Host Inflammatory Response to Mosquito Bites Enhances the Severity of Arbovirus Infection.

Aedes aegypti mosquitoes are responsible for transmitting many medically important viruses such as those that cause Zika and dengue. The inoculation of viruses into mosquito bite sites is an important and common stage of all mosquito-borne virus infections. We show, using Semliki Forest virus and Bunyamwera virus, that these viruses use this inflammatory niche to aid their replication and dissemination in vivo. Mosquito bites were characterized by an edema that retained virus at the inoculation site and an inflammatory influx of neutrophils that coordinated a localized innate immune program that inadvertently facilitated virus infection by encouraging the entry and infection of virus-permissive myeloid cells. Neutrophil depletion and therapeutic blockade of inflammasome activity suppressed inflammation and abrogated the ability of the bite to promote infection. This study identifies facets of mosquito bite inflammation that are important determinants of the subsequent systemic course and clinical outcome of virus infection.

Transfusion-Transmitted Cache Valley Virus Infection in a Kidney Transplant Recipient With Meningoencephalitis.

BACKGROUND: Cache Valley virus (CVV) is a mosquito-borne virus that is a rare cause of disease in humans. In the fall of 2020, a patient developed encephalitis 6 weeks following kidney transplantation and receipt of multiple blood transfusions. METHODS: After ruling out more common etiologies, metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) was performed. We reviewed the medical histories of the index kidney recipient, organ donor, and recipients of other organs from the same donor and conducted a blood traceback investigation to evaluate blood transfusion as a possible source of infection in the kidney recipient. We tested patient specimens using reverse-transcription polymerase chain reaction (RT-PCR), the plaque reduction neutralization test, cell culture, and whole-genome sequencing. RESULTS: CVV was detected in CSF from the index patient by mNGS, and this result was confirmed by RT-PCR, viral culture, and additional whole-genome sequencing. The organ donor and other organ recipients had no evidence of infection with CVV by molecular or serologic testing. Neutralizing antibodies against CVV were detected in serum from a donor of red blood cells received by the index patient immediately prior to transplant. CVV neutralizing antibodies were also detected in serum from a patient who received the co-component plasma from the same blood donation. CONCLUSIONS: Our investigation demonstrates probable CVV transmission through blood transfusion. Clinicians should consider arboviral infections in unexplained meningoencephalitis after blood transfusion or organ transplantation. The use of mNGS might facilitate detection of rare, unexpected infections, particularly in immunocompromised patients.

Digoxin is a potent inhibitor of Bunyamwera virus infection in cell culture.

Drug repurposing is a valuable source of new antivirals because many compounds used to treat a variety of pathologies can also inhibit viral infections. In this work, we have tested the antiviral capacity of four repurposed drugs to treat Bunyamwera virus (BUNV) infection in cell cultures. BUNV is the prototype of the Bunyavirales order, a large group of RNA viruses that includes important pathogens for humans, animals and plants. Mock- and BUNV-infected Vero and HEK293T cells were treated with non-toxic concentrations of digoxin, cyclosporin A, sunitinib and chloroquine. The four drugs inhibited BUNV infection with varying potency in Vero cells, and all except sunitinib also in HEK293T cells, with digoxin rendering the lowest half maximal inhibitory concentration (IC50). Since digoxin rendered the best results, we selected this drug for a more detailed study. Digoxin is an inhibitor of the Na+/K+ ATPase, a plasma membrane enzyme responsible for the energy-dependent exchange of cytoplasmic Na+ for extracellular K+ in mammalian cells and involved in many signalling pathways. Digoxin was shown to act at an early time point after viral entry reducing the expression of the viral proteins Gc and N. Effects on the cell cycle caused by BUNV and digoxin were also analysed. In Vero cells, digoxin favoured the transition from G1 phase of the cell cycle to S phase, an effect that might contribute to the anti-BUNV effect of digoxin in this cell type. Transmission electron microscopy showed that digoxin impedes the assembly of the characteristic spherules that harbour the BUNV replication complexes and the morphogenesis of new viral particles. Both BUNV and digoxin induce similar changes in the morphology of mitochondria that become more electron-dense and have swollen cristae. The alterations of this essential organelle might be one of the factors responsible for digoxin-induced inhibition of viral infection. Digoxin did not inhibit BUNV infection in BHK-21 cells that have a digoxin-resistant Na+/K+ ATPase, which suggests that the effects of the blockade of this enzyme is a key factor of the antiviral activity of digoxin in BUNV-infected Vero cells.

Role of Anopheles Mosquitoes in Cache Valley Virus Lineage Displacement, New York, USA.

Cache Valley virus (CVV) is a mosquitoborne virus that infects livestock and humans. We report results of surveillance for CVV in New York, USA, during 2000-2016; full-genome analysis of selected CVV isolates from sheep, horse, humans, and mosquitoes from New York and Canada; and phenotypic characterization of selected strains. We calculated infection rates by using the maximum-likelihood estimation method by year, region, month, and mosquito species. The highest maximum-likelihood estimations were for Anopheles spp. mosquitoes. Our phylogenetic analysis identified 2 lineages and found evidence of segment reassortment. Furthermore, our data suggest displacement of CVV lineage 1 by lineage 2 in New York and Canada. Finally, we showed increased vector competence of An. quadrimaculatus mosquitoes for lineage 2 strains of CVV compared with lineage 1 strains.

Reassortant Cache Valley Virus Associated With Acute Febrile, Nonneurologic Illness, Missouri.

An adult male from Missouri sought care for fever, fatigue, and gastrointestinal symptoms. He had leukopenia and thrombocytopenia and was treated for a presumed tickborne illness. His condition deteriorated with respiratory and renal failure, lactic acidosis, and hypotension. Next-generation sequencing and phylogenetic analysis identified a reassortant Cache Valley virus.

Comparative characterization of the reassortant Orthobunyavirus Ngari with putative parental viruses, Bunyamwera and Batai: in vitro characterization and ex vivo stability.

Bunyamwera (BUNV), Batai (BATV) and Ngari (NRIV) are mosquito-borne viruses that are members of the genus Orthobunyavirus in the order Bunyavirales. These three viruses are enveloped with single-stranded, negative-sense RNA genomes consiting of three segments, denoted as Small (S), Medium (M) and Large (L). Ngari is thought to be the natural reassortant progeny of Bunyamwera and Batai viruses. The relationship between these 'parental' viruses and the 'progeny' poses an interesting question, especially given that there is overlap in their respective transmission ecologies, but differences in their infection host ranges and pathogenesis. We compared the in vivo kinetics of these three viruses in a common laboratory system and found no significant difference in growth kinetics. There was, however, a tendency of BATV to have smaller plaques than either BUNV or NRIV. Furthermore, we determined that all three viruses are stable in extracellular conditions and retain infectivity for a week in non-cellular media, which has public health and biosafety implications. The study of this understudied group of viruses addresses a need for basic characterization of viruses that have not yet reached epidemic transmission intensity, but that have the potential due to their infectivity to both human and animal hosts. These results lay the groundwork for future studies of these neglected viruses of potential public and One Health importance.

Vector competence of Aedes vittatus (Bigot) mosquitoes from India for Japanese encephalitis, West Nile, Chandipura and Chittoor viruses.

BACKGROUND & OBJECTIVES: Aedes vittatus (Bigot), an anthropophilic mosquito, plays an important role in the maintenance and transmission of yellow fever (YF), dengue (DEN), chikungunya (CHIKV) and Zika (ZIK) viruses in Africa. In India, though natural isolation of none of these viruses was reported from the mosquito, experimental studies have shown vector competence to DEN and CHIK viruses. Despite wide prevalence in India, their potential in transmitting viruses of public health importance viz., Japanese encephalitis (JEV), West Nile (WNV), Chandipura (CHPV), Chittoor (CHITV) etc., has never been investigated. The objective of the present study is to determine the vector potential of the mosquito to these viruses. METHODS: Mosquitoes were infected by intra-thoracic inoculation as well as by oral feeding, and growth kinetics was determined. Virus dissemination to organs was investigated by determining virus in the harvested organs on specified days' post infection (PI). Vector competence was determined by detecting the virus in saliva. RESULTS: Intra thoracic inoculation has shown vector competence of the mosquito to JEV, WNV, CHIV and CHPV. However, using the oral route of infection, replication was observed with only WNV, JEV and CHITV. High degree of WNV replication (6.7log TCID50/ml) with rapid dissemination to wings, legs and salivary glands was seen from 5th day PI onwards. WNV was detected in saliva with a titer of 0.7log10 TCID50/ml on 5th day PI. JEV and CHITV replicated in the mosquito yielding 3log and 4log10 TCID50/ml on 5th and 10th day PI respectively, but virus was not detected in saliva till 15th day PI. INTERPRETATION & CONCLUSION: From the results it is difficult to indict the mosquito as a vector of the viruses studied. However, presence of WNV in saliva of the mosquito shows its potential as a bridge vector and poses a concern especially when virulent WNV strains are circulating in the country.

Interferon-Stimulated Gene (ISG)-Expression Screening Reveals the Specific Antibunyaviral Activity of ISG20.

Bunyaviruses pose a significant threat to human health, prosperity, and food security. In response to viral infections, interferons (IFNs) upregulate the expression of hundreds of interferon-stimulated genes (ISGs), whose cumulative action can potently inhibit the replication of bunyaviruses. We used a flow cytometry-based method to screen the ability of ∼500 unique ISGs from humans and rhesus macaques to inhibit the replication of Bunyamwera orthobunyavirus (BUNV), the prototype of both the Peribunyaviridae family and the Bunyavirales order. Candidates possessing antibunyaviral activity were further examined using a panel of divergent bunyaviruses. Interestingly, one candidate, ISG20, exhibited potent antibunyaviral activity against most viruses examined from the Peribunyaviridae, Hantaviridae, and Nairoviridae families, whereas phleboviruses (Phenuiviridae) largely escaped inhibition. Similar to the case against other viruses known to be targeted by ISG20, the antibunyaviral activity of ISG20 is dependent upon its functional RNase activity. Through use of an infectious virus-like particle (VLP) assay (based on the BUNV minigenome system), we confirmed that gene expression from all 3 viral segments is strongly inhibited by ISG20. Using in vitro evolution, we generated a substantially ISG20-resistant BUNV and mapped the determinants of ISG20 sensitivity/resistance. Taking all the data together, we report that ISG20 is a broad and potent antibunyaviral factor but that some bunyaviruses are remarkably ISG20 resistant. Thus, ISG20 sensitivity/resistance may influence the pathogenesis of bunyaviruses, many of which are emerging viruses of clinical or veterinary significance.IMPORTANCE There are hundreds of bunyaviruses, many of which cause life-threatening acute diseases in humans and livestock. The interferon (IFN) system is a key component of innate immunity, and type I IFNs limit bunyaviral propagation both in vitro and in vivo Type I IFN signaling results in the upregulation of hundreds of IFN-stimulated genes (ISGs), whose concerted action generates an "antiviral state." Although IFNs are critical in limiting bunyaviral replication and pathogenesis, much is still unknown about which ISGs inhibit bunyaviruses. Using ISG-expression screening, we examined the ability of ∼500 unique ISGs to inhibit Bunyamwera orthobunyavirus (BUNV), the prototypical bunyavirus. Using this approach, we identified ISG20, an interferon-stimulated exonuclease, as a potent inhibitor of BUNV. Interestingly, ISG20 possesses highly selective antibunyaviral activity, with multiple bunyaviruses being potently inhibited while some largely escape inhibition. We speculate that the ability of some bunyaviruses to escape ISG20 may influence their pathogenesis.

Five Emerging Neuroinvasive Arboviral Diseases: Cache Valley, Eastern Equine Encephalitis, Jamestown Canyon, Powassan, and Usutu.

There are many arthropod-borne viruses (arboviruses) capable of neuroinvasion, with West Nile virus being one of the most well known. In this review, we highlight five rarer emerging or reemerging arboviruses capable of neuroinvasion: Cache Valley, eastern equine encephalitis, Jamestown Canyon, Powassan, and Usutu viruses. Cache Valley and Jamestown Canyon viruses likely circulate throughout most of North America, while eastern equine encephalitis and Powassan viruses typically circulate in the eastern half. Usutu virus is not currently circulating in North America, but has the potential to be introduced in the future given similar climate, vectors, and host species to Europe (where it has been circulating). Health care providers should contact their state or local health departments with any questions regarding arboviral disease surveillance, diagnosis, treatment, or prevention. To prevent neuroinvasive arboviral diseases, use of insect repellent and other mosquito and tick bite prevention strategies are key.

Bunyamwera orthobunyavirus glycoprotein precursor is processed by cellular signal peptidase and signal peptide peptidase.

The M genome segment of Bunyamwera virus (BUNV)-the prototype of both the Bunyaviridae family and the Orthobunyavirus genus-encodes the glycoprotein precursor (GPC) that is proteolytically cleaved to yield two viral structural glycoproteins, Gn and Gc, and a nonstructural protein, NSm. The cleavage mechanism of orthobunyavirus GPCs and the host proteases involved have not been clarified. In this study, we investigated the processing of BUNV GPC and found that both NSm and Gc proteins were cleaved at their own internal signal peptides (SPs), in which NSm domain I functions as SP(NSm) and NSm domain V as SP(Gc) Moreover, the domain I was further processed by a host intramembrane-cleaving protease, signal peptide peptidase, and is required for cell fusion activities. Meanwhile, the NSm domain V (SP(Gc)) remains integral to NSm, rendering the NSm topology as a two-membrane-spanning integral membrane protein. We defined the cleavage sites and boundaries between the processed proteins as follows: Gn, from residue 17-312 or nearby residues; NSm, 332-477; and Gc, 478-1433. Our data clarified the mechanism of the precursor cleavage process, which is important for our understanding of viral glycoprotein biogenesis in the genus Orthobunyavirus and thus presents a useful target for intervention strategies.

Cache Valley Virus in Aedes japonicus japonicus Mosquitoes, Appalachian Region, United States.

We detected Cache Valley virus in Aedes japonicus, a widely distributed invasive mosquito species, in an Appalachian forest in the United States. The forest contained abundant white-tailed deer, a major host of the mosquito and virus. Vector competence trials indicated that Ae. j. japonicus mosquitoes can transmit this virus in this region.

A novel cause of chronic viral meningoencephalitis: Cache Valley virus.

OBJECTIVE: Immunodeficient patients are particularly vulnerable to neuroinvasive infections that can be challenging to diagnose. Metagenomic next generation sequencing can identify unusual or novel microbes and is therefore well suited for investigating the etiology of chronic meningoencephalitis in immunodeficient patients. METHODS: We present the case of a 34-year-old man with X-linked agammaglobulinemia from Australia suffering from 3 years of meningoencephalitis that defied an etiologic diagnosis despite extensive conventional testing, including a brain biopsy. Metagenomic next generation sequencing of his cerebrospinal fluid and brain biopsy tissue was performed to identify a causative pathogen. RESULTS: Sequences aligning to multiple Cache Valley virus genes were identified via metagenomic next generation sequencing. Reverse transcription polymerase chain reaction and immunohistochemistry subsequently confirmed the presence of Cache Valley virus in the brain biopsy tissue. INTERPRETATION: Cache Valley virus, a mosquito-borne orthobunyavirus, has only been identified in 3 immunocompetent North American patients with acute neuroinvasive disease. The reported severity ranges from a self-limiting meningitis to a rapidly fatal meningoencephalitis with multiorgan failure. The virus has never been known to cause a chronic systemic or neurologic infection in humans. Cache Valley virus has also never previously been detected on the Australian continent. Our research subject traveled to North and South Carolina and Michigan in the weeks prior to the onset of his illness. This report demonstrates that metagenomic next generation sequencing allows for unbiased pathogen identification, the early detection of emerging viruses as they spread to new locales, and the discovery of novel disease phenotypes. Ann Neurol 2017;82:105-114.

Vector competence of certain Culex and Aedes mosquitoes for the Chittoor virus, the Indian variant of the Batai virus.

Chittoor virus (CHITV), a mosquito-borne bunyavirus (Orthobunyavirus: Bunyaviridae) isolated in India, has been found to be antigenically close to the Batai virus (BATV), which has a wide distribution across Asia, Europe, and Africa. The latter virus causes influenza-like illness in humans and mild illness in sheep and goats. BATV has been involved in genetic reassortment with other bunyaviruses, generating novel genome combinations and causing severe clinical manifestations including hemorrhagic fever. Conversely, CHITV has never been associated with any major outbreaks in India, although neutralizing antibodies have been detected in humans and domestic animals. Repeated isolations and seroprevalence have prompted us to determine the vector competence of three important mosquito species, viz., Culex quinquefasciatus, Culex tritaeniorhynchus, and Aedes aegypti, for CHITV. The three mosquito species replicated CHITV to titers of 6.3, 5.0, and 5.2 log10 TCID50/mL, respectively, and maintained the virus for substantial periods. Both of the Culex species demonstrated vector competence, while A. aegypti did not. Horizontal transmission to infant mice was also demonstrated by both Culex species. Active circulation of the virus and the availability of both susceptible hosts and competent vector mosquitoes pose a serious threat to public health should there be a reassortment.

Seroprevalence of Cache Valley virus and related viruses in sheep and other livestock from Saskatchewan, Canada.

Cache Valley virus, an orthobunyavirus, is an important cause of ovine neonatal malformations. Information on the seroprevalence of this virus in Saskatchewan livestock populations is lacking. The objectives of this study were to determine the seroprevalence of Cache Valley virus and closely related viruses in sheep, cattle, goats, horses, and mule deer in Saskatchewan by performing a plaque-reduction neutralization test using Cache Valley virus. In total, sera from 130 sheep from 50 flocks were tested. Seroprevalence in sheep was 64.6% (84/130) and 94.0% (47/50) of flocks had 1 or more seropositive sheep. Antibodies to Cache Valley virus or closely related viruses were also detected in serum samples collected from cattle, goats, horses, and mule deer with seroprevalences of 20.0% (5/25), 33.3% (8/24), 69.0% (40/58), and 50.8% (33/65), respectively. These results suggest widespread exposure to Cache Valley virus or closely related viruses in domestic animals and mule deer in Saskatchewan.

Séroprevalence du virus de la Vallée Cache ou de virus connexes chez les moutons et d'autres animaux de cheptel en Saskatchewan, Canada. Le virus de la Vallée Cache, un orthobunyavirus, est une cause importante de malformations néonatales ovines. Il manque des renseignements sur la séroprévalence de ce virus dans les populations des cheptels de la Saskatchewan. Les objectifs de cette étude consistaient à déterminer la séroprévalence du virus de la Vallée Cache et des virus étroitement apparentés chez les moutons, les bovins, les chèvres, les chevaux et les cerfs mulets en Saskatchewan en réalisant un test de séro-neutralisation par réduction des plages en utilisant le virus de la Vallée Cache. Au total, le sérum provenant de 130 moutons dans 50 troupeaux a été testé. Chez les moutons, la séroprévalence était de 64,6 % (84/130) et 94,0 % (47/50) des troupeaux avaient un mouton ou plusieurs moutons séropositifs. Les anticorps pour le virus de la Vallée Cache ou les virus étroitement apparentés ont aussi été détectés dans les échantillons de sérum prélevés auprès des bovins, des chèvres, des chevaux et des cerfs mulets avec une séroprévalence de 20,0 % (5/25), de 33,3 % (8/24), de 69,0 % (40/58) et de 50,8 % (33/65), respectivement. Ces résultats suggèrent une vaste exposition au virus de la Vallée Cache ou à des virus étroitement apparentés chez les animaux domestiques et les cerfs mulets en Saskatchewan.(Traduit par Isabelle Vallières).

Modulation of Potassium Channels Inhibits Bunyavirus Infection.

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare, and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K(+)) channels to infect cells. Time of addition assays using K(+) channel modulating agents demonstrated that K(+) channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K(+) channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, two-pore domain K(+) channels (K2P) were identified as the K(+) channel family mediating BUNV K(+) channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

Characterization of Batai virus isolated from a domestic Muscovy duck (Cairina moschate).

Batai virus (BATV) belongs to the genus Orthobunyavirus of the family Bunyaviridae. It has been isolated from mosquitos, pigs, cattle, and humans throughout Africa, Asia, and Europe, and causes clinical signs in domestic animals and humans. Here, we report the isolation of BATV from a domestic duck flock. Genome sequence analysis revealed clustering of this isolate in the Africa-Asia lineage. The virus replicated in mosquitos and vertebrate host cells, showing different phenotypic characteristics, and showed the potential to infect mice. This is the first report of BATV in domestic birds and indicates the wide circulation of BATV in China.

Evolution of the Bunyamwera virus polymerase to accommodate deletions within genomic untranslated region sequences.

UNLABELLED: The untranslated regions (UTR) present at the ends of bunyavirus genome segments are required for essential steps in the virus life cycle and provide signals for encapsidation by nucleocapsid protein and the promoters for RNA transcription and replication as well as for mRNA transcription termination. For the prototype bunyavirus, Bunyamwera virus (BUNV), only the terminal 11 nucleotides (nt) of the segments are identical. Thereafter, the UTRs are highly variable both in length and in sequence. Furthermore, apart from the conserved termini, the UTRs of different viruses are highly variable. We previously generated recombinant BUNV carrying the minimal UTRs on all three segments that were attenuated for growth in cell culture. Following serial passage of these viruses, the viruses acquired increased fitness, and amino acid changes were observed to accumulate in the viral polymerase (L protein) of most mutant viruses, with the vast majority of the amino acid changes occurring in the C-terminal region. The function of this domain within L remains unknown, but by using a minigenome assay we showed that it might be involved in UTR recognition. Moreover, we identified an amino acid mutation within the polymerase that, when introduced into an otherwise wild-type BUNV, resulted in a virus with a temperature-sensitive phenotype. Viruses carrying temperature-sensitive mutations are good candidates for the design of live attenuated vaccines. We suggest that a combination of stable deletions of the UTRs together with the introduction of temperature-sensitive mutations in both the nucleocapsid and the polymerase could be used to design live attenuated vaccines against serious pathogens within the family Bunyaviridae. IMPORTANCE: Virus growth in tissue culture can be attenuated by introduction of mutations in both coding and noncoding sequences. We generated attenuated Bunyamwera viruses by deleting sequences within both the 3' and 5' untranslated regions (UTR) on each genome segment and showed that the viruses regained fitness following serial passage in cell culture. The fitter viruses had acquired amino acid changes predominantly in the C-terminal domain of the viral polymerase (L protein), and by using minigenome assays we showed that the mutant polymerases were better adapted to recognizing the mutant UTRs. We suggest that deletions within the UTRs should be incorporated along with other specific mutations, including deletion of the major virulence gene encoding the NSs protein and introduction of temperature-sensitive mutations, in the design of attenuated bunyaviruses that could have potential as vaccines.

Phylogenetic analysis of Bunyamwera and Ngari viruses (family Bunyaviridae, genus Orthobunyavirus) isolated in Kenya.

Orthobunyaviruses, tri-segmented, negative-sense RNA viruses, have long been associated with mild to severe human disease in Africa, but not haemorrhagic fever. However, during a Rift Valley fever outbreak in East Africa in 1997-1998, Ngari virus was isolated from two patients and antibody detected in several others with haemorrhagic fever. The isolates were used to identify Ngari virus as a natural Orthobunyavirus reassortant. Despite their potential to reassort and cause severe human disease, characterization of orthobunyaviruses is hampered by paucity of genetic sequences. Our objective was to obtain complete gene sequences of two Bunyamwera virus and three Ngari virus isolates from recent surveys in Kenya and to determine their phylogenetic positioning within the Bunyamwera serogroup. Newly sequenced Kenyan Bunyamwera virus isolates clustered closest to a Bunyamwera virus isolate from the same locality and a Central African Republic isolate indicating that similar strains may be circulating regionally. Recent Kenyan Ngari isolates were closest to the Ngari isolates associated with the 1997-1998 haemorrhagic fever outbreak. We observed a temporal/geographical relationship among Ngari isolates in all three gene segments suggesting a geographical/temporal association with genetic diversity. These sequences in addition to earlier sequences can be used for future analyses of this neglected but potentially deadly group of viruses.

Bunyamwera virus possesses a distinct nucleocapsid protein to facilitate genome encapsidation.

Bunyamwera virus (BUNV), which belongs to the genus Orthobunyavirus, is the prototypical virus of the Bunyaviridae family. Similar to other negative-sense single-stranded RNA viruses, bunyaviruses possess a nucleocapsid protein (NP) to facilitate genomic RNA encapsidation and virus replication. The structures of two NPs of members of different genera within the Bunyaviridae family have been reported. However, their structures, RNA-binding features, and functions beyond RNA binding significantly differ from one another. Here, we report the crystal structure of the BUNV NP-RNA complex. The polypeptide of the BUNV NP was found to possess a distinct fold among viral NPs. An N-terminal arm and a C-terminal tail were found to interact with neighboring NP protomers to form a tetrameric ring-shaped organization. Each protomer bound a 10-nt RNA molecule, which was acquired from the expression host, in the positively charged crevice between the N and C lobes. Inhomogeneous oligomerization was observed for the recombinant BUNV NP-RNA complex, which was similar to the Rift Valley fever virus NP-RNA complex. This result suggested that the flexibility of one NP protomer with adjacent protomers underlies the BUNV ribonucleoprotein complex (RNP) formation. Electron microscopy revealed that the monomer-sized NP-RNA complex was the building block of the natural BUNV RNP. Combined with previous results indicating that mutagenesis of the interprotomer or protein-RNA interface affects BUNV replication, our structure provides a great potential for understanding the mechanism underlying negative-sense single-stranded RNA RNP formation and enables the development of antiviral therapies targeting BUNV RNP formation.

Orthobunyavirus ultrastructure and the curious tripodal glycoprotein spike.

The genus Orthobunyavirus within the family Bunyaviridae constitutes an expanding group of emerging viruses, which threaten human and animal health. Despite the medical importance, little is known about orthobunyavirus structure, a prerequisite for understanding virus assembly and entry. Here, using electron cryo-tomography, we report the ultrastructure of Bunyamwera virus, the prototypic member of this genus. Whilst Bunyamwera virions are pleomorphic in shape, they display a locally ordered lattice of glycoprotein spikes. Each spike protrudes 18 nm from the viral membrane and becomes disordered upon introduction to an acidic environment. Using sub-tomogram averaging, we derived a three-dimensional model of the trimeric pre-fusion glycoprotein spike to 3-nm resolution. The glycoprotein spike consists mainly of the putative class-II fusion glycoprotein and exhibits a unique tripod-like arrangement. Protein-protein contacts between neighbouring spikes occur at membrane-proximal regions and intra-spike contacts at membrane-distal regions. This trimeric assembly deviates from previously observed fusion glycoprotein arrangements, suggesting a greater than anticipated repertoire of viral fusion glycoprotein oligomerization. Our study provides evidence of a pH-dependent conformational change that occurs during orthobunyaviral entry into host cells and a blueprint for the structure of this group of emerging pathogens.