| Newbury-1 virus,usually expressed by Nebovirus(NeV),is the sole member of genus Nebovirus in family Caliciviridae currently.NeV is a single-stranded positive-strand RNA virus and is a causative agent of calf diarrhea.Until now,NeV has been detected in 12 countries including the United States,the United Kingdom,Brazil,Turkey,Japan,etc.,which suggested that NeV has been widely distributed around the world.Based on the RdRp fragment sequence,NeV can be divided into three genotypes,NB-like,NA1-like and Dijon A216-like.Based on the complete VP1 sequence,NeV can be divided into two genotypes(genotype 1 and genotype 2),and genotype 1 can be further divided into four lineages(lineage 1~4).Until now,NeV has not been reported in China.The study aimed to investigate the prevalence and molecular characteristics of NeV in China,and the results were as follows;1.Molecular detection and genomic characteristics of NeV in dairy cows in some provinces of ChinaIn total of 307 fecal samples(237 diarrheic and 70 non-diarrheic fecal samples)were collected from 23 dairy farms in Xinjiang uygur autonomous region,Sichuan province,Liaoning province,Henan province,Shandong province and Shanxi province.NeV was detected by RT-PCR assay.The results showed that the detection rate of NeV in diarrheic samples(48.1%)was significantly higher than that of NeV in non-diarrheic samples(5.7%,P<0.001),and the farm positive rate was 95.7%(22/23).40 NeV RdRp fragments from 6 provinces shared 91.8%~100% nucleotide(nt)similarity between each other,and shared 75.6%~90.0% nt similarity with other RdRp fragments available in the Gen Bank database.Phylogenetic analysis based on RdRp fragment showed that the 40 strains in this study were closely related to NB-like strains but were all located in a unique large branch,showing a common evolutionary trend.28 NeV complete VP1 sequence from 5 provinces shared 69.0%~100% nt similarity and 73.2%~100% amino acid(aa)similarity between each other,and shared 65.5%~94.7% nt similarity and 69.9%~98.7 aa similarity with other complete VP1 sequence available in the Gen Bank database.Phylogenetic analysis based on the complete VP1 sequence showed that 27 NeV strains belong to genotype 1(21 strains of lineage 1,4 strains of lineage 3,2 strains of lineage 4),and remaining one strain belong to genotype 2,indicating that VP1 of NeV in Chinese dairy cows has high genetic diversity.The complete genomes of strains Bo/LZB-1/17/CH and Bo/YLA-2/17/CH were successfully sequenced,and they are both 7,453 bp in length.Two complete genomes in this study shared 88.9% nt similarity between each other,and shared 79.4%~87.5% nt similarity with other four known genomes of NeV.Phylogenetic analysis based on P34,NTPase,P30,VPg,3CLpro and RdRp proteins showed that strain Bo/LZB-1/CH/17 was most related to strain Bo/YLA-2/17/CH,but the significant genetic difference between the two strains was located in the complete VP1(the VP1 of strain Bo/LZB-1/CH/17 belong to genotype 2,but the VP1 of strain Bo/YLA-2/17/CH belong to genotype 1 lineage 3).Recombination analysis revealed that a recombination event occurred in the VP1 P1 A of strain Bo/YLA-2/17/CH,and a recombination event occurred at the 3' end of RdRp of strain Bo/LZB-1/CH/17.In conclusion,the study first confirmed the existence of NeV in China,and the virus has circulated widely in dairy cows in China and exhibited a unique evolution,contributing to prevent and control of dairy cow diarrhea in China.Moreover,the study first reported the complete genomes of genotype 2 and genotype 1 lineage 3,and it also first reported the recombination events of VP1 P1 A and RdRp 3' end,contributing to understand the molecular characteristics and genetic evolution of NeV.2.Molecular detection and genomic characteristics of NeV in yak354 diarrheic fecal samples of yak were collected from 55 herds in Tibet autonomous region,Sichuan province and Yunnan province for detecting NeV by RT-PCR.The results showed that the detection rate of NeV was 22.0%,and the herds positive rate was 69.1%.78 RdRp fragments of yak NeV from three regions shared 63.1%~100% nt similarity between each other,and shared 62.4%~97.2% nt similarity with other RdRp fragment available in the Gen Bank database.Phylogenetic analysis based on RdRp fragment showed that 69 NeV strains from yak were most related to NeV strains from dairy cows in China,all of which belong to NB-like genotype.The remaining 9 NeV strains were clustered into an independent branch,which were different from other strains of known RdRp genotypes.The result indicated that the RdRp of the 9 strains may represent a novel RdRp genotype,proposed to be SC-Yak.The complete genome of two strains(YAK/NRG-17/17/CH and YAK/HY1-2/18/CH)with potential novel RdRp genotype were successfully amplified with 7,459 bp and 7,460 bp in length,respectively,and the 5'-UTR were 78 bp and 79 bp,respectively,and the 3'-UTR of the two strains were 66 bp.The complete genome of strains YAK/NRG-17/17/CH and YAK/HY1-2/18/CH shared 90.2% nt similarity between each other,and only shared 68.1%~69.3% nt similarity with that of other six known NeV strains.Analysis of the complete VP1 suggested that the VP1 of the two strains may represent a novel VP1 genotype,proposed to be genotype 3.Phylogenetic analysis based on genome,VP1,RdRp,VP2,P34,NTPase,P30,VPg and 3CLpro proteins showed that strains YAK/NRG-17/17/CH and YAK/HY1-2/18/CH were clustered into an independent branch,indicating that the two strains represent a novel NeV.Interestingly,9(11.5%)NeV strains from 6 farms in two counties were screened as the novel NeV strains based VP1 and RdRp sequences,indicating that the novel NeV has spread in local region.In conclusion,the results first confirmed the existence of NeV in yak,and the virus has circulated widely in yak in China,providing an important reference for the prevention and control of yak diarrhea.Moreover,a novel NeV strain with novel VP1 genotype and novel RdRp genotype was identified based on its complete genome,contributing to to understand the genetic diversity of NeV.3.Establishment of a real-time RT-PCR assay for detecting NeVRT-PCR assays for detecting NeV were targeted to the 3' end of RdRp.Our previous results showed that the NeV RdRp from China has a unique evolution and multiple nucleotide mutations compared to other known NeV RdRp sequences,which may affect the detection rate of RT-PCR.The aim of this study was to establish a real-time RT-PCR assay for detecting NeV.A TB Green real-time RT-PCR assay was successfully established through designing primers targeted to RdRp fragment of NeV strains in China and optimizing the reaction conditions and system.The real-time RT-PCR assay showed a good linear relationship between 2.9×101 copies/?L and 2.9×109 copies/?L,linear correlation coefficient R2=0.9994,and amplification efficiency was 98%.This assay only specifically detected NeV,and did not detect other unrelated pathogens;the minimum detection limit was 29 copies/?L;the intra-and inter-coefficients of variation were 0.89%~1.89% and 0.83%~1.15%,respectively;In 45 diarrheic fecal samples,the detection rate(68.8%)of this assay was higher than SYBR Green real-time RT-PCR(28.8%),nested RT-PCR(44.4%)and conventional RT-PCR(13.3%)reported previously;all the positive samples detected by this assay including all the positive samples detected by other 3 RT-PCR assays,and these positive samples were confirmed to be NeV by sequencing.The real-time RT-PCR assay established in this study has a good specificity,stability and sensitivity,which provided a reliable method for detection and epidemiological investigation of NeV. |
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