Although metagenomic sequencing has revealed high numbers of viruses in mosquitoes sampled globally, our understanding of how their diversity and abundance varies in time and space, as well as by host species and gender, remains unclear. To address this we collected 23,109 mosquitoes over the course of 12 months from a bat-dwelling cave and a nearby village in Yunnan province, China. These samples were organized by mosquito species, mosquito gender and sampling time for meta-transcriptomic sequencing. A total of 162 eukaryotic virus species were identified, of which 101 were novel, including representatives of 17 RNA virus multi-family supergroups and four species of DNA virus from the families Parvoviridae, Circoviridae and Nudiviridae. In addition, two known vector-borne viruses – Japanese encephalitis virus and Banna virus – were found. Analyses of the entire virome revealed strikingly different viral compositions and abundance levels in warmer compared to colder months, a strong host structure at the level of mosquito species, and no substantial differences between those viruses harbored by male and female mosquitoes. At the scale of individual viruses, some were found to be ubiquitous throughout the year and across four mosquito species, while most of the other viruses were season and/or host specific. Collectively, this study reveals the diversity, dynamics and evolution of the mosquito virome at a single location and sheds new lights on the ecology of these important vector animals.

Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within bats at the level of individual animals, and hence the frequency of virus co-infection and inter-species transmission. Using an unbiased meta-transcriptomics approach we characterised the mammalian associated viruses present in 149 individual bats sampled from Yunnan province, China. This revealed a high frequency of virus co-infection and species spillover among the animals studied, with 12 viruses shared among different bat species, which in turn facilitates virus recombination and reassortment. Of note, we identified five viral species that are likely to be pathogenic to humans or livestock, including a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV-2 and SARS-CoV, with only five amino acid differences between its receptor-binding domain sequence and that of the earliest sequences of SARS-CoV-2. Functional analysis predicts that this recombinant coronavirus can utilize the human ACE2 receptor such that it is likely to be of high zoonotic risk. Our study highlights the common occurrence of inter-species transmission and co-infection of bat viruses, as well as their implications for virus emergence.

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.

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.

Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within bats at the level of individual animals, and hence the frequency of virus co-infection and inter-species transmission. Using an unbiased meta-transcriptomics approach we characterised the mammalian associated viruses present in 149 individual bats sampled from Yunnan province, China. This revealed a high frequency of virus co-infection and species spillover among the animals studied, with 12 viruses shared among different bat species, which in turn facilitates virus recombination and reassortment. Of note, we identified five viral species that are likely to be pathogenic to humans or livestock, including a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV-2 and SARS-CoV, with only five amino acid differences between its receptor-binding domain sequence and that of the earliest sequences of SARS-CoV-2. Functional analysis predicts that this recombinant coronavirus can utilize the human ACE2 receptor such that it is likely to be of high zoonotic risk. Our study highlights the common occurrence of inter-species transmission and co-infection of bat viruses, as well as their implications for virus emergence.