Generation and Characterisation of Monoclonal Antibodies against Nairobi Sheep Disease Virus Nucleoprotein.

Nairobi sheep disease (NSD), caused by the viral agent NSD virus (NSDV), is a haemorrhagic fever disease affecting and inducing high mortality in sheep and goat populations. NSDV belongs to the genus Orthonairovirus of the Nairoviridae family from the order Bunyavirales. Other viruses circulating in livestock such as Crimean-Congo haemorrhagic fever virus (CCHFV) and Dugbe virus (DUGV) are members of the same genus and are reported to share antigenic features. There are very few available materials to study NSDV infection both in vitro and in vivo. In the present work, we characterised two monoclonal antibodies generated in mice that recognise NSDV specifically but not CCHFV or DUGV, along with a potential use to define virus-infected cells, using flow cytometry. We believe this tool can be useful for research, but also NSDV diagnostics, especially through immunological staining.

Diagnosis and Pathogenesis of Nairobi Sheep Disease Orthonairovirus Infections in Sheep and Cattle.

Nairobi sheep disease orthonairovirus (NSDV) is a zoonotic tick-borne arbovirus, which causes severe gastroenteritis in small ruminants. To date, the virus is prevalent in East Africa and Asia. However, due to climate change, including the spread of transmitting tick vectors and increased animal movements, it is likely that the distribution range of NSDV is enlarging. In this project, sheep and cattle (hitherto classified as resistant to NSDV) were experimentally infected with NSDV for a comparative study of the species-specific pathogenesis. For this purpose, several new diagnostic assays (RT-qPCR, ELISA, iIFA, mVNT, PRNT) were developed, which will also be useful for future epidemiological investigations. All challenged sheep (three different doses groups) developed characteristic clinical signs, transient viremia and virus shedding-almost independent on the applied virus dose. Half of the sheep had to be euthanized due to severe clinical signs, including hemorrhagic diarrhea. In contrast, the course of infection in cattle was only subclinical. However, all ruminants showed seroconversion-implying that, indeed, both species are susceptible for NSDV. Hence, not only sheep but also cattle sera can be included in serological monitoring programs for the surveillance of NSDV occurrence and spread in the future.

Nairobi sheep disease virus/Ganjam virus.

Nairobi sheep disease virus (NSDV) is a tick-borne virus which causes a severe disease in sheep and goats, and has been responsible for several outbreaks of disease in East Africa. The virus is also found in the Indian subcontinent, where it is known as Ganjam virus. The virus only spreads through the feeding of competent infected ticks, and is therefore limited in its geographic distribution by the distribution of those ticks, Rhipicephalus appendiculata in Africa and Haemaphysalis intermedia in India. Animals bred in endemic areas do not normally develop disease, and the impact is therefore primarily on animals being moved for trade or breeding purposes. The disease caused by NSDV has similarities to several other ruminant diseases, and laboratory diagnosis is necessary for confirmation. There are published methods for diagnosis based on polymerase chain reaction, for virus growth in cell culture and for other simple diagnostic tests, though none has been commercialised. There is no established vaccine against NSDV, although cell-culture attenuated strains have been developed which show promise and could be put into field trials if it were deemed necessary. The virus is closely related to Crimean-Congo haemorrhagic fever virus, and studies on NSDV may therefore be useful in understanding this important human pathogen.

Antibodies to the core proteins of Nairobi sheep disease virus/Ganjam virus reveal details of the distribution of the proteins in infected cells and tissues.

Nairobi sheep disease virus (NSDV; also called Ganjam virus in India) is a bunyavirus of the genus Nairovirus. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV). Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N) and polymerase (L) and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain) it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus in vivo. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN), where NSDV appeared to target monocytes and/or macrophages.

The nairovirus nairobi sheep disease virus/ganjam virus induces the translocation of protein disulphide isomerase-like oxidoreductases from the endoplasmic reticulum to the cell surface and the extracellular space.

Nairobi sheep disease virus (NSDV) of the genus Nairovirus causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%; the virus is found in East and Central Africa, and in India, where the virus is called Ganjam virus. NSDV is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus, which also causes a haemorrhagic disease. As with other nairoviruses, replication of NSDV takes place in the cytoplasm and the new virus particles bud into the Golgi apparatus; however, the effect of viral replication on cellular compartments has not been studied extensively. We have found that the overall structure of the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment and the Golgi were unaffected by infection with NSDV. However, we observed that NSDV infection led to the loss of protein disulphide isomerase (PDI), an oxidoreductase present in the lumen of the endoplasmic reticulum (ER) and which assists during protein folding, from the ER. Further investigation showed that NSDV-infected cells have high levels of PDI at their surface, and PDI is also secreted into the culture medium of infected cells. Another chaperone from the PDI family, ERp57, was found to be similarly affected. Analysis of infected cells and expression of individual viral glycoproteins indicated that the NSDV PreGn glycoprotein is involved in redistribution of these soluble ER oxidoreductases. It has been suggested that extracellular PDI can activate integrins and tissue factor, which are involved respectively in pro-inflammatory responses and disseminated intravascular coagulation, both of which manifest in many viral haemorrhagic fevers. The discovery of enhanced PDI secretion from NSDV-infected cells may be an important finding for understanding the mechanisms underlying the pathogenicity of haemorrhagic nairoviruses.

Ganjam virus/Nairobi sheep disease virus induces a pro-inflammatory response in infected sheep.

Partly due to climate change, and partly due to changes of human habitat occupation, the impact of tick-borne viruses is increasing. Nairobi sheep disease virus (NSDV) and Ganjam virus (GV) are two names for the same virus, which causes disease in sheep and goats and is currently known to be circulating in India and East Africa. The virus is transmitted by ixodid ticks and causes a severe hemorrhagic disease. We have developed a real-time PCR assay for the virus genome and validated it in a pilot study of the pathogenicity induced by two different isolates of NSDV/GV. One isolate was highly adapted to tissue culture, grew in most cell lines tested, and was essentially apathogenic in sheep. The second isolate appeared to be poorly adapted to cell culture and retained pathogenicity in sheep. The real-time PCR assay for virus easily detected 4 copies or less of the viral genome, and allowed a quantitative measure of the virus in whole blood. Measurement of the changes in cytokine mRNAs showed similar changes to those observed in humans infected by the closely related virus Crimean Congo hemorrhagic fever virus.

Ganjam virus.

Ganjam virus (GANV), a member of genus Nairovirus of family Bunyavirdae is of considerable veterinary importance in India. Though, predominantly tick borne, GANV was also isolated from mosquitoes, man and sheep. Neutralizing and complement fixing antibodies to GANV have been detected in animal and human sera collected from different parts of the country. Thirty three strains of GANV have been isolated from India, mainly from Haemaphysalis ticks. The virus replicated in certain vertebrate and mosquito cell lines and found pathogenic to laboratory animals. One natural infection and five laboratory-acquired infections in men were also reported. GANV is antigenically related to Nairobi sheep disease virus (NSDV) of Africa, which is highly pathogenic for sheep and goats causing 70-90 per cent mortality among the susceptible population. Recent molecular studies have demonstrated that GANV is an Asian variant of NSDV and both these viruses are related to the dreaded Crimean Congo haemorrhagic fever (CCHF) group viruses. The versatility of the virus to replicate in different arthropod species, its ability to infect sheep, goat and man makes it an important zoonotic agent.

The high genetic variation of viruses of the genus Nairovirus reflects the diversity of their predominant tick hosts.

The genus Nairovirus (family Bunyaviridae) contains seven serogroups consisting of 34 predominantly tick-borne viruses, including several associated with severe human and livestock diseases [e.g., Crimean Congo hemorrhagic fever (CCHF) and Nairobi sheep disease (NSD), respectively]. Before this report, no comparative genetic studies or molecular detection assays had been developed for this virus genus. To characterize at least one representative from each of the seven serogroups, reverse transcriptase-polymerase chain reaction (RT-PCR) primers targeting the L polymerase-encoding region of the RNA genome of these viruses were successfully designed based on conserved amino acid motifs present in the predicted catalytic core region. Sequence analysis showed the nairoviruses to be a highly diverse group, exhibiting up to 39.4% and 46.0% nucleotide and amino acid identity differences, respectively. Virus genetic relationships correlated well with serologic groupings and with tick host associations. Hosts of these viruses include both the hard (family Ixodidae) and soft (family Argasidae) ticks. Virus phylogenetic analysis reveals two major monophyletic groups: hard tick and soft tick-vectored viruses. In addition, viruses vectored by Ornithodoros, Carios, and Argas genera ticks also form three separate monophyletic lineages. The striking similarities between tick and nairovirus phylogenies are consistent with possible coevolution of the viruses and their tick hosts. Fossil and phylogenetic data placing the hard tick-soft tick divergence between 120 and 92 million years ago suggest an ancient origin for viruses of the genus Nairovirus.

Nairobi sheep disease virus, an important tick-borne pathogen of sheep and goats in Africa, is also present in Asia.

Nairobi sheep disease (NSD) virus is the prototype of the tick-borne NSD serogroup, genus Nairovirus, family Bunyaviridae. It is highly pathogenic for sheep and goats, causes disease in humans, and is widespread throughout East Africa. Ganjam virus has caused disease in goats and humans in India. Due to their occurrence on different continents and association with different ticks, these viruses were considered distinct despite serologic cross-reactivity. Their S RNA genome segments and encoded nucleocapsid proteins were found to be 1590 nucleotides and 482 amino acids in length and differed by only 10 and 3% at nucleotide and amino acid levels, respectively. Genetic and serologic data demonstrate that Ganjam virus is an Asian variant of NSD virus. These viruses were phylogenetically more closely related to Hazara virus than Dugbe virus.

Nairobi sheep disease.

Nairobi sheep disease is probably the most pathogenic virus known for sheep and goats. It is transmitted by an Ixodid tick, both trans-stadially and transovarially and causes an acute gastroenteritis. In totally susceptible populations, mortality rates of over 90% regularly occur. The infection also causes abortion. The disease is known to occur in East Africa, Somalia and Rwanda. It may exist in the south east of Ethiopia. No evidence for its existence has been found in those parts of Africa where the principle vector tick, Rhipicephalus appendiculatus has a seasonal breeding cycle. Thus countries like Zambia, Zimbabwe and Botswana appear to be free from the disease.

General review of tick-borne diseases of sheep and goats world-wide.

A general review of the tick-borne diseases of sheep and goats is given, with the emphasis on those thought to be of greatest economic importance. These include babesiosis, theileriosis, cowdriosis, anaplasmosis, ehrlichiosis, Nairobi sheep diseases and tick paralysis. A commented list of tick-borne diseases and their vectors is presented. It is stressed that large gaps remain in our knowledge of the real importance in the field of many of these diseases, especially in local stock.

Investigation of tick-borne viruses as pathogens of humans in South Africa and evidence of Dugbe virus infection in a patient with prolonged thrombocytopenia.

In the course of investigating suspected cases of viral haemorrhagic fever in South Africa patients were encountered who had been bitten by ticks, but who lacked evidence of infection with Crimean-Congo haemorrhagic fever (CCHF) virus or non-viral tick-borne agents. Cattle sera were tested by enzyme-linked immunoassay to determine whether tick-borne viruses other than CCHF occur in the country. The prevalence of antibody in cattle sera was 905/2116 (42.8%) for CCHF virus, 70/1358 (5.2%) for Dugbe, 21/1358 (1.5%) for louping ill, 6/450 (1.3%) for West Nile, 7/1358 (0.5%) for Nairobi sheep disease, 3/625 (0.5%) for Kadam and 2/450 (0.4%) for Chenuda. No reactions were recorded with Hazara, Bahig, Bhanja, Thogoto and Dhori viruses. The CCHF findings confirmed previous observations that the virus is widely prevalent within the distribution range of ticks of the genus Hyalomma, while antibody activity to Dugbe antigen was detected only within the distribution range of the tick Amblyomma hebraeum. Cross-reactivity for the nairoviruses, Hazara, Nairobi sheep disease and Dugbe, was detected in serum samples from 3/72 human patients with confirmed CCHF infection, and serum from 1/162 other patients reacted monospecifically with Dugbe antigen. The latter patient suffered from febrile illness with prolonged thrombocytopenia.

Insuficiencia cardiaca congestiva derecha en becerras Holstein de reemplazo II: clínica y patología / Right congestive heart failure in replacement Holstein calves II: clinical signs and pathology

Se estudió un grupo de becerras Holstein criadas en confinamiento, para identificar los factores clínicos y patológicos que influyen en el desarrollo de la Insuficiencia Cardiaca Congestiva Derecha (ICCD). El principal padecimiento asociado fue la neumonía en el 81 porciento de los animales con ICCD, la cual se desarrolló independientemente del grado de extensión de las lesiones de pulmón. El análisis de los antecedentes clínicos de la neumonía no mostró una asociación significativa con el desarrollo de este síndrome (P>0.05). La hipertrofia en la capa muscular de las arterias pulmonares en animales in neumonía (19 porciento) comprueba el efecto de la altura sobre el desarrollo de la ICCD. La hipertensión pulmonar ocasionada por la altura tiene especial importancia en unidades de producción intensiva, donde la alta incidencia de problemas respiratorios permite el desarrollo del cuadro clínico de esta síndromem el cual podría agravarse si las unidades de producción se ubicaran en altitudes tan grandes como 2,200 metros sobre el nivel el mar. Se concluye que se deben introducir mejores métodos de prevención y control de neumonía, el principal factor de desencadena la ICCD considerando el efecto de la altura como factor predisponente.

Coding strategy of the S RNA segment of Dugbe virus (Nairovirus; Bunyaviridae).

The S RNA segment of Dugbe (DUG) virus (Nairovirus; Bunyaviridae) was sequenced from three overlapping cDNA clones and by primer extension. The S RNA is 1712 nucleotides in length and contains one large open reading frame (ORF) of 1326 nucleotides coding for a 49.4-kDa protein on viral complementary (vc) RNA. This protein in size corresponds to the DUG nucleocapsid (N) protein (P. Cash, 1985, J. Gen. Virol. 66, 141-148). The 49.4-kDa product was expressed as a fusion protein with beta-galactosidase in Escherichia coli cells and confirmed as DUG N protein by Western blotting with DUG N-specific monoclonal antibody. An additional ORF of 150 nucleotides coding for a possible 5.9-kDa protein is present in the +1 reading frame, 3' to the N protein ORF on vcRNA. DUG S segment mRNA was found to be essentially full length. No evidence was obtained for the existence of a smaller mRNA species that could code for a 5.9-kDa protein. Comparisons of the DUG S RNA sequence and predicted N protein amino acid sequence, with the respective sequences of snowshoe hare, La Crosse (bunyaviruses), Punta Toro, Sandfly fever Sicilian (phleboviruses), and Hantaan (hantavirus) viruses, failed to detect any sequence similarity, although the genomic structure of DUG S RNA is similar to that of the S RNA segment of Hantaan (HTN) virus.

RNA probes detect nucleotide sequence homology between members of two different nairovirus serogroups.

Cloned cDNA derived from the small (S) and medium (M) genomic RNA segments of Dugbe (DUG) virus, isolate ArD44313, a member of the Nairobi sheep disease (NSD) serogroup of nairoviruses (family, Bunyaviridae) was used to prepare 32P-labelled DNA and RNA probes. The S and M segments of six isolates of DUG virus all hybridised to both DNA and RNA probes, although the M segment of isolate KT281/75 reacted only weakly. Of nine other nairoviruses tested, representing all the six other serogroups within the Nairovirus genus, none hybridised to the DNA probes. However, under conditions of low stringency, the DUG S and M RNA probes hybridised to the respective S and M segments of Ganjam (GAN) virus (another member of the NSD serogroup). The DUG S RNA probe also hybridised to the S segments of Crimean-Congo haemorrhagic fever (CCHF) virus and Hazara (HAZ) virus (members of the CCHF serogroup). The indicated sequence relationships between DUG, GAN, CCHF and HAZ viruses show that the NSD serogroup is more closely related to members of the CCHF serogroup than it is to nairoviruses of the other five serogroups.

Physical and biological properties of Nairobi sheep disease virus.

Thermal and pH stability of Nairobi sheep disease (NSD) virus were studied. The 180th mouse brain passage lost infectivity at a higher rate than "wild" virus at 4 degrees C. At 37 degrees C and neutral pH, "wild" virus again was more stable than cell culture and mouse brain attenuated strains with half-life periods of 104, 87 and 51 min, respectively. At 0 degrees C the cell culture attenuated virus was most stable at pH 7.4 with an estimated half-life of 164 h. The density of the virus in sucrose gradients came to 1.195 g cm -3. Metabolic growth inhibition studies using a halogenated nucleoside, and staining of RNase and DNase-treated infected cell cultures with acridine orange, indicated that NSD virus has a single stranded RNA genome. The growth of the cell culture adapted virus was assayed in monolayers of BHK21/13 cells at low multiplicity of infection. Cell-associated virus (CAV) was first detected at 6 h post-inoculation (PI). The titre increased rapidly until CPE appeared at 48 h and declined after 72 h PI. Cell-free virus (CFV) was first detected at 10 h PI. The titre of CFV increased up to 72 h, but on average was two log units less than the CAV titre.