Epidemiology Of Multiple Viruses In Rectal Samples From Bats

Bat is big virus reservoir,which novel virus number is refreshing annually to add to the constitution of the storage.Digestive tract,an open orifices,connecting bat and the outside environment,offer a possibility for disease transmission.Previous studies reported adenovirus,herpesvirus,rotavirus,palpillomavirus and calicivirus in bats in other countries,however,the bat species collected in the reported studies is limited.Surveillance of virus in different bat species from different geographic region at regular interval,is essential to better understand the bat-virus relationship and the phylogenetic information of bat virus.Regarding that the epidemiological information of the above viruses in China is still lacking,we aim to conduct an epidemiological and phylogenetic study of adenovirus,herpesvirus,rotavirus,palpillomavirus,picornavirus and calicivirus in the fecal and rectal samples from bats collected in Guangzhou,Huizhou,Yunfu city in Guangdong province and Haikou city in Hainan province.Bat plays an important role in the virus storage and transmission.Previous studies have identified numerous virus in brain,intestine,lung,kidney,liver and blood sample from bats.Specific virus detection in previous studies were mainly depended on traditional methods,including cell culture isolation,electron microscope,serological test,PCR,ect.However,these methods have certain limitations for novel virus identification,especially when the information of the virus sequences is unknown.The application of the next generation sequencing-based viral metagenomics largely prompted the identification of novel viruses,which could help better understand the taxonomy and abundance information of the viruses in bats,and facilitate the identification of the emerging pathogens of infection.To date,seven studies of bat virome analysis have provided valuable data about the diversity of viruses in bats.Four viral metagenomics studies in China were conducted based on multiple pooled tissues RNA extraction,library construction and next generation sequencing.However,the pool sample detection might reduce the positive detection rate of certain viruses,and neglect the "real" infected tissue with viruses.Knowing the virus-enriched tissue could better understand the possible transmission routine of the viruses in bats.In addition,virus detection in individual sample takes heavy workload and is influenced by the sensitivity of conventional detection method.This might lower the efficacy of discovering the novel viruses.Hence,we aim to construct the method of viral metagenomic analysis in six bat fecal/rectum/rectal swab samples in southern China.1 Methods1.1 Bat samples collectionBetween July 2011 and August 2014,520 samples were collected from 9 sites including residential areas,city parks,abandoned houses and mine caves in Hainan(Haikou city)and Guangdong province(Huizhou,Guangzhou and Yunfu city).Rectal swabs or rectums of each bat were collected,and if available,fresh fecal samples,were harvested.Bat species identification was confirmed by amplification and sequencing of the cytochrome B(cytB)gene,in combination with morphological1.2 Molecular detection of six common viruses in batsThe RNAs of 520 bat fecal and retrum samples were extracted and reversed into cDNA.DNA extraction was conducted for 520 bat fecal/rectum/rectal swab samples.Nested-PCR and PCR of the cDNA and DNA were amplified to detect rotavirus,picornavirus,calicivirus,adenovirus,herpesvirus and palpilomavirus.The products of PCR were separated by electrophoresis on 1%agarose gels stained with ethidium bromide.Eligible PCR products were sequenced directly.The PCR products were analywith low concentration were gel purified for sequencing.Sequences were ananlyzed by multiple bioinformatics method,including multiple sequence alignment,phylogenetic tree construction and amino acid/nucleotide identity calculation.13 Viral metagenomics methodsSix pooled samples including GZ.CS(Cynopterus sphinx in Guangzhou),HN.MS(Miniopterus schreibersi in Hainan),HN.RL(Rousettus leschenaulti in Hainan),HZ.HL(Hipposideros larvatus in Huizhou),YF.RB(Rhinolophus blythi in Yunfu)and YF.SK(Scotophilus kuhlii in Yunfu)were generated based on the geographic and bat species characteristic.Each pooled sample including 10 to 15 bat fecal sample were treated with removing background nucleic acid,virus DNA/RNA extraction,double-stranded cDNA(dc-DNA)conversion,random PCR and sequencing.Based on the viral metagenomitcs results,eligible sequences of Retrovirus(ReVs)and adenovirus(AdVs)were preliminarily analyzed by multiple sequence alignment,phylogenetic tree construction and amino acid/nucleotide identity calculation.2 Results2.1 Molecular detection of six common viruses in batsOf 520 bat fecal/rectum/rectal swab samples,the positive detection rate of herpesvirus,adenovirus,papillomavirus and rotavirus is 73%、6.9%,2.1%and 0.76%,respectively.None of the calicivirus and picornavirus was found.Bat HVs in this study were clustered into two subfamilies of Betaherpesvirinae(4 isolates)and Gammaherpesvirinae(69 isolates).None of the Alphaherpesvirinae was detected in the present study.Phylogenetic analysis of HVs gB and DPOL genes revealed that a high molecular diversity of HVs was present in bats from different species and geographic regions.Our study indicated the co-evolution between bats and HVs.Thirty-six bat adenoviruses(AdVs)detected in this study belonged to the Mastadenovirus,sharing low amino acid identity with that from human and other animals(48.3-80.6%).Eleven bat papillomaviruses(PVs)shared high amino acid identity(94.5%-100%)with each other,compared with previously reported PVs from bat and other animals(34.5-58.9%).Four rotavirus A(RVAs)in Scotophilus kuhlii shared the highest amino acid identity in VP7 gene with a simian and dog RVA(80.2%),which could be assigned as a tentative G3 genotype strain.2.2 Metagenomic analysis of NGS resultsA total of 108,443,256 reads were obtained in this study,among which 18,261,633 were matched with viral sequences.The remaining 90,181,623 reads could be classified as cellular,archaea and other unclassified vial reads.13,466,976 reads could be assembled into contigs,with an average length of 182bp.There were 5496 contigs longer than 500bp,with an average length of 776bp.The datasizes between each groups ranged from 8,191,142 to 35,722,372 reads,with the biggest datasize of HN.RL and the smallest of YF.RB.There were 1412 contigs of Deltaretrovirus,which accouted for the most viral associated contigs.The annotation of these viral contigs were most similar to Pteropus vampyrus endogenous retrovirus group K pol gene,sharing the identity of 90%.Contigs of Phlebovirus-,Tospovirus-,Rotavirus A-,Mastarenavirus-,Picobirnavirus-,Simplexvirus-,Varicellovirus-,Mastadenovirus-,Circovirus-,Mamastrovirus-,Coronavirus-,Orthohepadnavirus-,Reoviridae,I_flavirus-,Brcacovirus-,Bracovirus-,Orthopoxvirus,Orthopoxvirus,Vesiculovirus-,Chrysovirus-and Phage shared the identity of more than 80%with the existing sequences in the database.The rest contigs including Alphapapillomavirus-,Deltarefrovirus-,Gammaretrovirus-,Alphare trovirus-,Lymphovirus-,Cytomegalovirus-,Ictalurivirus-,Chlorovirus-and Cucumovirus-shared the identity of less than 70%,when compared with the existing sequences.The abundance and tanxonomy of different viruses have their own geographic characteristics.2.3 A preliminary analysis of retrovirus(ReVs)and adenovirus(AdVs)The ReVs sequences of HN.RL group included polymerase/polyprotein(Pol)and envelope protein(Env),which shared the most similar identity of 78-90%with Pteropus vampyrus Re Vs.All of the retroviral contigs encoding Env protein contained stop codons within the translation region.The phylogenetic trees indicated that the longest pol sequence in this study clustered with Pteropus vampyrus endogenous ReVs,sharing the highest amino acid identity of 85.7%.The phylogenetic tree based on the E4orf1 genes indicated that contigs of of GZ.CS and HN.MS clustered with human AdV-C,sharing the identity of 94.1-100%.Further phylogenetic analysis of IVa2 gene revealed that contig of AdV in HN.MS fell within the lineage of Human adenovirus C,sharing the identity of 100%.3.Conclusions3.1 Scotophilus kuhlii,Hipposideros larvatus,Emballonuroidea and Cynopterus sphinx had been tested for the carriage of HVs for the first time,expanding the taxonomy of bat species in bats.The high prevalence rate,coupled with the low virulence and high diversity of HVs in bats,suggesting that there is a co-evolution between bats and HVs.In addition,Bovine herpesviruses 6 from Macavirus genus fell within SK/11HZ85,CS/13HN70 and RB/13YF87,sharing high identity in the DPOL and gB regions with bat HVs in our study,suggesting potential interspecies transmissions3.2 We identified diverse AdVs in bats based on the molecular detection and phylogenetic analysis,among which novel AdVs tested positive in Cynopterus sphinx for the first time.Our study results further indicated that bat AdVs and human AdVs are species-specific.There is no evidence of cross-species transmission of AdV between bats and humans based on the current data.Further studies that include more detailed genomic sequence information from bat and human AdVs,coupled with additional epidemiological investigation,might offer greater insights.3.3 PVs in Scotophilus kuhlii and Rhinolophus blythi were tested positive for the first time,which were classified as the tentative member of a novel,yet unnamed PV genus,according to the recent papillomavirus classification criteria.Highly species-specific and co-evoluted characteristic of PVs did not present in this study,as two Scotophilus kuhlii and nine Rhinolophus blythi PVs clustered in the same clade,sharing high similarity.3.4 Four RVAs identified in this study could be assigned as tentative G3 genotypes,based on the criteria of RCWG.3.5 None of the calicivirus and picornavirus tested positive in the 520 fecal/rectum/rectal swab samples in bats.This may have been due to limited sample sizes,insensitive detection method and low infected rate of CV/PiV in this study.3.6 The viral metagenomic method based on the bat fecal/rectum/rectal swab specimens were established.The virome of bat fecal/rectum/rectal swab specimens were identified,including viruses from vertebrate animals,insects,plants,archaea,fungi and alga.Possible novel viruses were found,including Alphapapillomavirus,Gammaretrovirus,Betaretrovirus,Lymphovirus,Cytomegalovirus,ect.Different viruses presented different geographic characteristics.3.7 A preliminary analysis of ReVs was conducted based on the pol gene,providing possible evidence of cross-species transmission between ReVs from Rousettus leschenaultii and Pteropus vampyrus.Distant phylogenetic relationship could be observed between multiple bat species,indicating that bats are suitable vectors for ReVs transmission.