Aedes aegypti Totivirus identified in mosquitoes in the Brazilian Amazon region.

The totiviridae family contains viruses with double-stranded RNA genomes of 4.6-7.0 kpb, which encode a capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), and they are approximately 40 nm in diameter with icosahedral symmetry. Totiviruses were first isolated from mosquitoes collected in Shaanxi Province (China). Here, we report a new Aedes aegypti Totivirus (AaTV) identified in mosquitoes from the Amazon rainforest. Mosquitoes (Diptera: Culicidae) were collected from a forest reserve belonging to the Amazon forest in the city of Macapá, Amapá state, Northern Brazil. A viral sequence with a 5748 nucleotide length that was nearly identical to Aedes aegypti Totivirus (AaTV), here named Aedes aegypti Totivirus BR59AP, was detected. A detailed molecular analysis was performed and shows that AaTV-BR59AP is highly related to the AaTV strain from the Caribbean region. We emphasize the importance of the characterization of new viruses in mosquitoes to deepen our understanding of viral diversity in insects and their potential role in disease.

A Simple Multiplex Reverse Transcription-PCR Method for the Diagnosis of L-A and M Totiviruses in Saccharomyces cerevisiae.

Killer yeasts and their toxins have many potential applications in environmental, medical, and industrial biotechnology. The killer phenotype in Saccharomyces cerevisiae relies on the cytoplasmic persistence of two dsRNA viruses, L-A and M. M encodes the toxin, and L-A provides proteins for expression, replication, and capsids for both viruses. Yeast screening and characterization of this trait are usually performed phenotypically based on their toxin production and immunity. In this study, we describe a simple and specific reverse transcription (RT) multiplex PCR assay for direct diagnosis of the dsRNA totivirus genomes associated with the killer trait in the S. cerevisiae yeast. This method obviates RNA purification steps and primer addition to the RT reaction. Using a mixture of specific primers at the PCR step, this multiplex RT-PCR protocol provided an accurate diagnosis of both L-A and M totivirus in all its known variants, L-A-1/M1, L-A-2/M2, L-A-28/M28, and L-A-lus/Mlus, found in infected killer yeasts. Using this method, the expected L-A-2/M2 totivirus associations in natural wine yeasts cells were identified but, importantly, asymptomatic L-A-2/M2 infected cells were found in addition to unexpected L-A-lus/M2 totiviral associations. IMPORTANCE The killer phenomenon in S. cerevisiae yeast cells provides the opportunity to study host-virus interactions in a eukaryotic model. Therefore, the development of simple methods for their detection significantly facilitates their study. The simplified multiplex RT-PCR protocol described here provides a useful and accurate tool for the genotypic characterization of yeast totiviruses in killer yeast cells. The killer trait depended on two dsRNA totiviruses, L-A and M. Each M dsRNA depends on a specific helper L-A virus. Thus, direct genotyping by the described method also provided valuable insights into L-A/M viral associations and their coadaptational events in nature.

The Diversity of Viral Community in Invasive Fruit Flies (Bactrocera and Zeugodacus) Revealed by Meta-transcriptomics.

RNA viruses are extremely diverse and rapidly evolving in various organisms. Our knowledge on viral evolution with interacted hosts in the manner of ecology is still limited. In the agricultural ecosystem, invasive insect species are posing a great threat to sustainable crop production. Among them, fruit flies (Diptera: Tephritidae Bactrocera and Zeugodacus) are destructive to fruits and vegetables, which are also closely related and often share similar ecological niches. Thus, they are ideal models for investigating RNA virome dynamics in host species. Using meta-transcriptomics, we found 39 viral sequences in samples from 12 fly species. These viral species represented the diversity of the viromes including Dicistroviridae, negev-like virus clades, Thika virus clades, Solemoviridae, Narnaviridae, Nodaviridae, Iflaviridae, Orthomyxoviridae, Bunyavirales, Partitiviridae, and Reoviridae. In particular, dicistrovirus, negev-like virus, orthomyxovirus, and orbivirus were common in over four of the fly species, which suggests a positive interaction between fly viromes that exist under the same ecological conditions. For most of the viruses, the virus-derived small RNAs displayed significantly high peaks in 21 nt and were symmetrically distributed throughout the viral genome. These results suggest that infection by these viruses can activate the host's RNAi immunity. Our study provides RNA virome diversity and evidence on their infection activity in ecologically associated invasive fruit fly species, which could help our understanding of interactions between complex species and viruses.

Identification and genetic analysis of a totivirus isolated from the Culex tritaeniorhynchus in northern China.

Totiviridae, a viral family of double-stranded RNA (dsRNA) viruses, contain a single dsRNA genome 4.6-7.0 kb in length. Totiviridae were initially only known to infect fungi and other eukaryotes as well as plants, but an increase in totiviruses has been detected in insects, mosquitoes, and bats. Here, we describe the isolation and characterization of a strain belonging to the family Totiviridae isolated from Culex tritaeniorhynchus in Kenli, China, in 2016. We isolated a totivirus from field-collected mosquitoes in China by cell culture in Aedes albopictus C6/36 cells, identified the virus by morphological observation and complete genome sequencing, and characterized it by phylogenetic analysis. Transmission electron microscopy identified icosahedral, non-enveloped virus particles with a mean diameter of 35-40 nm. The genome was 7612 bp in length, including two open reading frames (ORFs). ORF1 (5058 nt) encodes the capsid protein, while ORF2 (2216 nt) encodes the viral RNA-dependent RNA polymerase (RdRp). Nucleotide and amino acid homology analysis of isolate showed higher levels of sequence identity with isolate CTV_NJ2 (China, 2010) with 94.87% nucleic acid identity and 97.32% amino acid identity. The isolate was designated C. tritaeniorhynchus totivirus KL (CTV-KL). This is the first identification of a totivirus in a C. tritaeniorhynchus in northern China. Analysis of the virus's morphology, characteristic and genome organization will further enrich our understanding of the molecular and biological characteristics of dsRNA Totiviridae viruses.

Concentration of 2'C-methyladenosine triphosphate by Leishmania guyanensis enables specific inhibition of Leishmania RNA virus 1 via its RNA polymerase.

Leishmania is a widespread trypanosomatid protozoan parasite causing significant morbidity and mortality in humans. The endobiont dsRNA virus Leishmania RNA virus 1 (LRV1) chronically infects some strains, where it increases parasite numbers and virulence in murine leishmaniasis models, and correlates with increased treatment failure in human disease. Previously, we reported that 2'-C-methyladenosine (2CMA) potently inhibited LRV1 in Leishmania guyanensis (Lgy) and Leishmania braziliensis, leading to viral eradication at concentrations above 10 µm Here we probed the cellular mechanisms of 2CMA inhibition, involving metabolism, accumulation, and inhibition of the viral RNA-dependent RNA polymerase (RDRP). Activation to 2CMA triphosphate (2CMA-TP) was required, as 2CMA showed no inhibition of RDRP activity from virions purified on cesium chloride gradients. In contrast, 2CMA-TP showed IC50 values ranging from 150 to 910 µm, depending on the CsCl density of the virion (empty, ssRNA-, and dsRNA-containing). Lgy parasites incubated in vitro with 10 µm 2CMA accumulated 2CMA-TP to 410 µm, greater than the most sensitive RDRP IC50 measured. Quantitative modeling showed good agreement between the degree of LRV1 RDRP inhibition and LRV1 levels. These results establish that 2CMA activity is due to its conversion to 2CMA-TP, which accumulates to levels that inhibit RDRP and cause LRV1 loss. This attests to the impact of the Leishmania purine uptake and metabolism pathways, which allow even a weak RDRP inhibitor to effectively eradicate LRV1 at micromolar concentrations. Future RDRP inhibitors with increased potency may have potential therapeutic applications for ameliorating the increased Leishmania pathogenicity conferred by LRV1.

Diversity and distribution of Maize-associated totivirus strains from Tanzania.

Typically associated with fungal species, members of the viral family Totiviridae have recently been shown to be associated with plants, including important crop species, such as Carica papaya (papaya) and Zea mays (maize). Maize-associated totivirus (MATV) was first described in China and more recently in Ecuador, where it has been found to co-occur with other viruses known to elicit maize lethal necrosis disease (MLND). In a survey for maize-associated viruses, 35 samples were selected for Illumina HiSeq sequencing, from the Tanzanian maize producing regions of Mara, Arusha, Manyara, Kilimanjaro, Morogoro and Pwani. Libraries were prepared using an RNA-tag-seq methodology. Taxonomic classification of the resulting datasets showed that 6 of the 35 samples from the regions of Arusha, Kilimanjaro, Morogoro and Mara, contained reads that were assigned to MATV reference sequences. This was confirmed with PCR and Sanger sequencing. Read assembly of the six MATV-associated datasets yielded partial MATV genomes, two of which were selected for further characterization, using RACE. This yielded two full-length MATV genomes, one of which is divergent from other available MATV genomes.

The frenemies within: viruses, retrotransposons and plasmids that naturally infect Saccharomyces yeasts.

Viruses are a major focus of current research efforts because of their detrimental impact on humanity and their ubiquity within the environment. Bacteriophages have long been used to study host-virus interactions within microbes, but it is often forgotten that the single-celled eukaryote Saccharomyces cerevisiae and related species are infected with double-stranded RNA viruses, single-stranded RNA viruses, LTR-retrotransposons and double-stranded DNA plasmids. These intracellular nucleic acid elements have some similarities to higher eukaryotic viruses, i.e. yeast retrotransposons have an analogous lifecycle to retroviruses, the particle structure of yeast totiviruses resembles the capsid of reoviruses and segregation of yeast plasmids is analogous to segregation strategies used by viral episomes. The powerful experimental tools available to study the genetics, cell biology and evolution of S. cerevisiae are well suited to further our understanding of how cellular processes are hijacked by eukaryotic viruses, retrotransposons and plasmids. This article has been written to briefly introduce viruses, retrotransposons and plasmids that infect Saccharomyces yeasts, emphasize some important cellular proteins and machineries with which they interact, and suggest the evolutionary consequences of these interactions. Copyright © 2017 John Wiley & Sons, Ltd.

Association of the Endobiont Double-Stranded RNA Virus LRV1 With Treatment Failure for Human Leishmaniasis Caused by Leishmania braziliensis in Peru and Bolivia.

Cutaneous and mucosal leishmaniasis, caused in South America by Leishmania braziliensis, is difficult to cure by chemotherapy (primarily pentavalent antimonials [Sb(V)]). Treatment failure does not correlate well with resistance in vitro, and the factors responsible for treatment failure in patients are not well understood. Many isolates of L. braziliensis (>25%) contain a double-stranded RNA virus named Leishmaniavirus 1 (LRV1), which has also been reported in Leishmania guyanensis, for which an association with increased pathology, metastasis, and parasite replication was found in murine models. Here we probed the relationship of LRV1 to drug treatment success and disease in 97 L. braziliensis-infected patients from Peru and Bolivia. In vitro cultures were established, parasites were typed as L. braziliensis, and the presence of LRV1 was determined by reverse transcription-polymerase chain reaction, followed by sequence analysis. LRV1 was associated significantly with an increased risk of treatment failure (odds ratio, 3.99; P = .04). There was no significant association with intrinsic Sb(V) resistance among parasites, suggesting that treatment failure arises from LRV1-mediated effects on host metabolism and/or parasite survival. The association of LRV1 with clinical drug treatment failure could serve to guide more-effective treatment of tegumentary disease caused by L. braziliensis.

Mutant swarms of a totivirus-like entities are present in the red macroalga Chondrus crispus and have been partially transferred to the nuclear genome.

Chondrus crispus Stackhouse (Gigartinales) is a red seaweed found on North Atlantic rocky shores. Electrophoresis of RNA extracts showed a prominent band with a size of around 6,000 bp. Sequencing of the band revealed several sequences with similarity to totiviruses, double-stranded RNA viruses that normally infect fungi. This virus-like entity was named C. crispus virus (CcV). It should probably be regarded as an extreme viral quasispecies or a mutant swarm since low identity (<65%) was found between sequences. Totiviruses typically code for two genes: one capsid gene (gag) and one RNA-dependent RNA polymerase gene (pol) with a pseudoknot structure between the genes. Both the genes and the intergenic structures were found in the CcV sequences. A nonidentical gag gene was also found in the nuclear genome of C. crispus, with associated expressed sequence tags (EST) and upstream regulatory features. The gene was presumably horizontally transferred from the virus to the alga. Similar dsRNA bands were seen in extracts from different life cycle stages of C. crispus and from all geographic locations tested. In addition, similar bands were also observed in RNA extractions from other red algae; however, the significance of this apparently widespread phenomenon is unknown. Neither phenotype caused by the infection nor any virus particles or capsid proteins were identified; thus, the presence of viral particles has not been validated. These findings increase the known host range of totiviruses to include marine red algae.

Identification and Genome Characterization of a Novel Virus within the Genus Totivirus from Chinese Bayberry (Myrica rubra).

Chinese bayberry (Myrica rubra) is an economically significant fruit tree native to eastern Asia and widely planted in south-central China. However, studies about the viruses infecting M. rubra remain largely lacking. In the present study, we employed the metatranscriptomic method to identify viruses in M. rubra leaves exhibiting yellowing and irregular margin symptoms collected in Fuzhou, a city located in China's Fujian province in the year 2022. As a consequence, a novel member of the genus Totivirus was identified and tentatively named "Myrica rubra associated totivirus 1" (MRaTV1). The genome sequencing of MRaTV1 was determined by overlapping reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The two deduced proteins encoded by MRaTV1 have the highest amino acid (aa) sequence identity to the coat protein (CP) and RNA-dependent RNA polymerase (RdRP) of Panax notoginseng virus A (PNVA), a member of the genus Totivirus within the family Totiviridae, at 49.7% and 61.7%, respectively. According to the results of the phylogenetic tree and the species demarcation criteria of the International Committee on Taxonomy of Viruses (ICTV) for the genus Totivirus, MRaTV1 is considered a new member of the genus Totivirus.

Metagenomic of Liver Tissue Identified at Least Two Genera of Totivirus-like Viruses in Molossus molossus Bats.

The Totiviridae family of viruses has a unique genome consisting of double-stranded RNA with two open reading frames that encode the capsid protein (Cap) and the RNA-dependent RNA polymerase (RdRpol). Most virions in this family are isometric in shape, approximately 40 nm in diameter, and lack an envelope. There are five genera within this family, including Totivirus, Victorivirus, Giardiavirus, Leishmaniavirus, and Trichomonasvirus. While Totivirus and Victorivirus primarily infect fungi, Giardiavirus, Leishmaniavirus, and Trichomonasvirus infect diverse hosts, including protists, insects, and vertebrates. Recently, new totivirus-like species have been discovered in fish and plant hosts, and through metagenomic analysis, a novel totivirus-like virus (named Tianjin totivirus) has been isolated from bat guano. Interestingly, Tianjin totivirus causes cytopathic effects in insect cells but cannot grow in mammalian cells, suggesting that it infects insects consumed by insectivorous bats. In this study, we used next-generation sequencing and identified totivirus-like viruses in liver tissue from Molossus molossus bats in the Amazon region of Brazil. Comparative phylogenetic analysis based on the RNA-dependent RNA polymerase region revealed that the viruses identified in Molossus bats belong to two distinct phylogenetic clades, possibly comprising different genera within the Totiviridae family. Notably, the mean similarity between the Tianjin totivirus and the totiviruses identified in Molossus bats is less than 18%. These findings suggest that the diversity of totiviruses in bats is more extensive than previously recognized and highlight the potential for bats to serve as reservoirs for novel toti-like viruses.

Tracking Mycoviruses in Public RNAseq Datasets of Malassezia: Three Original Totiviruses Revealed.

Mycoviruses are viruses that selectively infect and multiply in fungal cells. Malassezia is the most abundant fungus on human skin and is associated with a variety of conditions, including atopic eczema, atopic dermatitis, dandruff, folliculitis, pityriasis versicolor, and seborrheic dermatitis. Here, we conducted mycovirome studies on 194 public transcriptomes of Malassezia (2,568,212,042 paired-end reads) screened against all available viral proteins. Transcriptomic data were assembled de novo resulting in 1,170,715 contigs and 2,995,306 open reading frames (ORFs) that were subsequently tracked for potential viral sequences. Eighty-eight virus-associated ORFs were detected in 68 contigs from 28 Sequence Read Archive (SRA) samples. Seventy-five and thirteen ORFs were retrieved from transcriptomes of Malassezia globosa and Malassezia restricta, respectively. Phylogenetic reconstructions revealed three new mycoviruses belonging to the Totivirus genus and named Malassezia globosa-associated-totivirus 1 (MgaTV1); Malassezia restricta-associated-totivirus 1 (MraTV1) and Malassezia restricta-associated-totivirus 2 (MraTV2). These viral candidates extend our understanding of the diversity and taxonomy of mycoviruses as well as their co-evolution with their fungal hosts. These results reflected the unexpected diversity of mycoviruses hidden in public databases. In conclusion, this study sheds light on the discovery of novel mycoviruses and opens the door to study their impact on disease caused by the host fungus Malassezia and globally, their implication in clinical skin disorders.

Molecular Characterization of Two Totiviruses from the Commensal Yeast Geotrichum candidum.

Mycoviruses can infect many of the major taxa of fungi including yeasts. Mycoviruses in the yeast fungus Geotrichum candidum are not well studied with only three G. candidum-associated viral species characterized to date, all of which belong to the Totiviridae genus Totivirus. In this study, we report the molecular characteristics of another two totiviruses co-infecting isolate Gc6 of G. candidum. The two totiviruses were tentatively named Geotrichum candidum totivirus 2 isolate Gc6 (GcTV2-Gc6) and Geotrichum candidum totivirus 4 isolate Gc6 (GcTV4-Gc6). Both viruses have the typical genome organization of totiviruses comprising two ORFs encoding capsid protein (CP) and RNA-dependent RNA polymerase (RdRp) at the N and C termini, respectively. The genomes of GcTV2-Gc6 and GcTV4-Gc6 are 4592 and 4530 bp long, respectively. Both viruses contain the-frameshifting elements and their proteins could be expressed as a single fusion protein. GcTV2-Gc6 is closely related to a totivirus isolated from the same host whereas GcTV4-Gc6 is related to insect-associated totiviruses. The phylogenetic analysis indicated that GcTV2-Gc6 and GcTV4-Gc6 belong to two different sister clades, I-A and I-B, respectively. It is interesting that all viruses identified from G. candidum belong to the genus Totivirus; however, this might be due to the lack of research reporting the characterization of mycoviruses from this fungal host. It is possible that the RNA interference (RNAi) mechanism cannot actively suppress totivirus accumulation in G. candidum Gc6.

The virome of the invasive Asian bush mosquito Aedes japonicus in Europe.

The Asian bush mosquito Aedes japonicus is rapidly invading North America and Europe. Due to its potential to transmit multiple pathogenic arthropod-borne (arbo)viruses including Zika virus, West Nile virus, and chikungunya virus, it is important to understand the biology of this vector mosquito in more detail. In addition to arboviruses, mosquitoes can also carry insect-specific viruses that are receiving increasing attention due to their potential effects on host physiology and arbovirus transmission. In this study, we characterized the collection of viruses, referred to as the virome, circulating in Ae. japonicus populations in the Netherlands and France. Applying a small RNA-based metagenomic approach to Ae. japonicus, we uncovered a distinct group of viruses present in samples from both the Netherlands and France. These included one known virus, Ae. japonicus narnavirus 1 (AejapNV1), and three new virus species that we named Ae. japonicus totivirus 1 (AejapTV1), Ae. japonicus anphevirus 1 (AejapAV1) and Ae. japonicus bunyavirus 1 (AejapBV1). We also discovered sequences that were presumably derived from two additional novel viruses: Ae. japonicus bunyavirus 2 (AejapBV2) and Ae. japonicus rhabdovirus 1 (AejapRV1). All six viruses induced strong RNA interference responses, including the production of twenty-one nucleotide-sized small interfering RNAs, a signature of active replication in the host. Notably, AejapBV1 and AejapBV2 belong to different viral families; however, no RNA-dependent RNA polymerase sequence has been found for AejapBV2. Intriguingly, our small RNA-based approach identified an ∼1-kb long ambigrammatic RNA that is associated with AejapNV1 as a secondary segment but showed no similarity to any sequence in public databases. We confirmed the presence of AejapNV1 primary and secondary segments, AejapTV1, AejapAV1, and AejapBV1 by reverse transcriptase polymerase chain reaction (PCR) in wild-caught Ae. japonicus mosquitoes. AejapNV1 and AejapTV1 were found at high prevalence (87-100 per cent) in adult females, adult males, and larvae. Using a small RNA-based, sequence-independent metagenomic strategy, we uncovered a conserved and prevalent virome among Ae. japonicus mosquito populations. The high prevalence of AejapNV1 and AejapTV1 across all tested mosquito life stages suggests that these viruses are intimately associated with Ae. japonicus.

A full-length infectious cDNA clone of a dsRNA totivirus-like virus.

Totivirus-like viruses are a group of non-segmented double-stranded (ds)RNA viruses with two open reading frames, which were recently discovered and provisionally assigned to the Totiviridae family. Unlike yeast and protozoan Totiviridae viruses, these totivirus-like viruses infect a diverse spectrum of metazoan hosts and currently have enormous impacts on fisheries and agriculture. We developed the first infectious full-length cDNA clone of a totivirus-like virus, the Omono River virus (OmRV), and produced infectious particles using an RNA-transcript-based method. Compared with the parent wild-type particles from nature, the infectious-cloning OmRV particles have presented strong cytopathic effects, infectivity and similar morphology. Thus far, the established system is one of the few reported systems for generating a non-segmented dsRNA virus cDNA clone.

The Characterization of a Novel Virus Discovered in the Yeast Pichia membranifaciens.

Mycoviruses are widely distributed across fungi, including the yeasts of the Saccharomycotina subphylum. This manuscript reports the first double-stranded RNA (dsRNA) virus isolated from Pichia membranifaciens. This novel virus has been named Pichia membranifaciens virus L-A (PmV-L-A) and is a member of the Totiviridae. PmV-L-A is 4579 bp in length, with RNA secondary structures similar to the packaging, replication, and frameshift signals of totiviruses that infect Saccharomycotina yeasts. PmV-L-A was found to be part of a monophyletic group within the I-A totiviruses, implying a shared ancestry between mycoviruses isolated from the Pichiaceae and Saccharomycetaceae yeasts. Energy-minimized AlphaFold2 molecular models of the PmV-L-A Gag protein revealed structural conservation with the Gag protein of Saccharomyces cerevisiae virus L-A (ScV-L-A). The predicted tertiary structure of the PmV-L-A Pol and other homologs provided a possible mechanism for totivirus RNA replication due to structural similarities with the RNA-dependent RNA polymerases of mammalian dsRNA viruses. Insights into the structure, function, and evolution of totiviruses gained from yeasts are essential because of their emerging role in animal disease and their parallels with mammalian viruses.

Expanding the Medfly Virome: Viral Diversity, Prevalence, and sRNA Profiling in Mass-Reared and Field-Derived Medflies.

The Mediterranean fruit fly (medfly), Ceratitis capitata, is an agricultural pest of a wide range of fruits. The advent of high-throughput sequencing has boosted the discovery of RNA viruses infecting insects. In this article, we aim to characterize the RNA virome and viral sRNA profile of medfly. By means of transcriptome mining, we expanded the medfly RNA virome to 13 viruses, including two novel positive ssRNA viruses and the first two novel dsRNA viruses reported for medfly. Our analysis across multiple laboratory-reared and field-collected medfly samples showed the presence of a core RNA virome comprised of Ceratitis capitata iflavirus 2 and Ceratitis capitata negev-like virus 1. Furthermore, field-collected flies showed a higher viral diversity in comparison to the laboratory-reared flies. Based on the small RNA sequencing, we detected small interfering RNAs mapping to all the viruses present in each sample, except for Ceratitis capitata nora virus. Although the identified RNA viruses do not cause obvious symptoms in medflies, the outcome of their interaction may still influence the medfly's fitness and ecology, becoming either a risk or an opportunity for mass-rearing and SIT applications.

Is the Intergenic Region of Aedes aegypti Totivirus a Recombination Hotspot?

The genus totivirus in the family Totiviridae contains double-stranded RNA viruses. Their genome has two open reading frames (ORFs) that encode capsid protein (CP) and RNA-dependent RNA polymerase (RdRp). The toti-like viruses recently identified in Anopheles sp. and Aedes aegypti mosquitoes (AaTV) share the same genome organization as other totiviruses. The AaTVs that have been described in distinct geographical regions are monophyletic. In this study, we show that AaTV sequences can be grouped into at least three phylogenetic clades (named A, B, and C). Clades A and B are composed of AaTV sequences from mosquitoes collected in the Caribbean region (Guadeloupe), and clade C contains sequences from the USA. These clades may represent AaTV lineages that are locally adapted to their host populations. We also identified three recombinant AaTV strains circulating in mosquitoes in Guadeloupe. Although these strains have different chimeric patterns, the position of the recombination breakpoint was identical in all strains. Interestingly, this breakpoint is located in a hairpin-like structure in the intergenic region of the AaTV genome. This RNA structure may stall RNA polymerase processivity and consequently induce template switching. In vitro studies should be conducted to further investigate the biological significance of AaTV's intergenic region as a recombination hotspot.

Characterization of Two Novel Toti-Like Viruses Co-infecting the Atlantic Blue Crab, Callinectes sapidus, in Its Northern Range of the United States.

The advancement of high throughput sequencing has greatly facilitated the exploration of viruses that infect marine hosts. For example, a number of putative virus genomes belonging to the Totiviridae family have been described in crustacean hosts. However, there has been no characterization of the most newly discovered putative viruses beyond description of their genomes. In this study, two novel double-stranded RNA (dsRNA) virus genomes were discovered in the Atlantic blue crab (Callinectes sapidus) and further investigated. Sequencing of both virus genomes revealed that they each encode RNA dependent RNA polymerase proteins (RdRps) with similarities to toti-like viruses. The viruses were tentatively named Callinectes sapidus toti-like virus 1 (CsTLV1) and Callinectes sapidus toti-like virus 2 (CsTLV2). Both genomes have typical elements required for -1 ribosomal frameshifting, which may induce the expression of an encoded ORF1-ORF2 (gag-pol) fusion protein. Phylogenetic analyses of CsTLV1 and CsTLV2 RdRp amino acid sequences suggested that they are members of two new genera in the family Totiviridae. The CsTLV1 and CsTLV2 genomes were detected in muscle, gill, and hepatopancreas of blue crabs by real-time reverse transcription quantitative PCR (RT-qPCR). The presence of ~40 nm totivirus-like viral particles in all three tissues was verified by transmission electron microscopy, and pathology associated with CsTLV1 and CsTLV2 infections were observed by histology. PCR assays showed the prevalence and geographic range of these viruses, to be restricted to the northeast United States sites sampled. The two virus genomes co-occurred in almost all cases, with the CsTLV2 genome being found on its own in 8.5% cases, and the CsTLV1 genome not yet found on its own. To our knowledge, this is the first report of toti-like viruses in C. sapidus. The information reported here provides the knowledge and tools to investigate transmission and potential pathogenicity of these viruses.

Characterization of Four Novel dsRNA Viruses Isolated from Mucor hiemalis Strains.

We previously screened the total nucleic acid extracts of 123 Mucor strains for the presence of dsRNA molecules without further molecular analyses. Here, we characterized five novel dsRNA genomes isolated from four different Mucor hiemalis strains with next-generation sequencing (NGS), namely Mucor hiemalis virus 1a (MhV1a) from WRL CN(M) 122; Mucor hiemalis virus 1b (MhV1b) from NRRL 3624; Mucor hiemalis virus 2 (MhV2) from NRRL 3616; and Mucor hiemalis virus 3 (MhV3) and Mucor hiemalis virus (MhV4) from NRRL 3617 strains. Genomes contain two open reading frames (ORF), which encode the coat protein (CP) and the RNA dependent RNA polymerase (RdRp), respectively. In MhV1a and MhV1b, it is predicted to be translated as a fusion protein via -1 ribosomal frameshift, while in MhV4 via a rare +1 (or-2) ribosomal frameshift. In MhV2 and MhV3, the presence of specific UAAUG pentanucleotide motif points to the fact for coupled translation termination and reinitialization. MhV1a, MhV2, and MhV3 are part of the clade representing the genus Victorivirus, while MhV4 is seated in Totivirus genus clade. The detected VLPs in Mucor strains were from 33 to 36 nm in diameter. Hybridization analysis revealed that the dsRNA molecules of MhV1a-MhV4 hybridized to the corresponding molecules.