Nairovirus polymerase mutations associated with the establishment of persistent infection in human cells.

Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne virus of the Orthonairovirus genus, persistently infects tick cells. It has been reported to establish persistent infection in non-human primates, but virological analysis has not yet been performed in human cells. Here, we investigated whether and how nairoviruses persistently infect human cells using Hazara orthonairovirus (HAZV), a surrogate model for CCHFV. We established a human cell line that was persistently infected with HAZV. Surprisingly, virions of persistently infected HAZV (HAZVpi) were not observed in the culture supernatants. There were five mutations (mut1, mut2, mut3, mut4, and mut5) in L protein of HAZVpi. Mutations in L protein of HAZVpi contribute to non-detection of virion in the supernatants. Lmut4 was found to cause low viral growth rate, despite its high polymerase activity. The low growth rate was restored by Lmut2, Lmut3, and Lmut5. The polymerase activity of Lmut1 was extremely low, and recombinant HAZV carrying Lmut1 (rHAZV/Lmut1) was not released into the supernatants. However, genomes of rHAZV/Lmut1 were retained in the infected cells. All mutations (Lmut1-5) found in L protein of HAZVpi were required for experimental reproduction of HAZVpi, and only Lmut1 and Lmut4 were insufficient. We demonstrated that point mutations in viral polymerase contribute to the establishment of persistent HAZV infection. Furthermore, innate immunity was found to be suppressed in HAZVpi-infected cells, which also potentially contributes to viral persistence. This is the first presentation of a possible mechanism behind how nairoviruses establish persistent infection in human cells. IMPORTANCE: We investigated whether and how nairoviruses persistently infect human cells, using Hazara orthonairovirus (HAZV), a surrogate model for Crimean-Congo hemorrhagic fever virus. We established a human cell line that was persistently infected with HAZV. Five mutations were found in L protein of persistently infected HAZV (HAZVpi): mut1, mut2, mut3, mut4, and mut5. Among them, Lmut1 and Lmut4 restricted viral growth by low polymerase activity and low growth rate, respectively, leading to inhibition of viral overgrowth. The restriction of viral growth caused by Lmut1 and Lmut4 was compensated by other mutations, including Lmut2, Lmut3, and Lmut5. Each of the mutations found in L protein of HAZVpi was concluded to cooperatively modulate viral growth, which facilitates the establishment of persistent infection. Suppression of innate immunity also potentially contributes to virus persistence. This is the first presentation of a possible mechanism behind how nairoviruses establish persistent infection in human cells.

Downregulation of transcription 1 hinders the replication of Dabie bandavirus by promoting the expression of TLR7, TLR8, and TLR9 signaling pathway.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a bunyavirus that causes SFTS, with a case fatality rate of up to 30 %. The innate immune system plays a crucial role in the defense against SFTSV; however, the impact of viral propagation of STFSV on the innate immune system remains unclear. Although proteomics analysis revealed that the expression of the downregulator of transcription 1 (DR1) increased after SFTSV infection, the specific change trend and the functional role of DR1 during viral infection remain unelucidated. In this study, we demonstrate that DR1 was highly expressed in response to SFTSV infection in HEK 293T cells using qRT-PCR and Western blot analysis. Furthermore, viral replication significantly increased the expression of various TLRs, especially TLR9. Our data indicated that DR1 positively regulated the expression of TLRs in HEK 293T cells, DR1 overexpression highly increased the expression of numerous TLRs, whereas RNAi-mediated DR1 silencing decreased TLR expression. Additionally, the myeloid differentiation primary response gene 88 (MyD88)-dependent or TIR-domain-containing adaptor inducing interferon-ß (TRIF)-dependent signaling pathways were highly up- and downregulated by the overexpression and silencing of DR1, respectively. Finally, we report that DR1 stimulates the expression of TLR7, TLR8, and TLR9, thereby upregulating the TRIF-dependent and MyD88-dependent signaling pathways during the SFTSV infection, attenuating viral replication, and enhancing the production of type I interferon and various inflammatory factors, including IL-1ß, IL-6, and IL-8. These results imply that DR1 defends against SFTSV replication by inducing the expression of TLR7, TLR8, and TLR9. Collectively, our findings revealed a novel role and mechanism of DR1 in mediating antiviral responses and innate immunity.

Calcium Influx Regulates the Replication of Several Negative-Strand RNA Viruses Including Severe Fever with Thrombocytopenia Syndrome Virus.

Negative-strand RNA viruses (NSVs) represent one of the most threatening groups of emerging viruses globally. Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic emerging virus that was initially reported in 2011 from China. Currently, no licensed vaccines or therapeutic agents have been approved for use against SFTSV. Here, L-type calcium channel blockers obtained from a U.S. Food and Drug Administration (FDA)-approved compound library were identified as effective anti-SFTSV compounds. Manidipine, a representative L-type calcium channel blocker, restricted SFTSV genome replication and exhibited inhibitory effects against other NSVs. The result from the immunofluorescent assay suggested that manidipine inhibited SFTSV N-induced inclusion body formation, which is believed to be important for the virus genome replication. We have shown that calcium possesses at least two different roles in regulating SFTSV genome replication. Inhibition of calcineurin, the activation of which is triggered by calcium influx, using FK506 or cyclosporine was shown to reduce SFTSV production, suggesting the important role of calcium signaling on SFTSV genome replication. In addition, we showed that globular actin, the conversion of which is facilitated by calcium from filamentous actin (actin depolymerization), supports SFTSV genome replication. We also observed an increased survival rate and a reduction of viral load in the spleen in a lethal mouse model of SFTSV infections after manidipine treatment. Overall, these results provide information regarding the importance of calcium for NSV replication and may thereby contribute to the development of broad-scale protective therapies against pathogenic NSVs. IMPORTANCE SFTS is an emerging infectious disease and has a high mortality rate of up to 30%. There are no licensed vaccines or antivirals against SFTS. In this article, L-type calcium channel blockers were identified as anti-SFTSV compounds through an FDA-approved compound library screen. Our results showed the involvement of L-type calcium channel as a common host factor for several different families of NSVs. The formation of an inclusion body, which is induced by SFTSV N, was inhibited by manidipine. Further experiments showed that SFTSV replication required the activation of calcineurin, a downstream effecter of the calcium channel. In addition, we identified that globular actin, the conversion of which is facilitated by calcium from filamentous actin, supports SFTSV genome replication. We also observed an increased survival rate in a lethal mouse model of SFTSV infection after manidipine treatment. These results facilitate both our understanding of the NSV replication mechanism and the development of novel anti-NSV treatment.

Oropouche orthobunyavirus infection is mediated by the cellular host factor Lrp1.

Oropouche orthobunyavirus (OROV; Peribunyaviridae) is a mosquito-transmitted virus that causes widespread human febrile illness in South America, with occasional progression to neurologic effects. Host factors mediating the cellular entry of OROV are undefined. Here, we show that OROV uses the host protein low-density lipoprotein-related protein 1 (Lrp1) for efficient cellular infection. Cells from evolutionarily distinct species lacking Lrp1 were less permissive to OROV infection than cells with Lrp1. Treatment of cells with either the high-affinity Lrp1 ligand receptor-associated protein (RAP) or recombinant ectodomain truncations of Lrp1 significantly reduced OROV infection. In addition, chimeric vesicular stomatitis virus (VSV) expressing OROV glycoproteins (VSV-OROV) bound to the Lrp1 ectodomain in vitro. Furthermore, we demonstrate the biological relevance of the OROV-Lrp1 interaction in a proof-of-concept mouse study in which treatment of mice with RAP at the time of infection reduced tissue viral load and promoted survival from an otherwise lethal infection. These results with OROV, along with the recent finding of Lrp1 as an entry factor for Rift Valley fever virus, highlight the broader significance of Lrp1 in cellular infection by diverse bunyaviruses. Shared strategies for entry, such as the critical function of Lrp1 defined here, provide a foundation for the development of pan-bunyaviral therapeutics.

A Viral NOD to Encephalitis.

In this issue, Gao and colleagues (J Virol 96:e00167-22, https://doi.org/10.1128/JVI.00167-22) dissect innate immune signaling in a microglial cell line infected with severe fever with thrombocytopenia syndrome virus (SFTSV). This virus has been designated a priority pathogen by the World Health Organization due to its capacity to induce a fatal cytokine storm. The study's findings attribute the pathogenesis to induction of the host inflammasome response by the SFTSV nonstructural protein.

Structural and Biochemical Basis for Development of Diketo Acid Inhibitors Targeting the Cap-Snatching Endonuclease of the Ebinur Lake Virus (Order: Bunyavirales).

The Bunyavirales contain many important human pathogens that lack an antiviral therapy. The cap-snatching endonuclease (EN) of segmented negative-strand RNA viruses is an attractive target for broad-spectrum antivirals due to its essential role in initiating viral transcription. L-742,001, a previously reported diketo acid inhibitor against influenza virus EN, demonstrated potent EN inhibition and antiviral activity on various bunyaviruses. However, the precise inhibitory mechanism of the compound is still poorly understood. We recently characterized a highly active EN from Ebinur Lake virus (EBIV), a newly identified member of the Orthobunyavirus genus, and obtained its high-resolution structures, paving the way for structure-guided inhibitor development. Here, nine L-742,001 derivatives were designed and synthesized de novo, and their structure-activity relationship with EBIV EN was studied. In vitro biochemical data showed that the compounds inhibited the EBIV EN activity with different levels and could be divided into three categories. Five representative compounds were selected for further cell-based antiviral assay, and the results largely agreed with those of the EN assays. Furthermore, the precise binding modes of L-742,001 and its derivatives in EN were revealed by determining the high-resolution crystal structures of EN-inhibitor complexes, which suggested that the p-chlorobenzene is essential for the inhibitory activity and the flexible phenyl has the greatest exploration potential. This study provides an important basis for the structure-based design and optimization of inhibitors targeting EN of segmented negative-strand RNA viruses. IMPORTANCE The Bunyavirales contain many important human pathogens such as Crimean-Congo hemorrhagic fever virus and Lassa virus that pose serious threats to public health; however, currently there are no specific antiviral drugs against these viruses. The diketo acid inhibitor L-742,001 is a potential drug as it inactivates the cap-snatching endonuclease (EN) encoded by bunyaviruses. Here, we designed and synthesized nine L-742,001 derivatives and assessed the structure-activity relationship using EN of the newly identified Ebinur Lake virus (EBIV) as a research model. Our results revealed that the p-chlorobenzene of this broad-spectrum EN inhibitor is crucial for the inhibitory activity and the flexible phenyl "arm" has the best potential for further optimization. As cap-snatching ENs are present not only in bunyaviruses but also in influenza viruses, our data provide important guidelines for the development of novel and more potent diketo acid-based antiviral drugs against those viruses.

Activation of the NLRP3 inflammasome and elevation of interleukin-1ß secretion in infection by sever fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging phlebovirus that causes a hemorrhagic fever known as the severe fever with thrombocytopenia syndrome (SFTS). Inflammasomes are a molecular platform that are assembled to process pro-caspase 1 and subsequently promote secretion of interleukin (IL)-1ß/IL-18 for proinflammatory responses induced upon infection. We hypothesize that inflammasome activation and pyroptosis induced in SFTS results in elevated levels of IL-1ß/IL-18 responsible for high fever and hemorrhage in the host, characteristic of SFTS. Here we report that IL-1ß secretion was elevated in SFTS patients and infected mice and IL-1ß levels appeared to be reversibly associated to disease severity and viral load in patients' blood. Increased caspase-1 activation, IL-1ß/IL-18 secretion, cell death, and processing of gasdermin D were detected, indicating that pyroptosis was induced in SFTSV-infected human peripheral blood monocytes (PBMCs). To characterize the mechanism of pyroptosis induction, we knocked down several NOD-like receptors (NLRs) with respective shRNAs in PBMCs and showed that the NLR family pyrin domain containing 3 (NLRP3) inflammasome was critical for processing pro-caspase-1 and pro-IL-1ß. Our data with specific inhibitors for NLRP3 and caspase-1 further showed that activation of the NLRP3 inflammasome was key to caspase-1 activation and IL-1ß secretion which may be inhibitory to viral replication in PBMCs infected with SFTSV. The findings in this study suggest that the activation of the NLPR3 inflammasome and pyroptosis, leading to IL-1ß/IL-18 secretion during the SFTSV infection, could play important roles in viral pathogenesis and host protection. Pyroptosis as part of innate immunity might be essential in proinflammatory responses and pathogenicty in humans infected with this novel phlebovirus.

Monoclonal antibodies to Cache Valley virus for serological diagnosis.

Cache Valley virus (CVV) is a mosquito-borne virus in the genus Orthobunyavirus, family Peribunyaviridae. It was first isolated from a Culiseta inorata mosquito in Cache Valley, Utah in 1956 and is known to circulate widely in the Americas. While only a handful of human cases have been reported since its discovery, it is the causative agent of fetal death and severe malformations in livestock. CVV has recently emerged as a potential viral pathogen causing severe disease in humans. Currently, the only serological assay available for diagnostic testing is plaque reduction neutralization test which takes several days to perform and requires biocontainment. To expand diagnostic capacity to detect CVV infections by immunoassays, 12 hybridoma clones secreting anti-CVV murine monoclonal antibodies (MAbs) were developed. All MAbs developed were found to be non-neutralizing and specific to the nucleoprotein of CVV. Cross-reactivity experiments with related orthobunyaviruses revealed several of the MAbs reacted with Tensaw, Fort Sherman, Tlacotalpan, Maguari, Playas, and Potosi viruses. Our data shows that MAbs CVV14, CVV15, CVV17, and CVV18 have high specific reactivity as a detector in an IgM antibody capture test with human sera.

SAFA initiates innate immunity against cytoplasmic RNA virus SFTSV infection.

Nuclear scaffold attachment factor A (SAFA) is a novel RNA sensor involved in sensing viral RNA in the nucleus and mediating antiviral immunity. Severe fever with thrombocytopenia syndrome virus (SFTSV) is a bunyavirus that causes SFTS with a high fatality rate of up to 30%. It remains elusive whether and how cytoplasmic SFTSV can be sensed by the RNA sensor SAFA. Here, we demonstrated that SAFA was able to detect SFTSV infection and mediate antiviral interferon and inflammatory responses. Transcription and expression levels of SAFA were strikingly upregulated under SFTSV infection. SAFA was retained in the cytoplasm by interaction with SFTSV nucleocapsid protein (NP). Importantly, SFTSV genomic RNA was recognized by cytoplasmic SAFA, which recruited and promoted activation of the STING-TBK1 signaling axis against SFTSV infection. Of note, the nuclear localization signal (NLS) domain of SAFA was important for interaction with SFTSV NP and recognition of SFTSV RNA in the cytoplasm. In conclusion, our study reveals a novel antiviral mechanism in which SAFA functions as a novel cytoplasmic RNA sensor that directly recognizes RNA virus SFTSV and mediates an antiviral response.

Characterization and reverse genetic establishment of cattle derived Akabane virus in China.

BACKGROUND: Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. RESULTS: An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. CONCLUSION: We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.

Oral susceptibility of aedine and culicine mosquitoes (Diptera: Culicidae) to Batai Orthobunyavirus.

BACKGROUND: A number of zoonotic mosquito-borne viruses have emerged in Europe in recent decades. Batai virus (BATV), a member of the genus Orthobunyavirus, is one example of a relatively newly emerged mosquito-borne virus, having been detected in mosquitoes and livestock. We conducted vector competency studies on three mosquito species at a low temperature to assess whether Aedes and Culex mosquito species are susceptible to infection with BATV. METHODS: Colonised lines of Aedes aegypti and Culex pipiens and a wild-caught species, Aedes detritus, were orally inoculated with BATV strain 53.2, originally isolated from mosquitoes trapped in Germany in 2009. Groups of blood-fed female mosquitoes were maintained at 20 °C for 7 or 14 days. Individual mosquitoes were screened for the presence of BATV in body, leg and saliva samples for evidence of infection, dissemination and transmission, respectively. BATV RNA was detected by reverse transcription-PCR, and positive results confirmed by virus isolation in Vero cells. RESULTS: Aedes detritus was highly susceptible to BATV, with an infection prevalence of ≥ 80% at both measurement time points. Disseminated infections were recorded in 30.7-41.6% of Ae. detritus, and evidence of virus transmission with BATV in saliva samples (n = 1, days post-infection: 14) was observed. Relatively lower rates of infection for Ae. aegypti and Cx. pipiens were observed, with no evidence of virus dissemination or transmission at either time point. CONCLUSIONS: This study shows that Ae. detritus may be a competent vector for BATV at 20 °C, whereas Ae. aegypti and Cx. pipiens were not competent. Critically, the extrinsic incubation period appears to be ≤ 7 days for Ae. detritus, which may increase the onward transmissibility potential of BATV in these populations.

Simbu Viruses' Infection of Livestock in Israel-A Transient Climatic Land.

Important lessons have been learned by the Israeli veterinary community regarding Simbu serogroup viruses infections. This serogroup of viruses might cause the births of neonatal malformation in susceptible ruminant's populations. Until 2012, only Akabane virus was connected with the births of malformed ruminants in Israel. However, serological and genomic detection tests, coupled with viral isolations, revealed that more than a single Simbu serogroup serotype could be present concurrently in the same farm or even in the same animal. From 2012 to date, Aino, Shuni, Shamunda, Satuperi, Peaton, Schmallenberg, and Sango viruses have been found in Israel either by serological or genomic investigation. Israel is located in the Eastern Mediterranean Basin, a terrestrial and climatic bridge between the three old continents. The Eastern Mediterranean shores benefit from both the tropical/subtropical and the continental climatic conditions. Therefore, the Eastern Mediterranean basin might serve as an optimal investigatory compound for several arboviral diseases, acting as a sentinel. This review summarizes updated information related to the presence of Simbu serogroup viruses in Israel.

The transmission ecology of Tahyna orthobunyavirus in Austria as revealed by longitudinal mosquito sampling and blood meal analysis in floodplain habitats.

BACKGROUND: Tahyna orthobunyavirus (TAHV) is a mosquito-borne virus that may cause mild flu-like symptoms or neurological symptoms in humans. It is historically associated with floodplain habitats in Central Europe, and the mammalophilic floodwater mosquito, Aedes vexans, is thought to be the principal vector. There are few contemporary reports of TAHV transmission ecology within mosquitoes or their vertebrate hosts, and virus infections are rarely reported (and probably seldom diagnosed). The objectives of this study were to survey the mosquito population for TAHV in three floodwater habitats and describe host usage by the predominant floodwater mosquito species to potentially define TAHV transmission at these foci. METHODS: We performed longitudinal mosquito sampling along three major rivers in eastern Austria to characterize the mosquito community in floodplain habitats, and tested for the presence of TAHV in pools of mosquitoes. We characterized TAHV rescued from mosquito pool homogenate by sequencing. We surveyed mosquito host selection by analyzing mosquito blood meals. RESULTS: We identified TAHV in two pools of Ae. vexans captured along the Leitha River. This mosquito, and other floodwater mosquitoes, used large mammals (red deer, roe deer, wild boar) as their hosts. The sequence of the rescued virus was remarkably similar to other TAHV isolates from the region, dating back to the first isolate of TAHV in 1958. CONCLUSIONS: In general, we confirmed that TAHV is most likely being transmitted by Ae. vexans, although the precise contribution of vertebrate-amplifying hosts to the ecological maintenance of the virus is unclear. The pattern of host selection matches the estimated exposure of the same large mammal species in the region to TAHV based on a recent serosurvey, but hares were also hosts at the site where TAHV was detected. We also confirm humans as hosts of two floodwater mosquito species, providing a potential mechanism for spillover of TAHV or other mosquito-borne viruses.

In Silico Structure-Based Design of Antiviral Peptides Targeting the Severe Fever with Thrombocytopenia Syndrome Virus Glycoprotein Gn.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus in Asia that causes severe disease. Despite its clinical importance, treatment options for SFTSV infection remains limited. The SFTSV glycoprotein Gn plays a major role in mediating virus entry into host cells and is therefore a potential antiviral target. In this study, we employed an in silico structure-based strategy to design novel cyclic antiviral peptides that target the SFTSV glycoprotein Gn. Among the cyclic peptides, HKU-P1 potently neutralizes the SFTSV virion. Combinatorial treatment with HKU-P1 and the broad-spectrum viral RNA-dependent RNA polymerase inhibitor favipiravir exhibited synergistic antiviral effects in vitro. The in silico peptide design platform in this study may facilitate the generation of novel antiviral peptides for other emerging viruses.

Novel murine models for studying Cache Valley virus pathogenesis and in utero transmission.

Cache Valley virus (CVV) is a prevalent emerging pathogen of significant importance to agricultural and human health in North America. Emergence in livestock can result in substantial agroeconomic losses resulting from the severe embryonic lethality associated with infection during pregnancy. Although CVV pathogenesis has been well described in ruminants, small animal models are still unavailable, which limits our ability to study its pathogenesis and perform preclinical testing of therapeutics. Herein, we explored CVV pathogenesis, tissue tropism, and disease outcomes in a variety of murine models, including immune -competent and -compromised animals. Our results show that development of CVV disease in mice is dependent on innate immune responses, and type I interferon signalling is essential for preventing infection in mice. IFN-αßR-/- mice infected with CVV present with significant disease and lethal infections, with minimal differences in age-dependent pathogenesis, suggesting this model is appropriate for pathogenesis-related, and short- and long-term therapeutic studies. We also developed a novel CVV in utero transmission model that showed high rates of transmission, spontaneous abortions, and congenital malformations during infection. CVV infection presents a wide tissue tropism, with significant amplification in liver, spleen, and placenta tissues. Immune-competent mice are generally resistant to infection, and only show disease in an age dependent manner. Given the high seropositivity rates in regions of North America, and the continuing geographic expansion of competent mosquito vectors, the risk of epidemic and epizootic emergence of CVV is high, and interventions are needed for this important pathogen.

Orthobunyaviruses: From Virus Binding to Penetration into Mammalian Host Cells.

With over 80 members worldwide, Orthobunyavirus is the largest genus in the Peribunyaviridae family. Orthobunyaviruses (OBVs) are arthropod-borne viruses that are structurally simple, with a trisegmented, negative-sense RNA genome and only four structural proteins. OBVs are potential agents of emerging and re-emerging diseases and overall represent a global threat to both public and veterinary health. The focus of this review is on the very first steps of OBV infection in mammalian hosts, from virus binding to penetration and release of the viral genome into the cytosol. Here, we address the most current knowledge and advances regarding OBV receptors, endocytosis, and fusion.

A new nairo-like virus associated with human febrile illness in China.

ABSTRACTSeveral nairo-like viruses have been discovered in ticks in recent years, but their relevance to public health remains unknown. Here, we found a patient who had a history of tick bite and suffered from a febrile illness was infected with a previously discovered RNA virus, Beiji nairovirus (BJNV), in the nairo-like virus group of the order Bunyavirales. We isolated the virus by cell culture assay. BJNV could induce cytopathic effects in the baby hamster kidney and human hepatocellular carcinoma cells. Negative-stain electron microscopy revealed enveloped and spherical viral particles, morphologically similar to those of nairoviruses. We identified 67 patients as BJNV infection in 2017-2018. The median age of patients was 48 years (interquartile range 41-53 years); the median incubation period was 7 days (interquartile range 3-12 days). Most patients were men (70%), and a few (10%) had underlying diseases. Common symptoms of infected patients included fever (100%), headache (99%), depression (63%), coma (63%), and fatigue (54%), myalgia or arthralgia (45%); two (3%) patients became critically ill and one died. BJNV could cause growth retardation, viremia and histopathological changes in infected suckling mice. BJNV was also detected in sheep, cattle, and multiple tick species. These findings demonstrated that the newly discovered nairo-like virus may be associated with a febrile illness, with the potential vectors of ticks and reservoirs of sheep and cattle, highlighting its public health significance and necessity of further investigation in the tick-endemic areas worldwide.

Evaluation of Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus Mosquitoes Competence to Oropouche virus Infection.

The emergence of new human viral pathogens and re-emergence of several diseases are of particular concern in the last decades. Oropouche orthobunyavirus (OROV) is an arbovirus endemic to South and Central America tropical regions, responsible to several epidemic events in the last decades. There is little information regarding the ability of OROV to be transmitted by urban/peri-urban mosquitoes, which has limited the predictability of the emergence of permanent urban transmission cycles. Here, we evaluated the ability of OROV to infect, replicate, and be transmitted by three anthropophilic and urban species of mosquitoes, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus. We show that OROV is able to infect and efficiently replicate when systemically injected in all three species tested, but not when orally ingested. Moreover, we find that, once OROV replication has occurred in the mosquito body, all three species were able to transmit the virus to immunocompromised mice during blood feeding. These data provide evidence that OROV is restricted by the midgut barrier of three major urban mosquito species, but, if this restriction is overcome, could be efficiently transmitted to vertebrate hosts. This poses a great risk for the emergence of permanent urban cycles and geographic expansion of OROV to other continents.

Host Cell Restriction Factors of Bunyaviruses and Viral Countermeasures.

The Bunyavirales order comprises more than 500 viruses (generally defined as bunyaviruses) classified into 12 families. Some of these are highly pathogenic viruses infecting different hosts, including humans, mammals, reptiles, arthropods, birds, and/or plants. Host cell sensing of infection activates the innate immune system that aims at inhibiting viral replication and propagation. Upon recognition of pathogen-associated molecular patterns (PAMPs) by cellular pattern recognition receptors (PRRs), numerous signaling cascades are activated, leading to the production of interferons (IFNs). IFNs act in an autocrine and paracrine manner to establish an antiviral state by inducing the expression of hundreds of IFN-stimulated genes (ISGs). Some of these ISGs are known to restrict bunyavirus infection. Along with other constitutively expressed host cellular factors with antiviral activity, these proteins (hereafter referred to as "restriction factors") target different steps of the viral cycle, including viral entry, genome transcription and replication, and virion egress. In reaction to this, bunyaviruses have developed strategies to circumvent this antiviral response, by avoiding cellular recognition of PAMPs, inhibiting IFN production or interfering with the IFN-mediated response. Herein, we review the current knowledge on host cellular factors that were shown to restrict infections by bunyaviruses. Moreover, we focus on the strategies developed by bunyaviruses in order to escape the antiviral state developed by the infected cells.

Recent Advances in Bunyavirus Glycoprotein Research: Precursor Processing, Receptor Binding and Structure.

The Bunyavirales order accommodates related viruses (bunyaviruses) with segmented, linear, single-stranded, negative- or ambi-sense RNA genomes. Their glycoproteins form capsomeric projections or spikes on the virion surface and play a crucial role in virus entry, assembly, morphogenesis. Bunyavirus glycoproteins are encoded by a single RNA segment as a polyprotein precursor that is co- and post-translationally cleaved by host cell enzymes to yield two mature glycoproteins, Gn and Gc (or GP1 and GP2 in arenaviruses). These glycoproteins undergo extensive N-linked glycosylation and despite their cleavage, remain associated to the virion to form an integral transmembrane glycoprotein complex. This review summarizes recent advances in our understanding of the molecular biology of bunyavirus glycoproteins, including their processing, structure, and known interactions with host factors that facilitate cell entry.