Alphavirus Identification in Neotropical Bats.

Alphaviruses (Togaviridae) are arthropod-borne viruses responsible for several emerging diseases, maintained in nature through transmission between hematophagous arthropod vectors and susceptible vertebrate hosts. Although bats harbor many species of viruses, their role as reservoir hosts in emergent zoonoses has been verified only in a few cases. With bats being the second most diverse order of mammals, their implication in arbovirus infections needs to be elucidated. Reports on arbovirus infections in bats are scarce, especially in South American indigenous species. In this work, we report the genomic detection and identification of two different alphaviruses in oral swabs from bats captured in Northern Uruguay. Phylogenetic analysis identified Río Negro virus (RNV) in two different species: Tadarida brasiliensis (n = 6) and Myotis spp. (n = 1) and eastern equine encephalitis virus (EEEV) in Myotis spp. (n = 2). Previous studies of our group identified RNV and EEEV in mosquitoes and horse serology, suggesting that they may be circulating in enzootic cycles in our country. Our findings reveal that bats can be infected by these arboviruses and that chiropterans could participate in the viral natural cycle as virus amplifiers or dead-end hosts. Further studies are warranted to elucidate the role of these mammals in the biological cycle of these alphaviruses in Uruguay.

Structural Insights into Alphavirus Assembly Revealed by the Cryo-EM Structure of Getah Virus.

Getah virus (GETV) is a member of the alphavirus genus, and it infects a variety of animal species, including horses, pigs, cattle, and foxes. Human infection with this virus has also been reported. The structure of GETV has not yet been determined. In this study, we report the cryo-EM structure of GETV at a resolution of 3.5 Å. This structure reveals conformational polymorphism of the envelope glycoproteins E1 and E2 at icosahedral 3-fold and quasi-3-fold axes, which is believed to be a necessary organization in forming a curvature surface of virions. In our density map, three extra densities are identified, one of which is believed a "pocket factor"; the other two are located by domain D of E2, and they may maintain the stability of E1/E2 heterodimers. We also identify three N-glycosylations at E1 N141, E2 N200, and E2 N262, which might be associated with receptor binding and membrane fusion. The resolving of the structure of GETV provides new insights into the structure and assembly of alphaviruses and lays a basis for studying the differences of biology and pathogenicity between arthritogenic and encephalitic alphaviruses.

Posaconazole inhibits multiple steps of the alphavirus replication cycle.

Repurposing drugs is a promising strategy to identify therapeutic interventions against novel and re-emerging viruses. Posaconazole is an antifungal drug used to treat invasive aspergillosis and candidiasis. Recently, posaconazole and its structural analog, itraconazole were shown to inhibit replication of multiple viruses by modifying intracellular cholesterol homeostasis. Here, we show that posaconazole inhibits replication of the alphaviruses Semliki Forest virus (SFV), Sindbis virus and chikungunya virus with EC50 values ranging from 1.4 µM to 9.5 µM. Posaconazole treatment led to a significant reduction of virus entry in an assay using a temperature-sensitive SFV mutant, but time-of-addition and RNA transfection assays indicated that posaconazole also inhibits post-entry stages of the viral replication cycle. Virus replication in the presence of posaconazole was partially rescued by the addition of exogenous cholesterol. A transferrin uptake assay revealed that posaconazole considerably slowed down cellular endocytosis. A single point mutation in the SFV E2 glycoprotein, H255R, provided partial resistance to posaconazole as well as to methyl-ß-cyclodextrin, corroborating the effect of posaconazole on cholesterol and viral entry. Our results indicate that posaconazole inhibits multiple steps of the alphavirus replication cycle and broaden the spectrum of viruses that can be targeted in vitro by posaconazole, which could be further explored as a therapeutic agent against emerging viruses.

Genomic Epidemiology of Salmonid Alphavirus in Norwegian Aquaculture Reveals Recent Subtype-2 Transmission Dynamics and Novel Subtype-3 Lineages.

Viral disease poses a major barrier to sustainable aquaculture, with outbreaks causing large economic losses and growing concerns for fish welfare. Genomic epidemiology can support disease control by providing rapid inferences on viral evolution and disease transmission. In this study, genomic epidemiology was used to investigate salmonid alphavirus (SAV), the causative agent of pancreas disease (PD) in Atlantic salmon. Our aim was to reconstruct SAV subtype-2 (SAV2) diversity and transmission dynamics in recent Norwegian aquaculture, including the origin of SAV2 in regions where this subtype is not tolerated under current legislation. Using nanopore sequencing, we captured ~90% of the SAV2 genome for n = 68 field isolates from 10 aquaculture production regions sampled between 2018 and 2020. Using time-calibrated phylogenetics, we infer that, following its introduction to Norway around 2010, SAV2 split into two clades (SAV2a and 2b) around 2013. While co-present at the same sites near the boundary of Møre og Romsdal and Trøndelag, SAV2a and 2b were generally detected in non-overlapping locations at more Southern and Northern latitudes, respectively. We provide evidence for recent SAV2 transmission over large distances, revealing a strong connection between Møre og Romsdal and SAV2 detected in 2019/20 in Rogaland. We also demonstrate separate introductions of SAV2a and 2b outside the SAV2 zone in Sognefjorden (Vestland), connected to samples from Møre og Romsdal and Trøndelag, respectively, and a likely 100 km Northward transmission of SAV2b within Trøndelag. Finally, we recovered genomes of SAV2a and SAV3 co-infecting single fish in Rogaland, involving novel SAV3 lineages that diverged from previously characterized strains >25 years ago. Overall, this study demonstrates useful applications of genomic epidemiology for tracking viral disease spread in aquaculture.

Complete genetic dissection and cell type-specific replication of old world alphaviruses, getah virus (GETV) and sagiyama virus (SAGV).

Getah virus (GETV), which was first isolated in Malaysia in 1955, and Sagiyama virus (SAGV), isolated in Japan in 1956, are members of the genus Alphavirus in the family Togaviridae. It is a consensus view that SAGV is a variant of GETV. In the present study, we determined the complete sequences of the prototype GETV MM2021 and SAGV M6-Mag132 genomic RNA extracted from plaque-purified viruses. The MM2021 genome was 11,692 nucleotides (nt) in length in the absence of 3' poly(A) tail, and the length of M6-Mag132 genome was 11,698 nt. Through sequence alignment of MM2021 and M6-Mag132, we located all the amino acid differences between these two strains, which were scattered in all the encoded proteins. Subsequently, we validated the close evolutionary relationship between GETV and SAGV by constructing phylogenetic trees based on either complete genomes or structural genomes. We eventually analyzed the growth kinetics of GETV and SAGV as well as other representative alphaviruses in various mammalian and insect cell lines. It was shown that human-oriented cell lines such as HEK-293T and Hela cells were relatively resistant to GETV and SAGV infection due to absence of proviral factors or species-specific barrier. On the other hand, both GETV and SAGV replicated efficiently in non-human cell lines. Our results provide essential genetic information for future epidemiological surveillance on Alphaviruses and lay the foundation for developing effective interventions against GETV and SAGV.

Virucidal and antiviral activities of pomegranate (Punica granatum) extract against the mosquito-borne Mayaro virus.

BACKGROUND: The arthropod-borne Mayaro virus (MAYV) causes "Mayaro fever," a disease of medical significance, primarily affecting individuals in permanent contact with forested areas in tropical South America. Recently, MAYV has attracted attention due to its likely urbanization. There are currently no licensed drugs against most mosquito-transmitted viruses. Punica granatum (pomegranate) fruits cultivated in Brazil have been subjected to phytochemical investigation for the identification and isolation of antiviral compounds. In the present study, we explored the antiviral activity of pomegranate extracts in Vero cells infected with Mayaro virus. METHODS: The ethanol extract and punicalagin of pomegranate were extracted solely from the shell and purified by chromatographic fractionation, and were chemically identified using spectroscopic techniques. The cytotoxicity of the purified compounds was measured by the dye uptake assay, while their antiviral activity was evaluated by a virus yield inhibition assay. RESULTS: Pomegranate ethanol extract (CC50 = 588.9, IC50 = 12.3) and a fraction containing punicalagin as major compound (CC50 = 441.5, IC50 = 28.2) were shown to have antiviral activity (SI 49 and 16, respectively) against Mayaro virus, an alphavirus. Immunofluorescence analysis showed the virucidal effect of pomegranate extract, and transmission electron microscopy (TEM) revealed damage in viral particles treated with this extract. CONCLUSIONS: The P. granatum extract is a promising source of antiviral compounds against the alphavirus MAYV and represents an excellent candidate for future studies with other enveloped RNA viruses.

Emergence of Salmonid Alphavirus Genotype 2 in Norway-Molecular Characterization of Viral Strains Circulating in Norway and Scotland.

Pancreas disease (PD) and sleeping disease (SD), caused by an alphavirus, are endemic in European salmonid aquaculture, causing significant mortality, reduced growth and poor flesh quality. In 2010, a new variant of salmonid alphavirus emerged in Norway, marine salmonid alphavirus genotype 2 (SAV2). As this genotype is highly prevalent in Scotland, transmission through well boat traffic was hypothesized as one possible source of infection. In this study, we performed full-length genome sequencing of SAV2 sampled between 2006 and 2012 in Norway and Scotland, and present the first comprehensive full-length characterization of Norwegian marine SAV2 strains. We analyze their relationship with selected Scottish SAV2 strains and explore the genetic diversity of SAV. Our results show that all Norwegian marine SAV2 share a recent last common ancestor with marine SAV2 circulating in Scotland and a higher level of genomic diversity among the Scottish marine SAV2 strains compared to strains from Norway. These findings support the hypothesis of a single introduction of SAV2 to Norway sometime from 2006-2010, followed by horizontal spread along the coast.

Development of a Sensitive Detection Method for Alphaviruses and Its Use as a Virus Neutralization Assay.

Alphaviruses have a single-stranded, positive-sense RNA genome that contains two open reading frames encoding either the non-structural or the structural genes. Upon infection, the genomic RNA is translated into the non-structural proteins (nsPs). NsPs are required for viral RNA replication and transcription driven from the subgenomic promoter (sgP). Transfection of an RNA encoding the luciferase gene under the control of the sgP into cells enabled the detection of replication-competent chikungunya virus (CHIKV) or Mayaro virus (MAYV) with high sensitivity as a function of the induced luciferase activity. This assay principle was additionally used to analyze virus-neutralizing antibodies in sera and might be an alternative to standard virus neutralization assays based on virus titration or the use of genetically modified tagged viruses.

The epidemiology of Mayaro virus in the Americas: A systematic review and key parameter estimates for outbreak modelling.

Mayaro virus (MAYV) is an arbovirus that is endemic to tropical forests in Central and South America, particularly within the Amazon basin. In recent years, concern has increased regarding MAYV's ability to invade urban areas and cause epidemics across the region. We conducted a systematic literature review to characterise the evolutionary history of MAYV, its transmission potential, and exposure patterns to the virus. We analysed data from the literature on MAYV infection to produce estimates of key epidemiological parameters, including the generation time and the basic reproduction number, R0. We also estimated the force-of-infection (FOI) in epidemic and endemic settings. Seventy-six publications met our inclusion criteria. Evidence of MAYV infection in humans, animals, or vectors was reported in 14 Latin American countries. Nine countries reported evidence of acute infection in humans confirmed by viral isolation or reverse transcription-PCR (RT-PCR). We identified at least five MAYV outbreaks. Seroprevalence from population based cross-sectional studies ranged from 21% to 72%. The estimated mean generation time of MAYV was 15.2 days (95% CrI: 11.7-19.8) with a standard deviation of 6.3 days (95% CrI: 4.2-9.5). The per-capita risk of MAYV infection (FOI) ranged between 0.01 and 0.05 per year. The mean R0 estimates ranged between 2.1 and 2.9 in the Amazon basin areas and between 1.1 and 1.3 in the regions outside of the Amazon basin. Although MAYV has been identified in urban vectors, there is not yet evidence of sustained urban transmission. MAYV's enzootic cycle could become established in forested areas within cities similar to yellow fever virus.

PERK Is Critical for Alphavirus Nonstructural Protein Translation.

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes encephalitis. Previous work indicated that VEEV infection induced early growth response 1 (EGR1) expression, leading to cell death via the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) arm of the unfolded protein response (UPR) pathway. Loss of PERK prevented EGR1 induction and decreased VEEV-induced death. The results presented within show that loss of PERK in human primary astrocytes dramatically reduced VEEV and eastern equine encephalitis virus (EEEV) infectious titers by 4-5 log10. Loss of PERK also suppressed VEEV replication in primary human pericytes and human umbilical vein endothelial cells, but it had no impact on VEEV replication in transformed U87MG and 293T cells. A significant reduction in VEEV RNA levels was observed as early as 3 h post-infection, but viral entry assays indicated that the loss of PERK minimally impacted VEEV entry. In contrast, the loss of PERK resulted in a dramatic reduction in viral nonstructural protein translation and negative-strand viral RNA production. The loss of PERK also reduced the production of Rift Valley fever virus and Zika virus infectious titers. These data indicate that PERK is an essential factor for the translation of alphavirus nonstructural proteins and impacts multiple RNA viruses, making it an exciting target for antiviral development.

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.

Characterization of an alphavirus isolated from a stranded harbor porpoise (Phocoena phocoena) from Alaska.

The family Togaviridae comprises several significant human and veterinary mosquito-borne pathogens. Two togaviruses (genus Alphavirus) have been previously identified in association with marine mammals, the southern elephant seal virus (SESV) and Eastern equine encephalitis virus (EEEV) from a fatal captive harbor seal infection. Herein we report the ultrastructural and phylogenomic characterization of a novel marine togavirus, the first isolated from a cetacean, an Alaskan harbor porpoise (Phocoena phocoena) displaying ulcerative dermatitis. A skin sample was processed for virus isolation on Vero.DogSLAMtag cells and cytopathic effects (CPE) were observed on primary isolation approximately 20 days post-infection. Transmission electron microscopy of the infected Vero.DogSLAMtag cells revealed typical alphavirus particles budding from both plasma and vacuolar membranes of infected cells. A next-generation sequencing approach was used to determine the near complete genome of the Alaskan harbor porpoise alphavirus (AHPV). Phylogenetic analysis supported the AHPV as the sister species to the SESV, forming a marine mammal alphavirus clade separate from the recognized alphavirus antigenic complexes. Genetic comparison of the protein coding sequence of the AHPV to other alphaviruses demonstrated amino acid identities ranging from 42.1-67.1%, with the highest identity to the SESV. Based on its genetic divergence, we propose the AHPV represents a novel alphavirus species, pending formal proposal to and ratification by the International Committee on Taxonomy of Viruses. The ecological and genetic characteristics of the AHPV and the SESV also suggest they represent a novel antigenic complex within the genus Alphavirus, which we propose to be named the Marine Mammal Virus Complex. The role of the AHPV in the associated harbor porpoise cutaneous pathology, if any, remains unclear. Further research is needed to determine AHPV's route(s) of transmission and potential vectors, host range, prevalence, and pathogenicity in cetaceans including harbour porpoises.

Host Factors and Pathways Involved in the Entry of Mosquito-Borne Alphaviruses.

Chikungunya virus (CHIKV) is an arthropod-borne virus that has re-emerged recently and has spread to previously unaffected regions, resulting in millions of infections worldwide. The genus Alphavirus, in the family Togaviridae, contains several members with a similar potential for epidemic emergence. In order for CHIKV to replicate in targeted cell types it is essential for the virus to enter these cells. In this review, we summarize our current understanding of the versatile and promiscuous steps in CHIKV binding and entry into human and mosquito host cells. We describe the different entry pathways, receptors, and attachment factors so far described for CHIKV and other mosquito-borne alphaviruses and discuss them in the context of tissue tropism and potential therapeutic targeting.

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.

Differential Alphavirus Defective RNA Diversity between Intracellular and Extracellular Compartments Is Driven by Subgenomic Recombination Events.

Alphaviruses are positive-sense RNA arboviruses that can cause either a chronic arthritis or a potentially lethal encephalitis. Like other RNA viruses, alphaviruses produce truncated, defective viral RNAs featuring large deletions during replication. These defective RNAs (D-RNAs) have primarily been isolated from virions after high-multiplicity-of-infection passaging. Here, we aimed to characterize both intracellular and packaged viral D-RNA populations during early-passage infections under the hypothesis that D-RNAs arise de novo intracellularly that may not be packaged and thus have remained undetected. To this end, we generated next-generation sequencing libraries using RNA derived from passage 1 (P1) stock chikungunya virus (CHIKV) 181/clone 25, intracellular virus, and P2 virions and analyzed samples for D-RNA expression, followed by diversity and differential expression analyses. We found that the diversity of D-RNA species is significantly higher for intracellular D-RNA populations than P2 virions and that specific populations of D-RNAs are differentially expressed between intracellular and extracellular compartments. Importantly, these trends were likewise observed in a murine model of CHIKV AF15561 infection, as well as in vitro studies using related Mayaro, Sindbis, and Aura viruses. Additionally, we identified a novel subtype of subgenomic D-RNA that is conserved across arthritogenic alphaviruses. D-RNAs specific to intracellular populations were defined by recombination events specifically in the subgenomic region, which were confirmed by direct RNA nanopore sequencing of intracellular CHIKV RNAs. Together, these studies show that only a portion of D-RNAs generated intracellularly are packaged and D-RNAs readily arise de novo in the absence of transmitted template.IMPORTANCE Our understanding of viral defective RNAs (D-RNAs), or truncated viral genomes, comes largely from passaging studies in tissue culture under artificial conditions and/or packaged viral RNAs. Here, we show that specific populations of alphavirus D-RNAs arise de novo and that they are not packaged into virions, thus imposing a transmission bottleneck and impeding their prior detection. This raises important questions about the roles of D-RNAs, both in nature and in tissue culture, during viral infection and whether their influence is constrained by packaging requirements. Further, during the course of these studies, we found a novel type of alphavirus D-RNA that is enriched intracellularly; dubbed subgenomic D-RNAs (sgD-RNAs), they are defined by deletion boundaries between the capsid-E3 region and the E1-3' untranslated region (UTR) and are common to chikungunya, Mayaro, Sindbis, and Aura viruses. These sgD-RNAs are enriched intracellularly and do not appear to be selectively packaged, and additionally, they may exist as subgenome-derived transcripts.

Phylogenetic and Timescale Analysis of Barmah Forest Virus as Inferred from Genome Sequence Analysis.

Barmah Forest virus (BFV) is a medically important mosquito-borne alphavirus endemic to Australia. Symptomatic disease can be a major cause of morbidity, associated with fever, rash, and debilitating arthralgia. BFV disease is similar to that caused by Ross River virus (RRV), the other major Australian alphavirus. Currently, just four BFV whole-genome sequences are available with no genome-scale phylogeny in existence to robustly characterise genetic diversity. Thirty novel genome sequences were derived for this study, for a final 34-taxon dataset sampled over a 44 year period. Three distinct BFV genotypes were characterised (G1-3) that have circulated in Australia and Papua New Guinea (PNG). Evidence of spatio-temporal co-circulation of G2 and G3 within regions of Australia was noted, including in the South West region of Western Australia (WA) during the first reported disease outbreaks in the state's history. Compared with RRV, the BFV population appeared more stable with less frequent emergence of novel lineages. Preliminary in vitro assessment of RRV and BFV replication kinetics found that RRV replicates at a significantly faster rate and to a higher, more persistent titre compared with BFV, perhaps indicating mosquitoes may be infectious with RRV for longer than with BFV. This investigation resolved a greater diversity of BFV, and a greater understanding of the evolutionary dynamics and history was attained.

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.

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.

An emerging public health threat: Mayaro virus increases its distribution in Peru.

BACKGROUND: The infection caused by Mayaro virus (MAYV), which presents as an acute febrile illness, is considered a neglected tropical disease. The virus is an endemic and emerging pathogen in South America and the Caribbean, responsible for occasional and poorly characterized outbreaks. Currently there is limited information about its expansion and risk areas. METHODS: A cross-sectional study was performed in 10 urban primary care health centers in the Cajamarca region of Peru from January to June 2017. A total of 359 patients with suspected febrile illness were assessed. RNA was extracted from serum samples, following which MAYV real-time reverse transcriptase PCR (RT-PCR) for the detection of the nsP1 gene was performed. RESULTS: MAYV was detected in 11.1% (40/359) of samples after RT-PCR amplification and confirmatory DNA sequencing. Most infections were detected in the adult population aged 18-39 years (40%) and 40-59 years (32.5%). Headache was the most frequent symptom in patients with MAYV infection (77.5%), followed by fever (72.5%), myalgia (55.0%), and arthralgia (50.0%). During the study, most of the MAYV cases were seen in May (47.5%) and April (35.0%), corresponding to the dry season (months without rain). CONCLUSIONS: This study is novel in describing the presence of MAYV in Cajamarca, an Andean region of Peru. Symptoms are non-specific and can be confused with those of other arbovirus or bacterial infections. Molecular biology methods such as RT-PCR allow the timely and accurate detection of MAYV and could thus be considered as a tool for surveillance in endemic areas.

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.