key: cord-0743541-voho91hc
authors: Nakagawa, Keisuke; Kumano, Hitomi; Kitamura, Yuko; Kuwata, Keisuke; Tanaka, Eiji; Fukushi, Hideto
title: Complete Genome Sequence of Bovine Coronavirus in Blood Diarrhea from Adult Cattle That Died from Winter Dysentery in Japan
date: 2021-10-21
journal: Microbiology resource announcements
DOI: 10.1128/mra.00807-21
sha: f87747b30eea23e0fd5dbc8ea3daefff0da46b2f
doc_id: 743541
cord_uid: voho91hc

We determined the complete genome sequence of bovine coronavirus (BCoV) recovered from bloody diarrhea from adult cattle that died from winter dysentery in 2020 in Japan. Information on the complete genome sequence of BCoV, which causes deadly diarrhea in adult cattle, has great potential for a better understanding of its pathogenicity.

B ovine coronavirus (BCoV) is responsible for respiratory disease and winter dysentery in cattle (1) . BCoV belongs to the order Nidovirales, family Coronaviridae, and the genus Betacoronavirus. BCoV consists of a nonsegmented, single-stranded, positive-sense RNA (2) . Zhang et al. showed that in the process of cell culture adaptation, an enteric BCoV strain accumulated mutations to resemble the corresponding respiratory BCoV isolate from the same animal (3), suggesting the importance of sequencing of the enteric BCoV genome without laboratory manipulation for a better understanding of the pathogenesis of enteric BCoV. We determined the complete genome sequence of a BCoV strain, termed GF2020, recovered from bloody diarrhea from adult cattle that died from winter dysentery in 2020 in Gifu Prefecture, Japan, without virus isolation in cell culture.

RNA was extracted from the bloody diarrhea using a Direct-zol RNA miniprep kit (Zymo Research). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) and random primers (Invitrogen). The whole viral genome, comprising six overlapping amplicons, except for 70 nucleotides (nt) at the 59 end and 97 nt at the 39 end, was generated from cDNA using KOD FX polymerase (Toyobo) and the sets of primers shown in Table 1 . From an equimolar mixture of 6 PCR products, sequencing libraries were prepared using the NEBNext Ultra II DNA library prep kit (NEB). The libraries were sequenced on a NovaSeq 6000 instrument (Illumina). A paired-end sequencing run with a 2 Â 150-nucleotide read length generated 9,654,684 raw reads. The raw reads were trimmed using Trimmomatic (4) and mapped to BCoV reference genome strain TCG-27 (GenBank accession number LC494186.1) using Bowtie2 (5) . All tools were run with default parameters unless otherwise specified.

The 59-terminal sequence of the genomic RNA of strain GF2020 was determined using the rapid amplification of cDNA ends (RACE) method (59-full RACE core set; TaKaRa Bio). The 39 end of the viral genome was amplified using specific primers and anchored oligo(dT) primers. Information about the primers is provided in Table 1 . The amplicon containing the 59-and 39-terminal sequences of the viral genome was sequenced using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) on an ABI PRISM 3100 DNA analyzer (Applied Biosystems).

The determined sequences were visually assembled using ApE (https://jorgensen.biology .utah.edu/wayned/ape/) with the NCBI reference sequence of isolate BCoV-ENT (GenBank accession number NC_003045.1), resulting in the following genome organization (Fig. 1) : 59 untranslated region (UTR)-ORF1a (polyprotein 1a)-ORF1b (polyprotein 1b)-ORF2 (32 kDa protein)-ORF3 (hemagglutinin esterase)-ORF4 (spike protein)-ORF5 (4.9 kDa protein)-ORF6 (4.8 kDa protein)-ORF7 (12.4 kDa protein)-ORF8 (small membrane protein)-ORF9 (membrane protein)-ORF10 (nucleocapsid protein)-ORF11 (internal protein)-39 UTR. The full-length genome sequence of strain GF2020 comprised 31,013 nucleotides with a G1C content of 37.0%. According to BLASTN analysis (6), the full-length genome sequence showed the highest nucleotide identity (99.44%) with BCoV strain TCG-27 (GenBank accession number LC494186.1), which was collected from a nasal swab from a dairy calf in Japan (7), supporting the notion that no genetic markers have been identified to discriminate BCoV from respiratory and enteric syndromes (1, 7) . Accumulation of information on the whole viral genome of enteric BCoV without laboratory manipulation will contribute to a better understanding of the virulence factors involved in BCoV-mediated winter dysentery in cattle. Data availability. The complete genome sequence of strain GF2020 has been deposited in GenBank under the accession number LC642814. The raw data were deposited under SRA accession number DRX301428, BioSample accession number SAMD00393674, and BioProject accession number PRJDB12037.

Bovine respiratory coronavirus

Nidovirales: a new order comprising Coronaviridae and Arteriviridae

Quasispecies of bovine enteric and respiratory coronaviruses based on complete genome sequences and genetic changes after tissue culture adaptation

Trimmomatic: a flexible trimmer for Illumina sequence data

Fast gapped-read alignment with Bowtie 2

Gapped BLAST and PSI-BLAST: a new generation of protein database search programs

Genomic characterization and phylogenetic classification of bovine coronaviruses through whole genome sequence analysis

We are grateful to the Gifu Prefectural Chuo Livestock Hygiene Service Center for sampling the bloody diarrhea from adult cattle that died from winter dysentery. This work was funded by a research grant from the Mishima Kaiun Memorial Foundation, Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (numbers 19K23706 and 20K15661), and a grant from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, for the Joint Research Program of the Research Center for Zoonosis Control, Hokkaido University.