Investigation On Bat Virome And Characterization Of Novel Bat Viruses In The Three Northeastern Provinces Of China

Bats are the second largest mammal in the world and distributed throughout all lands except the two poles. At the same time, they are also an important repository and could carry at least one hundred and seventy kinds of viruses, including some zoonotic viruses,such as rabies virus, hendra virus,ebola virus, bocavirus and so on. There are at least one hundred and twenty bat species from thirty bat genera from twelve families in China, and bats are widely distributed in many parts of China.Three northeast provinces of China, including Liaoning province, Jilin province and Heilongjiang province, adhere to the border of Russia and North Korea, which are important strategic position.The vast territory and the unique climate lead to the lack of the systematic data of bat.So far, Irkut virus from the lyssavirus,bocavirus and coronavirus were only found in three northeast provinces of China, so it’s important significance to systematically comprehensive survey the natural background and genetic polymorphism that bats carry viruses to fill gaps of bat viruses in three northeast provinces of China.Two hundred and four healthy bats, including Myotis ricketti, Rhinolophus ferrumequinum, Murina leucogaster and Vespertilio orientalis,were collected at the six cities from the sixteen cities of fieldtrips with bat ecological situation in 2013 and 2014.By viral metagenomic analysis, twenty-two viral families were found, which includes thirty-two viral species from twenty viral genera from vertebrate viruses such as phlebovirus, alphacoronavirus and betacoronavirus, human parainfluenza virus 4a, astrovirus and so on, and includes six insect viral families Baculoviridae, Dicistroviridae, Iflaviridae, Parvoviridae(Ambidensovirus), Polydnaviridae, Tetraviridae and so on, and includes one plant viral families, such as Phycodnaviridae.According to the results of viral metagenomics, astrovirus, parvovirus, vesiculovirus, orbivirus, bunyavirus, cirovirus, coronavirus and other significant viruses were identified and analyzed. The viral sequences annotated to astroviridae were found in the bats of Myotis ricketti and Rhinolophus ferrumequinum in Benxi city,Liaoning province, and had the 84% highest with Bhutan astrovirus MLB1. The viral sequences annotated to the vesiculovirus from rhabdoviridae were found in the bats of Myotis ricketti and Rhinolophus ferrumequinum in Benxi city,Liaoning province, and had the 64.3% highest with American bat vesiculovirus of TFFN-2013. Two bats of Rhinolophus ferrumequinum carried vesiculovirus from the six bats in in Xihu district of Benxi city in Liaoning Province were acquired and the positive rate was 33.3%. One bat of Rhinolophus ferrumequinum carried the vesiculovirus from the four bats in in Benxi county of Benxi city in Liaoning Province were acquired and the positive rate was 25.0%. All bats of Rhinolophus ferrumequinum carried the vesiculovirus from the two bats in in Hengren county of Benxi city in Liaoning Province were acquired and the positive rate was 100%. The identification analysis of picornavirus, we found viral sequence in bats of Myotis ricketti and Rhinolophus ferrumequinum in Tonghua city, Jilin Province annotated to the cardiovirus from picornaviridae, and had the 84% highest identity with African bat icavirus after the analysis. Nine bats of Myotis ricketti carried the cardiovirus from picornaviridae from the twenty-eight bats in Tonghua City, Jilin Province were acquired and the positive rate was 33.3%. Nineteen bats of Rhinolophus ferrumequinum carried the bocavirus from bocaparvovirus from parvoviridae from the twenty-nine bats in in Tonghua City, Jilin Province were acquired and the positive rate was 65.5%. Myotis ricketti and Rhinolophus ferrumequinum in Liaoning province, and Murina leucogaster in Jilin province, and Vespertilio orientalis in Heilongjiang province had viral sequences which annotated to dependoparvovirus from parvoviridae.These viruses had the highest identity as bat adeno-associated virus YNM with 84%, 89% and 87%, and they were the same branch in phylogenetic analysis.The identification analyswas of reovirus, we found that viral sequence in Benxi city, Liaoning Province annotated to the orbivirus from reoviridae, and shared the 79% highest identity with Eubenangee virus after the analyswas. Nine bats of Myotis ricketti carried the orbivirus from reoviridae from the twenty-nine bats in Benxi City, Liaoning Province were acquired and the positive rate was 33.3%. At the same time, this reovirus of orbivirus genus was first detected in bats in the world.In addition, by the specificity of PCR detection, we acquired two circoviruses of circovirus genus in the bats of Heilongjiang province, one was detected in the forty-three bats of Vespertilio orientalis in Acheng district of Harbin city in Heilongjiang Province, and positive rate was 4.7%. The other one was detected in the nine bats of Vespertilio orientalis in Saertu district of Daqing city in Heilongjiang Province, and positive rate was 11.1%.We acquired the two bats of Murina leucogaster carried the paramyxovirus of Rubulavirus genus from the forty bats in Tonghua City, Jilin Province, and the positive rate was 5%. We also acquired the eight bats of Rhinolophus ferrumequinum carried the bunyavirus(Phlebovirus) from the twenty-nine bats in Tonghua City, Jilin Province, and the positive rate was 27.6%. Two coronaviruses were detected in Tonghua City, Jilin Province, one coronavirus was detected in the bats of Rhinolophus ferrumequinum and the member of alphacoronavirus and the positive rate was 2.5%. Others was detected in the bats of Murina leucogaster and the member of betacoronavirus and the positive rate was 3.5%.We selected the circovirus, bunyavirus, paramyxovirus and coronavirus to further research in view of the above identification results. We acquired the 2117 nt and 2113 nt genome sequences which were firstly found in notheast China, and found a stem loop structure in the origin of replication after genome prediction exist in two circoviruses and structure of poly(T) also exist in two circoviruses, and found that the circovirus we got had the highest identity and the same branch in phylogenetic analysis with fox circovirus of VS7100003, they were all the member of circovirus. We found that the sequnces of bat bunyavirus we acquired not only the nucleotide sequence but amino acid sequence was highest identity with bat bunyavirus of RpBunya V/Shaanxi2011,which was the firstly found in northeast China.For the research of bat paramyxovirus, we got 5318 nt genomic sequence which was used to the analysis, and we found it was the highest identity and the same branch with human parainfluenza virus type 4, and also the member of rubulavirus, which was firstly found in northeast China.Two coronaviruses were detected, and the complete sequences were also acquired.We found that bat coronavirus of JTAC2 was the highest identity and the same branch with bat coronaviru of Neixiang-14, they were all the member of alphcoronavirus. Others of JTMC15 was the highest identity and the same branch with bat coronaviru of JL2012, they were all the member of betacoronavirus. However, we also found that this bat coronavirus of JTMC15 was lack of complete gene of 8 gene, which was the out of ordinary.This study firstly used the viral megagenomic analysis to comprehensively survey viruses bats carried, and a large number of novel bat viruses were discovered, and twelve vertebrate viruses were identified include astrovirus, parvovirus, orbivirus, picornavirus, vesiculovirus, circovirus, bunyavirus, paramyxovirus,coronavirus and so on.Four viruses contain circovirus, bunyavirus, paramyxovirus,coronavirus were used to furtherly analyze, and the complete sequences of circovirus and coronavirus were acquired. Many viruses were firstly found in three northeastern provinces of China, such as astrovirus, parvovirus, picornavirus, vesiculovirus, circovirus, bunyavirus, paramyxovirus, alphcoronavirus and so on. In adition, it reflected the advantage of viral megagenomic analysisi that we could find the viruses in some bats by the viral sequence of annotation.And viral megagenomic played a powerful tool to find the novel viruses. These viruses was enriched in the database of bat viruses, and was provided the base to know the situation that bats carried the viruses and the diversity of bat viruses,even to evaluate the threats to public health.At the same time,we also acquired the data of bat habitat which provided the basci data for the later understanding bat habitat environment.