
Naturally Infected Buffaloes at Kaluobia, Egypt ,Global Veterinaria 7 (3):
234-237.
54- Fayez Awadalla Salib* and Ahmed Hassan Osman, 2011. Incidence
of lumpy skin disease among Egyptian cattle in Giza Governorate, Egypt,
Veterinary World. Vol.4(4):162-167.

55- Felsenstein, J., 2001. Taking variation of evolutionary rates between


sites into account in inferring phylogenies. J. Mol.Evol., 53 : 447-455.

56- Fenner F, 1996. Poxviruses. In: Fields BN, Knipe DM, Howley PM
(eds), Fields virology third edn., vol 2. Chapter 84. Lippincott-Raven,
Philadelphia, PA.
57- Fenner, F., P. A. Bachmann, E. P. J. Gibbs, F. A. Murphy, M. J.
Studdert and D. O. White, 1987. Poxviridae. Veterinary Virology.
Academic Press, London.
Fenner's Veterinary virology, fourth edition, 2011.

163
58- G. Garia, b, , ,F. Biteau-Corollerb, C. LeGoffc, P. Caufourc, F. Rogerb,
2008. Evaluation of indirect fluorescent antibody test (IFAT) for the
diagnosis and screening of lumpy skin disease using Bayesian method.
Veterinary Microbiology Volume 129. Pages 269–280.
59- Gari, G., A. Waret-Szkuta, V.Grosbois, P.Jacquiet and F. Roger,
2010. Risk factors associated with observed clinical lumpy skin disease in
Ethiopia. Epidemiol. Infect. 138: 1657-1666.
60-Gemma C. Carter,1 Gaener Rodger,1,2 3 Brendan J. Murphy,1
Mansun Law,1,2 Oliver Krauss,2 4 Michael Hollinshead1,2 and Geoffrey
L. Smith,2003. Vaccinia virus cores are transported on microtubules,
Journal of General Virology, 84, 2443–2458.

61- Gezahegn Alemayehu & Girma Zewde & Berhanu Admassu, 2013.
Risk assessments of lumpy skin diseases in Borena bull market chain and
its implication for livelihoods and international trade. Trop Anim Health
Prod, 45:1153–1159.

62- Gulbahar MY, WC Davis, H Yuksel and M Cabalar, 2006.


Immunohistochemical evaluation of inflammatory infiltrate in the skin
and lung of lambs naturally infected with sheeppox virus. Vet Pathol, 43:
67-75.

63- Hell Creek Life © 1997-2010 Phillip Bigelow Revised 1/24/2010.

64- Hosamani, M.; Mondal, B.; Temburne, P. A.; Bandyopa, S. K.;


Singh, R. K. and2 Rasool, J. J., 2004. Differentiation of sheep pox and
goat pox virus sequence analysis and PCR-RELP of P32 gene. Virus
genes. Vol. 29,pp. 73-80.

65- http://www.ncbi.nlm.nih.gov/tools/primer-blast.

66- https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch.
67- https://moodle.ufsc.br/mod/resource/view.php?id=805619.

68.https://windward.hawaii.edu/paces/summerfiles/publications/qiagen.p
df_march.

164
69- https://www.emsdiasum.com/microscopy/.
70.https://www3.appliedbiosystems.com/cms/groups/mcb_marketing/.../c
ms_081527.pdf.

71.https://www3.appliedbiosystems.com/cms/groups/mcb_support/docu
ments/generaldocuments/cms_042772.pdf.

72-HunterP,WallaceD,2001. Lumpy skin disease in southern Africa: are


view of the disease and aspects of control. J S Afr Vet Assoc 72: 68–71.

73- Identification and Purification of a Local Isolate of Infectious


Laryngotracheitis Virus. Egyptian J. Vet. Sci. 39: 11-20.

74- Ireland D.C. & BINEPAL Y.S., 1998. Improved detection of


capripoxvirus in biopsy samples by PCR. J. Virol. Methods, 74, 1–7.
1a 2
75- Irons P C , Tuppurainen E S M , Venter E H,2005. Excretion of
lumpy skin disease virus in bull semen. Theriogenolog 63: 1290-1297.
76-J. B. Johnston and Grant McFadden, 2003. Poxvirus
Immunomodulatory Strategies: Current Perspectives, JOURNAL OF
VIROLOGY. Vol. 77, p. 6093–6100.

77- J. Brennera, , , M. Bellaicheb, E. Grossc, D. Eladd, Z. Ovedc, M.


Haimovitzc, A. Wassermanc, O. Friedguta, Y. Strama, V. Bumbarova, H.
Yadina, 2009. Appearance of skin lesions in cattle populations vaccinated
against lumpy skin disease: Statutory challenge. Vaccine.Volume 27,
Pages 1500–1503.

78- J. C. Lubinga, E. S. M. Tuppurainen, J. A. W. Coetzer, W. H. Stoltsz,


E. H. Venter ,2014. Evidence of lumpy skin disease virus over-wintering
by transstadial persistence in Amblyomma hebraeum and transovarial
persistence in Rhipicephalus decoloratus ticks. Experimental and Applied
Acarology . Volume 62, pp 77-90 .

165
79- J. C. Lubinga1,2,*, E. S. M. Tuppurainen3, R. Mahlare1, J. A. W.
Coetzer1, W. H. Stoltsz1 E. H. Venter, 2013. Evidence of Transstadial and
Mechanical Transmission of Lumpy Skin Disease Virus by Amblyomma
hebraeum Ticks. Transboundary and Emerging Diseases.

80- James A. House, Terrance M. Wilson, Sinan El Nakashly, Ikram A.


Karim,Ibrahim Ismail, Nabil El Danaf, Aly M.Moussa, Nazmi N.
Ayou,1990. The isolation of lumpy skin disease virus and bovine
herpesvirus- from cattle in Egypt, J Vet Diagn Invest 2:111-115.
81- K. A. Al-Salihi and I. Q. Hassan, 2015. Lumpy Skin Disease in Iraq:
Study of the Disease Emergence, Transboundary and Emerging Diseases.
62, 457–462.

82- Kitching, R. P. and C. Smale, 1986. Comparison of the external


dimensions of capripoxvirus isolates.Res. Vet. Sci., 41, 425-427.

83- Kitching, R. P.; Hammond, J. M. and Black, D. N., 1986. Studies on


the Major Common Precipitating Antigen of Capripoxvirus J. gen. Virol.
Vol. 67, pp. 139 – 148.
Laboratories, Inc.

84- Le Goff, C.; Lamien, C.E.; Fakhfakh, E.; Chadeyras, A.; Aba-
Adulugbad, E.; Libeau, G.; Tuppurainen, E.; Wallace, D.; Adam, T.;
Silber, R.; Gulyaz, V.; Madani, H.; Caufour, P.; Hamammi, S.; Diallo, A.
and Albina, E., 2009. Capripoxvirus G-protein-coupled chemokine
receptor,a host-range gene suitable for virus-animal origin
discrimination.J. Gen. Virol. Vol. 90, pp. 1967-1977.

85- M. El-Tholoth* and A. A. El-Kenawy, 2015. G-Protein-Coupled


Chemokine Receptor Gene in Lumpy Skin Disease Virus Isolates from
Cattle and Water Buffalo (Bubalus bubalis) in Egypt. Transboundary and
Emerging Diseases.

166
86- M.El-Haig.A.Selim and A.Shahira,2014. Efficiency of PCR as
Compared with Dot Blot Hybrdization Techniques in Diagnosis of
Lumpy Skin in Cattle in Egypt. International Journal of Virology 10 (2):
136-142.

87- MacLachlan, N. J. and Dubovi, E.J, 2011. Poxviridae. In


MacLachlan, N. J. and Dubovi, E.J editors, Fenner's veterinary virology.
4th ed. London ; Boston : Academic press,p.157-160.

88- Magori-Cohen, R., Y. Louzoun, Y. Herziger, E. Oron, A. Arazi, E.


Tuppurainen, N.Y. Shpige and E Klement, 2012. Mathematical modelling
and evaluation of the different routes of transmission of lumpy skin
disease virus. Veterinary Research, 43(1): 1297-1299.

89- Mahmoud M. ElHaig1, Abdelfatah Selim and Abdelwahab


Shahira,2013. Polymerase chain reaction and Dot blots hybridization
techniques for diagnosis of lumpy skin disease in cattle in Egypt, Jokull
journal , Vol 63, No. 6,

90- Mangana-Vougiouka, O.; Mark-oulatos, P.; Kaptopoulos, G.;


Nonikou, K.; Bakandritosos, N. and Papadopulos, P., 2000. "Sheep pox
virus id-entification from clinical specimens by PCR, cell culture,
immuno fluorescence and agar gel immuno precipitation assay." Mol.
Cell. Probes, (5): 305-310.

91- Matthews, R.E.F., 1982. Classification and nomenclature of viruses.


Inter virology 17:1-199.

92- Mercer J, Helenius A, 2008. Vaccinia virus uses macropinocytosis


and apoptotic mimicry to enter host cells. Science 320: 531–535.

93- Methods and techniques in virology, Pierre Payment and Michel


Trudel, 1993.

94-Mishra, S.S and B.B. Mallick, 1997. Detection of fowl pox virus using
immunoperoxidase test and fluorescent antibody technique Indian Vet. J.,
74: 199-202.

167
95- Mohamed Hassan Tageldin & David Brian Wallace & Gertruida
Hermanna Gerdes & John Fraser Putterill & Roelf Rudolph Greyling &
Maanda Noaxe Phosiwa & Rashied Mohammed Al Busaidy & Sultan
Issa Al Ismaaily, 2014. Lumpy skin disease of cattle: an emerging
problem in the Sultanate of Oman. Trop Anim Health Prod . 46:241–246.

96- Moss, B., 2006. Poxvirus entry and membrane fusion. Virology, 344,
48-54.

97- Mossman, K., Lee, S. F., Barry, M., Boshkov, L., McFadden, G,
1996. Disruption of M-T5, a novel myxoma virus gene member of
poxvirus host range superfamily, results in dramatic attenuation of
myxomatosis in infected European rabbits. J. Virol. 70:4394–4410.

98- Munz E K, Owen N C, 1966. Electron microscopic studies on lumpy


skin disease virus type ‘Neethling’. Onderstepoort Journal of Veterinary
Research 33: 3–8.
Nagi, A. A., 1990. Lumpy skin disease: cutaneous and testicular lesions.
Assiut. Vet. Med. J. 23, 90–99.
99- Nawathae,D.R.; Gibbs,E.P.J.; Asagba,M.O. and Lawman, M.J.P.,
1978. "Lumpy skin disease in Nigeria". Trop. Anim. Hlth. Pro., 10:49-54.
100- OIE, 2004. Manual of diagnostic tests and vaccines for terrestrial
animals. OIE Part 2, 1–17.

101- OIE, 2008. Lumpy Skin Disease (Neethling, Knopvelsiekte): A


technical Report. The Centre for Food Security and Public Health, Iowa
State University.
102- OIE, 2010. Terrestrial Manual of Lumpy Skin Disease, Chapter
2.4.14. Version adopted by the World Assembly of Delegates of the OIE
in May 2010, OIE, Paris.
103- OIE, 2016. Lumpy skin disease Chapter 2.4.13.

104- Omyma, M. El-Desawy,2008. Recent Isolation and identification of


lumpy skin disease virus from cattle in Egypt, Egypt. J. Comp. Path. &
Clinic. Path. Vol. 21 No. 1; 139- 147.

168
105- Osuagwuh U.I., Bagla V., Venter E.H., Annandale C.H., Irons P.C.,
2007.Absence of lumpy skin disease virus in semen of vaccinated bulls
following vaccination and subsequent experimental infection. Vaccine,
25:2238–2243.

106- Osuagwuh, Uchebuchi I, 2006.Semen quality and the excretion of


lumpy skin disease virus in semen following vaccination and
experimental challenge of vaccinated bulls.

107- P. D. Kara, C. L. Afonso, D. B. Wallace, G. F. Kutish, C. Abolnik,


Z. Lu, F. T. Vreede, L. C. F. Taljaard, A. Zsak, G. J. Viljoen, 2003.
Comparative sequence analysis of the South African vaccine strain and
two virulent field isolates of Lumpy skin disease virus. Archives of
Virology, Volume 148, pp 1335-1356.

108- P. T. Richard C. Condit, Nissin Moussatche, 2006. “In A Nutshell:


Structure and Assembly of the Vaccinia Virion,” Advances in Virus
Research, vol. 66, pp. 124–131.

109- Plowright, ViT., and R. D. FERRIS, 1959. Ether sensitivity of some


mammalian pox viruses. Virology 7, 357.

110- Prof. JAW Coetzer and Dr. Eeva Tuppurainen, 2004. Lumpy skin
disease, in: Infectious diseases of livestock, edited by Coetzer, J.A.W. &
Tustin, R.C. Cape Town: Oxford University Press Southern Africa,
2:1268-1276.
111- Prozesky L, Barnard BJ, 1982. A study of the pathology of lumpy
skin disease in cattle. Onderstepoort J Vet Res. 49(3):167-75.
112- 1Rao, T. V. and Bandyopadhyay, S. K., 2000. Comparative review
of goatpox and sheeppox and their diagnosis.Anim. Health Res. Rev., 1:
127 – 136.

113- Rao, T.V.S., Negi, B.S., Bansal, M.P., 1997. Development and
standardization of a rapid diagnostic test for sheep pox. Indian Journal of
Comparative Microbiology, Immunology and Infectious Diseases 18: 47–
51.

169
114- Rashid, P. M. A., Baba Sheikh, M. O., Raheem, Z. H. Marouf, A. S,
2016. Molecular characterisation of lumpy skin disease virus and
sheeppox virus based on p32 gene . Bulgarian Journal of Veterinary
Medicine, DOI: 10.15547.

115- Razek, A. ; Omar, B. and Magda, M. S., 2009. Preparation and


Evaluation of Lumpy Skin Disease Hyperimmune Serum Coniugated
with Fluorescein.International Journal of Virology; Volume: 5, Issue: 1,
Pp.: 44-48.

116- Rgbe Haftu, Lumpy Skin Disease (LSD): outbreak investigation,


isolation and molecular detection of LSDV in selected areas of eastern
shewa, ethiopia, 2012. degree of Masters Science in Veterinary
microbiology, Addis Ababa University, Ethiopia.

117- Rizkallah, S.S.,1994. "Further studies on sheep pox disease in


Egypt." Ph.D. Thesis (Infectious and Fish diseases). Faculty of Vet. Med.,
Cairo university.
118- Roberts KL, Smith GL. 2008. Vaccinia virus morphogenesis and
dissemination. Trends Microbiol. 16:472–479.

119- Rosel, J. L., P. L. Earl, J. P. Weir, and B. Moss,1986. Conserved


TAAATG sequence at the transcriptional and translational initiation sites
of vaccinia virus late genes deduced by structural and functional analysis
of the HindIII H genome fragment. J. Virol. 60:436-449.

120- S. M. Tamam, 2006. Isolation of Lumpy skin disease virus form


naturally infected cattle previously vaccinated with live attenuated sheep
poxvirus vaccine. BS. VET. MED. J. VOL. 16, p. 27-31.

121- S.S.A. Sharawi & I.H.A. Abd El-Rahim, 2011. The utility of
polymerase chain reaction for diagnosis of lumpy skin disease in cattle
and water buffaloes in Egypt, Rev. sci. tech. Off. int. Epiz., 30 (3), 821-
830.

170
122- Salib F., Osman A., 2011. Incidence of lumpy skin disease among
Egyptian cattle in Giza Governorate, Egypt. Vet. World 4162–167.

123- Sambrook, Fritsch, and Maniatis, 1989. Molecular Cloning:


Alaboratory manual 2nd ed., cold spring Harbor press, cold spring
Harbor, NewYork, vol.3, appendix B12.
124- Sameeh M Abutarbush, 2015.Hematological and serum biochemical
findings in clinical cases of cattle naturally infected with lumpy skin
disease, J Infect Dev Ctries, 9(3):283-288.

125- Shchelkunov, S. N., Safronov, P. F., Totmenin, A. V., Petrov, N. A.,


Ryazankina, O. I., Gutoro,V. V., Kotwal, G. J.,1998. The genome
sequence analysis of the left and right species-specific terminal region of
a cowpox virus strain reveals unique sequences and a cluster of intact
ORFs for immunomodulatory and host range proteins. Virology 243:432–
460.

126- Sherrylin Wainwright a, Ahmed El Idrissi et al, 2013. Emergence of


lumpy skin disease In the Eastern Mediterranean Basin countries, empress
watch, VOL 29. EMPRES-ANIMAL-HEALTH@fao.org.

127- Silvia Kreindel, Julio Pinto, Caryl Lockhart, Ahmed ElIdrissi, Eran
Raizman, 2015. Emergence of lumpy skin disease, express watch, VOL
33.

128- Smith, G.L., Vanderplasschen, A., Law, M., 2002. The formation
and function of extracellular enveloped vaccinia virus. J. Gen. Virol. 83,
2915 – 2931.

129- Sohair, I. Badr*; Ibrahim, E. M.* and Shahein M.A.,2008.


Virological and pathological studies on lumpy skin disease in naturally
infected cattle, Egypt. J. Comp. Path. & Clinic. Path. Vol. 21, 446- 465.
130- Sumasundaram Mathan Kumar, 2011. An outbreak of Lumpy Skin
Disease in a Holstein Dairy Herd in Oman: A Clinical Report. Asian
Journal of Animal and Veterinary Advances 6 (8): 851-859.

171
131- Thomas, A.D. and Mare, C.V.E.,1945. " Kopvesiekte". J.S.Afri. Vet.
ed. Assoc., 16:36-43
132- Thompson, J. D.; Higgins, D. G. andGibson, T. J.,1994. CLUSTAL
W: improving the sensitivity of progressive multiple sequence alignment
through sequence weighting positions-specific gap penalties and weight
matrix choice. Nucleic Acids Research, 22: 4673-4680.
133- Tiwari, A.K., Rao, T.V.S., Negi, B.S., 1996. Spot agglutination test
for the rapid diagnosis of goat pox. Tropical Animal Health and
Production, 28(3):213-215.

134- Tuppurainen E.S.M., Venter E.H. & Coetzer J.A.W., 2005. The
detection of lumpy skin disease virus in samples of experimentally
infected cattle using different diagnostic techniques. Onderstepoort J. Vet.
Res., 72, 153–164.
135-Tuppurainen, E.S.M. and C.A.L. Oura, 2012. Review: Lumpy Skin
Disease: An Emerging Threat to Europe, the Middle East and Asia. Tran
boundary and Emerging Diseases, 59(1): 40-48.
136- Van Rooyen, P.J.,E.K. Munz and K.E. Weiss, 1969. The optimal
condition for the multiplication of Neethling type LSDV in embryonated
eggs. Onderstepoort, J. Vet.Res., 36: 165-174.
137- W. Awadin1,2, H. Hussein1, Y. Elseady1, S. Babiuk3,4, H. Furuoka2,
2011. Detection of Lumpy Skin Disease Virus Antigen and Genomic
DNA in Formalin-Fixed Paraffin-Embedded Tissues from an Egyptian
Outbreak in 2006. Transboundary and Emerging Diseases. Volume 581,
pages 451–457.

138- W. M. Ahmed and Kawther S. Zaher,2008. Observations on lumpy


skin disease in local Egyptian cows with emphasis on its impact on
ovarian function, African Journal of Microbiology Research Vol. (2) pp.
252-257.

139- Walid S. Awad, Adel K. Ibrahim, Khaled Mahran, Khaled M.


Fararh, Mervet I. Abdel Moniem, 2010. Evaluation of different diagnostic
methods for diagnosis of Lumpy skin disease in cows. Tropical Animal
Health and Production .Volume 42, pp 777-783.

172
140- Weis1s, K. E., 1959, 1960, 1962, 1964, 1966, 1967. Unpublished
observations,
141- 1Weiss, K. E., and J. Broekman, 1965. Unpublished work.
142- WEI1SS, K. E., and S. M. GEYER, 1959. The effect of lactalbumin
hydrolysate on the cytopathogenesis of lumpy skin disease virus in tissue
culture. Bull. epizoot. Dis. Afr.7, 243.

143- Weiss, K. E.,1968. Lumpy skin disease virus. Virol. Monograph,


Vol. 3: pp. 111-31.

144- Wood roofe, G. M. and F. Fenner, 1962. Serological relationship


within the poxvirus group: an antigen common to all members of the group.
Virology, 16, 334-341.

145- Woods, J.A., 1988. "LSD, A review". Trop. Anim. Hlth. Prod., 20:
11-17.

146- Yehuda Stram, , Larisa Kuznetzova, Orly Friedgut, Boris Gelman,


Hagai Yadin, Marisol Rubinstein-Guini, 2008. The use of lumpy skin
disease virus genome termini for detection and phylogenetic analysis.
Journal of Virological Methods. Volume 151, Pages 225–229.

147- YS. Binepal, F.A. Ongadi and J.C. Chepkwony, 2001.Alternative


cell lines for the propagation of lumpy skin disease virus Onderstepoort
Journal of Veterinary Research, 68: 151-153.

148- Zelalem Abera, Hailu De 1 2 gefu, 3Getachew Gari and 4Zelalem


Ayana, 2015. Review on Epidemiology and Economic Importance of
Lumpy Skin Disease. International Journal of Basic and Applied
Virology 4(1): 08-21.

173
۱
۲
‫الملخص العربى‬

‫اثناء فترة ‪ 2014‬ظهرت حاالت لفيروس الجلد العقدى فى االبقار بمحافظة القليوبية حيث ان‬
‫الحيوانات كانت تعانى من اعراض مرضية جسيمة تختلف عن اعراض الفيروس التى كانت‬
‫تحدث سابقا مثل االختناق والموت بنسبة اعلى من نسبة الوفيات التى كانت تحدث سابقا‬
‫حيث اظهرت النتائج العلمية مايلى‪:‬‬

‫‪-۱‬اظهرت محاوالت عزل الفيروس من القشرة الجلدية من حاالت االبقار الحية بعد ثالث‬
‫تمريرات خالل الغشاء الكوريونى ظهور اصابة تنكرزية فى الغشاء الكوريونى وموت لالجنة التى كانت‬
‫تعانى من تورمات وانزفة وتورمات وانزفة على الكبد وانزفه وتورمات على القلب والتى كانت‬
‫واضحه فى اليوم السادس من الحقن فى التمريرة الثانية والثالثة·‬

‫على خاليا كلى المجترات لمادين‬


‫‪-۲‬اظهرت محاوالت عزل الفيروس من الغشاء الكوريونى المصاب‬
‫وديربى المادين داربى بعددثالث تمريرات استدارة وتجمع للخاليا المصابة فى صورة عناقيد‬
‫والتى كانت منتشرة على سطح الخاليا بالكامل وذلك بعد ‪ 72‬ساعة من حقن العينة والتى كانت‬
‫تزداد تدريجيا نهاية بالوقوع الكامل للخاليا·‬

‫‪ -۳‬اظهر الفحص الهستوباثولوجى للغشاء الكوريونى المصاب بعينات فيروس مرض الجلد‬
‫العقدى نموا كثيفا الى حد ما على خاليا طبقة االكتوديرم والميزوديرم مع وجود جسيمات كبيرة‬
‫للفيروس فى السيتوبالزم الخاص بالخاليا·‬

‫‪ -٤‬اظهر الفحص االلكترونى المجهرى الشكل البيضاوى المميز لعائلة فيروسات الجدرى فى الغشاء الكوريونى‬
‫المحقون بعينات فيروس مرض الجلد العقدى‪.‬‬

‫‪ -٥‬اظهر فحص معزول فيروس مرض الجلد العقدى باختبار التلزن الدموى باستخدام كرات الدم‬
‫الحمراء لالبقار والماعز واالغنام والدجاج على عدم قدرة الفيروس على احداث تالزن دموى‬
‫لهذه الخاليا·‬

‫‪ -٦‬اظهرالتعرف السيرولوجى على معزولة فيروس مرض الجلد العقدى فى الغشاء الكوريونى‬
‫وخاليا الكلى لمادين وداربى وميضات فلوريسية ذات لون اصفر مخضر بداخل سيتوبالزم‬
‫الخاليا والغشاء الكوريونى·‬

‫‪۳‬‬
‫‪-٧‬اظهر التعرف الجزيئى لفيروس مرض الجلد العقدى من العينات االصلية بالجلد والغشاء‬
‫الكوريونى والكبد الجنينى باستخدام تفاعل انزيم البلمرة المتسلسل على وجود ناتج جينى بمقدار‬
‫‪ 137‬والخاص بجين البروتين المماثل لغالف الفيروس·‬

‫‪ -٨‬حيث تم نشرتتابع معزول فيروس الجلد العقدى على الجين باالسماء وارقام االلتحاق التالية‬
‫فيروس الجلد العقدى‪-‬القاضى‪KU 760905-2014-‬‬
‫فيروس الجلد العقدى‪-‬القاضى‪KX236312- 2014-3‬‬
‫فيروس الجملد العقدى‪-‬القاضى ‪KX236313-2014-4‬‬
‫فيروس الجلد العقدى‪-‬القاضى‪KX236311-2014-2-‬‬
‫فيروس الجلد العقدى‪-‬القاضى‪(KX250367) -2014-5-‬‬
‫فيروس الجلد العقدى‪ -‬القاضى‪·(KX236310)-2014-1-‬‬

‫‪-۹‬اظهر تحليل الشجرة الجينية لمعزول فيروس الجلد العقدى من مصادره المختلفة مع معزوالت‬
‫فيروس الجلد العقدى وجدرى الغنم وجدرى الماعز المنشورين على الجين بنك انه يتشابه‬
‫بدرجات من ‪ %99-98‬مع هذه المعزوالت لهذه الفيروسات_ لذلك عترتنا الجديده من فيروس‬
‫الجلد العقدى اكثر تشابها لفيروس الجلد العقدى عن جدرى االغنام والماعز·‬

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