Crimean–Congo haemorrhagic fever virus (CCHFV) of the genus Orthonairovirus of the family

Nairoviridae causes a zoonotic disease in many countries of Asia, Africa, the Middle East and
south-eastern Europe. As the distribution of CCHFV coincides with the distribution of its main
vector, ticks of the genus Hyalomma, the spread of infected ticks into new, unaffected areas
facilitates the spread of the virus. The virus circulates in a tick–vertebrate–tick cycle, but can also
be transmitted horizontally and vertically within the tick population. Hyalomma ticks infest a wide
spectrum of different wildlife species, e.g. deer and hares, and free-ranging livestock animals, e.g.
goat, cattle, and sheep. Many birds are resistant to infection, but ostriches appear to be more
susceptible. Viraemia in livestock is short-lived, and of low intensity. These animals play a crucial
role in the life cycle of ticks, and in the transmission and amplification of the virus and are,
therefore, in the focus of veterinary public health. As animals do not develop clinical signs, CCHFV
infections have no effect on the economic burden regarding livestock animal production. In contrast
to animals, infections of humans can result in the development of a severe disease, Crimean–
Congo haemorrhagic fever (CCHF).

Every year, more than 1000 human CCHF cases are reported from Albania, Bulgaria, Kosovo and
Turkey. In other countries, infection rates and case numbers are largely unknown. Case fatality
rates of 5% (in Turkey) to 80% have been reported and may depend on the virus strain, education
of the local population and the effectiveness of public health interventions. At present, the
pathogenesis of the disease in humans is not well understood. Most people become infected by tick
bites and by crushing infected ticks, but infection is also possible through contact with blood and
other body fluids of viraemic animals. As CCHFV also has the potential to be transmitted directly
from human-to-human, nosocomial outbreaks might occur.

There is no approved CCHF vaccine available and therapy is restricted to treatment of the
symptoms. Health education and information on prevention and behavioural measures are most
important in order to enhance public risk perception and, therefore, decrease the probability of
infections. Thus the identification of endemic areas is crucial for focused and targeted
implementation of public health measures. Serological screening of ruminants allows CCHFV-
affected areas to be identified, as antibody prevalence in animals is a good indicator of local virus
circulation. Treatment with tick repellents can be quite effective in reducing the tick infestation of
animals. To protect laboratory staff, handling of CCHFV infectious materials should only be carried
out at an appropriate biocontainment level.

Identification of the agent: Only a single virus serotype is known to date although sequencing
analysis indicates considerable genetic diversity. CCHFV has morphological and physiochemical
properties typical of the family Nairoviridae. The virus has a single-stranded, negative-sense RNA
genome consisting of three segments: L (large), M (medium) and S (small), each of which is
contained in a separate nucleocapsid within the virion. The virus can be isolated from serum or
plasma samples collected during the febrile or viraemic stage of infection, or from liver of infected
animals. Primary isolations are made by inoculation of several tissue cultures, commonly African
green monkey kidney (Vero) cells or by intracerebral inoculation of suckling mice. For identification
and characterisation of the virus, conventional and real-time reverse transcriptase polymerase
chain reaction (PCR) can be used. As infections of animals remain clinically inapparent, the
likelihood of isolating virus from a viraemic animal is very low.
 Serological tests: Type-specific antibodies are demonstrable by indirect immunofluorescence test
or by IgG-sandwich and IgM-capture ELISA. Currently there are no commercial test systems
available for animal health; only a few in-house systems have been published or kits are used
replacing the conjugate provided in kit with one that is suitable for the animal species to be
screened for CCHFV-specific antibodies.

Requirements for vaccines: There is no vaccine available for animals.

Crimean-Congo haemorrhagic fever (CCHF) is a zoonotic disease caused by a tick-borne CCHF virus (CCHFV)
of the genus Orthonairovirus of the family Nairoviridae, order Bunyvirales. CCHFV possesses a negative-sense
RNA genome consisting of three segments, L (large), M (medium) and S (small) each contained in a separate
nucleocapsid within the virion. All orthonairoviruses are believed to be transmitted by either ixodid or argasid ticks,
and only three are known to be pathogenic to humans, namely CCHF, Dugbe and Nairobi sheep disease viruses
(Swanepoel & Burt, 2004; Swanepoel & Paweska, 2011; Whitehouse, 2004). Recently CCHFV was grown
successfully for the first time in several tick cell lines derived from both a natural vector (Hyalomma anatolicum)
and other tick species not implicated in natural transmission of the virus (Bell-Sakyiet al., 2012).

The virus from an outbreak of “Crimean haemorrhagic fever” in soldiers and peasants in the Crimean Peninsula in
1944 was not isolated or characterised until 1967. “Congo haemorrhagic fever” virus, isolated from a patient in the
former Zaire (now Democratic Republic of the Congo) in 1956, was shown in 1969 to be the same virus. As a
consequence the names of both countries have been used in combination to describe the disease (Hoogstraal,
1979). Distribution of the virus reflects the broad distribution of Hyalomma ticks, the predominant vector of the
virus (Avsic-Zupanc, 2007; Grard et al., 2011; Papa et al., 2011; Swanepoel & Paweska, 2011).

The natural cycle of CCHFV includes transovarial and transstadial transmission among ticks and a tick-
vertebrate-tick cycle involving a variety of wild and domestic animals. Infection can also be transferred between
infected and uninfected ticks during co-feeding on a host; so called ‘non-viraemic transmission’ phenomenon.
Hyalomma ticks feed on a variety of domestic ruminants (sheep, goats, and cattle), and wild herbivores, hares,
hedgehogs, and certain rodents. CCHFV infection in animals was reviewed by Nalca et al., (2007). Although
animal infections are generally subclinical, the associated viraemia levels are sufficient to enable virus
transmission to uninfected ticks (Swanepoel & Burt, 2004; Swanepoel & Paweska, 2011). Many birds are
resistant to infection, but ostriches appear to be more susceptible than other bird species (Swanepoel et al.,
1998). Although they do not appear to become viraemic, ground feeding birds may act as a vehicle for spread of
CCHFV infected ticks. Results from serological surveys conducted in Africa and Eurasia indicate extensive
circulation of the virus in livestock and wild vertebrates (Swanepoel & Burt, 2004).

Humans acquire infection from tick bites, or from contact with infected blood or tissues from livestock or human
patients. After incubation humans can develop a severe disease with a prehaemorrhagic phase, a haemorrhagic
phase, and a convalescence period. Haemorrhagic manifestations can range from petechiae to large
haematomas. Bleeding can be observed in the nose, gastrointestinal system, uterus and urinary tract, and the
respiratory tract, with a mortality rate ranging from 5% to 80% (Ergonul, 2006; Yen et al., 1985; Yilmaz et al.,
2008).The severity of CCHF in humans highlights the impact of this zoonotic disease on public health. Although
CCHFV has no economic impact on livestock animal production, the serological screening of animal serum
samples for CCHFV-specific antibodies is very important. As prevalence in animals is a good indicator for local
virus circulation, such investigations allow identification of high-risk areas for human infection (Mertens et al.,
2013). Slaughterhouse workers, veterinarians, stockmen and others involved with the livestock industry should be
made aware of the disease. They should take practical steps to limit or avoid xposure of naked skin to fresh blood
and other animal tissues, and to avoid tick bites and handling ticks. Experiences from South Africa demonstrated
that the use of repellents on animals before slaughter could reduce the numbers of infected slaughterhouse
workers (Swanepoel et al., 1998). The treatment of livestock in general can reduce the tick density among these
animals and thus reduce the risk of tick bite in animal handlers (Mertens et al., 2013). Such tick control by the use
of acaricides is possible to some extent, but may be difficult to implement under extensive farming conditions.
Inactivated mouse brain vaccine for the prevention of human infection has been used on a limited scale in
Eastern Europe and the former USSR (Swanepoel & Paweska, 2011).

Infectivity of CCHFV is destroyed by boiling or autoclaving and low concentrations of formalin or beta-
propriolactone. The virus is sensitive to lipid solvents. It is labile in infected tissues after death, presumably due to
a fall in pH, but infectivity is retained for a few days at ambient temperature in serum, and for up to 3 weeks at
4°C. Infectivity is stable at temperatures below –60°C (Swanepoel & Paweska, 2011). CCHFV is classed in Risk
Group 3 for human infection and should be handled with appropriate measures as described in Chapter 1.1.4
Biosafety and biosecurity: Standard for managing biological risk in the veterinary laboratory and animal facilities.
Biocontainment measures should be determined by risk analysis as described in Chapter 1.1.4 (Palmer, 2011;
Whitehouse, 2004).
 Purpose

Method Population Individual animal Confirmation Immune status in

Contribute to Prevalence of
freedom freedom from of clinical individual animals or
eradication infection –
from infection prior to cases in populations post-
policies surveillance
infection movement animals vaccination

Identification of the agent1

Real-time
– +++ – – – –
RT-PCR

Virus isolation
– – – – + –
in cell culture

Detection of immune response

IgG ELISA +++ + – – +++ –

Competitive
+++ + – – +++ –
ELISA

IgM ELISA – ++ – – – –

Key: +++ = recommended for this purpose; ++ recommended but has limitations;
+ = suitable in very limited circumstances; – = not appropriate for this purpose.
ELISA = enzyme-linked immunosorbent assay; RT-PCR = reverse-transcription polymerase chain reaction.

CCHFV infection causes only a mild fever in domestic and wild vertebrate animals with a detectable viraemia of
up to 2 weeks (Gonzalez et al., 1998; Gunes et al., 2011). Similarly infected ostriches develop only low and short-
lived viraemia and no clinical signs (Swanepoel & Burt, 2004). Therefore, recent infections in animals are rarely
diagnosed and methods such as polymerase chain reaction (PCR), virus isolation in cell culture and IgM detection
by enzyme-linked immunosorbent assay (ELISA) are mainly used in human CCHF diagnostic or in the special
case that an animal has to be classified as CCHFV free. For prevalence analysis and for determination of whether
CCHFV is circulating in a country, methods for the detection of IgG antibodies are preferred (Table 1). If there is
any possibility or suspicion that diagnostic samples could be contaminated with CCHFV, they should be handled
under an adequate biosafety level and all persons dealing with those samples should be aware of the possible
risk and should use personal protective equipment to avoid human infections.

For testing animals for viraemia, as well as in human clinical diagnosis, a rapid diagnosis can be achieved by
detection of viral nucleic acid in serum or plasma using conventional (Burt et al., 1998) or real-time reverse
transcriptase (RT-) PCR (Drosten et al., 2002; Duh et al., 2006; Wölfel et al., 2007), or by demonstration of viral
antigen (Shepherd et al., 1988). Specimens to be submitted for laboratory confirmation of CCHF include blood
and liver samples. Because of the risk of laboratory-acquired infections, work with CCHFV should be conducted in
appropriate biosafety facilities.

The virus can be isolated from serum and organ suspensions in a wide variety of cell cultures, including Vero,
LLC-MK2, SW-13, CER and BHK21 cells, and identified by immunofluorescence using specific antibodies.
Isolation and identification of virus can be achieved in 1–5 days, but cell cultures lack sensitivity and usually only
detect high concentrations of virus present in the blood. Intracerebral inoculation of sucking mice is more sensitive
than cell cultures for virus isolation, but is not recommended, on the grounds of animal welfare.

1 A combination of agent identification methods applied on the same clinical sample is recommended.
CCHFV can be isolated in mammalian cell cultures. Vero cells are commonly used, usually yielding an
isolate between 1 and 5 days post-inoculation (p.i). CCHFV is poorly cytopathic and thus infectivity is
titrated by demonstration of immunofluorescence in infected cells (Shepherd et al., 1986). SW-13 cell
line has also been used extensively for virus isolation, producing plaques within 4 days (p.i.).
Identification of a CCHFV isolate has to be confirmed by immunofluorescence or molecular techniques
(Burt et al., 1998; Shepherd et al., 1986).

i) Susceptible cell lines include Vero-E6, BHK-21, LLC-MK2 and SW-13 cells. Inoculate 80%
confluent monolayers of the preferred cell line with the specimen. The volume of specimen
to be used depends on the size of the culture vessel (i.e. 25 cm 2 culture flask or 6- or 24-
well tissue culture plate). The specimen volume should be sufficient to cover the cell
monolayer. Samples of insufficient volumes can be diluted with tissue culture medium to
prepare sufficient inoculation volume.
ii) Adsorb the specimen for 1 hour at 37°C.
iii) Remove inoculum. Add fresh tissue culture medium containing 2% fetal calf serum and
other required additives, as per specific medium and cell line requirements.
iv) Incubate at 37°C and 5% CO2 for 4–7 days.

v) Test supernatant for presence of CCHFV viral RNA using real-time RT-PCR as described
below, or perform immunofluorescence assay on cell scrapings.
vi) Isolates of CCHFV from clinical specimens cause no microscopically recognisable
cytopathic effects (CPE) in most of these cell lines.

Molecular-based diagnostic assays, such as RT-PCR, serve as the front-line tool in the diagnosis of
CCHF, as well as other viral haemorrhagic fevers (Drosten et al., 2003). The benefit of molecular
diagnostic assays is their rapidity compared to virus culture, often allowing a presumptive diagnosis to be
reported within a few hours after receiving a specimen (Burt et al., 1998). The RT-PCR is a sensitive
method for diagnosis, but because of genetic diversity of CCHFV, there might be some challenges with
regard to design of primers or probes that allow detection of all circulating strains of the virus. A real-time
RT-PCR that detects strains from different geographic locations has been recently described. The assay
was shown to be highly sensitive; it can detect as little as 1,164 viral RNA copies per ml of plasma (Wölfel
et al., 2007). A low-density microarray developed on the basis of the most up-to-date genome information
has been extensively validated in clinical specimens collected from confirmed cases of CCHF over
20 years by a WHO reference laboratory. It was shown to detect as few as 6.3 genome copies per
reaction (Wölfel et al., 2009).

A real-time RT-PCR which targets the S-segment of CCHFV genome and detects strains from different
geographic locations was developed by Wölfel et al. (2007). The method employs an in-vitro
transcribed RNA copy of the full S-segment as quantitative RNA standard. The assay is based on a
pair of primers and three probes. The template used in the assay is viral RNA extracted from the
specimen by using any standard viral RNA extraction method or commercially available kit. The
sequences and binding sites for the primers and three probes are as follows:

i) Forward primer
ii) Position 1068–1095
iii) Sequence 5’-CAA-GGG-GTA-CCA-AGA-AAA-TGA-AGA-AGG-C-3’

i) Reverse primer
ii) Position 1248–1223
iii) Sequence 5’-GCC-ACA-GGG-ATT-GTT-CCA-AAG-CAG-AC-3’
 i) Broad-range probe
ii) Position 1172–1198
iii) Sequence 5’-FAM-ATC-TAC-ATG-CAC-CCT-GCT-GTG-TTG-ACA-TAMRA-3’

i) Additional probe
ii) Position 1172–1198
iii) Sequence 5’-FAM-ATT-TAC-ATG-CAC-CCT-GCC-GTG-CTT-ACA-TAMRA-3’

i) Additional probe
ii) Position 1131–1106
iii) Sequence 5’-FAM-AGC-TTC-TTC-CCC-CAC-TTC-ATT-GGA-GT-TAMRA-3’

The assay was validated using reagents from a specific commercial kit, but these can be substituted
with any equivalent reagents from other commercial kits. The reaction is set up as follows. 25 µl
reaction volume consisting of 5 µl viral RNA, 1× concentration of PCR reaction buffer and enzymes
from any commercially available one-step RT-PCR kit, and 400 µmol dNTP, 800 ng non-acetylated
bovine serum albumin. The cycling parameters are as follows: 30 minutes at 50°C, 15 minutes at 95°C,
46× 15 seconds at 94°C, 30 seconds at 59°C, and 30 seconds at 72°C. Fluorescence acquisition
occurs at the 59°C step mode.

Virus neutralisation assays, generally considered to be highly specific, are rarely used for CCHFV diagnosis.
Members of the Nairovirus genus generally induce a weaker neutralising antibody response than members of
other genera of the family Bunyaviridae. Another drawback is the necessity to perform this assay in high biosafety
containment because it uses live virus (Burt et al., 1994; Rodriguez et al., 1997).

Currently, there are only a few CCHFV commercial kits for IgM or IgG by ELISA or immunofluorescence (IFA).
These are all designed for the human diagnostic market. However, it is possible to adapt these commercial
ELISAs and IFAs for serological testing in animals. In addition, some in-house ELISAs have been published for
the detection of CCHFV-specific antibodies in animals (Table 2).

ELISAs for CCHFV-specific IgM and IgG antibodies are specific and more sensitive than IFA. IgM antibodies in
livestock (sheep, goat and cattle) can be detected by using an IgM-capture ELISA. IgG antibodies can be
detected by an IgG-sandwich or indirect ELISA, and total antibodies can be detected by competition ELISA. The
benefit of competitive ELISA is the capacity to investigate different animal species, because they are host species
independent. The protocols of the published assays (Table 2) exemplify general procedures and protocols for
different kind of assays for the detection of CCHFV-specific antibodies. The limiting factor for the replication of
these protocols in other laboratories is the availability of antigens and (where relevant) specified monoclonal
antibodies. Most of the tests described for livestock and wild animals have not undergone a formal validation
process (Mertens et al., 2013). One of the biggest challenges for such validation studies is the availability of an
adequate number of positive well characterised control samples.

For information on the availability of reference reagents for use in veterinary diagnostic laboratories, contact the
OIE Reference Laboratories or the OIE Collaborating Centre for Zoonoses in Europe.
 Assay Target species Developer/producer of the assay

BDSL, Dreghorn, Scotland

IgM ELISA
commercial

Vector-Best, Novosibirsk, Russia

assays

IgG ELISA human Vector-Best, Novosibirsk, Russia

IgM IFA Euroimmun, Luebeck, Germany
IgG IFA Euroimmun, Luebeck, Germany
antigen capture ELISA virus Vector-Best, Novosibirsk, Russia
(Mourya et al., 2012)
(Garcia et al., 2006)
(Dowall et al., 2012)
IgM ELISA human
(Emmerich et al., 2010)
(Tang et al., 2003)
(Burt et al., 1994)
(Dowall et al., 2012)
(Garcia et al., 2006)
(Burt et al., 1994)
IgG ELISA human (Emmerich et al., 2010)
(Samudzi et al., 2012)
(Saijo et al., 2002a)
in-house assays

USAMRIID, Fort Detrick, USA

IgM IFA (Burt et al., 1994)
(Saijo et al., 2002b)
human
IgG IFA (Burt et al., 1994)
(Xia et al., 2011)
IgG Western-Blot assay human (Xia et al., 2011)
IgM ELISA sheep, Burt et al., 1993)
(Burt et al., 1993)
sheep (Qing et al., 2003)
(Garcia et al., 2006)
IgG ELISA
cattle (Garcia et al., 2006)
CDC, Atlanta, USA
animals
USAMRIID, Fort Detrick, USA
competitive ELISA species independent (Burt et al., 1993)
(Burt et al., 1993)
antigen capture ELISA virus
(Saijo et al., 2005)
IFA: immunofluorescence assay. ELISA: enzyme-linked immunosorbent assay
(Data and table modified from Mertens et al. 2013.)

There is no vaccine available for animals.

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13.

*
* *

NB: There is currently (2021) there were no OIE Reference Laboratories

for Crimean–Congo haemorrhagic fever (please consult the OIE Web site:
https://www.oie.int/en/what-we-offer/expertise-network/reference-laboratories/#ui-id-3).

NB: FIRST ADOPTED IN 2014.