Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress.

We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle, including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern.

Molecular Epidemiology of Mayaro Virus among Febrile Patients, Roraima State, Brazil, 2018-2021.

We detected Mayaro virus (MAYV) in 3.4% (28/822) of febrile patients tested during 2018-2021 from Roraima State, Brazil. We also isolated MAYV strains and confirmed that these cases were caused by genotype D. Improved surveillance is needed to better determine the burden of MAYV in the Amazon Region.

Comparative pathogenesis of three Mayaro virus genotypes in the cynomolgus macaque.

Mayaro virus (MAYV), a mosquito-borne alphavirus, is considered an emerging threat to public health with epidemic potential. Phylogenetic studies show the existence of three MAYV genotypes. In this study, we provide a preliminary analysis of the pathogenesis of all three MAYV genotypes in cynomolgus macaques (Macaca facicularis, Mauritian origin). Significant MAYV-specific RNAemia and viremia were detected during acute infection in animals challenged intravenously with the three MAYV genotypes, and strong neutralizing antibody responses were observed. MAYV RNA was detected at high levels in lymphoid tissues, joint muscle and synovia over 1 month after infection, suggesting that this model could serve as a promising tool in studying MAYV-induced chronic arthralgia, which can persist for years. Significant leucopenia was observed across all MAYV genotypes, peaking with RNAemia. Notable differences in the severity of acute RNAemia and composition of cytokine responses were observed among the three MAYV genotypes. Our model showed no outward signs of clinical disease, but several major endpoints for future MAYV pathology and intervention studies are described. Disruptions to normal blood cell counts and cytokine responses were markedly distinct from those observed in macaque models of CHIKV infection, underlining the importance of developing non-human primate models specific to MAYV infection.

Cotranslational folding stimulates programmed ribosomal frameshifting in the alphavirus structural polyprotein.

Viruses maximize their genetic coding capacity through a variety of biochemical mechanisms, including programmed ribosomal frameshifting (PRF), which facilitates the production of multiple proteins from a single mRNA transcript. PRF is typically stimulated by structural elements within the mRNA that generate mechanical tension between the transcript and ribosome. However, in this work, we show that the forces generated by the cotranslational folding of the nascent polypeptide chain can also enhance PRF. Using an array of biochemical, cellular, and computational techniques, we first demonstrate that the Sindbis virus structural polyprotein forms two competing topological isomers during its biosynthesis at the ribosome-translocon complex. We then show that the formation of one of these topological isomers is linked to PRF. Coarse-grained molecular dynamics simulations reveal that the translocon-mediated membrane integration of a transmembrane domain upstream from the ribosomal slip site generates a force on the nascent polypeptide chain that scales with observed frameshifting. Together, our results indicate that cotranslational folding of this viral protein generates a tension that stimulates PRF. To our knowledge, this constitutes the first example in which the conformational state of the nascent polypeptide chain has been linked to PRF. These findings raise the possibility that, in addition to RNA-mediated translational recoding, a variety of cotranslational folding or binding events may also stimulate PRF.

Genetically modified attenuated salmonid alphavirus: A potential strategy for immunization of Atlantic salmon.

Pancreas disease (PD) is a serious challenge in European salmonid aquaculture caused by salmonid alphavirus (SAV). In this study, we report the effect of immunization of Atlantic salmon with three attenuated infectious SAV3 strains with targeted mutations in a glycosylation site of the envelope E2 protein and/or in a nuclear localization signal in the capsid protein. In a pilot experiment, it was shown that the mutated viral strains replicated in fish, transmitted to naïve cohabitants and that the transmission had not altered the sequences. In the main experiment, the fish were immunized with the strains and challenged with SAV3 eight weeks after immunization. Immunization resulted in infection both in injected fish and 2 weeks later in the cohabitant fish, followed by a persistent but declining load of the mutated virus variants in the hearts. The immunized fish developed clinical signs and pathology consistent with PD prior to challenge. However, fish injected with the virus mutated in both E2 and capsid showed little clinical signs and had higher average weight gain than the groups immunized with the single mutated variants. The SAV strain used for challenge was not detected in the immunized fish indicating that these fish were protected against superinfection with SAV during the 12 weeks of the experiment.

A survey of RNA viruses in mosquitoes from Mozambique reveals novel genetic lineages of flaviviruses and phenuiviruses, as well as frequent flavivirus-like viral DNA forms in Mansonia.

BACKGROUND: Mosquito-borne diseases involving arboviruses represent expanding threats to sub-Saharan Africa imposing as considerable burden to human and veterinary public health. In Mozambique over one hundred species of potential arbovirus mosquito vectors have been identified, although their precise role in maintaining such viruses in circulation in the country remains to be elucidated. The aim of this study was to screen for the presence of flaviviruses, alphaviruses and bunyaviruses in mosquitoes from different regions of Mozambique. RESULTS: Our survey analyzed 14,519 mosquitoes, and the results obtained revealed genetically distinct insect-specific flaviviruses, detected in multiple species of mosquitoes from different genera. In addition, smaller flavivirus-like NS5 sequences, frequently detected in Mansonia seemed to correspond to defective viral sequences, present as viral DNA forms. Furthermore, three lineages of putative members of the Phenuiviridae family were also detected, two of which apparently corresponding to novel viral genetic lineages. CONCLUSION: This study reports for the first-time novel insect-specific flaviviruses and novel phenuiviruses, as well as frequent flavivirus-like viral DNA forms in several widely known vector species. This unique work represents recent investigation of virus screening conducted in mosquitoes from Mozambique and an important contribution to inform the establishment of a vector control program for arbovirus in the country and in the region.

Isolation and phylogenetic analysis of Getah virus from a commercial modified live vaccine against porcine reproductive and respiratory syndrome virus.

In the present study, Getah virus (GETV) isolate, GETV-V1, was isolated from a commercial PRRSV attenuated live vaccine (MLV), which has been widely used to immunize pigs against porcine reproductive and respiratory syndrome virus (PRRSV). Further analysis demonstrated that nine batches of the PRRSV MLV vaccine (three batches per year from 2017 to 2019) from the same manufacturer were all positive for GETV. Genomic analyses indicated that the GETV-V1 isolate shared the highest sequence identity with the GETV strain, 16-I-674, which was isolated from horses in Japan. The phylogenetic analysis based on the genomic sequences showed that the GETV-V1 strain was clustered with the Japanese GETV strains. Taken together, this is the first report of GETV contamination in live swine vaccines in China. Our findings demonstrate that immunization with commercial live vaccines might be a potential novel route of GETV transmission in swine. This highlights the need for more extensive monitoring of commercial live vaccines.

Novel positive-sense single-stranded RNA virus related to alphavirus-like viruses from Fusarium graminearum.

A putative novel positive-sense (+) RNA virus was detected in isolate CF16158 of the fungus Fusarium graminearum, the causal agent of Fusarium head blight and crown rot in wheat in China. The full genome of this virus was sequenced and characterized. The complete cDNA sequence is 7,051 nt long and contains four open reading frames (ORFs). ORF2 is predicted to encode helicase (Hel) and RNA-dependent RNA polymerase (RdRp) domains that are conserved among the alphavirus-like viruses. Pairwise comparisons and phylogenetic analysis of the deduced amino acid sequences of Hel and RdRp indicated that this (+) RNA mycovirus is a novel member of a new, yet to be established family of alphavirus-like viruses. Therefore, we named this virus "Fusarium graminearum alphavirus-like virus 1" (FgALV1). This is the first report of a full-length genomic sequence of a putative alphavirus-like virus in F. graminearum.

Divergent Barmah Forest Virus from Papua New Guinea.

We report a case of Barmah Forest virus infection in a child from Central Province, Papua New Guinea, who had no previous travel history. Genomic characterization of the virus showed divergent origin compared with viruses previously detected, supporting the hypothesis that the range of Barmah Forest virus extends beyond Australia.

Highly Pathogenic Swine Getah Virus in Blue Foxes, Eastern China, 2017.

We isolated Getah virus from infected foxes in Shandong Province, eastern China. We sequenced the complete Getah virus genome, and phylogenetic analysis revealed a close relationship with a highly pathogenic swine epidemic strain in China. Epidemiologic investigation showed that pigs might play a pivotal role in disease transmission to foxes.

First isolation and characterization of Getah virus from cattle in northeastern China.

BACKGROUND: Getah virus (GETV) is a neglected mosquito-borne Alphavirus that causes pyrexia, body rash, and leg oedema in horses and foetal death and reproductive disorders in pigs. Infected animals may play a critical role in the amplification and circulation of the virus. The present study aimed to investigate GETV infection in clinically infected cattle and vector mosquito species in northeastern China. RESULTS: Serum samples were collected from beef cattle that presented sudden onset of fever in forest grazing areas, and metagenomic sequencing was conducted, revealing 29 contigs from ten serum samples matching the GETV genome. Quantitative RT-PCR (RT-qPCR) was performed with GETV RNA from 48 beef cattle serum samples, showing that the overall prevalence of GETV in the beef cattle samples was 6.25% (3/48). Serological investigation indicated that GETV neutralizing antibodies were detected in 83.3% (40/48, 95% CI 67-100) of samples from the study region. The GETV JL1808 strain was isolated from clinically infected cattle showing fever. Sequence comparisons showed high identity with the HuN1 strain, a highly pathogenic swine epidemic isolate obtained in Hunan province in 2017, at the nucleotide level (99.5%) and at the deduced amino acid level (99.7-99.9%). The phylogenetic analysis of JL1808 clustered in Group III, and also revealed a close genetic relationship with the HuN1 strain. Additionally, about 12,000 mosquitoes were trapped in this region. The presence of GETV infection was detected in mosquitoes, suggesting that the minimum infection rate (MIR) was 1.50‰, with MIRs of 1.67‰ in Culex pseudovishnui, 1.60‰ in Culex tritaeniorhynchus, and 1.21‰ in Anopheles sinensis. CONCLUSIONS: To the best of our knowledge, this is the first report of GETV infection in cattle. These results demonstrated that a highly pathogenic, mosquito-borne swine GETV can infect and circulate in cattle, implying that it is necessary to conduct surveillance of GETV infection in animals in northeastern China.

Nanovirus-alphasatellite complex identified in Vicia cracca in the Rhône delta region of France.

Nanoviruses are multi-component plant-infecting single-stranded DNA viruses. Using a viral metagenomics-informed approach, a new nanovirus and two associated alphasatellite molecules have been identified in an uncultivated asymptomatic Vicia cracca plant in the Rhône region of France. This novel nanovirus genome includes eight genomic components (named DNA-R, DNA-S, DNA-M, DNA-C, DNA-N, DNA-U1, DNA-U2 and DNA-U4) and, across all components, shares < 66% pairwise sequence identity with other nanovirus genomes. The two associated alphasatellites share 62% identity with each other and < 81% identity will all other nanovirus-associated alphasatellites.

Getah virus epizootic among wild boars in Japan around 2012.

In 2014, an outbreak of Getah virus (GETV) infection occurred in Japan in a horse population that was inoculated with a vaccine against GETV. In this study, we investigated the seroprevalence of GETV infection among wild boars in Japan. Interestingly, the highest rate of anti-GETV-positive wild boars was observed in 2013, which gradually decreased during 2014-2016. The results suggested that GETV spread among wild boars around 2012, resulting in the 2014 outbreak.

Molecular characterization of faba bean necrotic yellows viruses in Tunisia.

Faba bean necrotic yellows virus (FBNYV) (genus Nanovirus; family Nanoviridae) has a genome comprising eight individually encapsidated circular single-stranded DNA components. It has frequently been found infecting faba bean (Vicia faba L.) and chickpea (Cicer arietinum L.) in association with satellite molecules (alphasatellites). Genome sequences of FBNYV from Azerbaijan, Egypt, Iran, Morocco, Spain and Syria have been determined previously and we now report the first five genome sequences of FBNYV and associated alphasatellites from faba bean sampled in Tunisia. In addition, we have determined the genome sequences of two additional FBNYV isolates from chickpea plants sampled in Syria and Iran. All individual FBNYV genome component sequences that were determined here share > 84% nucleotide sequence identity with FBNYV sequences available in public databases, with the DNA-M component displaying the highest degree of diversity. As with other studied nanoviruses, recombination and genome component reassortment occurs frequently both between FBNYV genomes and between genomes of nanoviruses belonging to other species.

Isolation of salmonid alphavirus subtype 6 from wild-caught ballan wrasse, Labrus bergylta (Ascanius).

The use of cleaner fish as a biological control for sea lice in Atlantic salmon aquaculture has increased in recent years. Wild-caught wrasse are commonly used as cleaner fish in Europe. In Ireland, samples of wrasse from each fishing area are screened for potential pathogens prior to their deployment into sea cages. Salmonid alphavirus was isolated from a pooled sample of ballan wrasse, showing no signs of disease, caught from the NW of Ireland. Partial sequencing of the E2 and nsP3 genes showed that it was closely related to the previously reported SAV subtype 6. This represents only the second isolation of this subtype and the first from a wild fish species, namely ballan wrasse.

Discovery of a novel alphavirus related to Eilat virus.

Most alphaviruses are transmitted by arthropods and infect vertebrate hosts. An exception is Eilat virus (EILV), the only described alphavirus with a host range restricted to insects. We established a new generic reverse transcription PCR assay for alphaviruses and tested 8860 tropical mosquitoes. We detected a novel alphavirus, tentatively named Taï Forest alphavirus (TALV), in Culex decens mosquitoes collected in Ivory Coast. The full genome was sequenced, and closest similarity was found to EILV. Pairwise amino acid identities to EILV ranged between 67 and 88 % for the corresponding proteins, suggesting that TALV defines a proposed new alphavirus species. Phylogenetic analyses placed TALV as a sister species to EILV with a basal relationship to the western equine encephalitis virus complex. In comparison to the highly abundant insect-specific flaviviruses, insect-specific alphaviruses seem to be rare. This new PCR assay can detect novel alphaviruses and may facilitate the identification of additional new alphaviruses.

Geospatial and temporal associations of Getah virus circulation among pigs and horses around the perimeter of outbreaks in Japanese racehorses in 2014 and 2015.

BACKGROUND: We studied a recent epizootic of Getah virus infection among pigs in the southern part of Ibaraki Prefecture and the northern part of Chiba Prefecture, Japan, focusing on its possible association with outbreaks in racehorses in 2014 and 2015. The genomic sequence of a Getah virus strain from an infected pig was analyzed to evaluate the degree of identity with the strains from horses. RESULTS: Sera were collected from pigs from September to December 2012 to 2015 in south Ibaraki (380 pigs in 29 batches), and from September to December 2010 to 2015 in north Chiba (538 pigs in 104 batches). They were examined by using a virus-neutralizing test for Getah virus. Seropositivity rates in 2012-2013 in south Ibaraki and 2010-2012 in north Chiba ranged from 0% to 1.6%. In south Ibaraki, seropositivity rates in 2014 (28.8%) and 2015 (65.0%) were significantly higher than those in the previous years (P < 0.01); 4/5 batches had positive sera in 2014 and 7/7 in 2015. In north Chiba, seropositivity rates in 2013 (14.1%), 2014 (17.8%), and 2015 (48.0%) were significantly higher than those in the previous years (P < 0.01); 6/27 batches had positive sera in 2013, 3/9 in 2014, and 5/5 in 2015. Complete genome analysis revealed that the virus isolated from an infected pig had 99.89% to 99.94% nucleotide identity to the strains isolated from horses during the outbreaks in 2014 and 2015. CONCLUSIONS: Serological surveillance of Getah virus in pigs revealed that the virus was circulating in south Ibaraki and north Chiba in 2014 and 2015; this was concomitant with the outbreaks in racehorses. The Getah virus strain isolated from a pig was closely related to the ones from horses during the 2014 and 2015 outbreaks. To our knowledge, this is the first convincing case of simultaneous circulation of Getah virus both among pigs and horses in specific areas.

Identification and characterization of alphavirus M1 as a selective oncolytic virus targeting ZAP-defective human cancers.

Oncolytic virotherapy is a growing treatment modality that uses replicating viruses as selective antineoplastic agents. Safety and efficacy considerations dictate that an ideal oncolytic agent would discriminate between normal and cancer cells on the basis of common genetic abnormalities in human cancers. Here, we identify a naturally occurring alphavirus (M1) as a novel selective killer targeting zinc-finger antiviral protein (ZAP)-deficient cancer cells. In vitro, in vivo, and ex vivo studies showed potent oncolytic efficacy and high tumor tropism of M1. We showed that the selectivity depends on ZAP deficiency by systematic identification. A large-scale multicenter pathology study using tissue microarrays reveals that ZAP is commonly deficient in human cancers, suggesting extensive application prospects for M1. Additionally, M1 killed cancer cells by inducing endoplasmic reticulum stress-mediated apoptosis. Our report provides novel insights into potentially personalized cancer therapy using oncolytic viruses.

Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses.

We describe a sensitive method for simultaneous detection of Oropouche and Oropouche-like viruses carrying the Oropouche S segment, as well as the Mayaro virus, using a multiplexed one-step reverse transcription real-time polymerase chain reaction (RT-qPCR). A chimeric plasmid containing both Mayaro and Oropouche targets was designed and evaluated for the in vitro production of transcribed RNA, which could be easily used as a non-infectious external control. To track false-negative results due to PCR inhibition or equipment malfunction, the MS2 bacteriophage was also included in the multiplex assay as an internal positive control. The specificity of the multiplex assay was evaluated by Primer-Blast analysis against the entire GenBank database, and further against a panel of 17 RNA arboviruses. The results indicated an accurate and highly sensitive assay with amplification efficiency greater than 98% for both targets, and a limit of detection between two and 20 copies per reaction. We believe that the assay described here will provide a tool for Mayaro and Oropouche virus detection, especially in areas where differential diagnosis of Dengue, Zika and Chikungunya viruses should be performed.

Rainbow trout (Oncorhynchus mykiss) muscle satellite cells are targets of salmonid alphavirus infection.

Sleeping disease in rainbow trout is characterized by an abnormal swimming behaviour of the fish which stay on their side at the bottom of the tanks. This sign is due to extensive necrosis and atrophy of red skeletal muscle induced by the sleeping disease virus (SDV), also called salmonid alphavirus 2. Infections of humans with arthritogenic alphaviruses, such as Chikungunya virus (CHIKV), are global causes of debilitating musculoskeletal diseases. The mechanisms by which the virus causes these pathologies are poorly understood due to the restrictive availability of animal models capable of reproducing the full spectrum of the disease. Nevertheless, it has been shown that CHIKV exhibits a particular tropism for muscle stem cells also known as satellite cells. Thus, SDV and its host constitute a relevant model to study in details the virus-induced muscle atrophy, the pathophysiological consequences of the infection of a particular cell-type in the skeletal muscle, and the regeneration of the muscle tissue in survivors together with the possible virus persistence. To study a putative SDV tropism for that particular cell type, we established an in vivo and ex vivo rainbow trout model of SDV-induced atrophy of the skeletal muscle. This experimental model allows reproducing the full panel of clinical signs observed during a natural infection since the transmission of the virus is arthropod-borne independent. The virus tropism in the muscle tissue was studied by immunohistochemistry together with the kinetics of the muscle atrophy, and the muscle regeneration post-infection was observed. In parallel, an ex vivo model of SDV infection of rainbow trout satellite cells was developed and virus replication and persistence in that particular cell type was followed up to 73 days post-infection. These results constitute the first observation of a specific SDV tropism for the muscle satellite cells.