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.

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.

Ovine fetal immune response to Cache Valley virus infection.

Cache Valley virus (CVV)-induced malformations have been previously reproduced in ovine fetuses. To evaluate the development of the antiviral response by the early, infected fetus, before the development of immunocompetency, ovine fetuses at 35 days of gestation were inoculated in utero with CVV and euthanized at 7, 10, 14, 21, and 28 days postinfection. The antiviral immune response in immature fetuses infected with CVV was evaluated. Gene expression associated with an innate, immune response was quantified by real-time quantitative PCR. The upregulated genes in infected fetuses included ISG15, Mx1, Mx2, IL-1, IL-6, TNF-α, TLR-7, and TLR-8. The amount of Mx1 protein, an interferon-stimulated GTPase capable of restricting growth of bunyaviruses, was elevated in the allantoic and amniotic fluid in infected fetuses. ISG15 protein expression was significantly increased in target tissues of infected animals. B lymphocytes and immunoglobulin-positive cells were detected in lymphoid tissues and in the meninges of infected animals. These results demonstrated that the infected ovine fetus is able to initiate an innate and adaptive immune response much earlier than previously known, which presumably contributes to viral clearance in infected animals.

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.

Evaluating the vector competence of Aedes simpsoni sl from Kenyan coast for Ngari and Bunyamwera viruses.

BACKGROUND: Bunyamwera(BUNV) and Ngari (NGIV) viruses are arboviruses of medical importance globally, the viruses are endemic in Africa, Aedes(Ae) aegypti and Anopheles(An) gambiae mosquitoes are currently competent vectors for BUNV and NGIV respectively. Both viruses have been isolated from humans and mosquitoes in various ecologies of Kenya. Understanding the risk patterns and spread of the viruses necessitate studies of vector competence in local vector population of Ae. simpsoni sl which is abundant in the coastal region. This study sought to assess the ability of Ae. Simpsoni sl mosquitoes abundant at the Coast of Kenya to transmit these viruses in experimental laboratory experiments. METHODS: Field collected larvae/pupae of Ae. Simpsoni sl mosquitoes from Rabai, Kilifi County, were reared to adults, the first filial generation (F0) females' mosquitoes were orally exposed to infectious blood meal with isolates of the viruses using the hemotek membrane feeder. The exposed mosquitoes were incubated under insectary conditions and sampled on day 7, 14 and 21days post infection to determine susceptibility to the virus infection using plaque assay. RESULTS: A total of 379 (Bunyamwera virus 255 and Ngari virus 124) Ae. simpsoni sl were orally exposed to infectious blood meal. Overall, the infection rate (IR) for BUNV and NGIV were 2.7 and 0.9% respectively. Dissemination occurred in 5 out 7 mosquitoes with mid-gut infection for Bunyamwera virus and 1 out of 2 mosquitoes with mid-gut infection for Ngari virus. Further, the transmission was observed in 1 out of 5 mosquitoes that had disseminated infection and no transmission was observed for Ngari virus in all days post infection (dpi). CONCLUSION: Our study shows that Ae. simpsoni sl. is a laboratory competent vector for Bunyamwera virus since it was able to transmit the virus through capillary feeding while NGIV infection was restricted to midgut infection and disseminated infection, these finding adds information on the epidemiology of the viruses and vector control plan.

The N-terminus of Bunyamwera orthobunyavirus NSs protein is essential for interferon antagonism.

Bunyamwera virus NSs protein is involved in the inhibition of cellular transcription and the interferon (IFN) response, and it interacts with the Med8 component of Mediator. A spontaneous mutant of a recombinant NSs-deleted Bunyamwera virus (rBUNdelNSs2) was identified and characterized. This mutant virus, termed mBUNNSs22, expresses a 21 aa N-terminally truncated form of NSs. Like rBUNdelNSs2, mBUNNSs22 is attenuated in IFN-deficient cells, and to a greater extent in IFN-competent cells. Both rBUNdelNSs2 and mBUNNSs22 are potent IFN inducers and their growth can be rescued by depleting cellular IRF3. Strikingly, despite encoding an NSs protein that contains the Med8 interaction domain, mBUNNSs22 fails to block RNA polymerase II activity during infection. Overall, our data suggest that both the interaction of NSs with Med8 and a novel unidentified function of the NSs N-terminus, seem necessary for Bunyamwera virus to counteract host antiviral responses.

Evaluation of the eastern cottontail Sylvilagus floridanus as an amplifying vertebrate host for Cache Valley virus (Bunyaviridae) in Indiana.

To evaluate the importance of eastern cottontails (Sylvilagus floridanus) as amplifying hosts for Cache Valley virus (CVV), we tested hunter-provided blood samples from northern Indiana for specific neutralizing (N) antibodies against this mosquito-borne bunya-virus. Samples were collected during the winter of 1994-95. Two seronegative eastern cottontails, captured in July 1995, were also infected with CVV by subcutaneous inoculation, and two others were infected by allowing CVV-infected mosquitoes to feed on them. The results indicate that eastern cottontails probably are not important amplifying hosts for CVV. The prevalence of N antibodies against CVV was low (6.0%, n=82) among the hunter-killed animals. Low viremia (<1.8 log10 plaque-forming units/ml) of short duration (1-3 days) were seen in three of four experimentally infected eastern cottontails. The viremias were insufficient for infecting Coquillettidia perturbans, a mosquito species commonly found naturally infected with CVV.

Detection of Usutu, Sindbis, and Batai Viruses in Mosquitoes (Diptera: Culicidae) Collected in Germany, 2011⁻2016.

Due to the emergence of non-endemic mosquito vectors and the recent outbreaks of mosquito-borne diseases, mosquito-borne pathogens are considered an increasing risk to public and animal health in Europe. To obtain a status quo regarding mosquito-borne viruses and their vectors in Germany, 97,648 mosquitoes collected from 2011 to 2016 throughout the country were screened for arboviruses. Mosquitoes were identified to species, pooled in groups of up to 50 individuals according to sampling location and date, and screened with different PCR assays for Flavi-, Alpha- and Orthobunyavirus RNA. Two pools tested positive for Usutu virus-RNA, two for Sindbis virus-RNA, and 24 for Batai virus-RNA. The pools consisted of Culex pipiens s.l., Culex modestus, Culex torrentium, Culiseta sp., Aedes vexans, Anopheles daciae, and Anopheles messeae mosquitoes and could be assigned to nine different collection sites, with seven of them located in northeastern Germany. Phylogenetic analyses of the viral RNA sequences showed relationships with strains of the viruses previously demonstrated in Germany. These findings confirm continuing mosquito-borne zoonotic arbovirus circulation even though only a rather small percentage of the screened samples tested positive. With respect to sampling sites and periods, virus circulation seems to be particularly intense in floodplains and after flooding events when mosquitoes develop in excessive numbers and where they have numerous avian hosts available to feed on.

Comparison of two zoonotic viruses from the order Bunyavirales.

A comparison of two geographicallly distinct viruses in the order Bunyavirales that are zoonotic and known to cause congenital abnormalities in ruminant livestock was performed. One of these viruses, Cache Valley fever virus, is found in the Americas and is primarily associated with disease in sheep. The other, Rift Valley fever virus, is found in Sub-Saharan Africa and is associated with disease in camels, cattle, goats and sheep. Neither virus has been associated with teratogenicity in humans to date. These two viruses are briefly reviewed and potential for genetic changes especially if introduced into new ecology that could affect pathogenicity are discussed.

Cache Valley and Potosi viruses (Bunyaviridae) in white-tailed deer (Odocoileus virginianus): experimental infections and antibody prevalence in natural populations.

Cache Valley virus (CVV) and Potosi virus (POTV) are two closely related mosquito-borne viruses (Bunyaviridae: Bunyamwera group) that appear to circulate in several regions of the United States, especially the Midwest. We determined the prevalence of specific neutralizing antibodies to both viruses in Indiana white-tailed deer and conducted infection experiments to assess whether deer could serve as an vertebrate-amplifying host. Cross-infection experiments also were carried out to investigate the level of antibody cross-reactivity and cross-protection between the two viruses. The seroprevalence rate was high for both CVV (> 66%) and POTV (> 43%) in adult deer statewide. Antibodies neutralizing CVV were more common among deer harvested in the northern part of Indiana whereas the prevalence of POTV antibodies suggested a more southern distribution for this virus. Experimental infections of captive deer showed that they may serve as amplifying hosts for either virus. Deer infected with CVV or POTV developed a 1-3-day viremia with 3.0 and 4.1 log10 plaque-forming units/ml mean peak titers, respectively. However, significant levels of antibody cross-reactivity between the two viruses were observed. Viremia was lower and shorter when animals immune to either CVV or POTV were cross-infected with the alternate virus and antibody responses following cross-infections resembled original antigenic sin with higher titers of antibodies against the primary agent.

Ovine fetal malformations induced by in utero inoculation with Main Drain, San Angelo, and LaCrosse viruses.

The teratogenic potential of three bunyaviruses, two California serogroup bunyaviruses, LaCrosse virus and San Angelo virus, and a Bunyamwera serogroup member, Main Drain virus, in sheep was studied following in utero inoculation of ewes in early gestation. Although Main Drain virus appeared to be most teratogenic, all three viruses induced a range of lesions including arthrogryposis, hydrocephalus, fetal death, axial skeletal deviations, anasarca, and oligohydramnios. The teratogenic effects of these viruses are identical to those described in ovine infections by Cache Valley and Akabane viruses. Demonstration of a common bunyaviral tropism for fetal tissue infection that results in congenital brain and musculoskeletal malformations provides evidence that human in utero infection by bunyaviruses could result in similar malformations in human infants.

Experimental infections of birds with Turlock virus.

Two experiments were conducted with five gallinaceous and one passerine bird species to determine their responses to Turlock (TUR) virus inoculations. Inoculation of TUR strain 847-32 into bobwhites, chukars, ring-necked pheasants, chickens, and Japanese quail did not product detectable viremias. The first four species, however, did respond with neutralizing antibody. Inoculation of chickens with strain 69V-1095 resulted in a viremia which lasted 5 days and had a peak mean titer of 2.0 log 10 PFU per 0.2 ml of blood on the third day after infection. The observation that viremic birds exhibited no noticeable virus-associated morbidity or mortality suggested that TUR virus does not have a detrimental effect on free-ranging populations of the avian hosts studied during this investigation.

[Pathogenic properties of the LEIV23 Astrakhan strain of Batai virus for primates]. / Patogennye svoistva dlia primatov virusa Batai, shtamm LEIV23 Astrakhan'.

Pathogenic properties of Batai virus, LEIV23 Astrakhañ strain, isolated from Aedes vexans mosquitoes in Astrakhañ region were studied comparatively. The degree of susceptibility to the virus of green monkeys and rodents (white mice, Syrian hamsters), and the pattern of lesions produced by the virus in organs of these animals were established. The virus was shown to have a comparatively wide host range affecting phylogenetically far distant animals. Monkeys were found to have virus-carrier state for 50 days (the observation period). The virus is pantropic. Apparently mammals may be virus hosts in nature, but human infection cannot be ruled out. This requires further study in an epidemiological experiment.

The transient nature of Bunyamwera orthobunyavirus NSs protein expression: effects of increased stability of NSs protein on virus replication.

The NSs proteins of bunyaviruses are the viral interferon antagonists, counteracting the host's antiviral response to infection. During high-multiplicity infection of cultured mammalian cells with Bunyamwera orthobunyavirus (BUNV), NSs is rapidly degraded after reaching peak levels of expression at 12hpi. Through the use of inhibitors this was shown to be the result of proteasomal degradation. A recombinant virus (rBUN4KR), in which all four lysine residues in NSs were replaced by arginine residues, expresses an NSs protein (NSs4KR) that is resistant to degradation, confirming that degradation is lysine-dependent. However, despite repeated attempts, no direct ubiquitylation of NSs in infected cells could be demonstrated. This suggests that degradation of NSs, although lysine-dependent, may be achieved through an indirect mechanism. Infection of cultured mammalian cells or mice indicated no disadvantage for the virus in having a non-degradable NSs protein: in fact rBUN4KR had a slight growth advantage over wtBUNV in interferon-competent cells, presumably due to the increased and prolonged presence of NSs. In cultured mosquito cells there was no difference in growth between wild-type BUNV and rBUN4KR, but surprisingly NSs4KR was not stabilised compared to the wild-type NSs protein.

Heterocellular induction of interferon by negative-sense RNA viruses.

The infection of cells by RNA viruses is associated with the recognition of virus PAMPs (pathogen-associated molecular patterns) and the production of type I interferon (IFN). To counter this, most, if not all, RNA viruses encode antagonists of the IFN system. Here we present data on the dynamics of IFN production and response during developing infections by paramyxoviruses, influenza A virus and bunyamwera virus. We show that only a limited number of infected cells are responsible for the production of IFN, and that this heterocellular production is a feature of the infecting virus as opposed to an intrinsic property of the cells.

Role of N-linked glycans on bunyamwera virus glycoproteins in intracellular trafficking, protein folding, and virus infectivity.

The membrane glycoproteins (Gn and Gc) of Bunyamwera virus (BUN, family Bunyaviridae) contain three potential sites for the attachment of N-linked glycans: one site (N60) on Gn and two (N624 and N1169) on Gc. We determined that all three sites are glycosylated. Digestion of the glycoproteins with endo-beta-N-acetylglucosaminidase H (endo H) or peptide:N-glycosidase F revealed that Gn and Gc differ significantly in their glycan status and that late in infection Gc glycans remain endo H sensitive. The roles of the N-glycans in intracellular trafficking of the glycoproteins to the Golgi, protein folding, and virus replication were investigated by mutational analysis and confocal immunofluorescence. Elimination of the glycan on Gn, by changing N60 to a Q residue, resulted in the protein misfolding and failure of both Gn and Gc proteins to traffic to the Golgi complex. We were unable to rescue a viable virus by reverse genetics from a cDNA containing the N60Q mutation. In contrast, mutant Gc proteins lacking glycans on either N624 or N1169, or both sites, were able to target to the Golgi. Gc proteins containing mutations N624Q and N1169Q acquired endo H resistance. Three viable N glycosylation-site-deficient viruses, lacking glycans on one site or both sites on Gc, were created by reverse genetics. The viability of these recombinant viruses and analysis of growth kinetics indicates that the glycans on Gc are not essential for BUN replication, but they do contribute to the efficiency of virus infection.

Attenuation of bunyavirus replication by rearrangement of viral coding and noncoding sequences.

Bunyamwera virus (BUN) is the prototype virus of the family Bunyaviridae. BUN has a tripartite negative-sense RNA genome comprising small (S), medium (M), and large (L) segments. Partially complementary untranslated regions (UTRs) flank the coding region of each segment. The terminal 11 nucleotides of these UTRs are conserved between the three segments, while the internal regions are unique. The UTRs direct replication and transcription of viral RNA and are sufficient to allow encapsidation of viral RNA into ribonucleoprotein complexes. To investigate the segment-specific functions of the UTRs, we have used reverse genetics to recover a recombinant virus (called BUN MLM) in which the L segment open reading frame (ORF) is flanked by the M segment UTRs. Compared to wild-type virus, BUN MLM virus shows growth attenuation in cultured mammalian cells and a slower disease progression in mice, produces small plaques, expresses reduced levels of L mRNA and L (RNA polymerase) protein, synthesizes less L genomic and antigenomic RNA, and has an increased particle-to-PFU ratio. Our data suggest that the packaging of BUN RNAs is not segment specific. In addition, the phenotype of BUN MLM virus supports the finding that BUN UTRs differ in their regulation of RNA synthesis but suggests that the interplay between each segment UTR and its cognate ORF may contribute to that regulation. Since BUN MLM virus is attenuated due to an essentially irreversible mutation, the rearrangement of UTRs is a feasible strategy for vaccine design for the more pathogenic members of the Bunyaviridae.

Formation of recombinants between snowshoe hare and La Crosse bunyaviruses.

Wild-type recombinants were obtained at high frequency from coinfections of BHK cells involving temperature-sensitive, conditional-lethal mutants of snowshoe hare (SSH) and La Crosse (LAC) bunyaviruses. Analyses of two of the recombinants indicated that they have the genome compositions SSH/LAC/SSH and SSH/LAC/LAC for their respective L, M, and S virion RNA species. This evidence, together with that for the genetic stability of the recombinants, indicates that they were derived by segment reassortment of the competent genome pieces of the parental viruses. The SSH/LAC/SSH recombinant appears, from polypeptide analysis, to have the SSH type of nucleocapsid protein (N), whereas the SSH/LAC/LAC recombinant has the LAC nucleocapsid protein, suggesting that the viral S RNA codes for the N protein.

Activation of PKR by Bunyamwera virus is independent of the viral interferon antagonist NSs.

Double-stranded RNA (dsRNA) is a by-product of viral RNA polymerase activity, and its recognition is one mechanism by which the innate immune system is activated. Cellular responses to dsRNA include induction of alpha/beta interferon (IFN) synthesis and activation of the enzyme PKR, which exerts its antiviral effect by phosphorylating the eukaryotic initiation factor eIF-2 alpha, thereby inhibiting translation. We have recently identified the nonstructural protein NSs of Bunyamwera virus (BUNV), the prototype of the family Bunyaviridae, as a virulence factor that blocks the induction of IFN by dsRNA. Here, we investigated the potential of NSs to inhibit PKR. We show that wild-type (wt) BUNV that expresses NSs triggered PKR-dependent phosphorylation of eIF-2 alpha to levels similar to those of a recombinant virus that does not express NSs (BUNdelNSs virus). Furthermore, the sensitivity of viruses in cell culture to IFN was independent of PKR and was not determined by NSs. PKR knockout mice, however, succumbed to infection approximately 1 day earlier than wt mice or mice deficient in expression of RNase L, another dsRNA-activated antiviral enzyme. Our data indicate that (i) bunyaviruses activate PKR, but are only marginally sensitive to its antiviral effect, and (ii) NSs is different from other IFN antagonists, since it inhibits dsRNA-dependent IFN induction but has no effect on the dsRNA-activated PKR and RNase L systems.