| Bovine norovirus(BNoV)belongs to the cup virus family,the RNA virus of norovirus genus,is one of the new pathogens of calf diarrhea in China.It has been widely prevalent in the breeding industry around the world,and has long been existed and rapidly spread in feces and digestive tract.BNoV high variability,more host and potential zoism risk is worth our in-depth study,its more in cows,beef cattle,and more recessive infection or mixed infection,although BNoV has been confirmed in China,but its epidemic in Yunnan region and molecular characteristics is still not clear,should cause the attention of the cattle industry.Therefore,an efficient detection method and molecular detection research for BNoV are established to provide a theoretical basis for the scientific prevention and control of BNoV.In this study,based on the analysis of the number of samples of BNoV conserved regions,BNoV common RT-PCR and Real-time RT-PCR methods were established to detect 655 cow dung stool samples collected from 15 cattle sites in 13 regions of Yunnan Province,and gene sequencing and genetic evolution analysis of some BNoV positive samples.The following results were obtained:(1)The common RT-PCR detection method for BNoV was established,and the minimum detection amount was 9.67×10~3copies/μL;25 positive samples were detected,with a positive rate of 3.82%.At the same time,the Real-time RT-PCR detection method of BNoV was established,and the lowest detection amount was9.67×10~0copies/μL;32 positive samples were detected,and the positive rate was4.89%.Both assays showed good specificity,sensitivity and stability.(2)BNoV detection results at different ages:BNoV detection rates of 1 week,1week to 1 month,1 month to 6 months and 1 year old were 15.00%(3/20),15.93%(18/113),1.87%(6/321)and 2.49%(5/201),respectively.The BNoV detection rates in diarrhoea samples and non-diarrhoea samples were 8.99%(25/278)and1.86%(7/377),respectively.The detection rate of BNoV in fecal samples of dairy cows,beef cattle and yak was 5.92%(32/540),0%(0/104)and 0%(0/11),respectively.(3)BNoV positive samples Rd Rp gene fragment amplification sequencing,6Rd Rp sequences,named BNoV-YN 01～06-2022,genetic evolution analysis with 33reference strains,shows that 6 Yunnan strains are in the same evolutionary branch,all belong to BNoV GIII.2 type;6 Yunnan strains have 94.0%～98.4%nucleotide identity.(4)13 specific primers were designed to perform whole gene amplification sequencing,named BNoV-YN01-2022 and BNoV-YN02-2022,7281bp and 7293 bp,including part of 5’UTR and 3’UTR and three complete open reading frames.With a typical BNoV genomic structure,a highly conserved nucleotide sequence of ATGAAGATGACTGA(5075 bp-5088 bp)is present at the junction of both the ORFl and ORF 2 overlapping regions.The genetic and evolution analysis with 33 strains showed that the nucleotide identity was 97.6%between the two strains,including GIII.2.According to the recombination prediction analysis of RDP 4 software,only BNoV-YN 02-2022 strains had genetic recombination in the ORF 3(VP2)region,and the parents were mainly Xinjiang and Norway in China.The prediction of GIII.2original strain Bo/Newbury2/1976/UK capsid protein and amino acid site mutation analysis show that the BNoV-YN01-2022 capsid protein is similar to the original strain capsid protein,BNoV-YN02-2022 with an altered capsid protein surface possibility,protein hydrophilic character,and antigen index,This alteration corresponds to amino acid site mutations,Mainly concentrated in the P hypervariable regions,The above results reveal that BNoV perhaps evolved through this recombination and mutational adaptation,Lay the foundation for the subsequent application of immunodetection methods,For a deeper study of the relationship between protein structure and function. |
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