Novel members of the order Picornavirales identified in freshwater from Guarapiranga reservoir in São Paulo.

The global challenge of water resource availability is exacerbated by anthropogenic influences that promote the emergence of pollutants. Among these pollutants are microbiological agents, including viruses, which are ubiquitous in the biosphere and play a pivotal role in both ecological balance and the occurrence of diseases in animals and plants. Consequently, monitoring viruses in water sources becomes indispensable for the establishment of effective prevention, promotion, and control strategies. Within this context, the study focuses on the identification of novel viruses belonging to the Picornavirales order in freshwater from the Guarapiranga Reservoir in the state of São Paulo, Brazil. The samples were subjected to viral metagenomics. Our analysis led to the characterization of four distinct sequences (GinkV-05, AquaV_10, MarV_14, and MarV_64), which exhibited significant divergence compared to other members of the Picornavirales order. This remarkable diversity prompted the identification of a potential new genus within the Marnaviridae family, tentatively named Ginkgonavirus. Additionally, we characterized four sequences in a very distinct clade and propose the recognition of a novel family (named Aquaviridae) within the Picornavirales order. Our findings contribute valuable insights into the previously uncharted diversity of Picornavirales present in water sources, shedding light on an important facet of viral ecology and evolution in aquatic environments.

Extensive Diversity of Viruses in Millipedes Collected in the Dong Nai Biosphere Reserve (Vietnam).

Advances in sequencing technologies and bioinformatics have led to breakthroughs in the study of virus biodiversity. Millipedes (Diplopoda, Myriapoda, Arthropoda) include more than 12,000 extant species, yet data on virus diversity in Diplopoda are scarce. This study aimed to explore the virome of the millipedes collected in the Dong Nai Biosphere Reserve in Vietnam. We studied 14 species of millipedes and managed to assemble and annotate the complete coding genomes of 16 novel viruses, the partial coding genomes of 10 more viruses, and several fragmented viral sequences, which may indicate the presence of about 54 more viruses in the studied samples. Among the complete and partial genomes, 27% were putative members of the order Picornavirales. Most of the discovered viruses were very distant from the viruses currently present in the relevant databases. At least eight viruses meet the criteria to be recognized as a new species by the International Committee on Taxonomy of Viruses, and, for two of them, a higher taxonomic status (genus and even family) can be suggested.

Three picorna-like viruses found associated with the spider mite, Tetranychus truncatus (Acari: Tetranychidae).

Herbivorous arthropods, such as mites and insects, host a variety of microorganisms that significantly influence their ecology and evolution. While insect viruses have been extensively studied, our understanding of the diversity and composition of mite viromes and the interactions with mite hosts remains limited. The Asian spider mite, Tetranychus truncatus Ehara (Acari: Tetranychidae), a major agricultural pest, has not yet been reported to harbor any viruses. Here, using publicly available RNA-Seq data, we identified and characterized three picorna-like viruses associated with T. truncatus: Tetranychus truncatus-associated iflavirus 1 (TtAIV-1), Tetranychus truncatus-associated picorna-like virus 1 (TtAV-1), and Tetranychus truncatus-associated picorna-like virus 2 (TtAV-2). TtAIV-1 has a typical Iflaviridae genome structure with a single ORF, representing the first iflavirus associated with the Tetranychus genus. TtAV-1 and TtAV-2 exhibit bicistronic arrangements similar to dicistroviruses and other picorna-like viruses, with complex secondary structures in their non-coding regions. Phylogenetic analysis places TtAIV-1 within Iflaviridae, possibly as a new species, while TtAV-1 and TtAV-2 form distinct clades within unclassified picorna-like viruses, suggesting new families within Picornavirales. We analyzed in silico the presence and abundance of these viruses in T. truncatus across four bioproject SRAs, mostly finding them co-associated, with viral reads reaching up to 30% of total reads. Their presence and abundance varied by mite treatment and origin, with no significant impact from Wolbachia infection or abamectin exposure, although TtAV-2 was absent in abamectin-treated mites. Temperature influenced virus abundance, and variations were observed among Chinese mite populations based on geography and host plant association. Our findings offer insights into picorna-like virus diversity and dynamics in T. truncatus, revealing potential roles in mite biology and suggesting applications for mite population control, thereby enhancing agricultural productivity and food security.

Diversity of Picorna-Like Viruses in the Teltow Canal, Berlin, Germany.

The viromes of freshwater bodies are underexplored. The Picornavirales order, with 371 acknowledged species, is one of the most expansive and diverse groups of eukaryotic RNA viruses. In this study, we add 513 picorna-like viruses to the assemblage of more than 2000 unassigned picorna-like viruses. Our set of the aquatic Picornavirales virome of the Teltow Canal in Berlin, Germany, consists of 239 complete and 274 partial genomes. This urban freshwater body is characterized by the predominance of marna-like viruses (30.8%) and dicistro-like viruses (19.1%), whereas picornaviruses, iflaviruses, solinvi-like viruses, polycipi-like viruses, and nora-like viruses are considerably less prevalent. Caliciviruses and secoviruses were absent in our sample. Although presenting characteristic domains of Picornavirales, more than 100 viruses (20.8%) could not be assigned to any of the 9 Picornavirales families. Thirty-three viruses of the Marnaviridae-mostly locarna-like viruses-exhibit a monocistronic genome layout. Besides a wealth of novel virus sequences, viruses with peculiar features are reported. Among these is a clade of untypeable marna-like viruses with dicistronic genomes, but with the capsid protein-encoding open reading frame located at the 5' part of their RNA. A virus with a similar genome layout but clustering with dicistroviruses was also observed. We further detected monocistronic viruses with a polymerase gene related to aparaviruses. The detection of Aichi virus and five novel posa-like viruses indicates a slight burden in municipal wastewater.

Insect-specific RNA virus affects the stylet penetration activity of brown citrus aphid (Aphis citricidus) to facilitate its transmission.

Sap-sucking insects often transmit plant viruses but also carry insect viruses, which infect insects but not plants. The impact of such insect viruses on insect host biology and ecology is largely unknown. Here, we identified a novel insect-specific virus carried by brown citrus aphid (Aphis citricidus), which we tentatively named Aphis citricidus picornavirus (AcPV). Phylogenetic analysis discovered a monophyletic cluster with AcPV and other unassigned viruses, suggesting that these viruses represent a new family in order Picornavirales. Systemic infection with AcPV triggered aphid antiviral immunity mediated by RNA interference, resulting in asymptomatic tolerance. Importantly, we found that AcPV was transmitted horizontally by secretion of the salivary gland into the feeding sites of plants. AcPV influenced aphid stylet behavior during feeding and increased the time required for intercellular penetration, thus promoting its transmission among aphids with plants as an intermediate site. The gene expression results suggested that this mechanism was linked with transcription of salivary protein genes and plant defense hormone signaling. Together, our results show that the horizontal transmission of AcPV in brown citrus aphids evolved in a manner similar to that of the circulative transmission of plant viruses by insect vectors, thus providing a new ecological perspective on the activity of insect-specific viruses found in aphids and improving the understanding of insect virus ecology.

Viromes of Tabanids from Russia.

Advances in sequencing technologies and bioinformatics have greatly enhanced our knowledge of virus biodiversity. Currently, the viromes of hematophagous invertebrates, such as mosquitoes and ixodid ticks, are being actively studied. Tabanidae (Diptera) are a widespread family, with members mostly known for their persistent hematophagous behavior. They transmit viral, bacterial, and other pathogens, both biologically and mechanically. However, tabanid viromes remain severely understudied. In this study, we used high-throughput sequencing to describe the viromes of several species in the Hybomitra, Tabanus, Chrysops, and Haematopota genera, which were collected in two distant parts of Russia: the Primorye Territory and Ryazan Region. We assembled fourteen full coding genomes of novel viruses, four partial coding genomes, as well as several fragmented viral sequences, which presumably belong to another twelve new viruses. All the discovered viruses were tested for their ability to replicate in mammalian porcine embryo kidney (PEK), tick HAE/CTVM8, and mosquito C6/36 cell lines. In total, 16 viruses were detected in at least one cell culture after three passages (for PEK and C6/36) or 3 weeks of persistence in HAE/CTVM8. However, in the majority of cases, qPCR showed a decline in virus load over time.

RNA Viruses Are Prevalent and Active Tenants of the Predatory Mite Phytoseiulus persimilis (Acari: Phytoseiidae).

Many arthropod species harbor a diverse range of viruses. While much is known about pathogenic viruses of some economically important insects and arthropods involved in disease transmission, viruses associated with mites have rarely been studied. The main objective of this study was to characterize the virome of Phytoseiulus persimilis (Phytoseiidae), a predatory mite commercially used worldwide for the biological control of the key pest Tetranychus urticae (Tetranichidae). A combination of de novo transcriptome assembly and virion sequencing, revealed that RNA viruses are highly prevalent and active tenants of commercial populations of P. persimilis, comprising on average 9% of the mite's total mRNA. Seventeen RNA viruses dominated the mite's virome (i.e., were highly transcribed) with over half (n = 10) belonging to the order Picornavirales, + ssRNA viruses that infect a large range of hosts, including arthropods. Screening of the 17 dominant virus sequences in P. persimilis and T. urticae revealed that three viruses (two Picornavirales of the families Iflaviridae and Dicistroviridae, and one unclassified Riboviria) are unique to P. persimilis and three others (two unclassified Picornavirales and one unclassified Riboviria) are present in both mite species. Most of the sequences were related to viruses previously documented in economically important arthropods, while others have rarely been documented before in arthropods. These findings demonstrate that P. persimilis, like many other arthropods, harbors a diverse RNA virome, which might affect the mite's physiology and consequently its efficiency as a biological control agent.

Black queen cell virus detected in Canadian mosquitoes.

Black queen cell virus (BQCV) is a ubiquitous honeybee virus and a significant pathogen to queen bee (Apis mellifera) larvae. However, many aspects of the virus remain poorly understood, including the transmission dynamics. In this study, we used next-generation sequencing to identify BQCV in Aedes vexans (n = 4,000) collected in 2019 and 2020 from Manitoba, Canada. We assembled de novo the nearly complete (>96%) genome sequence of the virus, which is the first available from North America and the first report of BQCV being harbored by mosquitoes. Phylogenetic tree reconstructions indicated that the genome had 95.5% sequence similarity to a BQCV isolate from Sweden. Sequences of a potential vector (Varroa destructor) and a microsporidian associated with BQCV (Nosema apis) were not identified in the mosquito samples, however, we did detect sequences of plant origin. We, therefore, hypothesize that the virus was indirectly acquired by mosquitoes foraging at the same nectar sources as honeybees.

Genome of a husavirus from Southern Brazil

ABSTRACT New viruses of the Picornavirales order have been discovered with the increase in the number of sequences obtained by high-throughput sequencing, as well as human stool-associated RNA virus (husavirus [HuV]), found in human stool samples. However, there is much to be clarified about HuV. Its cellular host, evolutionary history, and other biological characteristics are still unknown. Therefore, samples collected from human beings and environmental samples in a watershed in Southern Brazil were processed for the metagenomic library. Upon metagenomic analysis, we identified a HuV (husavirus LMM_67754 OP019707) genome with 8,846 bp, which was reported for the first time in Southern Brazil. The new genome presents only 37% of nucleotide identity with Brazilian strains and more than 90% with genomes from China, Vietnam, Venezuela, and the Netherlands. The HuV phylogeny presents significant differences among genomes, probably because multiple introductions of the virus may have occurred. Many questions still need to be answered about HuV. Therefore, more sequences and studies on this virus are necessary to improve the comprehension of the unknown origin of Picornavirales.

Virome Analysis of Normal and Growth Retardation Disease-Affected Macrobrachium rosenbergii.

The giant freshwater prawn, Macrobrachium rosenbergii, is an important aquaculture species in China. Growth retardation disease (GRD) is a common contagious disease in M. rosenbergii, resulting in slow growth and precocious puberty in prawns, and has caused growing economic losses in the M. rosenbergii industry. To investigate the viral diversity of M. rosenbergii and identify potentially high-risk viruses linked to GRD, virome analysis of the GRD-affected and normal M. rosenbergii was carried out using next-generation sequencing (NGS). A total of 327 contigs (>500 bp) were related to viral sequences belonging to 23 families/orders and a group of unclassified viruses. The majority of the viral contigs in M. rosenbergii belonged to the order Picornavirales, with the Solinviviridae family being the most abundant in both the diseased and normal groups. Furthermore, 16 RNA viral sequences with nearly complete genomes were characterized and phylogenetically analyzed, belonging to the families Solinviviridae, Flaviviridae, Polycipiviridae, Marnaviridae, and Dicistroviridae as well as three new clades of the order Picornavirales. Notably, the cross-species transmission of a picorna-like virus was observed between M. rosenbergii and plants. The "core virome" seemed to be present in the diseased and normal prawns. Still, a clear difference in viral abundance was observed between the two groups. These results showed that the broad diversity of viruses is present in M. rosenbergii and that the association between viruses and disease of M. rosenbergii needs to be further investigated. IMPORTANCE Growth retardation disease (GRD) has seriously affected the development and economic growth of the M. rosenbergii aquaculture industry. Our virome analysis showed that diverse viral sequences were present in M. rosenbergii, significantly expanding our knowledge of viral diversity in M. rosenbergii. Some differences in viral composition were noted between the diseased and normal prawns, indicating that some viruses become more abundant in occurrences or outbreaks of diseases. In the future, more research will be needed to determine which viruses pose a risk for M. rosenbergii. Our study provides important baseline information contributing to disease surveillance and risk assessment in M. rosenbergii aquaculture.

Structure of Human Enterovirus 70 and Its Inhibition by Capsid-Binding Compounds.

Enterovirus 70 (EV70) is a human pathogen belonging to the family Picornaviridae. EV70 is transmitted by eye secretions and causes acute hemorrhagic conjunctivitis, a serious eye disease. Despite the severity of the disease caused by EV70, its structure is unknown. Here, we present the structures of the EV70 virion, altered particle, and empty capsid determined by cryo-electron microscopy. The capsid of EV70 is composed of the subunits VP1, VP2, VP3, and VP4. The partially collapsed hydrophobic pocket located in VP1 of the EV70 virion is not occupied by a pocket factor, which is commonly present in other enteroviruses. Nevertheless, we show that the pocket can be targeted by the antiviral compounds WIN51711 and pleconaril, which block virus infection. The inhibitors prevent genome release by stabilizing EV70 particles. Knowledge of the structures of complexes of EV70 with inhibitors will enable the development of capsid-binding therapeutics against this virus. IMPORTANCE Globally distributed enterovirus 70 (EV70) causes local outbreaks of acute hemorrhagic conjunctivitis. The discharge from infected eyes enables the high-efficiency transmission of EV70 in overcrowded areas with low hygienic standards. Currently, only symptomatic treatments are available. We determined the structures of EV70 in its native form, the genome release intermediate, and the empty capsid resulting from genome release. Furthermore, we elucidated the structures of EV70 in complex with two inhibitors that block virus infection, and we describe the mechanism of their binding to the virus capsid. These results enable the development of therapeutics against EV70.

Honeybee Iflaviruses Pack Specific tRNA Fragments from Host Cells in Their Virions.

The Picornavirales include viruses that infect vertebrates, insects, and plants. It was believed that they pack only their genomic mRNA in the particles; thus, we envisaged these viruses as excellent model systems for studies of mRNA modifications. We used LC-MS to analyze digested RNA isolated from particles of the sacbrood and deformed wing iflaviruses as well as of the echovirus 18 and rhinovirus 2 picornaviruses. Whereas in the picornavirus RNAs we detected only N6 -methyladenosine and 2'-O-methylated nucleosides, the iflavirus RNAs contained a wide range of methylated nucleosides, such as 1-methyladenosine (m1 A) and 5-methylcytidine (m5 C). Mapping of m1 A and m5 C through RNA sequencing of the SBV and DWV RNAs revealed the presence of tRNA molecules. Both modifications were detected only in tRNA. Further analysis revealed that tRNAs are present in form of 3' and 5' fragments and they are packed selectively. Moreover, these tRNAs are typically packed by other viruses.

Field evaluation of Solenopsis invicta virus 3 against its host Solenopsis invicta.

Viruses have been used successfully as biocontrol agents against several insect pests but not ants. Laboratory tests have shown that Solenopsis invicta virus 3 (SINV-3) may be an effective natural control agent against its host, the red imported fire ant (Solenopsis invicta Buren). In this field trial, SINV-3 was released into 12 active S. invicta nests within a 0.088-hectare area in Florida and the impact on the ants monitored. SINV-3 was successfully transmitted, established, and multiplied within treated colonies reaching a maximum mean value of 8.71 × 108 ± 8.26 × 108 SINV-3 genome equivalents/worker ant 77 days after inoculation. SINV-3 was not detected in any of the nests in the control group. A 7-fold decrease in nests was observed in the SINV-3-treated group compared with the untreated control. A correspondingly significant decrease in S. invicta nest size also was observed over the course of the evaluation. Based on the nest rating scale, nest size among those treated with SINV-3 decreased from 3.92 ± 1.24 on day 0 to 1.67 ± 2.06 on day 77, which represents a 57.4% decrease in size. Conversely, the nest rating for the control group increased 9.3%, from 4.42 ± 1.24 on day 0 to 4.83 ± 2.12 on day 77. A follow-up survey of SINV-3-treated and -untreated plots conducted 9 months after initial treatment revealed that fire ant populations rebounded, but at a different rate. A total of 11 and 19 nests were detected in the SINV-3-treated and -untreated areas, respectively. SINV-3 was still detected in the treated area 1.8 years after the initial virus treatment and the virus had spread into the adjacent control plot. Results demonstrate that SINV-3 is an effective natural control agent against the invasive ant, S. invicta; the virus causes no known detrimental ecological impacts, is host specific, and sustained in the environment.

The Virome of Healthy Honey Bee Colonies: Ubiquitous Occurrence of Known and New Viruses in Bee Populations.

Honey bees are globally important pollinators threatened by many different pathogens, including viruses. We investigated the virome of honey bees collected at the end of the beekeeping season (August/September) in Czechia, a Central European country. Samples were examined in biological replicates to assess the homogeneity, stability, and composition of the virome inside a single hive. By choice of healthy workers from colonies, where Varroa destructor was under control, we could identify ubiquitous bee viruses. Deformed wing virus (DWV) was highly prevalent, even though the bees were healthy, without any noticeable disease signs. The overall virome composition (consisting of honey bee-, plant-, and bacterium-infecting viruses) was driven primarily by the hive and its location. However, honey bee-specific viruses showed an uneven distribution within the same hive. In addition, our results point to an unusual cooccurrence between two rhabdoviruses and reveal the presence of five distinct lineages of Lake Sinai viruses (LSVs) clustering with other LSV strains described globally. Comparison of our results with the virome of Australian honey bees, the last truly Varroa- and DWV-free population, showed a strong difference with respect to DWV and a set of diverse members of the Picornavirales, of which the latter were absent in our samples. We hypothesize that the occurrence of DWV introduced by Varroa strongly affects the virome structure despite the mite being under control. IMPORTANCE The Western honey bee, Apis mellifera, is a vital part of our ecosystem as well as cultural heritage. Annual colony losses endanger beekeeping. In this study, we examined healthy bees from the heart of Central Europe, where honey bee colonies have been commonly affected by varroosis over 5 decades. Our virome analysis showed the presence of ubiquitous viruses in colonies where the mite Varroa destructor was under control and no honey bee disease signs were observed. Compared to previous studies, an important part of our study was the analysis of multiple replicates from individual hives. Our overall results indicate that the virome structure (including bee-infecting viruses, plant-infecting viruses, and bacteriophages) is stable within hives; however, the bee-infecting viruses varied largely within interhive replicates, suggesting variation of honey bee viruses within individual bees. Of interest was the striking difference between the viromes of our 39 pools and 9 pools of honey bee viromes previously analyzed in Australia. It could be suggested that Varroa not only affects DWV spread in bee colonies but also affects diverse members of the Picornavirales, which were strongly decreased in Czech bees compared to the Varroa- and DWV-naive Australian bees.

Picorna-Like Viruses of the Havel River, Germany.

To improve the understanding of the virome diversity of riverine ecosystems in metropolitan areas, a metagenome analysis was performed with water collected in June 2018 from the river Havel in Berlin, Germany. After enrichment of virus particles and RNA extraction, paired-end Illumina sequencing was conducted and assignment to virus groups and families was performed. This paper focuses on picorna-like viruses, the most diverse and abundant group of viruses with impact on human, animal, and environmental health. Here, we describe altogether 166 viral sequences ranging in size from 1 to 11.5 kb. The 71 almost complete genomes are comprised of one candidate iflavirus, one picornavirus, two polycipiviruses, 27 marnaviruses, 27 dicistro-like viruses, and 13 untypeable viruses. Many partial picorna-like virus sequences up to 10.2 kb were also investigated. The sequences of the Havel picorna-like viruses represent genomes of seven of eight so far known Picornavirales families. Detection of numerous distantly related dicistroviruses suggests the existence of additional, yet unexplored virus groups with dicistronic genomes, including few viruses with unusual genome layout. Of special interest is a clade of dicistronic viruses with capsid protein-encoding sequences at the 5'-end of the genome. Also, monocistronic viruses with similarity of their polymerase and capsid proteins to those of dicistroviruses are interesting. A second protein with NTP-binding site present in the polyprotein of solinviviruses and related viruses needs further attention. The results underline the importance to study the viromes of fluvial ecosystems. So far acknowledged marnaviruses have been isolated from marine organisms. However, the present study and available sequence data suggest that rivers and limnic habitats are relevant ecosystems with circulation of marnaviruses as well as a plethora of unknown picorna-like viruses.

Putative Invertebrate, Plant, and Wastewater Derived ssRNA Viruses in Plankton of the Anthropogenically Impacted Anacostia River, District of Columbia, USA.

The Anacostia River is a highly impacted watershed in the Northeastern United States which experiences combined sewage outfall in downstream waters. We examined the composition of RNA viruses at three sites in the river using viral metagenomics. Viromes had well represented Picornaviruses, Tombusviruses, Wolframviruses, Nodaviruses, with fewer Tobamoviruses, Sobemoviruses, and Densoviruses (ssDNA). Phylogenetic ana-lyses of detected viruses provide evidence for putatively autochthonous and allochthonous invertebrate, plant, and vertebrate host origin. The number of viral genomes matching Ribovaria increased downstream, and assemblages were most disparate between distant sites, suggesting impacts of the combined sewage overflows at these sites. Additionally, we recovered a densovirus genome fragment which was highly similar to the Clinch ambidensovirus 1, which has been attributed to mass mortality of freshwater mussels in Northeastern America. Taken together, these data suggest that RNA viromes of the Anacostia River reflect autochthonous production of virus particles by benthic metazoan and plants, and inputs from terrestrial habitats including sewage.

Virome in Fecal Samples From Wild Giant Pandas (Ailuropoda Melanoleuca).

The giant panda (Ailuropoda melanoleuca) is one of the most endangered mammals in the world; anthropogenic habitat loss and poaching still threaten the survival of wild pandas. Viral infection has become one of the potential threats to the health of these animals, but the available information related to these infections is still limited. In order to detect possible vertebrate viruses, the virome in the fecal samples of seven wild giant pandas from Qinling Mountains was investigated by using the method of viral metagenomics. From the fecal virome of wild giant pandas, we determined six nearly complete genomes belonging to the order Picornavirales, two of which may be qualified as a novel virus family or genus. In addition, four complete genomes belonging to the Genomoviridae family were also fully characterized. This virological investigation has increased our understanding of the gut viral community in giant pandas. Whether these viruses detected in fecal samples can really infect giant panda needs further research.

A Novel Picornavirus Discovered in White Leg Shrimp Penaeus vannamei.

Global shrimp farming is increasingly threatened by various emerging viruses. In the present study, a novel picornavirus, Penaeus vannamei picornavirus (PvPV), was discovered in moribund White leg shrimp (Penaeus vannamei) collected from farm ponds in China in 2015. Similar to most picornaviruses, PvPV is non-enveloped RNA virus, with a particle diameter of approximately 30 nm. The sequence of the positive single-stranded RNA genome with a length of 10,550 nts was characterized by using genome sequencing and reverse transcription PCR. The existence of PvPV related proteins was further proved by confirmation of viral amino acid sequences, using mass spectrometry analysis. Phylogenetic analysis based on the full-length genomic sequence revealed that PvPV was more closely related to the Wenzhou shrimp virus 8 than to any other dicistroviruses in the order Picornavirales. Genomic sequence conservative domain prediction analysis showed that the PvPV genome encoded a large tegument protein UL36, which was unique among the known dicistroviruses and different from other dicistroviruses. According to these molecular features, we proposed that PvPV is a new species in the family Dicistroviridae. This study reported the first whole-genome sequence of a novel and distinct picornavirus in crustaceans, PvPV, and suggests that further studies of PvPV would be helpful in understanding its evolution and potential pathogenicity, as well as in developing diagnostic techniques.

Monsavirus in monkey rectal swab and throat swab specimens in China: Proposal for Posaliviridae as a new family in Picornavirales.

Posa-like viruses have been detected in the fecal samples of several host species and are considered unclassified members of Picornavirales. Here, we identified genomic fragments of novel posa-like viruses (monsaviruses) in monkey specimens through next generation sequencing and obtained 11 full-length genomes. This monsavirus shared 88.5-89.2% nucleotide similarity with the Tottori-HG1 strain (GenBank accession LC123275). In total, 713 nucleotide polymorphism sites were identified, indicating their persistent evolution during circulation. The genomic organization and phylogenetic relationship of monsavirus were determined. Subsequent phylogenetic analysis of the conserved replication block of Hel-Pro-RdRp and core RNA-dependent RNA polymerase domain-based analysis of posa-like viruses showed significant separation compared with other known families. Further, posa-like virus genomes possessed the classical replication block of picornavirus in the 5' part of genome and picorna-like capsid domains at the structural coding region of 3' part of genome. Based on these results, we proposed the new family Posaliviridae, within Picornavirales. Four genera, which showed 68.6-75.5% amino acid distances but similar genomic organization including the conserved replication block of Hel-Pro-RdRp, the same order of the genomic coding region, and picorna-like capsid domains, were identified. The flexible genomic organization strategy and a large evolutionary scale of Posaliviridae was explicit. This study provides novel information on monsaviruses and important taxonomic data for the family Posaliviridae.

Diverse RNA Viruses Discovered in Three Parasitoid Wasps of the Rice Weevil Sitophilus oryzae.

In this study, many virus-like fragments were obtained from transcriptomes of three wasp species, including Anisopteromalus calandrae (8), Lariophagus distinguendus (3), and Theocolax elegans (18), which can parasitize and control rice weevil Sitophilus oryzae, a serious insect pest of farm-stored grains. By further bioinformatic analysis and sequencing, we identified six novel RNA viruses with complete genomes and named them WWPSRV-1, WWPSRV-2, AcPSRV-1, AcNSRV-1, AcNSRV-2, and LdNSRV-1. PCR-based detection revealed that WWPSRV-1 and WWPSRV-2 had the possibility of interspecies virus transmission, especially WWPSRV-2, which was also present in the rice weevil adults. Phylogenetically, three out of these six viruses appeared to be members of order Picornavirales: WWPSRV-1 belonged to unassigned virus families of this order, whereas WWPSRV-2 and AcPSRV-1 belonged to families Iflaviridae and Dicistroviridae, respectively. The conserved picornavirus-typical domains helicase, protease, and RNA-dependent RNA polymerase could be found in the nonstructural protein encoded by the three viruses, whose genomes consisted of the different numbers of open reading frames (ORFs). The other three RNA viruses could be classified to order Mononegavirales: AcNSRV-1 and AcNSRV-2 belonged to family Lispiviridae, whereas LdNSRV-1 belonged to a big family Rhabdoviridae The genomes of the three viruses contained at least five ORFs, encoding deduced proteins in the following order: 3'-N-P-M-G-L-5'. All the ORFs were separated by conserved intergenic sequences which likely regulated the transcription termination and initiation. Our findings enhance the understanding of RNA viruses in weevil wasps and set the foundation for the future study of the association among weevils, weevil wasps, and RNA viruses.IMPORTANCE The enormous diversity of RNA viruses in insects is continuously validated. Parasitoid wasps, as biocontrol insects which are widely used against insect pests in agroecosystems, may also carry many "good" RNA viruses. Some RNA viruses in parasitoid wasps have been reported to affect the host wasps or the wasps' host. Here, six novel RNA viruses with complete genomes were identified in three parasitoid wasps of the rice weevil. One of these viruses was also detected in the rice weevil adults. Phylogenetically, WWPSRV-1 was the first unambiguous detection of Nora-like virus in insect parasitoids. WWPSRV-2 and AcPSRV-1 belong to families Iflaviridae and Dicistroviridae, some viruses of which can result in lethal infections in silkworms and honeybees. The other three RNA viruses belong to order Mononegavirales, which comprises many well-known insect-associated viruses.