Development of reverse genetics system for Guanarito virus: substitution of E1497K in the L protein of Guanarito virus S-26764 strain changes plaque phenotype and growth kinetics.

Guanarito virus (GTOV) is the causative agent of Venezuelan hemorrhagic fever. GTOV belongs to the genus Mammarenavirus, family Arenaviridae and has been classified as a Category A bioterrorism agent by the United States Centers for Disease Control and Prevention. Despite being a high-priority agent, vaccines and drugs against Venezuelan hemorrhagic fever are not available. GTOV S-26764, isolated from a non-fatal human case, produces an unclear cytopathic effect (CPE) in Vero cells, posing a significant obstacle to research and countermeasure development efforts. Vero cell-adapted GTOV S-26764 generated in this study produced clear CPE and demonstrated rapid growth and high yield in Vero cells compared to the original GTOV S-26764. We developed a reverse genetics system for GTOV to study amino acid changes acquired through Vero cell adaptation and leading to virus phenotype changes. The results demonstrated that E1497K in the L protein was responsible for the production of clear plaques as well as enhanced viral RNA replication and transcription efficiency. Vero cell-adapted GTOV S-26764, capable of generating CPE, will allow researchers to easily perform neutralization assays and anti-drug screening against GTOV. Moreover, the developed reverse genetics system will accelerate vaccine and antiviral drug development.IMPORTANCEGuanarito virus (GTOV) is a rodent-borne virus. GTOV causes fever, prostration, headache, arthralgia, cough, sore throat, nausea, vomiting, diarrhea, epistaxis, bleeding gums, menorrhagia, and melena in humans. The lethality rate is 23.1% or higher. Vero cell-adapted GTOV S-26764 shows a clear cytopathic effect (CPE), whereas the parental virus shows unclear CPE in Vero cells. We generated a reverse genetics system to rescue recombinant GTOVs and found that E1497K in the L protein was responsible for the formation of clear plaques as well as enhanced viral RNA replication and transcription efficiency. This reverse genetic system will accelerate vaccine and antiviral drug developments, and the findings of this study contribute to the understanding of the function of GTOV L as an RNA polymerase.

A review of emerging health threats from zoonotic New World mammarenaviruses.

Despite repeated spillover transmission and their potential to cause significant morbidity and mortality in human hosts, the New World mammarenaviruses remain largely understudied. These viruses are endemic to South America, with animal reservoir hosts covering large geographic areas and whose transmission ecology and spillover potential are driven in part by land use change and agriculture that put humans in regular contact with zoonotic hosts.We compiled published studies about Guanarito virus, Junin virus, Machupo virus, Chapare virus, Sabia virus, and Lymphocytic Choriomeningitis virus to review the state of knowledge about the viral hemorrhagic fevers caused by New World mammarenaviruses. We summarize what is known about rodent reservoirs, the conditions of spillover transmission for each of these pathogens, and the characteristics of human populations at greatest risk for hemorrhagic fever diseases. We also review the implications of repeated outbreaks and biosecurity concerns where these diseases are endemic, and steps that countries can take to strengthen surveillance and increase capacity of local healthcare systems. While there are unique risks posed by each of these six viruses, their ecological and epidemiological similarities suggest common steps to mitigate spillover transmission and better contain future outbreaks.

Mammarenavirus Genetic Diversity and Its Biological Implications.

Members of the family Arenaviridae are classified into four genera: Antennavirus, Hartmanivirus, Mammarenavirus, and Reptarenavirus. Reptarenaviruses and hartmaniviruses infect (captive) snakes and have been shown to cause boid inclusion body disease (BIBD). Antennaviruses have genomes consisting of 3, rather than 2, segments, and were discovered in actinopterygian fish by next-generation sequencing but no biological isolate has been reported yet. The hosts of mammarenaviruses are mainly rodents and infections are generally asymptomatic. Current knowledge about the biology of reptarenaviruses, hartmaniviruses, and antennaviruses is very limited and their zoonotic potential is unknown. In contrast, some mammarenaviruses are associated with zoonotic events that pose a threat to human health. This review will focus on mammarenavirus genetic diversity and its biological implications. Some mammarenaviruses including lymphocytic choriomeningitis virus (LCMV) are excellent experimental model systems for the investigation of acute and persistent viral infections, whereas others including Lassa (LASV) and Junin (JUNV) viruses, the causative agents of Lassa fever (LF) and Argentine hemorrhagic fever (AHF), respectively, are important human pathogens. Mammarenaviruses were thought to have high degree of intra-and inter-species amino acid sequence identities, but recent evidence has revealed a high degree of mammarenavirus genetic diversity in the field. Moreover, closely related mammarenavirus can display dramatic phenotypic differences in vivo. These findings support a role of genetic variability in mammarenavirus adaptability and pathogenesis. Here, we will review the molecular biology of mammarenaviruses, phylogeny, and evolution, as well as the quasispecies dynamics of mammarenavirus populations and their biological implications.

Peptide based vaccine designing against endemic causing mammarenavirus using reverse vaccinology approach.

The rodent-borne Arenavirus in humans has led to the emergence of regional endemic situations and has deeply emerged into pandemic-causing viruses. Arenavirus have a bisegmented ambisense RNA that produces four proteins: glycoprotein, nucleocapsid, RdRp and Z protein. The peptide-based vaccine targets the glycoprotein of the virus encountered by the immune system. Screening of B-Cell and T-Cell epitopes was done based on their immunological properties like antigenicity, allergenicity, toxicity and anti-inflammatory properties were performed. Selected epitopes were then clustered and epitopes were stitched using linker sequences. The immunological and physico-chemical properties of the vaccine construct was checked and modelled structure was validated by a 2-step MD simulation. The thermostability of the vaccine was checked followed by the immune simulation to test the immunogenicity of the vaccine upon introduction into the body over the course of the next 100 days and codon optimization was performed. Finally a 443 amino acid long peptide vaccine was designed which could provide protection against several members of the mammarenavirus family in a variety of population worldwide as denoted by the epitope conservancy and population coverage analysis. This study of designing a peptide vaccine targeting the glycoprotein of mammarenavirues may help develop novel therapeutics in near future.

ICTV Virus Taxonomy Profile: Arenaviridae 2023.

Arenaviridae is a family for ambisense RNA viruses with genomes of about 10.5 kb that infect mammals, snakes, and fish. The arenavirid genome consists of two or three single-stranded RNA segments and encodes a nucleoprotein (NP), a glycoprotein (GP) and a large (L) protein containing RNA-directed RNA polymerase (RdRP) domains; some arenavirids encode a zinc-binding protein (Z). This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Arenaviridae, which is available at www.ictv.global/report/arenaviridae.

Molecular characterization of a reptarenavirus detected in a Colombian Red-Tailed Boa (Boa constrictor imperator).

The global decline in biodiversity is a matter of great concern for members of the class Reptilia. Reptarenaviruses infect snakes, and have been linked to various clinical conditions, such as Boid Inclusion Body Disease (BIBD) in snakes belonging to the families Boidae and Pythonidae. However, there is a scarcity of information regarding reptarenaviruses found in snakes in both the United States and globally. This study aimed to contribute to the understanding of reptarenavirus diversity by molecularly characterizing a reptarenavirus detected in a Colombian Red-Tailed Boa (Boa constrictor imperator). Using a metagenomics approach, we successfully identified, and de novo assembled the whole genomic sequences of a reptarenavirus in a Colombian Red-Tailed Boa manifesting clinically relevant symptoms consistent with BIBD. The analysis showed that the Colombian Red-Tailed Boa in this study carried the University of Giessen virus (UGV-1) S or S6 (UGV/S6) segment and L genotype 7. The prevalence of the UGV/S6 genotype, in line with prior research findings, implies that this genotype may possess specific advantageous characteristics or adaptations that give it a competitive edge over other genotypes in the host population. This research underscores the importance of monitoring and characterizing viral pathogens in captive and wild snake populations. Knowledge of such viruses is crucial for the development of effective diagnostic methods, potential intervention strategies, and the conservation of vulnerable reptilian species. Additionally, our study provides valuable insights for future studies focusing on the evolutionary history, molecular epidemiology, and biological properties of reptarenaviruses in boas and other snake species.

A novel mammarenavirus (family Arenaviridae) in hedgehogs (Erinaceus roumanicus) in Europe.

In this study, a novel mammarenavirus (family Arenaviridae) was identified in a hedgehog (family Erinaceidae) in Hungary and genetically characterized. Mecsek Mountains virus (MEMV, OP191655, OP191656) was detected in nine (45%) out of 20 faecal specimens collected from a Northern white-breasted hedgehog (Erinaceus roumanicus). The L-segment proteins (RdRp and Z) and S-segment proteins (NP and GPC) of MEMV had 67.5%/70% and 74.6%/65.6% amino acid sequence identity, respectively, to the corresponding proteins of Alxa virus (species Mammarenavirus alashanense) identified recently in an anal swab from a three-toed jerboa (Dipus sagitta) in China. MEMV is the second known arenavirus endemic in Europe.

Pseudotyped Viruses for Mammarenavirus.

Mammarenaviruses are classified into New World arenaviruses (NW) and Old World arenaviruses (OW). The OW arenaviruses include the first discovered mammarenavirus-lymphocytic choriomeningitis virus (LCMV) and the highly lethal Lassa virus (LASV). Mammarenaviruses are transmitted to human by rodents, resulting in severe acute infections and hemorrhagic fever. Pseudotyped viruses have been widely used as a tool in the study of mammarenaviruses. HIV-1, SIV, FIV-based lentiviral vectors, VSV-based vectors, MLV-based vectors, and reverse genetic approaches have been applied in the construction of pseudotyped mammarenaviruses. Pseudotyped mammarenaviruses are commonly used in receptor research, neutralizing antibody detection, inhibitor screening, viral virulence studies, functional analysis of N-linked glycans, and studies of viral infection, endocytosis, and fusion mechanisms.

Towards specie-specific ensemble vaccine candidates against mammarenaviruses using optimized structural vaccinology pipeline and molecular modelling approaches.

Mammarena viruses are emerging pathogenic agents and cause hemorrhagic fevers in humans. These viruses accomplish host immune system evasion to replicate and spread in the host. There are only few available therapeutic options developed for Mammarena Virus (also called MMV). Currently, only a single candidate vaccine called Candid#1 is available against Junin virus. Similarly, the effective treatment Ribavirin is used only in Lassa fever treatments. Herein, immune-informatics pipeline has been used to annotate whole proteome of the seven human infecting Mammarena strains. The extensive immune based analysis reveals specie specific epitopes with a crucial role in immune response induction. This was achieved by construction of immunogenic epitopes (CTL "Cytotoxic T-Lymphocytes", HTL "Helper T-Lymphocytes", and B cell "B-Lymphocytes") based vaccine designs against seven different Mammarena virus species. Furthermore, validation of the vaccine constructs through exploring physiochemical properties was performed to confirm experimental feasibility. Additionally, in-silico cloning and receptor based immune simulation was performed to ensure induction of primary and secondary immune response. This was confirmed through expression of immune factors such as IL, cytokines, and antibodies. The current study provides with novel vaccine designs which needs further demonstrations through potential processing against MMVs. Future studies may be directed towards advanced evaluations to determine the efficacy and safety of the designed vaccines through further experimental procedures.

Identification of Reptarenaviruses, Hartmaniviruses, and a Novel Chuvirus in Captive Native Brazilian Boa Constrictors with Boid Inclusion Body Disease.

Boid inclusion body disease (BIBD) is a transmissible viral disease of captive snakes that causes severe losses in snake collections worldwide. It is caused by reptarenavirus infection, which can persist over several years without overt signs but is generally associated with the eventual death of the affected snakes. Thus far, reports have confirmed the existence of reptarenaviruses in captive snakes in North America, Europe, Asia, and Australia, but there is no evidence that it also occurs in wild snakes. BIBD affects boa species within the subfamily Boinae and pythons in the family Pythonidae, the habitats of which do not naturally overlap. Here, we studied Brazilian captive snakes with BIBD using a metatranscriptomic approach, and we report the identification of novel reptarenaviruses, hartmaniviruses, and a new species in the family Chuviridae The reptarenavirus L segments identified are divergent enough to represent six novel species, while we found only a single novel reptarenavirus S segment. Until now, hartmaniviruses had been identified only in European captive boas with BIBD, and the present results increase the number of known hartmaniviruses from four to six. The newly identified chuvirus showed 38.4%, 40.9%, and 48.1% amino acid identity to the nucleoprotein, glycoprotein, and RNA-dependent RNA polymerase, respectively, of its closest relative, Guangdong red-banded snake chuvirus-like virus. Although we cannot rule out the possibility that the found viruses originated from imported snakes, the results suggest that the viruses could circulate in indigenous snake populations.IMPORTANCE Boid inclusion body disease (BIBD), caused by reptarenavirus infection, affects captive snake populations worldwide, but the reservoir hosts of reptarenaviruses remain unknown. Here, we report the identification of novel reptarenaviruses, hartmaniviruses, and a chuvirus in captive Brazilian boas with BIBD. Three of the four snakes studied showed coinfection with all three viruses, and one of the snakes harbored three novel reptarenavirus L segments and one novel S segment. The samples originated from collections with Brazilian indigenous snakes only, which could indicate that these viruses circulate in wild snakes. The findings could further indicate that boid snakes are the natural reservoir of reptarena- and hartmaniviruses commonly found in captive snakes. The snakes infected with the novel chuvirus all suffered from BIBD; it is therefore not possible to comment on its potential pathogenicity and contribution to the observed changes in the present case material.

Development of Reverse Genetics for the Prototype New World Mammarenavirus Tacaribe Virus.

The New World mammarenavirus Tacaribe virus (TCRV) has been isolated from fruit bats, mosquitoes, and ticks, whereas all other known New World mammarenaviruses are maintained in rodents. TCRV has not been linked to human disease, but it has been shown to protect against Argentine hemorrhagic fever-like disease in marmosets infected with the New World mammarenavirus Junín virus (JUNV), indicating the potential of TCRV as a live-attenuated vaccine for the treatment of Argentine hemorrhagic fever. Implementation of TCRV as a live-attenuated vaccine or a vaccine vector would be facilitated by the establishment of reverse genetics systems for the genetic manipulation of the TCRV genome. In this study, we developed, for the first time, reverse genetics approaches for the generation of recombinant TCRV (rTCRV). We successfully rescued a wild-type (WT) rTCRV (a trisegmented form of TCRV expressing two reporter genes [r3TCRV]) and a bisegmented TCRV expressing a single reporter gene from a bicistronic viral mRNA (rTCRV/GFP). These reverse genetics approaches represent an excellent tool to investigate the biology of TCRV and to explore its potential use as a live-attenuated vaccine or a vaccine vector for the treatment of other viral infections. Notably, we identified a 39-nucleotide (nt) deletion (Δ39) in the noncoding intergenic region (IGR) of the viral large (L) segment that is required for optimal virus multiplication. Accordingly, an rTCRV containing this 39-nt deletion in the L-IGR (rTCRV/Δ39) exhibited decreased viral fitness in cultured cells, suggesting the feasibility of using this deletion in the L-IGR as an approach to attenuate TCRV, and potentially other mammarenaviruses, for their implementation as live-attenuated vaccines or vaccine vectors.IMPORTANCE To date, no Food and Drug Administration (FDA)-approved vaccines are available to combat hemorrhagic fever caused by mammarenavirus infections in humans. Treatment of mammarenavirus infections is limited to the off-label use of ribavirin, which is partially effective and associated with significant side effects. Tacaribe virus (TCRV), the prototype member of the New World mammarenaviruses, is nonpathogenic in humans but able to provide protection against Junín virus (JUNV), the causative agent of Argentine hemorrhagic fever, demonstrating the feasibility of using TCRV as a live-attenuated vaccine vector for the treatment of JUNV and potentially other viral infections. Here, we describe for the first time the feasibility of generating recombinant TCRV (rTCRV) using reverse genetics approaches, which paves the way to study the biology of TCRV and also its potential use as a live-attenuated vaccine or a vaccine vector for the treatment of mammarenavirus and/or other viral infections in humans.

Molecular detection and genetic characterization of Wenzhou virus in rodents in Guangzhou, China.

BACKGROUND: Wenzhou virus (WENV), a newly discovered mammarenavirus in rodents, is associated with fever and respiratory symptoms in humans. This study was aimed to detect and characterize the emerging virus in rodents in Guangzhou, China. RESULTS: A total of 100 small mammals, including 70 Rattus norvegicus, 22 Suncus murinus, 4 Bandicota indica, 3 Rattus flavipectus, and 1 Rattus losea, were captured in Guangzhou, and their brain tissues were collected and pooled for metagenomic analysis, which generated several contigs targeting the genome of WENV. Two R. norvegicus (2.9%) were further confirmed to be infected with WENV by RT-PCR. The complete genome (RnGZ37-2018 and RnGZ40-2018) shared 85.1-88.9% nt and 83.2-96.3% aa sequence identities to the Cambodian strains that have been shown to be associated with human disease. Phylogenetic analysis showed that all identified WENV could be grouped into four different lineages, and the two Guangzhou strains formed an independent clade. We also analyzed the potential recombinant events occurring in WENV strains. CONCLUSIONS: Our study showed a high genetic diversity of WENV strains in China, emphasizing the relevance of surveillance of this emerging mammarenavirus in both natural reservoirs and humans.

Characterization of Haartman Institute snake virus-1 (HISV-1) and HISV-like viruses-The representatives of genus Hartmanivirus, family Arenaviridae.

The family Arenaviridae comprises three genera, Mammarenavirus, Reptarenavirus and the most recently added Hartmanivirus. Arenaviruses have a bisegmented genome with ambisense coding strategy. For mammarenaviruses and reptarenaviruses the L segment encodes the Z protein (ZP) and the RNA-dependent RNA polymerase, and the S segment encodes the glycoprotein precursor and the nucleoprotein. Herein we report the full length genome and characterization of Haartman Institute snake virus-1 (HISV-1), the putative type species of hartmaniviruses. The L segment of HISV-1 lacks an open-reading frame for ZP, and our analysis of purified HISV-1 particles by SDS-PAGE and electron microscopy further support the lack of ZP. Since we originally identified HISV-1 in co-infection with a reptarenavirus, one could hypothesize that co-infecting reptarenavirus provides the ZP to complement HISV-1. However, we observed that co-infection does not markedly affect the amount of hartmanivirus or reptarenavirus RNA released from infected cells in vitro, indicating that HISV-1 does not benefit from reptarenavirus ZP. Furthermore, we succeeded in generating a pure HISV-1 isolate showing the virus to replicate without ZP. Immunofluorescence and ultrastructural studies demonstrate that, unlike reptarenaviruses, HISV-1 does not produce the intracellular inclusion bodies typical for the reptarenavirus-induced boid inclusion body disease (BIBD). While we observed HISV-1 to be slightly cytopathic for cultured boid cells, the histological and immunohistological investigation of HISV-positive snakes showed no evidence of a pathological effect. The histological analyses also revealed that hartmaniviruses, unlike reptarenaviruses, have a limited tissue tropism. By nucleic acid sequencing, de novo genome assembly, and phylogenetic analyses we identified additional four hartmanivirus species. Finally, we screened 71 individuals from a collection of snakes with BIBD by RT-PCR and found 44 to carry hartmaniviruses. These findings suggest that harmaniviruses are common in captive snake populations, but their relevance and pathogenic potential needs yet to be revealed.

ICTV Virus Taxonomy Profile: Arenaviridae.

Members of the family Arenaviridae produce enveloped virions containing genomes consisting of two or three single-stranded RNA segments totalling about 10.5 kb. Arenaviruses can infect mammals, including humans and other primates, snakes, and fish. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Arenaviridae, which is available at www.ictv.global/report/arenaviridae.

A Vaccine Platform against Arenaviruses Based on a Recombinant Hyperattenuated Mopeia Virus Expressing Heterologous Glycoproteins.

Several Old World and New World arenaviruses are responsible for severe endemic and epidemic hemorrhagic fevers, whereas other members of the Arenaviridae family are nonpathogenic. To date, no approved vaccines, antivirals, or specific treatments are available, except for Junín virus. However, protection of nonhuman primates against Lassa fever virus (LASV) is possible through the inoculation of the closely related but nonpathogenic Mopeia virus (MOPV) before challenge with LASV. We reasoned that this virus, modified by using reverse genetics, would represent the basis for the generation of a vaccine platform against LASV and other pathogenic arenaviruses. After showing evidence of exoribonuclease (ExoN) activity in NP of MOPV, we found that this activity was essential for multiplication in antigen-presenting cells. The introduction of multiple mutations in the ExoN site of MOPV NP generated a hyperattenuated strain (MOPVExoN6b) that is (i) genetically stable over passages, (ii) has increased immunogenic properties compared to those of MOPV, and (iii) still promotes a strong type I interferon (IFN) response. MOPVExoN6b was further modified to harbor the envelope glycoproteins of heterologous pathogenic arenaviruses, such as LASV or Lujo, Machupo, Guanarito, Chapare, or Sabia virus in order to broaden specific antigenicity while preserving the hyperattenuated characteristics of the parental strain. Our MOPV-based vaccine candidate for LASV, MOPEVACLASV, was used in a one-shot immunization assay in nonhuman primates and fully protected them from a lethal challenge with LASV. Thus, our hyperattenuated strain of MOPV constitutes a promising new live-attenuated vaccine platform to immunize against several, if not all, pathogenic arenaviruses.IMPORTANCE Arenaviruses are emerging pathogens transmitted to humans by rodents and responsible for endemic and epidemic hemorrhagic fevers of global concern. Nonspecific symptoms associated with the onset of infection make these viruses difficult to distinguish from other endemic pathogens. Moreover, the unavailability of rapid diagnosis in the field delays the identification of the virus and early care for treatment and favors spreading. The vaccination of exposed populations would be of great help to decrease morbidity and human-to-human transmission. Using reverse genetics, we generated a vaccine platform for pathogenic arenaviruses based on a modified and hyperattenuated strain of the nonpathogenic Mopeia virus and showed that the Lassa virus candidate fully protected nonhuman primates from a lethal challenge. These results showed that a rationally designed recombinant MOPV-based vaccine is safe, immunogenic, and efficacious in nonhuman primates.

Structural insights into reptarenavirus cap-snatching machinery.

Cap-snatching was first discovered in influenza virus. Structures of the involved domains of the influenza virus polymerase, namely the endonuclease in the PA subunit and the cap-binding domain in the PB2 subunit, have been solved. Cap-snatching endonucleases have also been demonstrated at the very N-terminus of the L proteins of mammarena-, orthobunya-, and hantaviruses. However, a cap-binding domain has not been identified in an arena- or bunyavirus L protein so far. We solved the structure of the 326 C-terminal residues of the L protein of California Academy of Sciences virus (CASV), a reptarenavirus, by X-ray crystallography. The individual domains of this 37-kDa fragment (L-Cterm) as well as the domain arrangement are structurally similar to the cap-binding and adjacent domains of influenza virus polymerase PB2 subunit, despite the absence of sequence homology, suggesting a common evolutionary origin. This enabled identification of a region in CASV L-Cterm with similarity to a cap-binding site; however, the typical sandwich of two aromatic residues was missing. Consistent with this, cap-binding to CASV L-Cterm could not be detected biochemically. In addition, we solved the crystal structure of the corresponding endonuclease in the N-terminus of CASV L protein. It shows a typical endonuclease fold with an active site configuration that is essentially identical to that of known mammarenavirus endonuclease structures. In conclusion, we provide evidence for a presumably functional cap-snatching endonuclease in the N-terminus and a degenerate cap-binding domain in the C-terminus of a reptarenavirus L protein. Implications of these findings for the cap-snatching mechanism in arenaviruses are discussed.

Taxonomy of the order Bunyavirales: second update 2018.

In October 2018, the order Bunyavirales was amended by inclusion of the family Arenaviridae, abolishment of three families, creation of three new families, 19 new genera, and 14 new species, and renaming of three genera and 22 species. This article presents the updated taxonomy of the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Aporé virus, a novel mammarenavirus (Bunyavirales: Arenaviridae) related to highly pathogenic virus from South America.

Here, we report the complete genome sequence of the Aporé virus (Bunyavirales: Arenaviridae), obtained from a wild rodent Oligoryzomys mattogrossae captured in Mato Grosso do Sul state, Brazil. The genome of this virus showed strong similarity to highly pathogenic mammarenavirus from South America.

Taxonomy of the family Arenaviridae and the order Bunyavirales: update 2018.

In 2018, the family Arenaviridae was expanded by inclusion of 1 new genus and 5 novel species. At the same time, the recently established order Bunyavirales was expanded by 3 species. This article presents the updated taxonomy of the family Arenaviridae and the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.

Arenavirus Coinfections Are Common in Snakes with Boid Inclusion Body Disease.

Recently, novel arenaviruses were found in snakes with boid inclusion body disease (BIBD); these form the new genus Reptarenavirus within the family Arenaviridae. We used next-generation sequencing and de novo sequence assembly to investigate reptarenavirus isolates from our previous study. Four of the six isolates and all of the samples from snakes with BIBD contained at least two reptarenavirus species. The viruses sequenced comprise four novel reptarenavirus species and a representative of a new arenavirus genus.