Detection And Molecular Characteristics Of Bovine Nebovirus In Some Provinces Of China

Nebovirus(NeV),a spherical and non-enveloped,single-stranded RNA virus,belongs to genus Nebovirus of family Caliciviridae,which is one of the important pathogens causing diarrhea in calves.Since it was discovered in Britain in 1976,it has been detected in 13 countries,with a wide geographical distribution.From 2018 to 2019,our team confirmed that the virus has been widely circulating among dairy cows and yak in China with a unique evolution trend.To date,three RdRp types(NB-like,NA1-like and SC-yak)have been identified based on the RdRp nucleotide sequences,and three VP1 types(genotype 1-genotype 3)have been identified based on all available complete VP1 amino acid sequences,where genotype 1 was further sub-classified into four phylogenetic lineages(genotype 1.1-genotype 1.4).The RdRp genotypes of endemic NeV strains in China were NB-like genotype and SC-yak genotype.The genotypes of VP1 include genotype 1(including genotype 1.1,1.3 and1.4),genotype 2 and genotype 3,indicating that there is abundant genetic diversity in China.Because of NeV is an emerging causative agent in China,the prevalence and molecular characteristics of NeV deserve further investigation.The purpose of this study was to monitor and investigate the prevalence of NeV in China and analyze its molecular characteristics,and the results as shown as follows:1.Detection and molecular characteristics of NeV in some provinces of China The purpose of this study was to investigate the prevalence and molecular characteristics of NeV in some provinces of China.177 diarrhea fecel samples of dairy calves from 20 cattle farms in 4 provinces and 99 diarrhea fecel samples of yak from 8 pastures in Sichuan province from 2019 to 2020 were detected by RT-PCR assay.The results showed that the positive rates of NeV in dairy calves from Ningxia Hui Autonomous Region,Henan,Sichuan and Yunnan were 11.32%,8.62%,46.88%and 38.24%,respectively.The positive rates of NeV in yaks from Sichuan Province were 4.04%.Phylogenetic analysis of 4 RdRp gene fragments(396bp)from yaks showed that the 4 NeV fragments clustered into three large branches.One strain clustered into the NB-like genotype,one strain clustered into the SC-Yak genotype,and the remaining two strains were clustered with four yak NeV strains from Norgay county(MK452075-MK452078)into an independent branch that was distinct from known strains of genotypes,indicating they may represent a novel RdRp genotype.21 complete VP1 and RdRp sequence of positive samples of NeV was successfully sequenced.Phylogenetic analysis of 21 complete VP1 amino acids showed that 4strains clustered into genotype 1.2 and the remaining 17 strains clustered into genotype 1.2.Phylogenetic analysis of 21 complete RdRp nucleotide showed that 21 strains clustered into NB-like genotype.This study successfully obtained 4 complete VP1 and RdRp sequences of genotype 1.2 strains and 17 complete VP1 and RdRp sequences of genotype 1.1 strains of dairy calves samples,which is firstly detection of VP1 1.2 genotype NeV strains in dairy calves in China.A possible novel RdRp genotype strain was found in yaks,indicating the NeV RdRp gene fragment of yaks in China had rich genetic diversity.These results provided useful information for understanding the molecular characteristics and genetic evolution of NeV in China.2.Genomic characterization of a nebovirus strain with a novel RdRp genotype In the previous study,two positive samples with the potential novel RdRp genotype were identified in yaks.In order to further understand the molecular characteristics of this novel RdRp genotype strain,genome amplification was performed on the two positive samples,and one complete genome of NeV was successfully sequenced and named YAK/NRG-A9/19/CH.The results showed that the length of the genome of YAK/NRG-A9/19/CH is 7454 nt in length,with 53.17% G + C content.The ORF1 length of YAK/NRG-A9/19/CH strain was shorter than other NeV genomes,and the5’-UTR and 3’-UTR of YAK/NRG-A9/19/CH strain were different from other NeV genomes,with 80 nt and 65 nt in length,respectively.The complete genome of YAK/NRG-A9/19/CH shared 68.3%～79.7% nt identity with the other 8 NeV genomes in Gen Bank.In a phylogenetic analysis of NeV genomes,strain YAK/NRG-A9/19/CH is closely related to SC-yak strains but is located in a unique branch.Otherwise,the VP1 of the YAK/NRG-A9/19/CH strain belongs to genotype 3.Compared with the other two genotype 3 strains,YAK/NRG-A9/19/CH strain shared 84.9% and 88.8% aa identity in P2 domain,with 18 unique amino acid mutations.One interesting characteristics was found in the genome: the RdRp sequence of YAK/NRG-A9/19/CH strain differed greatly from known RdRp genotypes(NB-like,NA1-like and SC-yak),and genetic analysis of its complete RdRp sequence showed that this strain is located in a unique branch,indicating it may represent a novel RdRp genotype,suggesting named SC-yak-like 1.These results of this study provide useful information for further understanding the molecular characteristics and genetic evolution of NeV.