Epidemiological Investigation,Isolation And Identification Of Yak Enterovirus And Transcriptomics Research Of Infection MDBK Cells

Bovine enteroviruses(BEVs)were members of the family Picornaviridae and genus Enterovirus(EVs).BEVs were non-enveloped icosahedral viruses that enclosed a single copy of positive-sense RNA.BEVs were presented in a variety of animals and could cause diseases of respiratory system and digestive system.BEVs usually caused co-infection or secondary infection with other viruses and bacteria,which led to the boost of various animal diseases.The aim of this study was to establish an RT-PCR method for the epidemiological investigation of EVs in yak diarrhea and healthy samples in some areas of the Qinghai-Tibet Plateau,and to identify an EV strain from one yak diarrhea sample.The complete genome sequence of the strain was obtained by PCR amplification,and analyzed its genetic evolution relationship.The transcriptomics analysis was used to study the mRNA transcription expression regulation of the strain in the viral initiation and persistent infection in MDBK cells,and to analyze the interaction between pathogeny and cells according to the mRNA level.The results were as follows: 1.Epidemic virological investigation and isolation of EVs in yakIn order to investigate the prevalence of EVs in 235 yak diarrhea samples and 116 yak healthy samples from Tibet,Qinghai,Sichuan and Yunnan,this study established a RT-PCR method for the test of EVs in yak feces.The method had the advantages of high sensitivity and low detection limit of 0.0876 pg/μL(2.65×103 copies),and had good stability and specificity.The results were as follows: a large-scale surveillance study indicated that the prevalence of EVs in yaks was 31.06%(95% CI = 25.2%-37.4%)in 235 animals with diarrhea and 24.14%(95% CI = 16.7%-33%)in 116 healthy yaks.However,there were no significant differences in virus prevalence between diarrheal and healthy samples.Interestingly,in the Tibet region,diarrheal feces had a higher incidence of EVs than feces of healthy yaks(odd ratios = 6.03,95% CI = 1.93%-18.86%),indicating that the incidence of EVs was potentially correlated with the clinical symptom of yak diarrhea.An EV was successfully isolated from one yak diarrheal fecal sample.The purified virus was obtained and typical CPEs were appeared at 16-18 h.The TCID50 was 10-7.02/mL.Electron microscopy showed that the size of the strain was 25-28 nm,which was spherical and conformed to the reported EVs.The strain was named as SWUN-AB001.These results would provide new data support for domestic EVs epidemiology.2.Genome sequencing and analysis of the yak EV SWUN-AB001 strainAccording to the 14 BEVs genome sequences published in GenBank,we designed 15 pairs of primers by Primer 5.0 software.The complete genome sequence of SWUN-AB001 was amplified by PCR,which were assembled and spliced by BioXM software and DNAMAN software.The complete genome of SWUN-AB001 was 7,382 bp in length and the content of G+C% was 51.1%.The structural characteristics were consistent with the reported EVs.Phylogenetic analysis of ORF,5’UTR,VP1,P1,P2 and P3 region were established by MEGA 6.06 software.The SWUN-AB001 was located in a unique lineage,and the strain had the closest genetic relationship with BEV-F strain PS87(GenBank accession NO.DQ092795).The amino acid and nucleotide homology shared 72.8%/76.9%(P1),77.6%/91.9%(P2),79.1%/94.8%(P3)and 71.1%/79.2%(VP1)with BEV-F strain PS87.Within Enterovirus genus,VP1 nucleotide and amino acid sequences provided an important source of information for EV classification,especially for the differentiation of serotype or genotype.For demarcation of a new serotype,values of <75% nucleotide identity and <88% amino acid homology have been employed as yardsticks.Therefore,the virus was believed as a candidate new type EV-F7 based on International Committee on Taxonomy of Viruses(ICTV).Further analysis showed that one possible interserotypic and intraserotypic recombination event occurred in VP1 region and the predicated recombination breakpoints were mapped at nucleotide positions 2594 and 2625 by the GARD.The SWUN-AB001 virus was identified as a new subtype in EVs,which could help to further study the epidemiological characteristics of EVs in the Qinghai-Tibet Plateau.3.Transcription analysis to yak EV-F7 SWUN-AB001 strain infection in MDBK cellsIn order to further analyze the pathogenic mechanisms of yak EV-F7 SWUN-AB001 strain,this study investigated transcription mRNA expression of the viral initiation and persistent infection in MDBK cells.In this study,to select the viral initiation and persistent infection periods,MDBK cells were infected with SWUN-AB001 strain at a MOI of 0.01 for 6 h,12 h,24 h and 36 h by immunofluorescence assays,and the accuracy was verified by qRT-PCR.The RNA-Seq was used to analyze the transcription of MDBK cells were infected in the viral initiation and persistent infection periods,and to study differential expressed genes in different infection times.The accuracy of differential expressed genes was verified by qRT-PCR.The immunofluorescence results showed that infection of MDBK cells in 12 h initiation and 24 h persistent infection.This study used qRT-PCR to verify the viral initiation and persistent infection from differences in viral content.This experiment obtained the average of 23,951,730 and 23,952,446 clean reads in EV-12 h and EV-24 h libraries of infection,and average of 23,952,280 and 23,952,647 clean reads in Con-12 h and Con-24 h libraries of control groups.In 12 h viral initiation,only 19 genes showed unique significant differential expressed,including 12 up-regulated and 7 down-regulated genes.A total of 1050 differential expressed genes were identified during 24 h persistent infection in MDBK cells,including 103 up-regulated genes and 947 down-regulated genes.In many differential expressed genes,a variety of cytokines and chemokines were found during viral infection,including IL-6,IL-11,LIF,GM-CSF and CSF2.The qRT-PCR analysis confirmed the tendency detected by the mRNA sequencing analysis.The 12 differential expressed genes were overlapped that expression genes of 10 were up-regulated and 2 were down-regulated between 12 h and 24 h infected SWUN-AB001 in MDBK cells.Furthermore,GO enrichment analysis showed that only 14 differential expressed genes had GO annotations in 12 h infection,which were mainly mapped to biological processes.There were a total of 697 unigenes had GO annotations in 24 h infection,which were mainly mapped to molecular functions,cellular components and biological processes.KEGG analysis on differential expressed genes revealed that only the 4 and 2 KEGG pathways were assigned in up-regulated and down-regulated genes in 12 h infection.In 24 h infection,down-regulated differential expressed genes were assigned to 33 KEGG pathways,including disease,metabolism,and organismal systems;the up-regulated differential expressed genes were assigned to 31 KEGG pathways and mainly focused on diseases and signal transduction,including MAPK involving in viral replication.The western blot results showed that yak EV-F7 infection had obvious increase of phosphorylation of JNK/SAPK and p38 MAPK in 24 h infection.The results indicated that yak EV-F7 infection activated MAPK pathways in MDBK and might leads to increase of viral yield and proinfammatory cytokine secretions.Therefore,these results provided a basis for further study of infection mechanisms of yak EV-F7 and potential molecular relationship between viral and host components.