Search

Emerging zoonoses of wildlife origin caused by previously unknown agents are one of the most important challenges for human health. The Qinghai-Tibet Plateau represents a unique ecological niche with diverse wildlife that harbours several human pathogens and numerous previously uncharacterized pathogens. In this study, we identified and characterized a novel arenavirus (namely, plateau pika virus, PPV) from plateau pikas ( Ochotona curzoniae ) on the Qinghai-Tibet Plateau by virome analysis. Isolated PPV strains could replicate in several mammalian cells. We further investigated PPV pathogenesis using animal models. PPV administered via an intraventricular route caused trembling and sudden death in IFNαβR -/- mice, and pathological inflammatory lesions in brain tissue were observed. According to a retrospective serological survey in the geographical region where PPV was isolated, PPV-specific IgG antibodies were detected in 8 (2.4%) of 335 outpatients with available sera. Phylogenetic analyses revealed that this virus was clearly separated from previously reported New and Old World mammarenaviruses. Under the co-speciation framework, the estimated divergence time of PPV was 77-88 million years ago (MYA), earlier than that of OW and NW mammarenaviruses (26-34 MYA).

Jingchuvirales is an order of negative-sense RNA viruses with genomes of 9.1-15.3 kb that have been associated with arachnids, barnacles, crustaceans, insects, fish and reptiles in Africa, Asia, Australia, Europe, North America and South America. The jingchuviral genome has two to four open reading frames (ORFs) that encode a glycoprotein (GP), a nucleoprotein (NP), a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain, and/or proteins of unknown function. Viruses in the order are only known from their genome sequences. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the order Jingchuvirales and on the families Aliusviridae , Chuviridae , Crepuscuviridae , Myriaviridae and Natareviridae , which are available at ictv.global/report/jingchuvirales, ictv.global/report/aliusviridae, ictv.global/report/chuviridae, ictv.global/report/crepuscuviridae, ictv.global/report/myriaviridae and ictv.global/report/natareviridae, respectively.

Since 2018, the outbreaks of genotype II African swine fever virus (ASFV) in China and several eastern Asian countries have caused a huge impact on the local swine industry, resulting in huge economic losses. However, little is known about the origin, genomic diversity, evolutionary features, and epidemiological history of the genotype II ASFV. Here, 14 high-quality complete genomes of ASFVs were generated via sequencing of samples collected from China over the course of 3 years, followed by phylogenetic and phylodynamic analyses. The strains identified were relatively homogeneous, with a total of 52 SNPs and 11 indels compared with the prototype strain HLJ/2018, among which there were four exceptionally large deletions (620-18,023 nt). Evolutionary analyses revealed that ASFV strains distributed in eastern Asia formed a monophyly and a 'star-like' structure centered around the prototype strain, suggesting a single origin. Additionally, phylogenetic network analysis and ancestral reconstruction of geographic state indicated that genotype II ASFV strains in eastern Asia likely originated from Western Europe. Overall, these results contribute to the understanding of the history and current status of genotype II ASFV strains in eastern Asian, which could be of considerable importance in disease control and prevention., Competing Interests: None declared., (© The Author(s) 2023. Published by Oxford University Press.)

Despite their widespread distribution and remarkable antiquity no RNA viruses definitively associated with the domain Archaea have been identified. In contrast, 17 families of DNA viruses are known to infect archaea. In an attempt to uncover more of the elusive archaeal virosphere, we investigated the metatranscriptomes of hypersaline lakes that are a rich source of archaea. We sequenced RNA extracted from water filter samples of Lake Tyrrell (Victoria, Australia) and cultures seeded from four lakes in Antarctica. To identify highly divergent viruses in these data, we employed a variety of search tools, including Hidden Markov models (HMMs) and position-specific scoring matrices (PSSMs). From this, we identified 12 highly divergent, RNA virus-like candidate sequences from the virus phyla Artverviricota , Duplornaviricota , Kitrinoviricota, Negarnaviricota , and Pisuviricota , including those with similarity to the RNA-dependent RNA polymerase (RdRp). An additional analysis with an artificial intelligence (AI)-based approach that utilises both sequence and structural information identified seven putative and highly divergent RdRp sequences of uncertain phylogenetic position. A sequence matching the Pisuviricota from Deep Lake in Antarctica had the strongest RNA virus signal. Analyses of the dinucleotide representation of the virus-like candidates in comparison to that of potential host species were in some cases compatible with an association to archaeal or bacterial hosts. Notably, however, the use of archaeal CRISPR spacers as a BLAST database failed to detect any RNA viruses. We also described DNA viruses from the families Pleolipoviridae , Sphaerolipoviridae , Halspiviridae , and the class Caudoviricetes . Although we were unable to provide definitive evidence the existence of an RNA virus of archaea in these hypersaline lakes, this study lays the foundations for further investigations of highly divergent RNA viruses in natural environments., Competing Interests: None declared., (© The Author(s) 2023. Published by Oxford University Press.)

Nelson Bay reovirus (NBV) is an emerging zoonotic virus that can cause acute respiratory disease in humans. These viruses are mainly discovered in Oceania, Africa, and Asia, and bats have been identified as their main animal reservoir. However, despite recent expansion of diversity for NBVs, the transmission dynamics and evolutionary history of NBVs are still unclear. This study successfully isolated two NBV strains (MLBC1302 and MLBC1313) from blood-sucking bat fly specimens ( Eucampsipoda sundaica ) and one (WDBP1716) from the spleen specimen of a fruit bat (Rousettus leschenaultii), which were collected at the China-Myanmar border area of Yunnan Province. Syncytia cytopathic effects (CPE) were observed in BHK-21 and Vero E6 cells infected with the three strains at 48 h postinfection. Electron micrographs of ultrathin sections showed numerous spherical virions with a diameter of approximately 70 nm in the cytoplasm of infected cells. The complete genome nucleotide sequence of the viruses was determined by metatranscriptomic sequencing of infected cells. Phylogenetic analysis demonstrated that the novel strains were closely related to Cangyuan orthoreovirus, Melaka orthoreovirus, and human-infecting Pteropine orthoreovirus HK23629/07. Simplot analysis revealed the strains originated from complex genomic reassortment among different NBVs, suggesting the viruses experienced a high reassortment rate. In addition, strains successfully isolated from bat flies also implied that blood-sucking arthropods might serve as potential transmission vectors. IMPORTANCE Bats are the reservoir of many viral pathogens with strong pathogenicity, including NBVs. Nevertheless, it is unclear whether arthropod vectors are involved in transmitting NBVs. In this study, we successfully isolated two NBV strains from bat flies collected from the body surface of bats, which implies that they may be vectors for virus transmission between bats. While the potential threat to humans remains to be determined, evolutionary analyses involving different segments revealed that the novel strains had complex reassortment histories, with S1, S2, and M1 segments highly similar to human pathogens. Further experiments are required to determine whether more NBVs are vectored by bat flies, their potential threat to humans, and transmission dynamics., Competing Interests: The authors declare no conflict of interest.

Continually emerging SARS-CoV-2 variants of concern that can evade immune defenses are driving recurrent epidemic waves of COVID-19 globally. However, the impact of measures to contain the virus and their effect on lineage diversity dynamics are poorly understood. Here, we jointly analyzed international travel, public health and social measures (PHSM), COVID-19 vaccine rollout, SARS-CoV-2 lineage diversity, and the case growth rate (GR) from March 2020 to September 2022 across 63 countries. We showed that despite worldwide vaccine rollout, PHSM are effective in mitigating epidemic waves and lineage diversity. An increase of 10,000 monthly travelers in a single country-to-country route between endemic countries corresponds to a 5.5% (95% CI: 2.9 to 8.2%) rise in local lineage diversity. After accounting for PHSM, natural immunity from previous infections, and waning immunity, we discovered a negative association between the GR of cases and adjusted vaccine coverage (AVC). We also observed a complex relationship between lineage diversity and vaccine rollout. Specifically, we found a significant negative association between lineage diversity and AVC at both low and high levels but not significant at the medium level. Our study deepens the understanding of population immunity and lineage dynamics for future pandemic preparedness and responsiveness.

Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within individual bats, and hence the frequency of virus co-infection and spillover among them. We characterize the mammal-associated viruses in 149 individual bats sampled from Yunnan province, China, using an unbiased meta-transcriptomics approach. This reveals a high frequency of virus co-infection (simultaneous infection of bat individuals by multiple viral species) and spillover among the animals studied, which may in turn facilitate virus recombination and reassortment. Of note, we identify five viral species that are likely to be pathogenic to humans or livestock, based on phylogenetic relatedness to known pathogens or in vitro receptor binding assays. This includes a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV and SARS-CoV-2. In vitro assays indicate that this recombinant virus can utilize the human ACE2 receptor such that it is likely to be of increased emergence risk. Our study highlights the common occurrence of co-infection and spillover of bat viruses and their implications for virus emergence., (© 2023. The Author(s).)

SARS-CoV Spike (S) protein shares considerable homology with SARS-CoV-2 S, especially in the conserved S2 subunit (S2). S protein mediates coronavirus receptor binding and membrane fusion, and the latter activity can greatly influence coronavirus infection. We observed that SARS-CoV S is less effective in inducing membrane fusion compared with SARS-CoV-2 S. We identify that S813T mutation is sufficient in S2 interfering with the cleavage of SARS-CoV-2 S by TMPRSS2, reducing spike fusogenicity and pseudoparticle entry. Conversely, the mutation of T813S in SARS-CoV S increased fusion ability and viral replication. Our data suggested that residue 813 in the S was critical for the proteolytic activation, and the change from threonine to serine at 813 position might be an evolutionary feature adopted by SARS-2-related viruses. This finding deepened the understanding of Spike fusogenicity and could provide a new perspective for exploring Sarbecovirus' evolution., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is ongoing and multiple studies have elucidated its pathogenesis, however, the related- microbiome imbalance caused by SARS-CoV-2 is still not clear. In this study, we have comprehensively compared the microbiome composition and associated function alterations in the oropharyngeal swabs of healthy controls and coronavirus disease 2019 (COVID-19) patients with moderate or severe symptoms by metatranscriptomic sequencing. We did observe a reduced microbiome alpha-diversity but significant enrichment of opportunistic microorganisms in patients with COVID-19 compared with healthy controls, and the microbial homeostasis was rebuilt following the recovery of COVID-19 patients. Correspondingly, less functional genes in multiple biological processes and weakened metabolic pathways such as carbohydrate metabolism, energy metabolism were also observed in COVID-19 patients. We only found higher relative abundance of limited genera such as Lachnoanaerobaculum between severe patients and moderate patients while no worthy-noting microbiome diversity and function alteration were observed. Finally, we noticed that the co-occurrence of antibiotic resistance and virulence was closely related to the microbiome alteration caused by SRAS-CoV-2. Overall, our findings demonstrate that microbial dysbiosis may enhance the pathogenesis of SARS-CoV-2 and the antibiotics treatment should be critically considered., (© 2023 Wiley Periodicals LLC.)

Background: Viruses play critical roles in the marine environment because of their interactions with an extremely broad range of potential hosts. Many studies of viruses in seawater have been published, but viruses that inhabit marine animals have been largely neglected. Oysters are keystone species in coastal ecosystems, yet as filter-feeding bivalves with very large roosting numbers and species co-habitation, it is not clear what role they play in marine virus transmission and coastal microbiome regulation., Results: Here, we report a Dataset of Oyster Virome (DOV) that contains 728,784 nonredundant viral operational taxonomic unit contigs (≥ 800 bp) and 3473 high-quality viral genomes, enabling the first comprehensive overview of both DNA and RNA viral communities in the oyster Crassostrea hongkongensis. We discovered tremendous diversity among novel viruses that inhabit this oyster using multiple approaches, including reads recruitment, viral operational taxonomic units, and high-quality virus genomes. Our results show that these viruses are very different from viruses in the oceans or other habitats. In particular, the high diversity of novel circoviruses that we found in the oysters indicates that oysters may be potential hotspots for circoviruses. Notably, the viruses that were enriched in oysters are not random but are well-organized communities that can respond to changes in the health state of the host and the external environment at both compositional and functional levels., Conclusions: In this study, we generated a first "knowledge landscape" of the oyster virome, which has increased the number of known oyster-related viruses by tens of thousands. Our results suggest that oysters provide a unique habitat that is different from that of seawater, and highlight the importance of filter-feeding bivalves for marine virus exploration as well as their essential but still invisible roles in regulating marine ecosystems. Video Abstract., (© 2023. The Author(s).)

Dynamic changes of the paired heavy and light chain B cell receptor (BCR) repertoire provide an essential insight into understanding the humoral immune response post-SARS-CoV-2 infection and vaccination. However, differences between the endogenous paired BCR repertoire kinetics in SARS-CoV-2 infection and previously recovered/naïve subjects treated with the inactivated vaccine remain largely unknown. We performed single-cell V(D)J sequencing of B cells from six healthy donors with three shots of inactivated SARS-CoV-2 vaccine (BBIBP-CorV), five people who received the BBIBP-CorV vaccine after having recovered from COVID-19, five unvaccinated COVID-19 recovered patients and then integrated with public data of B cells from four SARS-CoV-2-infected subjects. We discovered that BCR variable (V) genes were more prominently used in the SARS-CoV-2 exposed groups (both in the group with active infection and in the group that had recovered) than in the vaccinated groups. The VH gene that expanded the most after SARS-CoV-2 infection was IGHV3-33, while IGHV3-23 in the vaccinated groups. SARS-CoV-2-infected group enhanced more BCR clonal expansion and somatic hypermutation than the vaccinated healthy group. A small proportion of public clonotypes were shared between the SARS-CoV-2 infected, vaccinated healthy, and recovered groups. Moreover, several public antibodies had been identified against SARS-CoV-2 spike protein. We comprehensively characterize the paired heavy and light chain BCR repertoire from SARS-CoV-2 infection to vaccination, providing further guidance for the development of the next-generation precision vaccine.

Phlebotomus chinensis sandfly is a neglected insect vector in China that is well-known for carrying Leishmania . Recent studies have expanded its pathogen repertoire with two novel arthropod-borne phleboviruses capable of infecting humans and animals. Despite these discoveries, our knowledge of the general pathogen diversity and overall microbiome composition of this vector species is still very limited. Here we carried out a meta-transcriptomics analysis that revealed the actively replicating/transcribing RNA viruses, DNA viruses, bacteria, and eukaryotic microbes, namely, the "total microbiome", of several sandfly populations in China. Strikingly, "microbiome" made up 1.8% of total non-ribosomal RNA and comprised more than 87 species, among which 70 were novel, including divergent members of the genera Flavivirus and of the family Trypanosomatidae. Importantly, among these microbes we were able to reveal four distinguished types of human and/or mammalian pathogens, including two phleboviruses (hedi and wuxiang viruses), one novel Spotted fever group rickettsia, as well as a member of Leishmania donovani complex, among which hedi virus and Leishmania each had > 50% pool prevalence rate and relatively high abundance levels. Our study also showed the ubiquitous presence of an endosymbiont, namely Wolbachia , although no anti-viral or anti-pathogen effects were detected based on our data. In summary, our results uncovered the much un-explored diversity of microbes harboured by sandflies in China and demonstrated that high pathogen diversity and abundance are currently present in multiple populations, implying disease potential for exposed local human population or domestic animals.

In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Eighteen novel RNA viruses were found in Crassostrea hongkongensis . Phylogenic analysis shows evidence of recombination between major genes of viruses. Picobirnaviruses are ubiquitous and abundant in oysters., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. iMeta published by John Wiley & Sons Australia, Ltd on behalf of iMeta Science.)

Previous studies have identified multiple viruses in dead or severely diseased pangolins, but descriptions of the virome in healthy pangolins are lacking. This poses a greater risk of cross-species transmission due to poor preventive awareness and frequent interactions with breeders. In this study, we investigated the viral composition of 34 pangolins with no signs of disease at the time of sampling and characterized a large number of arthropod-associated viruses belonging to 11 families and vertebrate viruses belonging to eight families, including those with pathogenic potential in humans and animals. Several important vertebrate viruses were identified in the pangolins, including parvovirus, pestivirus, and picobirnavirus. The picobirnavirus was clustered with human and grey teal picobirnaviruses. Viruses with cross-species transmission ability were also identified, including circovirus, rotavirus, and astrovirus. Our study revealed that pangolins are frequently exposed to arthropod-associated viruses in the wild and can carry many vertebrate viruses under natural conditions. This study provides important insights into the virome of pangolins, underscoring the importance of monitoring potential pathogens in healthy pangolins to prevent outbreaks of infectious diseases in domesticated animals and humans.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases affecting the pig-raising industry. The PRRS virus (PRRSV) has high genetic diversity, partly owing to viral recombination. Some individual recombinant type 2 PRRSV (PRRSV-2) strains have been detected; however, the sequence composition characteristics of recombination hot spots and potential driving forces for recombinant PRRSV-2 are still unreported. Therefore, all available genomic sequences of PRRSV-2 ( n  = 949, including 29 genomes sequenced in this study) from 11 countries from 1991 to 2021 were collected and analyzed. The results revealed that the dominant major recombinant parent has been converted from lineage 3 (L3) to L1 since 2012. The recombination hot spots were located at nucleotides (nt) 7900 to 8200 (in NSP9, encoding viral RNA-dependent RNA polymerase) and nt 12500 to nt 13300 (in ORF2-ORF4, mean ORF2 to ORF4); no AU-rich characteristics were found in the recombination hot spots. Based on infectious clones of L1 and L8 PRRSV-2, recombinant PRRSVs were generated by switching complete or partial NSP9 (harboring the recombination hot spot). The results showed that recombinant PRRSVs based on the L1 backbone, but not the L8 backbone, acquired a higher replication capacity in pig primary alveolar macrophages. These findings will help to understand the reason behind the dominance of L1-based recombination in PRRSV-2 strains and provide new clues for an in-depth study of the recombination mechanism of PRRSV-2. IMPORTANCE Recombination is an important driver of the genetic shifts that are tightly linked to the evolution of RNA viruses. Viral recombination contributes substantially to the emergence of new variants, alterations in virulence, and pathogenesis. PRRSV is genetically diverse, partly because of extensive recombination. In this study, we analyzed interlineage recombination based on available genomic sequences of PRRSV-2 from 1991 to 2021. The study revealed the temporal and geographical distribution of recombinant PRRSVs and the recombination hot spot's location and showed that artificially constructed recombinant PRRSVs (harboring a high-frequency region) had more viral genomic copies than their parental virus, indicating that dominant recombination was shaped by a tendency to benefit viral replication. This finding will enrich our understanding of PRRSV recombination and provide new clues for an in-depth study of the recombination mechanism.

RNA viruses (realm: Riboviria), including RNA phages and eukaryote-infecting RNA viruses, are essential components of marine ecosystems. A large number of marine RNA viruses have been discovered in the last two decades because of the rapid development of next-generation sequencing (NGS) technology. Indeed, the combination of NGS and state-of-the-art meta-omics methods (viromics, the study of all viruses in a specific environment) has led to a fundamental understanding of the taxonomy and genetic diversity of RNA viruses in the sea, suggesting the complex ecological roles played by RNA viruses in this complex ecosystem. Furthermore, comparisons of viromes in the context of highly variable marine niches reveal the biogeographic patterns and ecological impact of marine RNA viruses, whose role in global ecology is becoming increasingly clearer. In this review, we summarize the characteristics of the global marine RNA virosphere and outline the taxonomic hierarchy of RNA viruses with a specific focus on their ancient evolutionary history. We also review the development of methodology and the major progress resulting from its applications in RNA viromics. The aim of this review is not only to provide an in-depth understanding of multifaceted aspects of marine RNA viruses, but to offer future perspectives on developing a better methodology for discovery, and exploring the evolutionary origin and major ecological significance of marine RNA virosphere., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. iMeta published by John Wiley & Sons Australia, Ltd on behalf of iMeta Science.)

African swine fever (ASF) outbreak have caused tremendous economic loss to the pig industry in China since its emergence in August 2018. Previous studies revealed that many published sequences are not suitable for detailed analyses due to the lack of data regarding quality parameters and methodology, and outdated annotations. Thus, high-quality genomes of highly pathogenic strains that can be used as references for early Chinese ASF outbreaks are still lacking, and little is known about the features of intra-host variants of ASF virus (ASFV). In this study, a full genome sequencing of clinical samples from the first ASF outbreak in Guangdong in 2018 was performed using MGI (MGI Tech Co., Ltd., Shenzhen, China) and Nanopore sequencing platforms, followed by Sanger sequencing to verify the variations. With 22 sequencing corrections, we obtained a high-quality genome of one of the earliest virulent isolates, GZ201801_2. After proofreading, we improved (add or modify) the annotations of this isolate using the whole genome alignment with Georgia 2007/1. Based on the complete genome sequence, we constructed the methylation profiles of early ASFV strains in China and predicted the potential 5mC and 6mA methylation sites, which are likely involved in metabolism, transcription, and replication. Additionally, the intra-host single nucleotide variant distribution and mutant allele frequency in the clinical samples of early strain were determined for the first time and found a strong preference for A and T substitution mutation, non-synonymous mutations, and mutations that resulted in amino acid substitutions into Lysine. In conclusion, this study provides a high-quality genome sequence, updated genome annotation, methylation profile, and mutation spectrum of early ASFV strains in China, thereby providing a reference basis for further studies on the evolution, transmission, and virulence of ASFV., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xu, Gao, Kuang, Xing, Wang, Cao, Xu, Liu, Huang, Zheng, Gong, Wang, Shi, Zhang and Sun.)

Pangolins are the most trafficked wild animal in the world according to the World Wildlife Fund. The discovery of SARS-CoV-2-related coronaviruses in Malayan pangolins has piqued interest in the viromes of these wild, scaly-skinned mammals. We sequenced the viromes of 161 pangolins that were smuggled into China and assembled 28 vertebrate-associated viruses, 21 of which have not been previously reported in vertebrates. We named 16 members of Hunnivirus, Pestivirus and Copiparvovirus pangolin-associated viruses. We report that the L-protein has been lost from all hunniviruses identified in pangolins. Sequences of four human-associated viruses were detected in pangolin viromes, including respiratory syncytial virus, Orthopneumovirus, Rotavirus A and Mammalian orthoreovirus. The genomic sequences of five mammal-associated and three tick-associated viruses were also present. Notably, a coronavirus related to HKU4-CoV, which was originally found in bats, was identified. The presence of these viruses in smuggled pangolins identifies these mammals as a potential source of emergent pathogenic viruses., (© 2022. The Author(s).)

The family Rhabdoviridae comprises viruses with negative-sense (-) RNA genomes of 10-16 kb. Virions are typically enveloped with bullet-shaped or bacilliform morphology but can also be non-enveloped filaments. Rhabdoviruses infect plants or animals, including mammals, birds, reptiles, amphibians or fish, as well as arthropods, which serve as single hosts or act as biological vectors for transmission to animals or plants. Rhabdoviruses include important pathogens of humans, livestock, fish or agricultural crops. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Rhabdoviridae , which is available at ictv.global/report/rhabdoviridae.

The family Rhabdoviridae contain important human and mammalian pathogens that are vectored by different arthropod species. The ground supernatants of mosquitoes were used to inoculate in BHK-21 and C6/36 cells for virus isolation. Then, the viral complete genome sequence was obtained and used for phylogenetic analysis. In this study, we observed a cytopathic effect (CPE) in mosquito cells (C6/36) and rod-like virion after inoculating a pool of Armigeres subalbatus samples collected in Shanxi Province, China, in 2019 (SX1916). Meta-transcriptomics sequencing revealed the presence of two distinctive rhabdoviruses with similar abundance levels, namely, Shanxi Armigeres subalbatus rhabdovirus (SXARV) and Shanxi Arboretum virus (SXABTV). Despite the fact that the SXARV genome (9590 nt) was much shorter than that of SXABTV (11,480 nt), both belonged to the Almendravirus group within Rhabdoviridae whose genomes encoded five proteins (N, P, M, G, and L) and a small hydrophobin (U1) and the difference in lengths is mainly caused by a substantially shorter N protein encoded by SXARV. On the phylogenetic tree, SXABTV was closely related (90.7% amino acid identity at L protein) with the Arboretum virus isolated from Psorophora albigenu mosquitoes in Peru in 2014, whereas SXARV was distantly related to Rio Chico virus (63.3% amino acid identity), a genetic distance large enough to be defined as a new species within Rhabdoviridae . Collectively, we report a simultaneous isolation of two related rhabdoviruses from Armigeres subalbatus that marked the circulation of almendraviruses in Shanxi, China.

Background: The global pork industry is continuously affected by infectious diseases that can result in large-scale mortality, trade restrictions, and major reductions in production. Nevertheless, the cause of many infectious diseases in pigs remains unclear, largely because commonly used diagnostic tools fail to capture the full diversity of potential pathogens and because pathogen co-infection is common., Results: We used a meta-transcriptomic approach to systematically characterize the pathogens in 136 clinical cases representing different disease syndromes in pigs, as well as in 12 non-diseased controls. This enabled us to simultaneously determine the diversity, abundance, genomic information, and detailed epidemiological history of a wide range of potential pathogens. We identified 34 species of RNA viruses, nine species of DNA viruses, seven species of bacteria, and three species of fungi, including two novel divergent members of the genus Pneumocystis. While most of these pathogens were only apparent in diseased animals or were at higher abundance in diseased animals than in healthy animals, others were present in healthy controls, suggesting opportunistic infections. Importantly, most of the cases examined here were characterized by co-infection with more than two species of viral, bacterial, or fungal pathogens, some with highly correlated occurrence and abundance levels. Examination of clinical signs and necropsy results in the context of relevant pathogens revealed that a multiple-pathogen model was better associated with the data than a single-pathogen model was., Conclusions: Our data demonstrate that most of the pig diseases examined were better explained by the presence of multiple rather than single pathogens and that infection with one pathogen can facilitate infection or increase the prevalence/abundance of another. Consequently, it is generally preferable to consider the cause of a disease based on a panel of co-infecting pathogens rather than on individual infectious agents. Video abstract., (© 2022. The Author(s).)

Pegivirus (family Flaviviridae) is a genus of small enveloped RNA viruses that mainly causes blood infections in various mammals including human. Herein, we carried out an extensive survey of pegiviruses from a wide range of wild animals mainly sampled in the Qinghai-Tibet Plateau of China. Three novel pegiviruses, namely Passer montanus pegivirus, Leucosticte brandti pegivirus and Montifringilla taczanowskii pegivirus, were identified from different wild birds, and one new rodent pegivirus, namely Phaiomys leucurus pegivirus, was identified from Blyth's vole. Interestingly, the pegiviruses of non-mammalian origin discovered in this study substantially broaden the host range of Pegivirus to avian species. Co-evolutionary analysis showed virus-host co-divergence over long evolutionary timescales, and indicated that pegiviruses largely followed a virus-host co-divergence relationship. Overall, this work extends the biodiversity of the Pegivirus genus to those infecting wild birds and hence revises the host range and evolutionary history of genus Pegivirus., (Copyright © 2022 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Game animals are wildlife species traded and consumed as food and are potential reservoirs for SARS-CoV and SARS-CoV-2. We performed a meta-transcriptomic analysis of 1,941 game animals, representing 18 species and five mammalian orders, sampled across China. From this, we identified 102 mammalian-infecting viruses, with 65 described for the first time. Twenty-one viruses were considered as potentially high risk to humans and domestic animals. Civets (Paguma larvata) carried the highest number of potentially high-risk viruses. We inferred the transmission of bat-associated coronavirus from bats to civets, as well as cross-species jumps of coronaviruses from bats to hedgehogs, from birds to porcupines, and from dogs to raccoon dogs. Of note, we identified avian Influenza A virus H9N2 in civets and Asian badgers, with the latter displaying respiratory symptoms, as well as cases of likely human-to-wildlife virus transmission. These data highlight the importance of game animals as potential drivers of disease emergence., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In March 2021, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by four families (Aliusviridae, Crepuscuviridae, Myriaviridae, and Natareviridae), three subfamilies (Alpharhabdovirinae, Betarhabdovirinae, and Gammarhabdovirinae), 42 genera, and 200 species. Thirty-nine species were renamed and/or moved and seven species were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far. Due to the high infectivity and pathogenicity of this virus, all studies on the live virus are strictly confined in the biosafety level 3 (BSL3) laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious. In this study, we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome (BAC) vector with deletion of the spike (S) gene. Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein. Therefore, such a replicon system is not infectious and can be used in ordinary biological laboratories. We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus. By mutational analysis of nsp12 and nsp14, we showed that the RNA polymerase, exonuclease, and cap N7 methyltransferase play essential roles in genome replication and sgRNA production. We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors. Thus, such a one-plasmid system is biosafe and convenient to use, which will benefit both fundamental research and development of antiviral drugs., (© 2021. Wuhan Institute of Virology, CAS.)

SARS-CoV-2, the causative agent for COVID-19, infect human mainly via respiratory tract, which is heavily inhabited by local microbiota. However, the interaction between SARS-CoV-2 and nasopharyngeal microbiota, and the association with metabolome has not been well characterized. Here, metabolomic analysis of blood, urine, and nasopharyngeal swabs from a group of COVID-19 and non-COVID-19 patients, and metagenomic analysis of pharyngeal samples were used to identify the key features of COVID-19. Results showed lactic acid, l-proline, and chlorogenic acid methyl ester (CME) were significantly reduced in the sera of COVID-19 patients compared with non-COVID-19 ones. Nasopharyngeal commensal bacteria including Gemella morbillorum , Gemella haemolysans and Leptotrichia hofstadii were notably depleted in the pharynges of COVID-19 patients, while Prevotella histicola , Streptococcus sanguinis , and Veillonella dispar were relatively increased. The abundance of G. haemolysans and L. hofstadii were significantly positively associated with serum CME, which might be an anti-SARS-CoV-2 bacterial metabolite. This study provides important information to explore the linkage between nasopharyngeal microbiota and disease susceptibility. The findings were based on a very limited number of patients enrolled in this study; a larger size of cohort will be appreciated for further investigation., Competing Interests: The authors declare that they have no conflicts of interest., (© 2021 The Authors.)

Despite the ongoing interest in virus discovery, little is known about the factors that shape communities of viruses within individual hosts. Here, we address how virus communities might be impacted by the age of the hosts they infect, using total RNA sequencing to reveal the RNA viromes of different age groups of Ruddy Turnstones (Arenaria interpres). From oropharyngeal and cloacal swabs we identified 14 viruses likely infecting birds, 11 of which were novel, including members of the Reoviridae, Astroviridae, and Picornaviridae. Strikingly, 12 viruses identified were from juvenile birds sampled in the first year of their life, compared to only two viruses in adult birds. Both viral abundance and alpha diversity were marginally higher in juvenile than adult birds. As well as informing studies of virus ecology, that host age might be associated with viral composition is an important consideration for the future surveillance of novel and emerging viruses., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

B cell response plays a critical role against SARS-CoV-2 infection. However, little is known about the diversity and frequency of the paired SARS-CoV-2 antigen-specific BCR repertoire after SARS-CoV-2 infection. Here, we performed single-cell RNA sequencing and VDJ sequencing using the memory and plasma B cells isolated from five convalescent COVID-19 patients, and analyzed the spectrum and transcriptional heterogeneity of antibody immune responses. Via linking BCR to antigen specificity through sequencing (LIBRA-seq), we identified a distinct activated memory B cell subgroup (CD11c high CD95 high ) had a higher proportion of SARS-CoV-2 antigen-labeled cells compared with memory B cells. Our results revealed the diversity of paired BCR repertoire and the non-stochastic pairing of SARS-CoV-2 antigen-specific immunoglobulin heavy and light chains after SARS-CoV-2 infection. The public antibody clonotypes were shared by distinct convalescent individuals. Moreover, several antibodies isolated by LIBRA-seq showed high binding affinity against SARS-CoV-2 receptor-binding domain (RBD) or nucleoprotein (NP) via ELISA assay. Two RBD-reactive antibodies C14646P3S and C2767P3S isolated by LIBRA-seq exhibited high neutralizing activities against both pseudotyped and authentic SARS-CoV-2 viruses in vitro. Our study provides fundamental insights into B cell response following SARS-CoV-2 infection at the single-cell level.

We isolated 17 viral strains capable of causing cytopathic effects in mammalian cells and death in neonatal mice from sand flies in China. Phylogenetic analysis showed that these strains belonged to the genus Phlebovirus. These findings highlight the need to control this potentially emerging virus to help safeguard public health.

Influenza viruses (FLUV) cause high morbidity and mortality annually in the world and pose a serious threat to the public health. Wuhan, as an important transportation hub in China, has a dense population and suitable climate, which also lays a major hidden danger for the outbreak of influenza. To survey and characterize the seasonal FLUV in Wuhan during 2016-2019, we collected 44,738 throat swabs, among which 15.5% were influenza A (FLUAV) positive, 6.1% influenza B (FLUBV) and 0.3% co-infection. By monitoring FLUV in each month from June 2016 to May 2019, different with the previously seasonality pattern, only a single influenza peak was appeared in winter of 2017-2018 and 2018-2019, respectively. These data indicated that the complex circulation pattern of seasonal influenza in Wuhan. In addition, we found the age group was skewed towards 5-14 years group whose activity were mostly school based, which suggested school may be an important place for influenza outbreaks. Meanwhile, phylogenic analysis revealed that two subtypes (subclade 3C.2a2 and 3C.2a1b) of A(H3N2) were circulating in Wuhan and there was an obvious transition in 2018 because the two subclades were detected simultaneously. Furthermore, by estimating the vaccine effectiveness, we found that the vaccine strain of FLUAV didn't seem to match very well the current epidemic strain, especially A(H3N2). Hence, more accurate prediction of seasonal outbreak is essential for vaccine design. Taken together, our results provided the current information about seasonal FLUV in Wuhan which form the basis for vaccine updating.

Background: Since its discovery, SARS-CoV-2 has been spread throughout China before becoming a global pandemic. In Beijing, family clusters are the main mode of human-human transmission accounting for 57.6% of the total confirmed cases., Method: We present the epidemiological and clinical features of the clusters of three large and one small families., Result: Our results revealed that SARS-CoV-2 is transmitted quickly through contact with index case, and a total of 22/24 infections were observed. Among those infected, 20/22 had mild symptoms and only two had moderate to severe clinical manifestations. Children in the families generally showed milder symptoms. The incubation period varied from 2 to 13 days, and the shedding of virus from the upper respiratory tract lasted from 5 to over 30 days. A prolonged period of virus shedding (>30 days) in upper respiratory tract was observed in 6/24 cases., Conclusion: SARS-CoV-2 is transmitted quickly in the form of family clusters. While the infection rate is high within the cluster, the disease manifestations, latent period, and virus shedding period varied greatly. We therefore recommend rigorously testing contacts even during the no-symptom phase and consider whether viral shedding has ceased before stopping isolation measures for an individual., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Despite its isolation and extreme climate, Antarctica is home to diverse fauna and associated microorganisms. It has been proposed that the most iconic Antarctic animal, the penguin, experiences low pathogen pressure, accounting for their disease susceptibility in foreign environments. There is, however, a limited understanding of virome diversity in Antarctic species, the extent of in situ virus evolution, or how it relates to that in other geographic regions. To assess whether penguins have limited microbial diversity we determined the RNA viromes of three species of penguins and their ticks sampled on the Antarctic peninsula. Using total RNA sequencing we identified 107 viral species, comprising likely penguin associated viruses (n = 13), penguin diet and microbiome associated viruses (n = 82), and tick viruses (n = 8), two of which may have the potential to infect penguins. Notably, the level of virome diversity revealed in penguins is comparable to that seen in Australian waterbirds, including many of the same viral families. These data run counter to the idea that penguins are subject to lower pathogen pressure. The repeated detection of specific viruses in Antarctic penguins also suggests that rather than being simply spill-over hosts, these animals may act as key virus reservoirs.

Mites are notorious for being vectors transmitting infectious pathogens and source of allergens causing allergic conditions in animals and humans. However, despite their huge impact on public health, the virome of mites remains unknown. Here we characterized the virus diversity and abundance of 14 species of medically important mites based on total RNA sequencing data sets generated in this study as well as those deposited in the Sequence Read Archive (SRA) database. A total of 47 genetically distinct viruses were identified and classified into 17 virus families or virus super-groups, and the viral sequences accounted for as much as 29.67% of total non-rRNA transcriptome in one mite library. The most commonly identified viruses are members of Picornavirales , among which we revealed more than one type of viruses that are evolutionarily related to dicistronic viruses but contain a single open reading frame, thus likely representing a recent example of host (i.e., mite)-related parallel evolution from dicistronic to monocistronic genomic form within the family Dicistroviridae To our best knowledge, this is the first time to perform comprehensive and systematic screening of RNA virome in medically important mites including house dust mites (HDM). Overall, the RNA virome identified here provides not only significant insights into the diversity and evolution of RNA viruses in mites, but also a solid knowledge base for studying their roles in human diseases. IMPORTANCE Mites are important group of arthropods that are associated with a variety of human diseases including scrub typhus and asthma. However, it remains unclear whether or not mites carry viruses that might play a role in human infections or allergic disease. In this study, we used a total transcriptomics approach to characterize and compare the complete RNA virome within mites that are relevant to human health and diseases. Specifically, our data revealed a large diversity, a high abundance, and a flexible genomic evolution for these viruses. Although most of the viruses identified here are unknown to associate with human infectious disease, the abundant presence of viral RNAs may play an immunomodulatory role in the development of allergic reactions such as asthma during environmental exposure to mite allergens, and therefore provide important insights into the mite-induced allergy and preparation of mite allergen vaccines., (Copyright © 2020 American Society for Microbiology.)

The advent of unbiased metagenomic virus discovery has revolutionized studies of virus biodiversity and evolution. Despite this, our knowledge of the virosphere, including in mammalian species, remains limited. We used unbiased metagenomic sequencing to identify RNA viruses in European field voles and rabbits. Accordingly, we identified a number of novel RNA viruses including astrovirus, rotavirus A, picorna-like virus and a morbilli-like paramyxovirus. In addition, we identified a sobemovirus and a novel luteovirus that likely originated from the rabbit diet. These newly discovered viruses were often divergent from those previously described. The novel astrovirus was most closely related to a virus sampled from the rodent-eating European roller bird ( Coracias garrulous ). PCR screening revealed that the novel morbilli-like paramyxovirus in the UK field vole had a prevalence of approximately 4%, and shared common ancestry with other rodent morbilli-like viruses sampled globally. Two novel rotavirus A sequences were detected in a UK field vole and a French rabbit, the latter with a prevalence of 5%. Finally, a highly divergent picorna-like virus found in the gut of the French rabbit virus was only ~35% similar to an arilivirus at the amino acid level, suggesting the presence of a novel viral genus within the Picornaviridae .

Background: Australian brushtail possums ( Trichosurus vulpecula ) are an introduced pest species in New Zealand, but native to Australia where they are protected for biodiversity conservation. Wobbly possum disease (WPD) is a fatal neurological disease of Australian brushtail possums described in New Zealand populations that has been associated with infection by the arterivirus ( Arteriviridae ) wobbly possum disease virus (WPDV-NZ). Clinically, WPD-infected possums present with chronic meningoencephalitis, choroiditis and multifocal neurological symptoms including ataxia, incoordination, and abnormal gait., Methods: We conducted a retrospective investigation to characterise WPD in native Australian brushtail possums, and used a bulk meta-transcriptomic approach (i.e. total RNA-sequencing) to investigate its potential viral aetiology. PCR assays were developed for case diagnosis and full genome recovery in the face of extensive genetic variation., Results: We identified genetically distinct lineages of arteriviruses from archival tissues of WPD-infected possums in Australia, termed wobbly possum disease virus AU1 and AU2. Phylogenetically, WPDV-AU1 and WPDV-AU2 shared only ~ 70% nucleotide similarity to each other and the WPDV-NZ strain, suggestive of a relatively ancient divergence. Notably, we also identified a novel and divergent hepacivirus ( Flaviviridae ) - the first in a marsupial - in both WPD-infected and uninfected possums, indicative of virus co-infection., Conclusions: We have identified marsupial-specific lineages of arteriviruses in mainland Australia that are genetically distinct from that in New Zealand, in some cases co-infecting animals with a novel hepacivirus. Our study provides new insight into the hidden genetic diversity of arteriviruses, the capacity for virus co-infection, and highlights the utility of meta-transcriptomics for disease investigation in a One Health context., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

We isolated Tamdy virus (TAMV; strain XJ01/TAMV/China/2018) from Hyalomma asiaticum ticks infesting Bactrian camels in Xinjiang, China, in 2018. The genome of the strain showed high nucleotide similarity with previously described TAMV strains from Asia. Our study highlights the potential threat of TAMV to public health in China.

Wolbachia is an endosymbiotic bacterium that can block viral infections in arthropods, generating interest in its potential to control the spread of mosquito-borne disease. Drosophila melanogaster is model organism for Wolbachia infection, and the w Mel strain of Wolbachia can improve host survival following viral infection. However, it is unclear whether w Mel induces anti-viral blocking against the broader native virome of D. melanogaster , or whether the major effect of Wolbachia is a reduction in viral abundance rather than viral clearance. We examined the effect of Wolbachia on viral abundance by comparing the total transcriptome of w Mel-positive and w Mel-negative D. melanogaster populations sampled from six locations in Australia. In addition, we examined the impact of w Mel on individual flies by obtaining transcriptome data from 20 w Mel-positive and 20 w Mel-negative D. melanogaster from the location (Melbourne) with highest density of w Mel. These data revealed high viral abundance in both Wolbachia -positive and -negative populations and individuals. Notably, none of the viral species identified, representing RNA viruses from at least nine families/floating genera, showed evidence of protection by w Mel. Although the viral loads of picorna-like viruses are reduced by w Mel under experimental conditions, we observed no such effect here. These data show that D. melanogaster can harbour abundant RNA viruses regardless of its Wolbachia status and imply that the interaction between Wolbachia and viruses in nature is more complex than simple blocking., (© 2018 The Author(s).)

Unlabelled: Viruses of the family Flaviviridae are important pathogens of humans and other animals and are currently classified into four genera. To better understand their diversity, evolutionary history, and genomic flexibility, we used transcriptome sequencing (RNA-seq) to search for the viruses related to the Flaviviridae in a range of potential invertebrate and vertebrate hosts. Accordingly, we recovered the full genomes of five segmented jingmenviruses and 12 distant relatives of the known Flaviviridae ("flavi-like" viruses) from a range of arthropod species. Although these viruses are highly divergent, they share a similar genomic plan and common ancestry with the Flaviviridae in the NS3 and NS5 regions. Remarkably, although these viruses fill in major gaps in the phylogenetic diversity of the Flaviviridae, genomic comparisons reveal important changes in genome structure, genome size, and replication/gene regulation strategy during evolutionary history. In addition, the wide diversity of flavi-like viruses found in invertebrates, as well as their deep phylogenetic positions, suggests that they may represent the ancestral forms from which the vertebrate-infecting viruses evolved. For the vertebrate viruses, we expanded the previously mammal-only pegivirus-hepacivirus group to include a virus from the graceful catshark (Proscyllium habereri), which in turn implies that these viruses possess a larger host range than is currently known. In sum, our data show that the Flaviviridae infect a far wider range of hosts and exhibit greater diversity in genome structure than previously anticipated., Importance: The family Flaviviridae of RNA viruses contains several notorious human pathogens, including dengue virus, West Nile virus, and hepatitis C virus. To date, however, our understanding of the biodiversity and evolution of the Flaviviridae has largely been directed toward vertebrate hosts and their blood-feeding arthropod vectors. Therefore, we investigated an expanded group of potential arthropod and vertebrate host species that have generally been ignored by surveillance programs. Remarkably, these species contained diverse flaviviruses and related viruses that are characterized by major changes in genome size and genome structure, such that these traits are more flexible than previously thought. More generally, these data suggest that arthropods may be the ultimate reservoir of the Flaviviridae and related viruses, harboring considerable genetic and phenotypic diversity. In sum, this study revises the traditional view on the evolutionary history, host range, and genomic structures of a major group of RNA viruses., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)