Metagenomic analysis of individual mosquito viromes reveals the geographical patterns and drivers of viral diversity.

Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Here, using a meta-transcriptomic approach, we determined the viromes of 2,438 individual mosquitoes (81 species), spanning ~4,000 km along latitudes and longitudes in China. From these data we identified 393 viral species associated with mosquitoes, including 7 (putative) species of arthropod-borne viruses (that is, arboviruses). We identified potential mosquito species and geographic hotspots of viral diversity and arbovirus occurrence, and demonstrated that the composition of individual mosquito viromes was strongly associated with host phylogeny. Our data revealed a large number of viruses shared among mosquito species or genera, enhancing our understanding of the host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, perhaps reflecting long-distance mosquito dispersal. Together, these results greatly expand the known mosquito virome, linked viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the biogeography and diversity of viruses in insect vectors.

Metagenomic analysis of individual mosquitos reveals the ecology of insect viruses.

Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Using a meta-transcriptomic approach, we analysed the virome of 2,438 individual mosquitos (79 species), spanning ~4000 km along latitudes and longitudes in China. From these data we identified 393 core viral species associated with mosquitos, including seven (putative) arbovirus species. We identified potential species and geographic hotspots of viral richness and arbovirus occurrence, and demonstrated that host phylogeny had a strong impact on the composition of individual mosquito viromes. Our data revealed a large number of viruses shared among mosquito species or genera, expanding our knowledge of host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, possibly facilitated by long-distance mosquito migrations. Together, our results greatly expand the known mosquito virome, linked the viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the ecology of viruses of insect vectors.

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.

Tick (Acari: Ixodoidea) fauna and zoogeographic division of Jiangxi Province, China.

Tick fauna and zoogeographic distribution of Jiangxi Province remain largely unknown due to the lack of data on distribution, occurrence, and host associations of ticks. Considering this, we collected 1,817 individual samples from natural hosts, humans, and vegetation in 18 counties/districts throughout Jiangxi Province, China, from 2015 to 2021. These 1,817 individuals were found to 13 tick species, 4 genera, and 1 family. In addition, the tick sample data from 8 sampling localities (counties and districts) reported in previous studies were also included in our data. A total of 4,021 individuals, including our sample collection and the previously reported data, were assigned to at least 18 species, 6 genera, and 2 families. One newly recorded species Dermacentor sp. (near D. steini Schulze) was found; three misidentified species (Ixodes acuminatus, Haemaphysalis spinigera, and Haemaphysalis verticalis) reported previously were deleted; and one misidentified species Dermacentor auratus Supino was revised as Dermacentor steini Schulze. In addition, we divided the tick fauna in Jiangxi Province into 5 zoogeographic areas and assigned the 18 tick species collected from 26 localities to these 5 zoogeographic areas. To summarize, our findings provide valuable information on the distribution, tick-host associations, and zoogeographic division of ticks in Jiangxi Province, China. Their molecular characterizations, phylogenetic relationships, and tick-borne pathogens that they may transmit should be further explored.

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.

Total infectome characterization of respiratory infections in pre-COVID-19 Wuhan, China.

At the end of 2019 Wuhan witnessed an outbreak of "atypical pneumonia" that later developed into a global pandemic. Metagenomic sequencing rapidly revealed the causative agent of this outbreak to be a novel coronavirus denoted SARS-CoV-2. To provide a snapshot of the pathogens in pneumonia-associated respiratory samples from Wuhan prior to the emergence of SARS-CoV-2, we collected bronchoalveolar lavage fluid samples from 408 patients presenting with pneumonia and acute respiratory infections at the Central Hospital of Wuhan between 2016 and 2017. Unbiased total RNA sequencing was performed to reveal their "total infectome", including viruses, bacteria and fungi. We identified 35 pathogen species, comprising 13 RNA viruses, 3 DNA viruses, 16 bacteria and 3 fungi, often at high abundance and including multiple co-infections (13.5%). SARS-CoV-2 was not present. These data depict a stable core infectome comprising common respiratory pathogens such as rhinoviruses and influenza viruses, an atypical respiratory virus (EV-D68), and a single case of a sporadic zoonotic pathogen-Chlamydia psittaci. Samples from patients experiencing respiratory disease on average had higher pathogen abundance than healthy controls. Phylogenetic analyses of individual pathogens revealed multiple origins and global transmission histories, highlighting the connectedness of the Wuhan population. This study provides a comprehensive overview of the pathogens associated with acute respiratory infections and pneumonia, which were more diverse and complex than obtained using targeted PCR or qPCR approaches. These data also suggest that SARS-CoV-2 or closely related viruses were absent from Wuhan in 2016-2017.

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.

Large-Scale Comparative Analyses of Tick Genomes Elucidate Their Genetic Diversity and Vector Capacities.

Among arthropod vectors, ticks transmit the most diverse human and animal pathogens, leading to an increasing number of new challenges worldwide. Here we sequenced and assembled high-quality genomes of six ixodid tick species and further resequenced 678 tick specimens to understand three key aspects of ticks: genetic diversity, population structure, and pathogen distribution. We explored the genetic basis common to ticks, including heme and hemoglobin digestion, iron metabolism, and reactive oxygen species, and unveiled for the first time that genetic structure and pathogen composition in different tick species are mainly shaped by ecological and geographic factors. We further identified species-specific determinants associated with different host ranges, life cycles, and distributions. The findings of this study are an invaluable resource for research and control of ticks and tick-borne diseases.

Author Correction: A new coronavirus associated with human respiratory disease in China.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A new coronavirus associated with human respiratory disease in China.

Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health1-3. Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 January 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 December 2019. Epidemiological investigations have suggested that the outbreak was associated with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 December 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing4 of a sample of bronchoalveolar lavage fluid from the patient identified a new RNA virus strain from the family Coronaviridae, which is designated here 'WH-Human 1' coronavirus (and has also been referred to as '2019-nCoV'). Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China5. This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans.

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 of rickettsiales bacteria in ticks from Wuhan, China.

Rickettsiales bacteria are important agents of (re)emerging infectious diseases, with ticks playing a key role in their evolution and transmission. We collected 1079 hard ticks belonging to five species (Ixodes sinensis, Rhipicephalus microplus, Haemaphysalis flava, Haemaphysalis hystricis and Haemaphysalis longicornis) from cattle and goats in Wuhan city, Hubei province, China. The dominant tick species was H. longicornis (578, 53.57%), followed by R. microplus (354, 32.81%), H. hystricis (62, 5.75%), H. flava (57, 5.28%), and I. sinensis (28, 2.59%). Rickettsiales bacteria were identified in these ticks by amplifying the Rickettsiales 16S rRNA (rrs), citrate synthase (gltA), and heat shock protein (groEL) genes. The rrs gene of Rickettsiales was positive in 32 (2.97%) ticks, including 2 cases of co-infection, with 4 (0.69%) in H. longicornis, 15 (4.24%) in R. microplus, 7 (12.28%) in H. flava, 1 (1.61%) in H. hystricis, and 5 (17.86%) in I. sinensis ticks. Phylogenetic analysis revealed the presence of six recognized and seven Candidatus species of Rickettsiaceae, Anaplasmataceae and Candidatus Midichloriaceae. Notably, one lineage within both Ehrlichia and Candidatus Midichloriaceae was distinct from any known Rickettsiales, suggesting the presence of potentially novel species of Rickettsiales bacteria. In sum, these data reveal an extensive diversity of Rickettsiales in ticks from Wuhan, highlighting the need to understand Rickettsiales infection in local animals and humans.

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.

Mosquito (Diptera: Culicidae) Habitat Surveillance by Android Mobile Devices in Guangzhou, China.

In 2014, Guangzhou City, South China, suffered from its worst outbreak of dengue fever in decades. Larval mosquito habitat surveillance was carried out by using android mobile devices in four study sites in May 2015. The habitats with larval mosquitoes were recorded as photo waypoints in OruxMaps or in videos. The total number of potential mosquito habitats was 342, of which 166 (49%) were found to have mosquito larvae or pupae. Small containers were the most abundant potential habitats, accounting for 26% of the total number. More mosquito larvae and pupae, were found in small containers than in other objects holding water, for example, potted or hydroponic plants (p < 0.05). Mosquito larvae were collected from all plastic road barriers, used tires, and underground water. Aedes albopictus larvae were found from small and large containers, stumps, among others. The overall route index (RI) was 11.3, which was 14.2 times higher than the grade C criteria of the National Patriotic Health Campaign Committee (NPHCC), China. The higher RIs were found from the bird and flower markets, schools, and underground parking lots. The results indicated that Android mobile devices are a convenient and useful tool for surveillance of mosquito habitats, and the enhancement of source reduction may benefit the prevention and control of dengue vector mosquitoes.

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.

Diversity and evolution of avian influenza viruses in live poultry markets, free-range poultry and wild wetland birds in China.

The wide circulation of novel avian influenza viruses (AIVs) highlights the risk of pandemic influenza emergence in China. To investigate the prevalence and genetic diversity of AIVs in different ecological contexts, we surveyed AIVs in live poultry markets (LPMs), free-range poultry and the wetland habitats of wild birds in Zhejiang and Hubei provinces. Notably, LPMs contained the highest frequency of AIV infection, and the greatest number of subtypes (n = 9) and subtype co-infections (n = 14), as well as frequent reassortment, suggesting that they play an active role in fuelling AIV transmission. AIV-positive samples were also identified in wild birds in both provinces and free-range poultry in one sampling site close to a wetland region in Hubei. H9N2, H7N9 and H5N1 were the most commonly sampled subtypes in the LPMs from Zhejiang, whilst H5N6 and H9N2 were the dominant subtypes in the LPMs from Hubei. Phylogenetic analyses of the whole-genome sequences of 43 AIVs revealed that three reassortant H5 subtypes were circulating in LMPs in both geographical regions. Notably, the viruses sampled from the wetland regions and free-range poultry contained complex reassortants, for which the origins of some segments were unclear. Overall, our study highlights the extent of AIV genetic diversity in two highly populated parts of central and south-eastern China, particularly in LPMs, and emphasizes the need for continual surveillance.