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.

Identification of a new orthonairovirus associated with human febrile illness in China.

The genus Orthonairovirus, which is part of the family Nairoviridae, includes the important tick-transmitted pathogens Crimean-Congo hemorrhagic fever virus and Nairobi sheep disease virus, as well as many other poorly characterized viruses found in ticks, birds and mammals1,2. In this study, we identified a new orthonairovirus, Songling virus (SGLV), from patients who reported being bitten by ticks in Heilongjiang Province in northeastern China. SGLV shared similar genomic and morphological features with orthonairoviruses and phylogenetically formed a unique clade in Tamdy orthonairovirus of the Nairoviridae family. The isolated SGLV induced cytopathic effects in human hepatoma cells in vitro. SGLV infection was confirmed in 42 hospitalized patients analyzed between 2017 and 2018, with the main clinical manifestations being headache, fever, depression, fatigue and dizziness. More than two-thirds (69%) of patients generated virus-specific antibody responses in the acute phase. Taken together, these results suggest that this newly discovered orthonairovirus is associated with human febrile illness in China.

Hazara Nairovirus Requires COPI Components in both Arf1-Dependent and Arf1-Independent Stages of Its Replication Cycle.

Hazara nairovirus (HAZV) is an enveloped trisegmented negative-strand RNA virus classified within the Nairoviridae family of the Bunyavirales order and a member of the same subtype as Crimean-Congo hemorrhagic fever virus, responsible for fatal human disease. Nairoviral subversion of cellular trafficking pathways to permit viral entry, gene expression, assembly, and egress is poorly understood. Here, we generated a recombinant HAZV expressing enhanced green fluorescent protein and used live-cell fluorescent imaging to screen an siRNA library targeting genes involved in cellular trafficking networks, the first such screen for a nairovirus. The screen revealed prominent roles for subunits of the coat protein 1 (COPI)-vesicle coatomer, which regulates retrograde trafficking of cargo between the Golgi apparatus and the endoplasmic reticulum, as well as intra-Golgi transport. We show the requirement of COPI-coatomer subunits impacted at least two stages of the HAZV replication cycle: an early stage prior to and including gene expression and also a later stage during assembly and egress of infectious virus, with COPI-knockdown reducing titers by approximately 1,000-fold. Treatment of HAZV-infected cells with brefeldin A (BFA), an inhibitor of Arf1 activation required for COPI coatomer formation, revealed that this late COPI-dependent stage was Arf1 dependent, consistent with the established role of Arf1 in COPI vesicle formation. In contrast, the early COPI-dependent stage was Arf1 independent, with neither BFA treatment nor siRNA-mediated ARF1 knockdown affecting HAZV gene expression. HAZV exploitation of COPI components in a noncanonical Arf1-independent process suggests that COPI coatomer components may perform roles unrelated to vesicle formation, adding further complexity to our understanding of cargo-mediated transport.IMPORTANCE Nairoviruses are tick-borne enveloped RNA viruses that include several pathogens responsible for fatal disease in humans and animals. Here, we analyzed host genes involved in trafficking networks to examine their involvement in nairovirus replication. We revealed important roles for genes that express multiple components of the COPI complex, which regulates transport of Golgi apparatus-resident cargos. COPI components influenced at least two stages of the nairovirus replication cycle: an early stage prior to and including gene expression and also a later stage during assembly of infectious virus, with COPI knockdown reducing titers by approximately 1,000-fold. Importantly, while the late stage was Arf1 dependent, as expected for canonical COPI vesicle formation, the early stage was found to be Arf1 independent, suggestive of a previously unreported function of COPI unrelated to vesicle formation. Collectively, these data improve our understanding of nairovirus host-pathogen interactions and suggest a new Arf1-independent role for components of the COPI coatomer complex.

Probing the impact of nairovirus genomic diversity on viral ovarian tumor domain protease (vOTU) structure and deubiquitinase activity.

Post-translational modification of host and viral proteins by ubiquitin (Ub) and Ub-like proteins, such as interferon stimulated gene product 15 (ISG15), plays a key role in response to infection. Viruses have been increasingly identified that contain proteases possessing deubiquitinase (DUB) and/or deISGylase functions. This includes viruses in the Nairoviridae family that encode a viral homologue of the ovarian tumor protease (vOTU). vOTU activity was recently demonstrated to be critical for replication of the often-fatal Crimean-Congo hemorrhagic fever virus, with DUB activity suppressing the type I interferon responses and deISGylase activity broadly removing ISG15 conjugated proteins. There are currently about 40 known nairoviruses classified into fourteen species. Recent genomic characterization has revealed a high degree of diversity, with vOTUs showing less than 25% amino acids identities within the family. Previous investigations have been limited to only a few closely related nairoviruses, leaving it unclear what impact this diversity has on vOTU function. To probe the effects of vOTU diversity on enzyme activity and specificity, we assessed representative vOTUs spanning the Nairoviridae family towards Ub and ISG15 fluorogenic substrates. This revealed great variation in enzymatic activity and specific substrate preferences. A subset of the vOTUs were further assayed against eight biologically relevant di-Ub substrates, uncovering both common trends and distinct preferences of poly-Ub linkages by vOTUs. Four novel X-ray crystal structures were obtained that provide a biochemical rationale for vOTU substrate preferences and elucidate structural features that distinguish the vOTUs, including a motif in the Hughes orthonairovirus species that has not been previously observed in OTU domains. Additionally, structure-informed mutagenesis provided the first direct evidence of a second site involved in di-Ub binding for vOTUs. These results provide new insight into nairovirus evolution and pathogenesis, and further enhances the development of tools for therapeutic purposes.

Tofla virus: A newly identified Nairovirus of the Crimean-Congo hemorrhagic fever group isolated from ticks in Japan.

Ixodid ticks transmit several important viral pathogens. We isolated a new virus (Tofla virus: TFLV) from Heamaphysalis flava and Heamaphysalis formsensis in Japan. The full-genome sequences revealed that TFLV belonged to the genus Nairovirus, family Bunyaviridae. Phylogenetic analyses and neutralization tests suggested that TFLV is closely related to the Hazara virus and that it is classified into the Crimean-Congo hemorrhagic fever group. TFLV caused lethal infection in IFNAR KO mice. The TFLV-infected mice exhibited a gastrointestinal disorder, and positron emission tomography-computed tomography images showed a significant uptake of (18)F-fluorodeoxyglucose in the intestinal tract. TFLV was able to infect and propagate in cultured cells of African green monkey-derived Vero E6 cells and human-derived SK-N-SH, T98-G and HEK-293 cells. Although TFLV infections in humans and animals are currently unknown, our findings may provide clues to understand the potential infectivity and to develop of pre-emptive countermeasures against this new tick-borne Nairovirus.

Diversity of ubiquitin and ISG15 specificity among nairoviruses' viral ovarian tumor domain proteases.

Nairoviruses are responsible for numerous diseases that affect both humans and animal. Recent work has implicated the viral ovarian tumor domain (vOTU) as a possible nairovirus virulence factor due to its ability to edit ubiquitin (Ub) bound to cellular proteins and, at least in the case of Crimean-Congo hemorrhagic fever virus (CCHFV), to cleave the Ub-like protein interferon-stimulated gene 15 (ISG15), a protein involved in the regulation of host immunity. The prospective roles of vOTUs in immune evasion have generated several questions concerning whether vOTUs act through a preserved specificity for Ub- and ISG15-conjugated proteins and where that specificity may originate. To gain insight into the substrate specificity of vOTUs, enzymological studies were conducted on vOTUs from Dugbe, CCHFV, and Erve nairoviruses. These studies revealed that vOTUs originating from different nairoviruses display a significant divergence in their preference toward Ub and ISG15. In addition, a recently identified vOTU from turnip yellow mosaic tymovirus was evaluated to elucidate any possible similarities between vOTUs originating from different viral families. Although possessing a similar preference for certain polymeric Ub moieties, its activity toward Ub in general was significantly less then those of nairoviruses. Lastly, the X-ray crystallographic structure of the vOTU from the Dugbe nairovirus was obtained in complex with Ub to reveal structural commonalities of vOTUs originating from nairoviruses. The structure suggests that divergences between nairovirus vOTUs specificity originate at the primary structural level. Comparison of this structure to that originating from CCHFV identified key residues that infer the substrate specificity of vOTUs.

Differential activation profiles of Crimean-Congo hemorrhagic fever virus- and Dugbe virus-infected antigen-presenting cells.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic, tick-borne member of the family Bunyaviridae and the genus Nairovirus. To better elucidate the pathogenesis of CCHFV, we analysed the host innate immune response induced in antigen-presenting cells (APCs) infected in vitro by CCHFV. Monocyte-derived dendritic cells (DCs) and macrophages (MPs) were both shown to be permissive for CCHFV and to replicate the virus, as monitored by genomic and antigenomic strand quantification. Virus replication was, however, controlled, corroborating an efficient alpha interferon-induced response. The upregulation of CD-83 and CD-86 indicated that CCHFV induced a partial maturation of DCs, which were also shown to activate the secretion of interleukin (IL)-6 and IL-8, but no tumour necrosis factor alpha (TNF-alpha). On the other hand, in MPs, CCHFV infection elicited a high IL-6 and TNF-alpha response and a moderate chemokine response. Nevertheless, when we compared these APC responses with those seen after infection with Dugbe virus (DUGV), a mildly pathogenic virus genetically close to CCHFV, we found that, in spite of some similarities, DUGV induced a higher cytokine/chemokine response in MPs. These results suggest that CCHFV is able to inhibit the activation of inflammatory mediators selectively in infection in vitro and that these differences could be relevant in pathogenesis.

Pathogenesis of Dugbe virus infection in wild-type and interferon-deficient mice.

In 129 mice, infection with the nairovirus Dugbe virus (DUGV) was lethal following intracerebral but not intraperitoneal inoculation. Following both routes of inoculation, immunostaining of tissue sections demonstrated virus-positive cells in the brain, indicating that DUGV is neuroinvasive in mice. Many brain areas were affected and neurones were the main cell type infected. Infected cells showed punctate accumulations of viral nucleoprotein in the cytoplasm, indicative of virus replication sites. Immunostaining for activated caspase 3 demonstrated no evidence of apoptosis. The type I interferon (IFN) system plays a significant role in defence against DUGV, as 129 IFN-alpha/beta R(-/-) mice died rapidly following both intraperitoneal and intracerebral inoculations. Studies were undertaken to determine whether the IFN-inducible proteins, protein kinase R (PKR) and MxA, were important for protection; neither PKR nor constitutively expressed human MxA played significant roles.

[Erve and Eyach: two viruses isolated in France, neuropathogenic for man and widely distributed in Western Europe]. / Erve et Eyach: deux virus isolés en France, neuropathogènes chez l'homme et à diffusion ouest-européenne.

Two arboviruses have been isolated from Mayenne, western France, in 1981-1982, during systematic field surveys: Eyach virus, a cultivirus from Ixodes ricinus and I. ventalloi ticks, and Erve virus, a nairovirus from the tissues of white-toothed shrews, Crocidura russula. For many years, these two viruses were considered as "orphan viruses" without pathogenic power for man, since it became evident that they circulate actively in Europe among populations of wild small and large mammals. Moreover, these viruses are able to infect human beings and were responsible for severe neurological disorders. The presently known geographical distribution of these agents includes France, Germany, Netherlands and Czech Republic.

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.