Molecular Epidemiological Investigation Of Viruses In Potential Animal Hosts Of Natural Focus Diseases Such As Bats

[Background]Emerging Infectious Diseases(EIDs)have occurred globally since the century,from SARS to the current COVID-19 outbreak,most of which are zoonotic,with approximately 70%or more caused by pathogens of wildlife origin.In order to quantify and reduce the risk of pathogen spillover,active surveillance of viruses carried by wildlife has been strengthened globally,and with the rise of viral metagenomics based on high-throughput sequencing,the way in which viruses are discovered has changed.As the second largest mammal in terms of species and number after rodent with biological and ecological characteristics such as long-distance migration,long life span,swarming activities and strong immunity,bats are capable of carrying a variety of human pathogens such as Ebola virus,Marburg virus,Henipaviruses and coronaviruses,making them important hosts for zoonotic viruses.Guangdong Province is located in the subtropics,with a dense population and abundant animal resources,and is an epidemic site for many natural focus diseases.Therefore,monitoring the viruses carried by bats in the region is essential to effectively prevent the spillover of bat-derived zoonotic viruses and to prevent and control emerging infectious diseases.On the other hand,in the monitoring of viruses carried by vertebrates,there is often a sampling bias for mammals and birds,while little is known about the viruses carried by the lower vertebrates-fish,amphibians and reptiles-which account for more than 75%of vertebrates.Recent viral metagenomics studies have identified several novel viruses in fish,amphibians and reptiles,such as influenza-like viruses,coronaviruses and picornaviruses,etc.Meanwhile,these animals are inevitably in close contact with humans,pets,livestock and other wild animals during breeding,with the potential risk of spreading natural focus diseases.Therefore,this study monitors the virus profiles carried by amphibians and fish from several regions in China to further enrich the virus resource base of lower vertebrates and provide a theoretical basis for virus-host co-divergence.[Objective]1.Based on high-throughput sequencing of viral metagenomics,understanding the diversity of viruses carried by bats in Guangdong Province,conducting PCR assays for important viruses to grasp their prevalence,and understanding the genetic evolutionary characteristics of viruses through phylogenetic analysis can help provide guidance for the prevention and control of zoonotic viruses and emerging infectious diseases of bat origin,which has far-reaching public health significance.2.To understand the composition of virus communities and new virus carriage in amphibians and fish through virus metagenomics sequencing,to characterize the genome of important viruses and perform phylogenetic analysis,to try to isolate viruses and to understand the distribution of infection of important viruses by PCR assay,which is important to prevent food-borne transmission as well as the risk of potential natural focus diseases.[Methods]1.A total of 120 bats were collected from caves in Huaiji County,Zhaoqing City and Dongyuan County,Heyuan City,Guangdong Province in 2015 and 2019,respectively,mixed into 10 libraries and then high-throughput sequenced using the MGISEQ-200RS sequencing platform to obtain virus spectrum.PCR was used to detect infections of pathogens closely related to human pathogenesis in bats,and a series of gene sequence and phylogenetic analyses were performed for important pathogens.2.7 species of amphibians and 2 species of fish were collected in 6 provinces/municipalities of Beijing,Hubei,Sichuan,Jilin,Liaoning and Yunnan Province from October 2021 to August 2022,and the tissue of lung,gut,liver and kidney of amphibians were mixed into 35 libraries,and the tissue of gill,gut and liver of fish were mixed into 6 libraries,respectively,for high-throughput sequencing to obtain RNA virus spectrum.The viral genomes were obtained by PCR method amplification,and bioinformatics software was used to predict the viral genome structure and function,homology analysis and phylogenetic analysis.We also designed specific primers for PCR validation and detection of important viruses to understand the distribution of virus infection in amphibians or fish.The ACTK-1 cell line was used to isolate new viruses identified in amphibians to understand their biological properties.[Results]1.Molecular epidemiological characteristics of virus diversity carried by bats in Guangdong Province1.1 Sampling results of batsIn this study,a total of 120 anal swabs of bats were collected in Guangdong Province,and bats were classified into 3 families,5 genera and 10 species,among which the H.armiger accounted for the largest number(63.3%)and horseshoe bats account for the largest number of species(n=5).H.armiger was the dominant bat population in the area.1.2 Virus spectrum carried by bats in Guangdong ProvinceViral metagenomics sequencing of bat anal swabs yielded 107.1 GB of nucleotide data,and a total of 18 viral families were annotated,with mammalian viral families including Astroviridae,Caliciviridae,Picornaviridae,Flaviviridae,Adenoviridae,Circoviridae,Hepadnaviridae,Parvoviridae,Poxviridae and Vilyaviridae,with circoviruses accounting for the highest proportion(29.6%).There were some differences in the types of viruses carried by bats in different regions,and bats from Heyuan had a broader viral diversity.1.3 Molecular epidemiological characterization of four important viruses in batsCoronaviruses,astroviruses,adenoviruses and hepatitis B viruses were detected in anal swabs from 120 bats,influenza viruses and paramyxoviruses were negative.Bats of the genera Rhinolophus and Hipposideros carried the broadest spectrum of viruses.(1)Coronavirus:A novel bat coronavirus strain was detected in each of R.thomasi and H.pomona,sharing more than 97%nucleotide homology with SADS-CoV and bat coronavirus HKU2,respectively,each clustered in the evolutionary tree.(2)Astrovirus:The highest rate of astroviruses infection(46.7%)was detected in all eight bat species,and the difference in the rate of astroviruses infection in different bat species was statistically significant(χ2=30.526,P<0.005).12 novel bat astroviruses showed less than 90%amino acid identity among each other and located in different phylogenetic positions,showing a high degree of genetic heterogeneity.(3)Adenovirus:A new bat adenovirus strain was detected in R.pearsonii and H.armiger,and a 37,642 bp bat adenovirus(Bat adenovirus strain A8)was also obtained by metagenomics sequencing and assembly.The phylogenetic tree of strain A8 suggests that this virus forms a branch with the Chinese and Japanese bat adenoviruses.(4)Hepatitis B virus:A total of 48 hepatitis B virus positive samples were detected in six species of bats,and the differences in hepatitis B virus infection rates among different species of bats were not statistically significant.Combining the metagenomics sequencing results and PCR assays,a total of five novel bat hepatitis B viruses were identified,among which the H.armiger could carry both virus strains 2019-A9 and 2015-HaRa-H31,and 2015-HaRa-H31 could infect both H.armiger and R.affinis,which occupy different positions in the phylogenetic tree of the genus Orthohepadnavirus and have extensive genetic diversity and suggest possible cross-species transmission.2.Molecular epidemiological characteristics of the diversity of RNA viruses carried by amphibians2.1 Sampling results of amphibiansIn this study,a total of 236 amphibians were collected in six provinces/municipalities in China,which were classified into four families,seven genera and seven species,among which the Rana dybowskii accounted for the highest percentage(49.6%),followed by the Microhyla fissipes 27.1%.2.2 RNA virus spectrum carried by amphibiansA total of 392.9 GB of nucleotide data were measured from 35 libraries of amphibians,and 33 RNA viruses from 12 virus orders/families were identified,of which 27 were new RNA viruses,such as influenza virus,coronavirus,astrovirus,rhabdovirus and bunyaviruses,and the Asiatic toad carried the richest diversity of RNA viruses.It is the first time that phenuiviruses and arenavirus were discovered in amphibians.2.3 Molecular epidemiological characterization of important viruses in amphibians(1)A novel influenza-like virus and a novel coronavirus,both of which have been reported previously,were found in Microhyla fissipes tadpoles and were also not detected in Microhyla fissipes adult.(2)A 6,994 nt long bastrovirus genome(AtBastV/GCCDC11/2022)was identified in the gut of Asiatic toad,and three open reading frames(ORFs)were predicted.The phylogenetic analysis suggested that the non-structural protein encoded by ORF1 and the coat protein encoded by ORF2 had some homology with hepatitis E virus and astrovirus respectively.The virus was detected in seven Sichuan Asiatic toads,with a positive rate of 77.8%(7/9),while it was not detected in Jilin Asiatic toads or other amphibians.(3)The L fragments of six new bunyaviruses were identified in the lung of the Sichuan Asiatic toads,of which AtPhenV1/GCCDC12,AtPhenV2/GCCDC13,AtBunyV2 and AtBuny V4 are members of the family Phenuiviridae.It is the first time that phenuiviruses were identified in amphibians.AtPhenVl/GCCDC12 was only detected in the lung of Sichuan Asiatic toads,and AtPhenV2/GCCDC13 was detected in the lung of Asiatic toads in both Sichuan and Jilin.(4)A novel strain of arenavirus(RdArenV)was identified in the lung of Rana dybowskii,forming an evolutionary branch independent of the arenaviruses that infect mammals,reptiles and fish,and matching the evolutionary relationship with the host,suggesting that this is a new genus of amphibian arenaviruses.This is the first time that arenavirus has been detected in amphibians,and PCR results showed that the virus was detected from only one Rana dybowskii,with a positive rate of 0.85%(1/117).(5)In this study,attempts were made to isolate AtBastV/GCCDC11,AtPhenV1/GCCDC 12 and AtPhenV2/GCCDC 1 3 using the ACTK-1 cell line,but no cytopathic effect was observed after three generations of blind transmission,and the PCR results were all negative.3.Molecular epidemiological characteristics of the diversity of RNA viruses carried by fish3.1 Sampling results of fishA total of 152 trouts were collected from a farm in Beijing and classified into two subspecies of rainbow trout and golden trout.3.2 RNA virus spectrum carried by fishA total of 48.5 GB of nucleotide data was measured in six fish libraries and 10 RNA viruses from five virus families were identified,including infectious hematopoietic necrosis virus(IHNV),which poses a serious threat to fish farming,as well as novel astrovirus and calicivirus.3.3 Molecular epidemiological characterization of important viruses in fish(1)An IHNV strain,named BJ2022,was identified in trout.Gene phylogenetic analysis showed that strain BJ2022 shares more than 98%nucleotide identity with domestic isolates CJ13,HLJ-09 and HLJ1702,all belonging to the J genotype,with similar detection rates in rainbow trout and golden trout.(3)Nearly full-length novel astrovirus and calicivirus genomes,named GtAstV and GtCaliV1 respectively,were identified in the gut of golden trout.GtAstV clusters with fish astroviruses,forming a sister group to the genera Avastrovirus and with similar detection rates in rainbow trout and golden trout.GtCaliVl clusters with other fish and amphibian caliciviruses,forming a sister group to the genus Norovirus,with high infection rates in golden and rainbow trout.[Conclusion]In this study,we investigated the diversity of viruses carried by bats,amphibians and fish using viral metagenomics sequencing,and understood the prevalence of important viruses by PCR assays.These results have enriched our understanding of the diversity of viruses carried by bats as zoonotic hosts and provided fundamental data for assessing the cross-species transmission of related bat-derived zoonotic viruses.At the same time,exploring the viral spectrum of amphibians and fish,which are potential animal hosts of natural epidemic diseases,will greatly enrich the resource base of lower vertebrate viruses and help to prevent food-borne transmission and potential risk of natural focus diseases,as well as provide a theoretical basis for elucidating the evolutionary history of vertebrate viruses-hosts,which has important public health implications.