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\begin{document}

\title{Complete genome and sequence and phylogenetic analyses of the first
bovine norovirus strain identified in northern China}



\author[1]{Meng-Jia Wang}%
\author[2]{Jinghui Fan}%
\author[1]{Hui-Xia Fan}%
\author[1]{Ying  Liu}%
\author[1]{Zhi-Hao Peng}%
\author[1]{Yu-Zhong Ma}%
\author[1]{Qing-Yan  Li}%
\author[1]{Yu-Zhu  Zuo}%
\affil[1]{Affiliation not available}%
\affil[2]{Hebei Agricultural University}%


\vspace{-1em}



  
  \date{May 08, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Bovine norovirus (BNoV) is a recently identified calicivirus in the
genus Norovirus, which can cause enteric disease with clinical signs
including vomiting, diarrhea, dehydration, and mortality in neonatal
calves. BNoV has been reported in different countries all over the
world. We collected 39 fecal, blood, and tissue samples from calves with
diarrhea from Hebei in northern China in 2019. The presence of BNoV was
determined by PCR only two samples (5.1\%) were positive. The complete
genome of the newly identified strain Bo/CH/HB/BD/2019 was successfully
sequenced (GenBank accession number: MN480761). Based on the existing
gene sequences in the GenBank database, evolutionary trees were
constructed. Sequence analysis showed that the nucleotide sequence
homology of the Bo/CH/HB/BD/2019 genome with BNoV genomes in GenBank was
84.0--92.4\%. Bo/CH/HB/BD/2019 has a GIII.2 genotype, and further
analysis of its VP1 gene revealed four amino acid substitutions, i.e.,
225C, 246T, 624T, and 945T. To the best of our knowledge, this is the
first time the complete genome of a BNoV isolate from northern China has
been sequenced.%
\end{abstract}%



\sloppy


\textbf{Complete genome and sequence and phylogenetic analyses of the
first bovine norovirus strain identified in northern China}

Meng-Jia
Wang\textsuperscript{a,1}\emph{https://orcid.org/0000-0003-2493-0723},
Jing-Hui Fan\textsuperscript{a,b,1}, Hui-Xia Fan\textsuperscript{c,1},
Ying Liu\textsuperscript{a}, Zhi-Hao Peng\textsuperscript{a}, Yu-Zhong
Ma \textsuperscript{a,b}, Qing-Yan Li \textsuperscript{a,*}, Yu-Zhu
Zuo\textsuperscript{a,b,*}

\textsuperscript{1}These authors contributed equally to this work.

\textsuperscript{a}College of Veterinary Medicine, Hebei Agricultural
University, Baoding 071001, People's Republic of China.

\textsuperscript{b}Hebei Veterinary Biotechnology Innovation Center,
Baoding 071001, People's Republic of China.

\textsuperscript{c}Information science and technology, Hebei
Agricultural University, Baoding 071001, People's Republic of China.

*Author for all correspondence: Qing-Yan Li; Yu-Zhu Zuo

Tel.: +86-312-7528581; fax: +86-312-7520275

E-mail address: zuoyuzhu@163.com (Yu-Zhu Zuo)

\textbf{Summary}

Bovine norovirus (BNoV) is a recently identified calicivirus in the
genus\emph{Norovirus} , which can cause enteric disease with clinical
signs including vomiting, diarrhea, dehydration, and mortality in
neonatal calves. BNoV has been reported in different countries all over
the world. We collected 39 fecal, blood, and tissue samples from calves
with diarrhea from Hebei in northern China in 2019. The presence of BNoV
was determined by PCR only two samples (5.1\%) were positive. The
complete genome of the newly identified strain Bo/CH/HB/BD/2019 was
successfully sequenced (GenBank accession number: MN480761). Based on
the existing gene sequences in the GenBank database, evolutionary trees
were constructed.

Sequence analysis showed that the nucleotide sequence homology of the
Bo/CH/HB/BD/2019 genome with BNoV genomes in GenBank was 84.0--92.4\%.
Bo/CH/HB/BD/2019 has a GIII.2 genotype, and further analysis of
its\emph{VP1} gene revealed four amino acid substitutions, i.e., 225C,
246T, 624T, and 945T. To the best of our knowledge, this is the first
time the complete genome of a BNoV isolate from northern China has been
sequenced.

Keywords: Bovine norovirus, calf diarrhea, complete genome, genomic
sequence, phylogenetic analysis

\textbf{Introduction}

Noroviruses (NoVs) are important pathogens causing gastroenteritis in
children and young animals. Infection with NoV in children and young
animals can cause intestinal lesions and post-infectious diarrhea
(Smiley,Chang,Hayes,Vinje, \& Saif, 2002; Bridger,Hall, \& Brown, 1984;
Woode \& Bridger, 1978). In humans, NoV is one of the most important
etiological agents of gastroenteritis; however, little is known about
NoV causing diarrhea in cattle.

NoVs are small non-enveloped, positive-sense single-stranded RNA
viruses. According to the diversity of the \emph{VP1} amino acid (aa)
sequence, NoVs are divided into seven genogroups (GI to GVII). Bovine
NoVs (BNoVs) belong to GIII and are further divided into two different
genotypes, i.e., genotype 1 (Bo/Jena/1980/DE) and genotype 2
(Bo/newbury2/1976/UK), which were first identified in Germany (Otto et
al., 2011) and the UK (Jung,Scheuer,Zhang,Wang, \& Saif, 2014). The BNoV
genome, which has a size of 7.5--7.7 kb, contains three open reading
frames (ORFs). Starting from the 5' end of the genome, ORF1 encodes a
non-structural protein, including an enzyme responsible for
transcription, viral genome replication, and accurate initiation of RNA
synthesis by RNA-dependent RNA polymerase (RdRp). RdRp prevents the
virus from losing its genetic information (Deval,Jin,Chuang, \& Kao,
2017; Lee,Chung, \& Kim, 2017). ORF2 encodes the major capsid protein
\emph{VP1} . It is involved in receptor recognition and host specificity
(Chen et al., 2004).Viral evasion of antibody neutralization results in
frequent variations in the\emph{VP1} aa sequences, which are also
associated with the emergence of new epidemic strains (Lochridge \&
Hardy, 2007; Lindesmith et al., 2013). ORF3 encodes the minor capsid
protein \emph{VP2} , a hypervariable protein that may play a role in
maintaining the stability of NoV particles (Lin,Fengling,Lianzhu,Yuxiu,
\& Yanhua, 2014).

Previous studies have reported the detection of BNoV in many countries
including the UK (Woode \& Bridger, 1978; Bridger,Hall, \& Brown, 1984),
Germany (Gunther,Otto, \& Heilmann, 1984), the Netherlands (van der Poel
et al., 2003; van Der Poel et al., 2000), the USA (Cho et al., 2013;
Wise et al., 2004), New Zealand (Wolf et al., 2007), South Korea (Park
et al., 2007), Norway (Jor,Myrmel, \& Jonassen, 2010), France
(Kaplon,Guenau,Asdrubal,Pothier, \& Ambert-Balay, 2011), Turkey
(Kaplon,Guenau,Asdrubal,Pothier, \& Ambert-Balay, 2011), and Tunisia
(Hassine-Zaafrane et al., 2012). According to previous reports, BNoV was
first detected in Southern China in 2018 (Wang,Yue, \& Tang, 2019), and
it was detected in central~China in 2019 (Shi,Wang,Xu,Zhang, \& Lan,
2019).

BNoV has become a threat to the global cattle industry, and it may bring
huge economic losses to the cattle industry in China. Although BNoV is
prevalent globally, the study of its pathogenicity, epidemiology, and
molecular biology is only in the preliminary stage. Up to now, GenBank
contained only seven complete BNoV genome sequences; two are GIII.1
strains (GenBank accession numbers: MK159169 and AJ011099) and five are
GIII.2 strains (GenBank accession numbers: JX145650, AY126474, AF097917,
MN122335.1, and EU794907). In China, only two BNoV genomes have been
completely sequenced, i.e., one from southern China and one from central
China. There are not enough data to establish the genetic relationship
and evolutionary rate of BNoVs in China. At present, the virus has not
been successfully isolated, hindering the development of an effective
vaccine. We studied the molecular profile of the virus and analyzed its
genetic variation, in order to provide a reference for the prevention
and control of viral spread in China.

We identified a BNoV strain, named Bo/CH/HB/BD/2019, from Hebei, in
northern China, and conducted phylogenetic analysis of the entire genome
and the \emph{VP1} region. BNoV may be highly prevalent in China, but
there are few epidemiological data on the prevalence of the virus. This
study lays a foundation for the further exploration of the molecular
biological characteristics of BNoV and the prevalence of BNoV in China.

\textbf{Materials and methods}

\textbf{Sample information}

Between April 2019 and September 2019, we collected and analyzed 39
samples from five different farms with calf diarrhea in Hebei, China,
including 30 diarrheal fecal samples, 5 blood samples and 4 tissue
samples (lung, liver, kidney, heart). Samples were screened for BNoV by
nested PCR.

Nucleotide \textbf{extraction and PCR detection of BNoV}

Total RNA was extracted by TRIzol reagent following the manufacturer's
instructions (Invitrogen, Carlsbad, CA, USA). Reverse transcription was
performed using the PrimeScript\textsuperscript{TM}1st strand cDNA
Synthesis Kit (TaKaRa Biotechnology, Dalian, China) with an oligo dT
primer. Nested PCR primers (F, AGTTAYTTTTCCTTYTAYGGBGA; R,
AGTGTCTCTGTCAGTCATCTTCAT (Smiley,Hoet,Traven,Tsunemitsu, \& Saif, 2003);
and nF, GTCGACGGYCTKGTSTTCCT; nR, CACAGCGACAAATCATGAAA (Park et al.,
2006)) targeting the \emph{RdRp} gene were used with the following
reaction conditions: 94\selectlanguage{ngerman}°C for 5 min, followed by 35 cycles at 94°C for
30 s, 54°C for 30 s and 72°C for 40 s, with a final extension at 72°C
for 10 min. The reaction generated a 326 bp PCR product. In addition,
three other diarrhea-related enteric viruses, i.e., bovine viral
diarrhea virus, bovine rotavirus, and bovine coronavirus, were detected
using previously reported molecular methods for the evaluation of
possible co-infection with BNoV.

\textbf{Sequencing and phylogenetic analysis of the complete genome of
the Bo/CH/HB/BD/2019 strain}

Based on the GenBank sequences, we designed six pairs of
specific~primers using Primer Premier 5 software. The reference
sequences were AY126472.2, JX145650.1, EU794907, AF097917.5, MK159169.1
and NC\_029645.1. The complete BNoV genome of the strain
Bo/CH/HB/BD/2019 was amplified using six sequence-specific primer pairs.

Reactions were performed in a total volume of 20 \selectlanguage{greek}μ\selectlanguage{english}L (2 \selectlanguage{greek}μ\selectlanguage{english}L of cDNA, 2 \selectlanguage{greek}μ\selectlanguage{english}L
of 10\selectlanguage{ngerman}× PCR Buffer, 3 \selectlanguage{greek}μ\selectlanguage{english}L of 2.5 mM dNTP, 1 \selectlanguage{greek}μ\selectlanguage{english}L of each primer, 0.5 \selectlanguage{greek}μ\selectlanguage{english}L of
Taq polymerase, and 10 \selectlanguage{greek}μ\selectlanguage{english}L of ddH\textsubscript{2}O). The whole genome of
BNoV strain Bo/CH/HB/BD/2019 was amplified by RT-PCR (oligonucleotide
primers are presented in Table 1). All PCR products were purified with
the PCR Purification Miniprep Kit (BIOMIGA, Shanghai, China) following
the manufacturer's instructions. The PCR products were cloned into the
pMD19-T cloning vector (TaKaRa Biotechnology, Dalian, China). The
positive recombinant plasmids were verified by restriction enzyme
digestion and sequenced by Sangon Biotech (Beijing, China). The raw
genomic sequence fragments were imported to SeqMan in DNAStar (DNAStar,
Inc., Madison, WI, USA) for assembly and annotation. The complete BNoV
genome sequence has been deposited in GenBank (accession number:
MN480761). Sequencing and phylogenetic analysis of the complete genome
of the Bo/CH/HB/BD/2019 strain was performed using the Clustal W program
in DNA Star software. Then, 10,000 bootstrap replication was performed
using MEGA 7 software, and the phylogenetic tree was constructed by the
neighbor-joining method.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Primer & Sequence (5'-3') & Product size(bp) & Annealing temperature
()\tabularnewline
\midrule
\endhead
BNoV1F & TGAATGAAGACTTTGACG & 1465 & 52\tabularnewline
BNoV1R & CGCACCCGATTAGACAT & &\tabularnewline
BNoV2F & CCTCGGKCARTATGGDA & 1304 & 53\tabularnewline
BNoV2R & CTTGGATGGCAGTGGTH & &\tabularnewline
BNoV3F & TGTGACCACTGCCATCCA & 1638 & 52\tabularnewline
BNoV3R & GCCACCAGCTCATCCTT & &\tabularnewline
BNoV4F & TCCCTGGACAAAACTACC & 1151 & 49\tabularnewline
BNoV4R & CCTTCCCACAGTGACAGAT & &\tabularnewline
BNoV5F & GAYGAYCCGARYGARAC & 1563 & 49\tabularnewline
BNoV5R & GAAACGAAATGGGTAATC & &\tabularnewline
BNoV6F & GATTACCCATTTBGTYTC & 924 & 42\tabularnewline
BNoV6R & AACACTGCTCACTATTTC & &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Results and discussion}

BNoV has been detected in many countries. This disease was first
detected in China in 2018, and has been reported in central and southern
China. Here, we detected a new BNoV strain, named Bo/CH/HB/BD/2019
(GenBank accession number: MN480761), in Hebei, in northern China, and
sequenced its whole genome. We collected 39 samples, two (5.1\%) of
which were BNoV positive. One sample was co-infected with bovine
rotavirus. The BNoV detection rate was similar to those reported in
Belgium (7.5\%) (Mauroy et al., 2009), Argentina, (3.3 \%) (Ferragut et
al., 2016), and South Korea (2.8\%) (Park et al., 2008), and much lower
than those reported in the USA (72.1\%) (Smiley,Hoet,Traven,Tsunemitsu,
\& Saif, 2003), Norway (49.6\%) (Jor,Myrmel, \& Jonassen, 2010b) and
Iran (39.5\%) (Pourasgari et al., 2018), and also lower than those
reported in central China (25\%) and south China (20.4\%)
(Shi,Wang,Xu,Zhang, \& Lan, 2019; Wang,Yue, \& Tang, 2019). Although it
has been confirmed that BNoV is prevalent in China, few studies and
epidemiological data are available on BNoV in China.

We sequenced and analyzed the Bo/CH/HB/BD/2019 genome, and learned that
its genome is 7320 nucleotides (nt) and includes three ORFs, a 21 nt 5'
untranslated region, and a 49 nt 3' untranslated region. ORF1 is located
from 22 nt to 5076 nt and encodes a 1685 aa non-structural protein. ORF2
is located from 5063 nt to 6631 nt and encodes the 523 aa \emph{VP1}
protein. ORF3 is located from 6621 nt to 7271 nt and encodes the 217 aa
\emph{VP2}protein. ORF1, ORF2, and ORF3 overlap each other by 14 nt and
11 nt. Based on the complete BNoV sequences available in the GenBank
database, we compared the~isolated strain Bo/CH/HB/BD/2019 with seven
isolates from other regions of the world. The sequence similarity
between Bo/CH/HB/BD/2019 and the other isolates ranged from
84.0--92.4\%. The homology between the isolates and all complete
sequences in the GenBank database ranged from 69.9\% to 91.8\% for
\emph{VP1} and from 67.9\% to 91.2\% for \emph{VP2} (Table 2),
indicating that BNoV genes are diverse and hence require further
exploration.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Strain\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
GenBank accession no\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Geographic location\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
genotype\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\% Identity Complete Genome\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\% Identity VP1\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\% Identity VP2\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Bo/Newbury2/1976/UK\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
AF097917.5\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
United Kingdom\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
GIII.2\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
84.3\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
86.9\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
84.6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Bo/Dumfries/94/UK\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
AY126474.2\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
United Kingdom\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
GIII.2\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
84.3\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
85.8\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
85.3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Bo/GIII/B309/2003/BEL\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
EU794907\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Belgium\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
GIII.2\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
84.0\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
84.3\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
84.8\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Bo/GIII.2/Adam/2006/No\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
JX145650.1\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Norway\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
GIII.2\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
85.9\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
85.9\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
86.2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Bo/BET-17/18/CH\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
MK159169.1\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
China Sichuan\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
GIII.1\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
80.1\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
69.9\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
67.9\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Bo/HN-1/2018/China\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
MN122335.1\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
China Henan\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
GIII.2\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
92.4\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
91.8\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
91.2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Bo/GIII.2/Adam/2006/No\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
NC\_029645.1\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Norway\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
GIII.2\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
85.9\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
85.9\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
86.2\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Phylogenetic trees of different hosts and genotypes were constructed
based on the complete Bo/CH/HB/BD/2019 genome sequence and the complete
genomes in the GenBank database (Figure 1). Figure 1a shows that bovine
isolates and human isolates are far related, so there is no potential
risk of cross-infection at present. However, BNoV (GIII) serum
antibodies have been detected in humans (Widdowson et al., 2005) and
human NoV sequences have been detected in bovine fecal samples (Menon et
al., 2013), and interspecific transmission may occur in the future.
Figure 1b shows that all BNoV isolates belonged to the GIII genotype.
The Bo/CH/HB/BD/2019 strain is more closely related to the other two
Chinese strains than to those from other countries, and is hence
clustered in the Chinese BNoV branch in the phylogenetic tree. The
isolated strain was more closely related to the Bo/HN-1/2018/China
strain from Henan (central China) (GenBank accession number: MN122335.1)
than to the Bo/BET-17/18/CH strain from Sichuan (southern China)
(GenBank accession number: MK159169.1). This suggests that there may be
a different evolutionary process underlying the spread of BNoV in China.
Unfortunately, few data are available on the genetic evolution and
prevalence of BNoV in China.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

The BNoV \emph{RdRp} gene contains highly conserved regions. \emph{VP1}
is the major structural component of caliciviruses and is involved in
receptor recognition, host specificity, strain antigenic diversity, and
immunogenicity (Chen et al., 2004). \emph{VP2} is highly variable and
can contain different types of mutations (Bok et al., 2009;
Seah,Gunesekere,Marshall, \& Wright, 1999). \emph{VP2} interacts
with\emph{VP1} and, in turn, enhances the expression of capsid proteins
(Vongpunsawad,Venkataram, \& Estes, 2013). Due to the slightly different
topologies between ORF2 and ORF3, different BNoV \emph{VP2} gene may have
resulted from distinct evolutionary strategies (Kamel et al., 2009).
Phylogenetic trees were constructed based on the BNoV \emph{VP1}
and\emph{RdRp} genes (Figure 2). The strain isolated in the present
study and CH-HNSC-2018 were clustered in one branch, while strains
isolated from Sichuan, southern China, were clustered in another branch,
indicating that the BNoV strains in China are diverse. Different strains
with different genotypes exist in China, making the prevention and
control of viral spread difficult (Figure 2a). The sequence and
phylogenetic analyses showed that Bo/CH/HB/BD/2019 belongs to GIII.2
BNoV (Figure 2b).

The \emph{VP1} protein includes a shell (S) domain, which is highly
conserved among different NoVs, and a protruding (P) domain, with
N-terminal P1 and C-terminal P1 and P2 parts (Chen et al., 2013). The
highly variable region of the sequence (residues 279--406) forms the
externally located P2 subdomain, whereas the central subdomain P1
(residues 226--278 and the C-terminal 124 residues) is located between
the S and P2 domains (Venkataram,Hardy, \& Estes, 2000). Comparing with
previous studies, in both China and other countries, we found four new
aa substitutions in \emph{VP1} , i.e., 225C, 246T, 624T, and 945T. The
225C residue is located in the conserved region and 246T is located in
the P1 subdomain in the highly variable region. This may be an
adaptation that is unique to BNoV circulating in cattle farms in Hebei,
China. Whether these mutations in Bo/CH/HB/BD/2019 lead to changes in
virulence, pathogenicity, and antigenicity remains to be explored.

In conclusion, in this study we isolated the Bo/CH/HB/BD/2019 strain
from Hebei, sequenced its genome, and identified its genes. We analyzed
its sequence homology with other BNoV strains and constructed
phylogenetic trees to analyze its genetic evolution. Our results provide
a reference for the development of a BNoV vaccine to prevent and control
viral spread. Nevertheless, more data are still required, so in the
future it will be necessary to further monitor and research BNoV and its
subtypes.

\textbf{Acknowledgments}

This research was supported by the Program of the Modern Agriculture
Industry Technology System Foundation of Hebei Province (HBCT2018120406,
HBCT2018130405) and the Hebei Key Research and Development Program
(19226611D).

\textbf{Conflict of interest}

The authors declare no conflict of interest.

\textbf{Ethical statement}

The authors confirm that the ethical policies of the journal, as noted on
the journal's author guidelines page, have been adhered to and the
appropriate ethical review committee approval has been received. The US
National Research Council's guidelines for the Care and Use of
Laboratory Animals were followed.

\textbf{All data generated or analyzed during this study are included in
this article.}

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