key: cord-0899479-em71g0g6 authors: Guo, Zijing; He, Qifu; Yue, Hua; Zhang, Bin; Tang, Cheng title: First detection of Nebovirus and Norovirus from cattle in China date: 2017-10-23 journal: Arch Virol DOI: 10.1007/s00705-017-3616-6 sha: c68bd70255e119366379e34bd50b9f875938853d doc_id: 899479 cord_uid: em71g0g6 Neboviruses and genogroup III noroviruses (NoVsGIII) are causative agents of calf diarrhea. The purpose of this study was to investigate the presence of neboviruses and noroviruses in cattle in China. Twenty-eight diarrhea fecal samples collected from 5 different farms were analyzed by RT-PCR. The results showed that 3 nebovirus positive samples were detected on 2 farms, with two strains being related to Bo/DijonA216/06/FR strain and the other one clustering with NB-like strains. Meanwhile, 3 norovirus positive samples were detected on 3 farms, all of which belonged to genotype 1. Our results confirmed the presence of neboviruses and NoVsGIII in China for the first time, and supported the presence of a novel “DijonA216-like” nebovirus genotype. using the Primescript TM reverse transcription kit (TaKaRa, China) and stored at -20 °C. Primers targeting the nebovirus genome were designed (using Primer 5.0 software) based on virus metagenomics sequencing data [GenBank accession number SUB2762873] obtained from a previously acquired calf diarrhea sample, which included nebovirus, as well as the nebovirus nucleotide sequences in GenBank. The primer sequences were as follows; F: 5'-CAG CCC GTC TGG GTG AAT -3'; R: 5'-CCA GCG TTA GCG TTC CAG -3'. The amplified fragment was 524 bp, composing part of the RdRp (1 bp to 297 bp) and the capsid gene (298 bp to 524 bp). For NoVsGIII, primer sets CBECU-F/CBECU-R were used to amplify 532 bp of the RdRp ORF of the virus according to previous reports [30] . Reverse transcription polymerase chain reaction (RT-PCR) was performed with Premix Taq™ (TaKaRa, China), following the manufacturer's protocol. The PCR amplification products were gel-purified using a gel extraction kit (OMEGA, USA) and then cloned into the pMD19-T vector and transformed into Escherichia coli DH5α competent cells. The recombinant plasmids were extracted using a plasmid extraction kit (OMEGA, USA) and sent to Sangon Biotech (China, Chengdu) for sequencing. Among the 28 fecal samples from cattle, 3 diarrhea samples from 2 farms were positive for nebovirus (accession numbers in GenBank: MF182624-MF182626 for strains LZB-1, YLA-1 and YLA-2 respectively). Meanwhile, 3 diarrhea samples from 3 farms were positive for NoVsGIII (accession numbers in GenBank: MF182621-MF182623 for strains HBC-1, SCD-1 and YLA-3, respectively). YLA-1, YLA-2 and YLA-3 were all discovered at the same farm, which indicated there was co-circulation of nebovirus and norovirus at this farm. Diarrhea is one of the commonest diseases of calves in China, which leads to serious economic losses. Bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus have been identified as major diarrhea causing viruses. However, there is little attention to neboviruses and noroviruses in terms of calf diarrhea. Although the number of samples was limited, the study confirmed for the first time the presence of neboviruses and NoVsGIII in China, which has significant implications for the diagnosis and control of calf diarrhea in China. The partial nucleotide sequences of the neboviruses and noroviruses identified in this study were analyzed using the MegAlign software to calculate sequence identities to other strains in GenBank. MEGA 6.0 was used to perform multiple sequence alignment and subsequently to build a neighbour-joining phylogenetic tree with bootstrap support. The 524 bp of the 3 neboviruses sequences in this study shared 88.4% to 97.7% nt identity to each other and 77.1% to 92.9% nt identity with neboviruses sequences in GenBank. The YLA-1 and LZB-1 strains shared 84.7% nt identity with Bo/DijonA216/06/FR, 81.5-85.1% nt identity with NB-like strains and 77.9-80.0% nt identity with NA1like strains. The YLA-2 strain shared 90.1-92.9% nt identity with NB-like strains, 87.4-88.4% nt identity with NA1like strains and 77.1% nt identity with Bo/DijonA216/06/ FR. Phylogenetic analysis demonstrated that two of the three strains clustered with Bo/DijonA216/06/FR isolated in France (Fig. 1) , which supported the presence of a novel "DijonA216-like" genotype in China. The other strain clustered with the NB-like strains. An alignment of the cloned 524 bp fragment amplified from neboviruses showed that YLA-1 and LZB-1 had similar nucleotide organization to Bo/Dijon216/06/FR. Interestingly, this genotype of nebovirus shared regular nucleotide mutations within the partial capsid sequence (312 bp to 412 bp within the cloned sequences) when compared with the other two genotypes of nebovirus (Fig. 2) . This data further supports the results whereby YLA-1 and LZB-1 clustered phylogenetically with Bo/DijonA216/06/ FR . Analysis of the 532 bp fragment representing the NoVs-GIII isolates indicated that the 3 NoVsGIII sequences shared 91.7% to 98.5% nt identity with each other and 75.3% to 89.0% nt identity with NoVsGIII sequences in GenBank. The 3 NoVsGIII strains in this study shared 84.8-88.9% nt identity with genotype 1 and 76.1%-80.8% nt identity with genotype 2. Phylogenetic analysis confirmed that all 3 strains were genotype 1. Interestingly, the 3 sequences clustered into an independent branch (Fig. 3) . Previous studies have suggested that genotype 2 is the main genotype worldwide, and genotype 1 is a minor circulating genotype [7, 10, 27] . It is therefore valuable to further investigate the prevalence and genotypes of NoVsGIII in China. In conclusion, this study has confirmed for the first time the presence of neboviruses and NoVsGIII in China, which will have significant implications for the diagnosis and control of calf diarrhea in China. The two nebovirus strains in this study clustered with Bo/DijonA216/06/FR, which supports the presence of a novel "DijonA216-like" nebovirus genotype in China. Norwalk-like calicivirus genes in farm animals Characterization of an enteropathogenic bovine calicivirus representing a potentially new calicivirus genus Infection of calves with bovine norovirus GIII.1 strain jena virus: an experimental model to study the pathogenesis of norovirus infection Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses Characterization of a calici-like virus (Newbury agent) found in association with astrovirus in bovine diarrhea Isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves Possible novel nebovirus genotype in cattle Molecular detection and characterization of unclassified bovine enteric caliciviruses in South Korea Evidence for recombination in neboviruses Molecular prevalence of bovine noroviruses and neboviruses detected in central-eastern Tunisia Identification of a bovine enteric calicivirus, Kirklareli virus, distantly related to neboviruses, in calves with enteritis in Turkey Genomic characterization of the unclassified bovine enteric virus newbury agent-1 (Newbury1) endorses a new genus in the family Caliciviridae First detection and molecular characterization of Nebovirus in Brazil Pathogenesis of GIII.2 bovine Norovirus, CV186-OH/00/US strain in gnotobiotic calves Molecular detection of enteric viruses from diarrheic calves in Egypt Studies of epidemiology and seroprevalence of bovine noroviruses in Germany Human noroviruses in Swine and Cattle Epidemiology of norwalk-like virus infections in cattle in The Netherlands Molecular characterization of noroviruses detected in diarrheic stools of Michigan and Wisconsin dairy calves: circulation of two distinct subgroups Retrospective study of noroviruses in samples of diarrhoea from cattle, using the Veterinary Laboratories Agency's Farmfile database Molecular identification of bovine enteric calciviruses in Venezuela Sensitive multiplex real-time reverse transcription-PCR assay for the detection of human and animal noroviruses in clinical and environmental samples Epidemiological study of bovine norovirus infection by RT-PCR and a VLP-based antibody ELISA Detection of genotype 1 and 2 bovine noroviruses in Hungary Detection and molecular characterisation of noroviruses and sapoviruses in asymptomatic swine and cattle in Slovenian farms First report on the phylogeny of bovine norovirus in Turkey A pilot survey of bovine norovirus in northern Italy Molecular detection of bovine Noroviruses in Argentinean dairy calves: Circulation of a tentative new genotype Case-control study of microbiological etiology associated with calf diarrhea Reverse transcription-PCR assays for detection of bovine enteric caliciviruses (BEC) and analysis of the genetic relationships among BEC and human caliciviruses Acknowledgements This work was funded by the 13th Five-Year