Host traits shape virome composition and virus transmission in wild small mammals.

Bats, rodents, and shrews are the most important animal sources of human infectious diseases. However, the evolution and transmission of viruses among them remain largely unexplored. Through the meta-transcriptomic sequencing of internal organ and fecal samples from 2,443 wild bats, rodents, and shrews sampled from four Chinese habitats, we identified 669 viruses, including 534 novel viruses, thereby greatly expanding the mammalian virome. Our analysis revealed high levels of phylogenetic diversity, identified cross-species virus transmission events, elucidated virus origins, and identified cases of invertebrate viruses in mammalian hosts. Host order and sample size were the most important factors impacting virome composition and patterns of virus spillover. Shrews harbored a high richness of viruses, including many invertebrate-associated viruses with multi-organ distributions, whereas rodents carried viruses with a greater capacity for host jumping. These data highlight the remarkable diversity of mammalian viruses in local habitats and their ability to emerge in new hosts.

RNA viromes from terrestrial sites across China expand environmental viral diversity.

Environmental RNA viruses are ubiquitous and diverse, and probably have important ecological and biogeochemical impacts. Understanding the global diversity of RNA viruses is limited by sampling biases, dependence on cell culture and PCR for virus discovery, and a focus on viruses pathogenic to humans or economically important animals and plants. To address this knowledge gap, we generated metatranscriptomic sequence data from 32 diverse environments in 16 provinces and regions of China. We identified 6,624 putatively novel virus operational taxonomic units from soil, sediment and faecal samples, greatly expanding known diversity of the RNA virosphere. These newly identified viruses included positive-sense, negative-sense and double-strand RNA viruses from at least 62 families. Sediments and animal faeces were rich sources of viruses. Virome compositions were affected by local environmental factors, including organic content and eukaryote species abundance. Notably, environmental factors had a greater impact on the abundance and diversity of plant, fungal and bacterial viruses than of animal viromes. Our data confirm that RNA viruses are an integral part of both terrestrial and aquatic ecosystems.

Coronaviruses in wild animals sampled in and around Wuhan at the beginning of COVID-19 emergence.

Over the last several decades, no emerging virus has had a profound impact on the world as the SARS-CoV-2 that emerged at the end of 2019 has done. To know where severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originated from and how it jumped into human population, we immediately started a surveillance investigation in wild mammals in and around Wuhan when we determined the agent. Herein, coronaviruses were screened in the lung, liver, and intestinal tissue samples from fifteen raccoon dogs, seven Siberian weasels, three hog badgers, and three Reeves's muntjacs collected in Wuhan and 334 bats collected around Wuhan. Consequently, eight alphacoronaviruses were identified in raccoon dogs, while nine betacoronaviruses were found in bats. Notably, the newly discovered alphacoronaviruses shared a high whole-genome sequence similarity (97.9 per cent) with the canine coronavirus (CCoV) strain 2020/7 sampled from domestic dog in the UK. Some betacoronaviruses identified here were closely related to previously known bat SARS-CoV-related viruses sampled from Hubei province and its neighbors, while the remaining betacoronaviruses exhibited a close evolutionary relationship with SARS-CoV-related bat viruses in the RdRp gene tree and clustered together with SARS-CoV-2-related bat coronaviruses in the M, N and S gene trees, but with relatively low similarity. Additionally, these newly discovered betacoronaviruses seem unlikely to bind angiotensin-converting enzyme 2 because of the deletions in the two key regions of their receptor-binding motifs. Finally, we did not find SARS-CoV-2 or its progenitor virus in these animal samples. Due to the high circulation of CCoVs in raccoon dogs in Wuhan, more scientific efforts are warranted to better understand their diversity and evolution in China and the possibility of a potential human agent.

Extensive genetic diversity and host range of rodent-borne coronaviruses.

To better understand the genetic diversity, host associations and evolution of coronaviruses (CoVs) in China we analyzed a total of 696 rodents encompassing 16 different species sampled from Zhejiang and Yunnan provinces. Based on reverse transcriptase PCR-based CoV screening of fecal samples and subsequent sequence analysis of the RNA-dependent RNA polymerase gene, we identified CoVs in diverse rodent species, comprising Apodemus agrarius, Apodemus chevrieri, Apodemus latronum, Bandicota indica, Eothenomys cachinus, Eothenomys miletus, Rattus andamanensis, Rattus norvegicus, and Rattus tanezumi. CoVs were particularly commonplace in A. chevrieri, with a detection rate of 12.44 per cent (24/193). Genetic and phylogenetic analysis revealed the presence of three groups of CoVs carried by a range of rodents that were closely related to the Lucheng Rn rat CoV (LRNV), China Rattus CoV HKU24 (ChRCoV_HKU24), and Longquan Rl rat CoV (LRLV) identified previously. One newly identified A. chevrieri-associated virus closely related to LRNV lacked an NS2 gene. This virus had a similar genetic organization to AcCoV-JC34, recently discovered in the same rodent species in Yunnan, suggesting that it represents a new viral subtype. Notably, additional variants of LRNV were identified that contained putative non-structural (NS)2b genes located downstream of the NS2 gene that were likely derived from the host genome. Recombination events were also identified in the open reading frame (ORF) 1a gene of Lijiang-71. In sum, these data reveal the substantial genetic diversity and genomic complexity of rodent-borne CoVs, and extend our knowledge of these major wildlife virus reservoirs.

Diverse and abundant resistome in terrestrial and aquatic vertebrates revealed by transcriptional analysis.

Despite increasing evidence that antibiotic resistant pathogens are shared among humans and animals, the diversity, abundance and patterns of spread of antibiotic resistance genes (ARGs) in wildlife remains unclear. We identified 194 ARGs associated with phenotypic resistance to 13 types of antibiotic in meta-transcriptomic data generated from a broad range of lower vertebrates residing in both terrestrial and aquatic habitats. These ARGs, confirmed by PCR, included those that shared high sequence similarity to clinical isolates of public health concern. Notably, the lower vertebrate resistome varied by ecological niche of the host sampled. The resistomes in marine fish shared high similarity and were characterized by very high abundance, distinct from that observed in other habitats. An assessment of ARG mobility found that ARGs in marine fish were frequently co-localized with mobile elements, indicating that they were likely spread by horizontal gene transfer. Together, these data reveal the remarkable diversity and transcriptional levels of ARGs in lower vertebrates, and suggest that these wildlife species might play an important role in the global spread of ARGs.

Outbreak of COVID-19 in a family, Wenzhou, China.

Since December 2019, China has experienced a widespread outbreak of COVID-19. However, at the early stage of outbreak, investigations revealed a variety of patterns resulting in the transmission of COVID-19. Thus, it is essential to understand the transmission types and the potential for sustained human-to-human transmission. Moreover, the information regarding the characteristics of transmission helps in coordinating the current screening programme, and controlling and containing measures, and also, helps in deciding the appropriate quarantine duration. Thus, this investigation reports an outbreak of COVID-19 in a family residing in Wenzhou, Zhejiang, China during the month of January-February 2020.

Newly identified viral genomes in pangolins with fatal disease.

Epizootic pathogens pose a major threat to many wildlife species, particularly in the context of rapidly changing environments. Pangolins (order Pholidota) are highly threatened mammals, in large part due to the trade in illegal wildlife. During July to August 2018 four sick wild pangolins (three Manis javanica and one Manis pentadactyla) exhibiting a variety of clinical symptoms were rescued by the Jinhua Wildlife Protection Station in Zhejiang province, China. Although three of these animals died, fortunately one recovered after 2 weeks of symptomatic treatment. Using meta-transcriptomics combined with reverse transcription polymerase chain reaction (RT-PCR), we identified two novel RNA viruses in two of the dead pangolins. Genomic analysis revealed that these viruses were most closely related to pestiviruses and coltiviruses, although still highly genetically distinct, with more than 48 and 25 per cent sequence divergence at the amino acid level, respectively. We named these Dongyang pangolin virus (DYPV) and Lishui pangolin virus (LSPV) based on the sampling site and hosts. Although coltiviruses (LSPV) are known to be transmitted by ticks, we found no evidence of LSPV in ticks sampled close to where the pangolins were collected. In addition, although DYPV was present in nymph ticks (Amblyomma javanense) collected from a diseased pangolin, they were not found in the local tick population. Epidemiological investigation revealed that both novel viruses might have been imported following the illegal international trade of pangolins. Hence, these data indicate that illegal wildlife trafficking not only threatens the status of pangolin populations, but may also spread epizootic pathogens.

Diversity and circulation of Jingmen tick virus in ticks and mammals.

Since its initial identification in ticks in 2010, Jingmen tick virus (JMTV) has been described in cattle, rodents, and primates. To better understand the diversity, evolution, and transmission of JMTV, we sampled 215 ticks, 104 cattle bloods, 216 bats, and 119 rodents in Wenzhou city, Zhejiang Province, China as well as 240 bats from Guizhou and Henan Provinces. JMTV was identified in 107 ticks (from two species), 54 bats (eleven species), 8 rodents (three species), and 10 cattle, with prevalence levels of 49.8, 11.8, 6.7, and 9.6 per cent, respectively, suggesting that bats may be a natural reservoir of JMTV. Phylogenetic analyses revealed that all the newly identified JMTVs were closely related to each other and to previously described viruses. Additionally, all tick and mammalian JMTV sampled in Wenzhou shared a consistent genomic structure, suggesting that the virus can cocirculate between ticks and mammals without observable variation in genome organization. All JMTVs sampled globally could be divided into two phylogenetic groups, Mantel tests suggested that geographic isolation, rather than host species, may be the main driver of JMTV diversity. However, the exact geographical origin of JMTV was difficult to determine, suggesting that this virus has a complex evolutionary history.

Identification of Norovirus and Human Parechovirus in Patients With Hand, Foot and Mouth Disease Syndrome.

BACKGROUND: Hand, foot and mouth disease (HFMD) is caused mostly by enteroviruses. However, other viral agents also can cause similar syndromes, and hence, the infections they cause are often misdiagnosed clinically. To determine non-enterovirus etiologic agents in HFMD-like cases, we screened enterovirus-negative samples collected from the patients who were clinically diagnosed as HFMD in China. METHODS: Two hundred enterovirus-negative samples were collected previously in Wenzhou city of Zhejiang province, China. Both high throughput sequencing and RT-PCR were used to screen viral agents. In addition, their clinical features were analyzed. RESULTS: Norovirus (NoV) and human parechovirus (HPeV) were identified from 22 (11.00%) and 9 (4.50%) samples, respectively. In addition, the complete genome sequences were recovered from 4 NoV-positive samples, and the VP1/3Dpol gene sequences were recovered from 5 HPeV-positive samples. Phylogenetic analyses of the NoV sequences revealed that they were closely related to those circulated in other regions of China. Notably, 4 genotypes of HPeVs, including HPeV-1, HPeV-4, HPeV-5 and HPeV-14, were found, indicating high genetic diversity of the virus. Frequent recombination between various genotypes was also observed in the HPeVs. Although most of the patients presented with the clinical features of HFMD, 4 patients infected with NoV GII.4 and 3 patients infected with HPeV-1 (1) and HPeV-4 (2) were characterized with diarrhea. Finally, tonsillitis, convulsion and granulocytopenia were observed in 1 NoV GII.4 patient, while liver dysfunction was found in 1 NoV GII.17 patient. CONCLUSIONS: These data reveal the variety of agents in the cases clinically diagnosed as HFMD.

Discovery of a highly divergent hepadnavirus in shrews from China.

Limited sampling means that relatively little is known about the diversity and evolutionary history of mammalian members of the Hepadnaviridae (genus Orthohepadnavirus). An important case in point are shrews, the fourth largest group of mammals, but for which there is limited knowledge on the role they play in viral evolution and emergence. Here, we report the discovery of a novel shrew hepadnavirus. The newly discovered virus, denoted shrew hepatitis B virus (SHBV), is divergent to be considered a new species of Orthohepadnavirus. Phylogenetic analysis revealed that these viruses were usually most closely related to TBHBV (tent-making bat hepatitis B virus), known to be able to infect human hepatocytes, and had a similar genome structure, although SHBV fell in a more basal position in the surface protein phylogeny. In sum, these data suggest that shrews are natural hosts for hepadnaviruses and may have played an important role in their long-term evolution.

Author Correction: The evolutionary history of vertebrate RNA viruses.

Change history: In this Article, author Li Liu should be associated with affiliation number 5 (College of Marine Sciences, South China Agricultural University, Guangzhou, Guangdong, China), rather than affiliation number 4 (Wenzhou Center for Disease Control and Prevention, Wenzhou, Zhejiang, China). This has been corrected online.

The evolutionary history of vertebrate RNA viruses.

Our understanding of the diversity and evolution of vertebrate RNA viruses is largely limited to those found in mammalian and avian hosts and associated with overt disease. Here, using a large-scale meta-transcriptomic approach, we discover 214 vertebrate-associated viruses in reptiles, amphibians, lungfish, ray-finned fish, cartilaginous fish and jawless fish. The newly discovered viruses appear in every family or genus of RNA virus associated with vertebrate infection, including those containing human pathogens such as influenza virus, the Arenaviridae and Filoviridae families, and have branching orders that broadly reflected the phylogenetic history of their hosts. We establish a long evolutionary history for most groups of vertebrate RNA virus, and support this by evaluating evolutionary timescales using dated orthologous endogenous virus elements. We also identify new vertebrate-specific RNA viruses and genome architectures, and re-evaluate the evolution of vector-borne RNA viruses. In summary, this study reveals diverse virus-host associations across the entire evolutionary history of the vertebrates.

Extensive diversity and evolution of hepadnaviruses in bats in China.

To better understand the evolution of hepadnaviruses, we sampled bats from Guizhou, Henan and Zhejiang provinces, China, and rodents from Zhejiang province. Genetically diverse hepadnaviruses were identified in a broad range of bat species, with an overall prevalence of 13.3%. In contrast, no rodent hepadnaviruses were identified. The newly discovered bat hepadnaviruses fell into two distinct phylogenetic groups. The viruses within the first group exhibited high diversity, with some closely related to viruses previously identified in Yunnan province. Strikingly, the newly discovered viruses sampled from Jiyuan city in the second phylogenetic group were most closely related to those found in bats from West Africa, suggestive of a long-term association between bats and hepadnaviruses. A co-phylogenetic analysis revealed frequent cross-species transmission among bats from different species, genera, and families. Overall, these data suggest that there are likely few barriers to the cross-species transmission of bat hepadnaviruses.

Discovery of a Highly Divergent Coronavirus in the Asian House Shrew from China Illuminates the Origin of the Alphacoronaviruses.

Although shrews are one of the largest groups of mammals, little is known about their role in the evolution and transmission of viral pathogens, including coronaviruses (CoVs). We captured 266 Asian house shrews (Suncus murinus) in Jiangxi and Zhejiang Provinces, China, during 2013 to 2015. CoV RNA was detected in 24 Asian house shrews, with an overall prevalence of 9.02%. Complete viral genome sequences were successfully recovered from the RNA-positive samples. The newly discovered shrew CoV fell into four lineages reflecting their geographic origins, indicative of largely allopatric evolution. Notably, these viruses were most closely related to alphacoronaviruses but sufficiently divergent that they should be considered a novel member of the genus Alphacoronavirus, which we denote Wénchéng shrew virus (WESV). Phylogenetic analysis revealed that WESV was a highly divergent member of the alphacoronaviruses and, more dramatically, that the S gene of WESV fell in a cluster that was genetically distinct from that of known coronaviruses. The divergent position of WESV suggests that coronaviruses have a long association with Asian house shrews. In addition, the genome of WESV contains a distinct NS7 gene that exhibits no sequence similarity to genes of any known viruses. Together, these data suggest that shrews are natural reservoirs for coronaviruses and may have played an important and long-term role in CoV evolution.IMPORTANCE The subfamily Coronavirinae contains several notorious human and animal pathogens, including severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, and porcine epidemic diarrhea virus. Because of their genetic diversity and phylogenetic relationships, it has been proposed that the alphacoronaviruses likely have their ultimate ancestry in the viruses residing in bats. Here, we describe a novel alphacoronavirus (Wénchéng shrew virus [WESV]) that was sampled from Asian house shrews in China. Notably, WESV is a highly divergent member of the alphacoronaviruses and possesses an S gene that is genetically distinct from those of all known coronaviruses. In addition, the genome of WESV contains a distinct NS7 gene that exhibits no sequence similarity to those of any known viruses. Together, these data suggest that shrews are important and longstanding hosts for coronaviruses that merit additional research and surveillance.

Extensive diversity of coronaviruses in bats from China.

To help reveal the diversity and evolution of bat coronaviruses we collected 1067 bats from 21 species in China. A total of 73 coronaviruses (32 alphacoronaviruses and 41 betacoronaviruses) were identified in these bats, with an overall prevalence of 6.84%. All newly-identified betacoronaviruses were SARS-related Rhinolophus bat coronaviruses (SARSr-Rh-BatCoV). Importantly, with the exception of the S gene, the genome sequences of the SARSr-Rh-BatCoVs sampled in Guizhou province were closely related to SARS-related human coronavirus. Additionally, the newly-identified alphacoronaviruses exhibited high genetic diversity and some may represent novel species. Our phylogenetic analyses also provided insights into the transmission of these viruses among bat species, revealing a general clustering by geographic location rather than by bat species. Inter-species transmission among bats from the same genus was also commonplace in both the alphacoronaviruses and betacoronaviruses. Overall, these data suggest that high contact rates among specific bat species enable the acquisition and spread of coronaviruses.

Diversity, evolution and population dynamics of avian influenza viruses circulating in the live poultry markets in China.

Live poultry markets (LPMs) are an important source of novel avian influenza viruses (AIV). During 2015-2016 we surveyed AIV diversity in ten LPMs in Hubei, Zhejiang and Jiangxi provinces, China. A high diversity and prevalence of AIVs (totaling 12 subtypes) was observed in LPMs in these provinces. Strikingly, however, the subtypes discovered during 2015-2016 were markedly different to those reported by us in these same localities one year previously, suggesting a dynamic shift in viral genetic diversity over the course of a single year. Phylogenetic analyses revealed frequent reassortment, including between high and low pathogenic AIV subtypes and among those that circulate in domestic and wild birds. Notably, the novel H5N6 reassortant virus, which contains a set of H9N2-like internal genes, was prevalent in all three regions surveyed. Overall, these data highlight the profound changes in genetic diversity and in patterns of reassortment in those AIVs that circulate in LPMs.

Extensive genetic diversity of Rickettsiales bacteria in multiple mosquito species.

Rickettsiales are important zoonotic pathogens, causing severe disease in humans globally. Although mosquitoes are an important vector for diverse pathogens, with the exception of members of the genus Wolbachia little is known about their role in the transmission of Rickettsiales. Herein, Rickettsiales were identified by PCR in five species of mosquitoes (Anopheles sinensis, Armigeres subalbatus, Aedes albopictus, Culex quinquefasciatus and Cu. tritaeniorhynchus) collected from three Chinese provinces during 2014-2015. Subsequent phylogenetic analyses of the rrs, groEL and gltA genes revealed the presence of Anaplasma, Ehrlichia, Candidatus Neoehrlichia, and Rickettsia bacteria in mosquitoes, comprising nine documented and five tentative species bacteria, as well as three symbionts/endosybionts. In addition, bacteria were identified in mosquito eggs, larvae, and pupae sampled from aquatic environments. Hence, these data suggest that Rickettsiales circulate widely in mosquitoes in nature. Also of note was that Ehrlichia and Rickettsia bacteria were detected in each life stage of laboratory cultured mosquitoes, suggesting that Rickettsiales may be maintained in mosquitoes through both transstadial and transovarial transmission. In sum, these data indicate that mosquitoes may have played an important role in the transmission and evolution of Rickettsiales in nature.

Redefining the invertebrate RNA virosphere.

Current knowledge of RNA virus biodiversity is both biased and fragmentary, reflecting a focus on culturable or disease-causing agents. Here we profile the transcriptomes of over 220 invertebrate species sampled across nine animal phyla and report the discovery of 1,445 RNA viruses, including some that are sufficiently divergent to comprise new families. The identified viruses fill major gaps in the RNA virus phylogeny and reveal an evolutionary history that is characterized by both host switching and co-divergence. The invertebrate virome also reveals remarkable genomic flexibility that includes frequent recombination, lateral gene transfer among viruses and hosts, gene gain and loss, and complex genomic rearrangements. Together, these data present a view of the RNA virosphere that is more phylogenetically and genomically diverse than that depicted in current classification schemes and provide a more solid foundation for studies in virus ecology and evolution.

Taxonomy of the order Mononegavirales: update 2016.

In 2016, the order Mononegavirales was emended through the addition of two new families (Mymonaviridae and Sunviridae), the elevation of the paramyxoviral subfamily Pneumovirinae to family status (Pneumoviridae), the addition of five free-floating genera (Anphevirus, Arlivirus, Chengtivirus, Crustavirus, and Wastrivirus), and several other changes at the genus and species levels. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Identification of novel and diverse rotaviruses in rodents and insectivores, and evidence of cross-species transmission into humans.

Rotaviruses are an important cause of severe diarrheal illness in children globally. We characterized rotaviruses sampled in humans, insectivores (shrews) and rodents from urban and rural regions of Zhejiang province, China. Phylogenetic analyses revealed seven genotypic constellations of human rotaviruses with six different combinations of G and P genotypes - G3P[8] (50.06%), G9P[8] (36.16%), G1P[8] (8.92%), G2P[4] (4.63%), G3P[3] (0.12%), and G3P[9] (0.12%). In rodents and shrews sampled from the same locality we identified a novel genotype constellation (G32-P[46]-I24-R18-C17-M17-A28-N17-T19-E24-H19), a novel P genotype (P[45]), and two different AU-1-like rotaviruses associated with a G3P[3] genotype combination. Of particular note was a novel rotavirus from a human patient that was closely related to viruses sampled from rodents in the same region, indicative of a local species jump. In sum, these data are suggestive of the cross-species transmission of rodent rotaviruses into humans and for reassortment among human and animal rotaviruses.