| Bovine viral diarrhea virus（BVDV）is a positive-stranded RNA virus belonging to the genus Pestivirus of the Flaviviridae family,affects many animals,which caused respiratory and digestive diseases.It is also common in herds and its surrounding environment.BVDV can cause persistent infection and immunosuppression.The serum antibodies of persistent infected animals are negative,but they are poisonous and detoxify for life,thereby resulting in major economic losses in the cattle and other animal industries.In the Qinghai-Tibet Plateau,yak infected BVDV can causes diarrhea,reduced fertility,growth retardation and secondary infection with other diseases.However,the prevalence of BVDV in yak in the Tibetan Plateau is not fully understood.Therefore,it is of great significance to master the epidemic situation of BVDV and the genomic characteristics of yak BVDV to prevent and control the disease.In this study,a molecular epidemiological investigation of BVDV was performed in diarrheal diarrhea and clinically healthy fecal samples from some regions of the Tibetan Plateau by RT-PCR.BVDV strains were isolated from fecal samples and the whole gene were obtained by RT-PCR,the results obtained are as follows:1.Molecular epidemiological survey and phylogenetic analysis of Yak BVDV in parts of the Qinghai-Tibetan PlateauTo investigate the prevalence and dominant subtype of BVDV in yaks,a total of390 fecal samples were collected from yaks with diarrhea and clinically healthy in the Qinghai-Tibetan Plateau（Tibet,Yunnan,Qinghai,and Sichuan）from 2015 to 2016.RT-PCR was used to target the 5’untranslated region（UTR）,which determined a prevalence of 25.52%（95%confidence interval（CI）=19.5%-32.39%）in 192 animals with diarrhea,and 13.13%（95%CI=8.8%-18.6%）in 198 clinically healthy Yaks.This suggested that BVDV infection was a potential factor of diarrhea in yaks in the Qinghai-Tibet Plateau.To identify the subtype of BVDV of the Qinghai-Tibet Plateau from 2015-2016,we randomly selected 35 positive samples（14 from clinically healthy yaks and 21from animals with diarrhea）for PCR amplification and the sequencing results were compared with the reference sequences in GenBank and constructed based on 5’-UTR,Npro and E2 genes.The 5’-UTR shared 71.6%-100%nt identities with each other,69.1%-90.9%with other BVDVs.The Npro shared 71.6%-100%nt identities with each other,69.1%-90.9%with other BVDV strains.The nucleotide and amino acid sequences of Npro gene were 72.8%-100%and 74.7%-99.3%,respectively.The nucleotide and amino acid homology of Npro gene sequence with other BVDV strains were 74.9%-87.5 and82.3%-89.0%,respectively.The nucleotide and amino acid sequences of E2 gene were 68.2%-99.9%and 66.8%-99.5%,respectively.The nucleotide and amino acid homology of E2 gene sequence with other BVDV strains were 68.2%-97.8%and67.4%-96.0%respectively.The phylogenetic tree was constructed based on 5’-UTR showed that samples were branched into an independent phylogenetic cluster and were more closely related to BVDV-1d strain 10JJ-SKR（Fig.2a）,indicating that the 31 samples belonged to BVDV-1d.Samples of both the Z3 and Z6 isolates were grouped within the BVDV-1a strain GS5 cluster,but were located in a unique lineage,whereas samples of both CB7and FB3 isolates were closely related to BVDV-1b stain Osloss.Thus,these findings demonstrated that the 35 BVDV-positive samples belonged to BVDV-1a（n=2）,BVDV-1b（n=2）,and BVDV-1d（n=31）.We showed that phylogenetic analysis of Npro nucleotide acid sequences and other BVDVs produced similar findings to the analysis of the 5’-UTR analysis.The results show that BVDV-1d is the predominant subtype of BVDV among calves in parts of the Tibetan Plateau from 2015 to 2016.2.Isolation and identification of two strains of cytopathogenic BVDV strainsTo isolate the virus,thirty-five of BVDV-positive feces were processed and used to inoculate the MDBK cells.After 3 generations of blind transmission,all samples were positive by RT-PCR.After 7 passages,both specimens of Z6 from Sichuan region（BVDV-1a）and DJ2 from Qinghai province（BVDV-1d）were observed the typical CPEs in about 70 hr.It showed that the infected cells had a rounded appearance with an increased refraction in the early stage of the lesion.Viruses were purified three times by plaque assay and RT-PCR test positive for BVDV.The isolated viruses were initially identified as two cytopathic（cp）BVDV strains SMU-Z6/1a/SC/2016 and SMU-DJ2/1d/QH/2015.The titers of the two isolates were calculated by the Reed-Muench method.The results showed that the TCID50/100μL of the 9 passages strains of the two isolates SMU-Z6/1a/SC/2016 and SMU-DJ2/1d/QH/2015 were 10-8.11/100μL and 10-5.81/100μL,respectively.To confirm the isolated virus,the indirect immunofluorescence assay（IFA）was performed by BVDV polyclonal antibody（VMRD,USA）.The specific immunofluorescent signals were detected in the cytoplasm of infected cells using BVDV positive serum antibody,whereas no immunofluorescent signal was detected in the mock MDBK cells.The isolated viruses were designated as SMU-Z6/1a/SC/2016 and SMU-DJ2/1d/QH/2015,which belonged to cytopathic（cp）BVDV strains.3.Genome amplification and phylogenetic analysis of yak BVDV isolates SMU-Z6/1a/SC/2016 and SMU-DJ2/1d/QH/2015To further understand the genomic information of the purified viruses,we designed 14 different pairs of primers to amplify the SMU-Z6/1a/SC/2016 and SMU-DJ2/1d/QH/2015.The complete genome of SMU-Z6/1a/SC/2016 was 12,178bp in length,with a 47.0%G+C content.Based on the sequence analysis,the virus had a large ORF of 11,703bp,which encoded a 3,884 aa long polyprotein precursor,and was flanked by a 300 bp long 5’-UTR and a 175 bp long 3’-UTR.In addition,the SMU-DJ2/1d/QH/2015 was 12,255 bp in length,with a 45.6%G+C content,which contained one ORF encoding a 3,897 aa,and was flanked by a 374 bp long 5’-UTR and a 190 bp long 3’-UTR.The genome organization was similar to other BVDV,containing 4 structural proteins and 7 non-structural proteins.Compared to that of other BVDV strains,the pairwise nt/aa identities of SMU-Z6/1a/SC/2016 and SMU-DJ2/1d/QH/2015 genomes ranged from 68.4%-89.8%/67.1%-84.4%and71.8%-97.3%/70.7%-90.5%for ORF,respectively.In the BVDV genome,the E2 glycoprotein coding region is considered as one of the least conserved regions and plays a key role in inducing an immune response against viral infections.Compared to the coding sequence of BVDV-1a strains,the E2glycoprotein coding region of SMU-Z6/1a/SC/2016 exhibited the highest degree of divergence and shared 78.6-88.8%nt/72.9-78.3%aa identities.In SMU-DJ2/1d/QH/2015,the E2 had 92.7-97.8%nt/95.7-96%aa identities to other BVDV-1d strains.These results suggested that the antigenicity of SMU-Z6/1a/SC/2016 might have the obvious difference to other BVDV-1a strains,indicating that the virus may be a novel virus.Furthermore,the NS3 region of SMU-Z6/1a/SC/2016 exhibited obvious variety compared to other BVDV-1a.We identified 109 nucleotide acid mutations in the NS3 gene of SMU-Z6/1a/SC/2016 strain,consisting of 49synonymous mutations and 62 non-synonymous mutations,leading to 47 aa changes and one aa deletion.To determine the genetic relationship of the two isolates,phylogenetic analysis was performed by analyzing the complete genomic and E2 sequences of two strains.The results indicated that SMU-Z6/1a/SC/2016 was clustered within the BVDV-1a strain GS5 cluster,and SWU-DJ2 was grouped within the BVDV-1d strain BJ1201cluster but were located in a unique lineage.The homologous relationship between the SMU-Z6/1a/SC/2016 isolate and the BVDV-1a OregonC24V and NADL was on the same branch,while the BVDV OregonC24V and NADL strains were both highly virulent CP type BVDV standard virus strains. |
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