Metagenomic Identification And Molecular Characteristics Of Viral Community In Diarrheal Feces Of Tibetan Pigs

Tibetan pigs are a unique plateau local pig breeds in the agricultural areas and semi-agricultural and semi-pastoral areas of the Qinghai-Tibet Plateau at an altitude of 2500 m-4300 m.Tibetan pigs are lean minipig breeds with more primitive grazing and feeding,have extremely strong adaptability to harsh climates at high altitudes,and are resistant to disease and roughage.Tibetan pigs graze in the field all year round,and in the process of mixed feeding with cattle and sheep,each animal species contacts each other,which provides an opportunity for cross infection of the virus to a certain extent.At the same time,in the daily grazing process,close contact with the population,it is also possible to cause the cyclic transmission of some zoonotic diseases between humans and animals.In this study,we first used metagenomics to identify the virus community in the diarrheal feces of Tibetan pigs,then analyzed the molecular characteristics of related viruses in the diarrheal feces of Tibetan pigs,and isolated and identified PEDV from Tibetan pigs.The results obtained were as follows:1.Identification of viral community in diarrheal feces of Tibetan pig by metagenomics.In order to further understand the viral community in diarrheal feces of Tibetan pigs,146 diarrheal feces samples were collected from 16 Tibetan pig farms in Ganzi Tibetan autonomous prefecture,Sichuan province,and the samples were mixed into a pool sample.RNA was purified and synthesized into cDNA.TruSeq Illumina sequencing was used to analyze the sequences and molecular characteristics of the diarrhea-associated viruses.The results showed that the virus species in the fecal samples of diarrhea included 19 viruses from 11 families.They were mainly linear and cyclic small DNA viruses,such as porcine stool-associated circular virus 7,porcine adenovirus and porcine bocavirus(PBoV).There are three diarrheal viral pathogens,such as porcine epidemic diarrhea virus(PEDV),porcine circovirus type2(PCV-2)and bovine viral diarrhea virus type 1(BVDV),as well as three new pathogens,including porcine bufavirus(PBuV),rabovirus and pasivirus.The complete or nearly full-length genome of PPV-6,PCV-2 and PBoV-2 and complete ORF2 gene sequence of HEV were assembled by SOAP software.The phylogenetic results showed that PPV-6 and PCV-2 had a relatively close genetic and evolutionary relationship with the reference strain,while PBoV-2 had a relatively distant genetic and evolutionary relationship with the reference strain,which were gathered into a single cluster,and may be a completely new genotype.In this study,it was found that the viruses in diarrhea feces of Tibetan pigs are complex and varied,and may be cross-infected with other animals and humans,which has important public health significance.Therefore,the study will also provide a theoretical basis for prevention and control of diarrhea in Tibetan pigs.2.The molecular characterization of PCV-2 and PBuV from Tibetan pigs.In order to further investigate the detection rate of PCV-2 in diarrhea feces of Tibetan pigs,146 samples were collected and the detection rate was 10.96%(16/146,95%CI:6.4%-17.2%).Three complete PCV-2 sequences,in length both of 1767 bp,were successfully amplified from the positive samples,and Gen Bank numbers were MK990814-MK990816,shared 98.4%-99.5%nucleotide identity with each other,and shared 96.9%-99.9%nucleotide identity with the reference strains.Moreover,the three strains were closely related to the epidemic PCV-2 strains in the Sichuan area.These results showed that the existence of PCV-2 in the Tibetan pig population,which may be associated with the introduction and improvement of Tibetan pig quality and the trade with the mainland,needs to be confirmed by further study.In this study,PCV-2 was first detected in Tibetan pigs,and analyzed the complete genomes,showing a unique trend of genetic evolution,which enriched the PCV-2virus spectrum and provided some information for understanding the genetic evolution of PCV-2.Bufavirus(BuV),an emerging zoonotic virus,has been reported in many mammals and humans.In order to further understand the prevalence of PBuV in Tibetan pigs,146 diarrheal fecal samples were collected from Tibetan pigs,6.85%(10/146,95%CI:3.3%-12.2%)of the diarrhoeic samples were detected as PBuV-positive by RT-PCR assay.10 PBuV NS1 gene sequences were successfully obtained,shared 95.6%-96.3%nucleotide identity with each other,and shared90.7%-99.7%nucleotide identity with the reference strains of pig source.Two complete PBuV genomes(GenBank numbers were MN868670-MN868671)were successfully sequenced with 4189 bp in length,shared 96.0%nucleotide identity with each other and 94.9%-98.3%nucleotide identity with the reference strains of pig source,and shared 52.2%-63.6%nucleotide identity with the reference strains of other species.In this study,PBuV was first detected in Tibetan pigs,and amplified the NS1 gene and the complete genomes sequences,found a unique trend of genetic evolution and aboundantabundant genetic diversity for PBuV in Tibetan pigs.It is worthy of continuous monitoring and special attention to control,which enriched the basic research data of PBuV,and provided a reference for further study of the genetic evolution trend and pathogenic mechanism of PBuV.3.The molecular characteristics,isolation and identification of PEDV from Tibetan pigs.The purpose of this study was to investigate the prevalence and genetic evolution of PEDV in Tibetan pigs.Here,193 fecal samples were collected from Tibetan pigs on13 farms in the Qinghai-Tibet Plateau,China.38.34%(74/193,95%CI:31.5%-45.6%)of the samples were detected as PEDV-positive by RT-PCR assay.The detection rate of PEDV was 42.64%(55/129)in 129 diarrhea samples,while the detection rate of PEDV was 29.69%(19/64)in clinically healthy pigs.Four complete PEDV genomes(Gen Bank numbers were MK140811-MK140814)were successfully sequenced with 28022 bp-28036 bp in length.The genomes of the four strains shared 98.7%-98.9%nucleotide identity and 98.2%-98.6%amino acid identity with other reported complete genome sequences of PEDV.Significantly,the S gene of the four strains in this study shared 93.4%-94.9%amino acid identity with the strain CV777,which were the lowest of the seven ORFs of PEDV.Otherwise,a recombination event was identified within the region of S2,ORF3 and E genes in the four strains in this study.This is the firstly report of the crossover regional recombination event in PEDV genome.These results not only enhance the understanding of PEDV genetic evolution,but also indicate that PEDV variants have been emerging in Tibetan pigs.In order to better understand the molecular characteristics and genetic evolution of PEDV from Tibetan pig,one PEDV strain,resulting in cytopathic effect(CPE)in Vero cells,was successfully isolated,named SMU-TP-H3.The virus was identified by RT-PCR assay and indirect immunofluorescence.The virus titer of the strain after plaque purification was 10^5.3TCID₅₀/mL.Therefore,the PEDV of Tibetan pigs isolated in this study,contribute to further understanding the molecular characteristics and genetic evolution of PEDV of Tibetan pigs,and provide an important resource for further studying the biological characteristics of PEDV from Tibetan pigs.