LSDV
LSDV
LSDV
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Dr_Gehad Elkady
Huazhong Agricultural University-College of Veterinary Medicine_ Wuhan_China
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Benha University
By
For
The degree of M. V. Sc (Virology)
Under supervision of
Professor of Virology
Department of Virology
Benha University
Department of Virology
Benha University
Department of Virology
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.
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.
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
3.8.1. SAMPLING OF LSDV…………………………………………………………………………………………58
4.1. MATERIAL…………………………………………………………………………………………………………………79
4.2. METHODS………………………………………………………………………………………………………………..102
5. RESULTS…………………………………………………………………………………………………………………………118
7. SUMMARY ……………………………………………………………………………………………………………………153
8. REFERENCES …………………………………………………………………………………………………………………156
9. ARABIC SUMMARY………………………………………………………………………………………………………..2
4
LIST OF ABBREVIATIONS
EV Enveloped Virus
5
EEV Extracellular Enveloped Virus
NP Nucleoprotein
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
10
LIST OF FIGURES
FIGURE.NO TITLE
FIGURE_1 Morphological structure LSDV
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
12
13
ABSTRACT
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
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
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).
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).
26
Table (1): DNA replication and nucleotide metabolism genes.
27
6 LD082_L-R 74375..7503 =1 uracil DNA AAN02650 GI:225956
1 glycosylase .1 17
28
Table (2): RNA transcription and modification genes.
3 LD049_L- 42985..45015 =1 putative RNA helicase AAN02617 GI:225955 similar to vaccinia virus
R .1 84 strain Copenhagen I8R,
NPH-II
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
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
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
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
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
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
34
Table (3): structure and assembly genes
Start
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
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
12 LD057_R- 50183..51304 =1 putative virion core AAN02625 GI:225955 similar to vaccinia virus
L protein .1 92 strain Copenhagen G7L
37
ORF CDS Codo Product protein_id db_xref note
complement n
start
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
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
40
ORF CDS Codo protein_id db_xref note
complement n
Product
start
31 LD123_L- 114067..1145 =1 putative EEV protein AAN02691 GI:225956 similar to vaccinia virus
R 82 .1 58 strain Copenhagen A34R
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
42
4 LD008_R- 4841..5668 =1 putative soluble AAN02574 GI:225955 similar to myxoma virus
L interferon gamma .1 41
M007
receptor
43
9 LD015_R- 10234..10719 =1 putative AAN02581 GI:225955
L interleukin-18 .1 48
binding protein
44
ORF CDS Codon Product protein_id db_xref NOTE
complement start
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
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
1-ANKYRIN REPEAT
Start
47
4 LD148_L- 142116..143459 =1 ankyrin repeat AAN02716.1 GI:22595683
R protein
2-KELCH LIKE
start
48
frameshifted homolog of
LSDV019
3-A52R LIKE
Start
49
3 LD136_L- 129464..129925 =1 hypothetical AAN02704.1 GI:22595671
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).
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).
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.
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).
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):
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 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).
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).
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).
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).
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).
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).
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).
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).
60
2011; Tageldin et al., 2014), ovoid in shape, with rounded ends and characteristic
ball of wool appearance (Aziza et al., 2015).
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).
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).
62
3.8.3.2. Serological Techniques:
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)
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).
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:
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
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:
69
Query 1 AAATTATATACGTAAATAACATACCTGCTTAAAACCATAGTAATTTAGAATTCAAATCCa 60
||||||||||||||||||||||||||||| |||||||||||||||||||||||||||| |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||||||||| ||||||||||||||||||||||||||||||||||||||||||
/ gene="LD074 "
/ gene="LD074 "
/ codon_start=1
/ protein_id="AAN02642.1 "
/ db_xref="GI:22595609 "
70
Score = 235 bits (127), Expect = 2e-58, Identities = 159/174 (91%), Gaps =
4/174 (2%), Strand=Plus/Plus
Query 1 AAATTATATACGTAAATAACATACCTGCTTAAAACCATAGTAATTTAGAATTCAAATCCA 60
||||||||||||||||||||||||||||| |||||||||||||||||||||||||||| |
|||||| | ||||||||||||||||||||||||||||||||||||||||||
/ gene="LD074 "
/ gene="LD074 "
/ codon_start=1
/ protein_id="AAN02642.1 "
/ db_xref="GI:22595609 "
71
Score = 612 bits (331), Expect = 1e-176, Identities = 331/331 (100%), Gaps =
0/331 (0%), Strand=Plus/Plus
Query 1 AAGAGCATTACATAATCCAGAAAAATATTCTGTAAAATTTTCAACACCTCCTGATTTTTC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||
72
/ gene="LD075 ", CDS complement(65982..68378 )
Query 1 CTTAGTACAGTTAGTAGCGCAACCATGTATAATAGTAGCAGTAATATTACCACTATAGCT 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
73
Query 289 GGATTATTTGGAAATATAATTGTGTTAACTGTTCTTCGTAAATATAAGATAAAAACAATA 348
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||
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:
75
1- VIROSTEALTH: SLEEPING WITH THE ENEMY
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:
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.
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:
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.
2- antibiotic.
3- antifungal .
2. gentamycin 1 ml
3. mycostatin 100-200µl
4. mixing well.
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:
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.
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.
- 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:
87
It was adjusted to pH 7.3 by adding N/10 hydrochloric acid. (was supplied
by EMS, Washington, USA)
- 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:
88
4.1.8.5.1. Acetone 100% (C3H6O, 58.08 g/mol, CAS #: 67-64-1):
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).
2- Tissue cutter.
4- Grid.
5- Grid holder.
7- Grid forceps.
9- Grid fixer.
90
4.1.9. Material for histopathological examination: (Bancroft and
Gamble, 2002)
Haemmatoxylin crystals 1g
Absolute alcohol 10 ml
Ammonium or Potassium alum sulphate 20 g
Mercuric oxide 0.5 g
Distilled water 200 ml
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.
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.
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:
4.1.11.3. Acetone:
1 PBS: 9 glycerin was used for mounting the infected cells fixed on
the cover slips, it was obtained from elnasser for chemical industries.
93
4.1.11.7. Equipments:
1- Incubator.
2. Vortexer.
6. PBS, pH 7.2.
94
7. Ethanol (96–100%).
4.1.13.3. Primers:
95
AMOUNT OF OLIGO
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.
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
96
4.1.13.4. Equipments
3- Micro centrifuge.
5- Ice buckets.
6- Latex gloves.
7- Spectrophotometer.
4.1.14. Material used for analysis of PCR product using Agarose Gel
Electrophoresis
97
- Preparation of EDTA 1 mM, pH 8.
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.
Laboratories, Inc.
98
4.1.14.3. Ethidium bromide: (Qiagen)
4.1.14.4. Gel Pilot DNA Loading Dye, 5x: (Cat. No. 239901, Qiagen,
USA)
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).
3- UV transilluminator (Biometra)®.
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:
For details P/N 4374408, Product P/N 4376487, Insert P/N 4376151,
REV
100
2. Reagents Preserved at -20 ºC:
3- Big Dye® Terminator V3.1- Cycle Sequencing Kit (1,000 rxns), P/N
4337456.
4.1.15.5.2. Equipments:
101
4.2 Methods:
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
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.
3- Good washing for 3 times of the cell sheet using maintenance media
without serum.
11- Tightly closing the prescriptions, roux bottle, tubes and plates.
104
13- Incubation at 37ºCat dry incubator and daily examination till sheet
formation.
5: No of secondary squares.
𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑙𝑖𝑣𝑒 𝑐𝑒𝑙𝑙𝑠
Cell viability = x100
𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑑𝑒𝑎𝑑 𝑐𝑒𝑙𝑙𝑠
105
2- Examination of the cell sheet under inverted microscope for 60–70 %
confluency.
5- Swirl the prescriptions, tubes and plates, and incubate at 37⁰C for 30 min
for adsorption time.
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.
106
2- The samples washed in sodium cacodylate buffer for 45 minutes with 3
changes, each one for 15 minutes.
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.
107
Tissue Blocks
Semithin Section Under Ultracut for selection
The semi thin section
of tissue by tissue cutter for ultracut section
2 µm
108
4.2.5.2. Histopathological examination:
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):
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.
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.
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:
using QIAamp® DNA Mini and Blood Mini Handbook kit. (Qiagen,
USA) according the instructions of the kit manufacturer.
4.2.6.2. PCR:
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.
The reaction was carried out in applied biosystem, Amp 9700 PCR
system, ramp speed 9700.
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:
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.
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.
13. The gel was then examined using ultraviolet trans-illuminator at a wave
length of 590 nm and photographed.
114
manufacturer in QIAquick® PCR Purification Kit (Cat. No. 28104 and
28106),http://2012.igem.org/wiki/images/a/a3/QIAquick_PCR-
purification.pdf
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:
Loading the sample into 96 well- plate with septa was done as
following:
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.
117
118
5-RESULTS
119
(Table _ 6 ): Alterations on CAM and Embryo of SPF-ECE inoculated
The signs were observed 6days post-inoculation, S1: first skin suspected viral sample,
S2: second skin suspected viral sample.
120
A
B
C
121
A
nd
The positive CAM of the 2 passage of suspected samples was
prepared and inoculated on MDBK cell line. It was observed that infected
together to form clusters that scattered all over the monolayer within
72hr post inoculation and gradually increased till 70-80 % of sheet was
(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.
125
5. 3. Non Serological identification of suspected LSD virus
isolates:
(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.
cytoplasm. The characteristic virus particle was released from the cellular
127
1
2
3
4
sheep, goat and chicken RBCs cleared that the isolate was non HA produce
(photo_8).
129
5. 4. Serological identification of suspected LSD virus isolate
using IFAT:
infected CAM and infected MDBK monolayer with CAM and embryo liver
and heart suspensions using specific hyper immune serum against LSD
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
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:
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
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).
133
1 2 3 4 5
L
100bp
Faint
bands
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
TTTCCAGGAAATATTTTTTTCTGGATTATGTAATGCTCTTTGTACAAAAGAGGCAAAAAGT
TCTATTGCGAAACACTTTAGTTTATGGAAATCGTATGCCGATGCGAA
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
GCTGGTTAGAAGGTAAATTTCTGGATTATGTAATGCTCTTTGTACAAAAGAGGCAAAAAGTT
CTATTGCGAAACACTTTAGTTTATGGAAATCGTATGCCGATGCGA
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:
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.
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
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).
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.
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.
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 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).
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.
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).
152
153
7- SUMMARY
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.
154
6-Serological identification of suspected LSDV isolate on CAM and
infected MDBK monolayer revealed specific intracytoplasmic yellowish
green fluorescence emission.
155
156
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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·
-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
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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·
٤