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\begin{document}

\title{Detection and genetic characteristic of porcine bocavirus in central
China}



\author[1]{Lan-Lan Zheng}%
\author[1]{Jian-Tao Cui}%
\author[1]{Han Qiao}%
\author[1]{Xin-Sheng Li}%
\author[1]{Xiaokang Li}%
\author[1]{Hongying Chen}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
To investigate the epidemic profile and genetic diversity of porcine
bocavirus (PBoV), 281 clinical samples including 236 intestinal tissue
samples and 45 fecal samples were collected from diarrheal piglets in 37
different pig farms of central China, and two SYBR Green I-based
quantitative PCR assays were developed to detect PBoV1/2 and PBoV3/4/5
respectively. The results showed the detection limits of two assays were
1.66 \selectlanguage{ngerman}× 101 genome copies/\selectlanguage{greek}μ\selectlanguage{english}l of PBoV1/2 and 3.3 \selectlanguage{ngerman}× 101 copies/\selectlanguage{greek}µ\selectlanguage{english}L of PBoV
3/4/5. 148 (52.67\%) of the 281 clinical samples were positive for
PBoV1/2, 117 (41.63\%) were positive for PBoV3/4/5, 55 (19.57\%) were
positive for both PBoV1/2 and PBoV3/4/5, and 86.49\% (32/37) of the pig
farms were positive for PBoV. Subsequently, complete genomic sequences
of two PBoV strains (designated CH/HNZM and PBoV-TY) from two different
farms were sequenced. The phylogenetic analysis demonstrated that the
two PBoV strains obtained in this study belonged to the PBoV2 group and
had a close relationship with other 12 PBoV2 strains, but differed
genetically from PBoV1, PBoV3/4/5 and 7 other bocaviruses. CH/HNZM and
PBoV-TY were closely related to the PBoV strain GD18 (KJ755666) which
may be derived from PBoV strains 0912/2012 (MH558677) and 57AT-HU
(KF206160) through the recombination analysis. Compared with reference
strain ZJD (HM053694)-China, a higher amino acid variation was found in
the NS1 protein of CH/HNZM and PBoV-TY. These results extend our
understanding of the molecular epidemiology and evolution of PBoV.%
\end{abstract}%



\sloppy


\textbf{Detection and genetic characteristic of porcine bocavirus in
central China}

Lan-Lan Zheng\textsuperscript{1}, Jian-Tao Cui\textsuperscript{1}, Han
Qiao\textsuperscript{2}, Xin-Sheng Li\textsuperscript{1}, Xiao-Kang
Li\textsuperscript{3}, Hong-Ying Chen\textsuperscript{1}

\textsuperscript{1}Zhengzhou Key Laboratory for Pig Disease Prevention
and Control, College of Animal Science and Veterinary Medicine, Henan
Agricultural University, Nongye Road 63\#, Zhengzhou 450002, Henan
Province, People's Republic of China.

\textsuperscript{2}College of Life Science, South China Agricultural
University, Guangzhou 510642, Guangdong Province, People's Republic of
China.

\textsuperscript{3}College of Animal Science and Technology, Henan
University of Science and Technology, Luoyang 471000, Henan Province,
People's Republic of China.

\textbf{Corresponding author} : Hong-Ying Chen, E-mail: chhy927@163.com.
Mailing address: College of Animal Science and Veterinary Medicine,
Henan Agricultural University, Zhengdong New District Longzi
Lake\textsuperscript{\#}15, 450046 Zhengzhou, Henan Province, People's
Republic of China. Tel: +86 371 55369208; fax: +86 371 55369208.
Xiaokang Li, E-mail: xiaokangli00@163.com. Mailing address: College of
Animal Science and Technology, Henan University of Science and
Technology, 263 Kaiyuan Avenue, 471000 Luoyang, Henan Province, People's
Republic of China. Tel: +86 379 64563979; fax: +86 379 64563979.

\textbf{Abstract:}To investigate the epidemic profile and genetic
diversity of porcine bocavirus (PBoV), 281 clinical samples including
236~intestinal tissue samples and 45 fecal samples were collected from
diarrheal piglets in 37 different pig farms of central China, and two
SYBR Green I-based quantitative PCR assays were developed to detect
PBoV1/2 and PBoV3/4/5 respectively. The results showed the detection
limits of two assays were 1.66 \selectlanguage{ngerman}× 10\textsuperscript{1} genome copies/\selectlanguage{greek}μ\selectlanguage{english}l
of PBoV1/2 and 3.3 \selectlanguage{ngerman}× 10\textsuperscript{1} copies/\selectlanguage{greek}µ\selectlanguage{english}L of PBoV 3/4/5. 148
(52.67\%) of the 281 clinical samples were positive for PBoV1/2, 117
(41.63\%) were positive for PBoV3/4/5, 55 (19.57\%) were positive for
both PBoV1/2 and PBoV3/4/5, and 86.49\% (32/37) of the pig farms were
positive for PBoV. Subsequently, complete genomic sequences of two PBoV
strains (designated CH/HNZM and PBoV-TY) from two different farms were
sequenced. The phylogenetic analysis demonstrated that the two PBoV
strains obtained in this study belonged to the PBoV2 group and had a
close relationship with other 12 PBoV2 strains, but differed genetically
from PBoV1, PBoV3/4/5 and 7 other bocaviruses. CH/HNZM and PBoV-TY were
closely related to the PBoV strain GD18 (KJ755666) which may be derived
from PBoV strains 0912/2012 (MH558677) and 57AT-HU (KF206160) through
the recombination analysis. Compared with reference strain ZJD
(HM053694)-China, a higher amino acid variation was found in the NS1
protein of CH/HNZM and PBoV-TY. These results extend our understanding
of the molecular epidemiology and evolution of PBoV.

\textbf{Key words:} porcine bocavirus, complete genome sequence,
phylogenetic analysis, evolutionary analysis

1 \textbf{INTRODUCTION}

Bocavirus is a novel classified genus in the family
of\emph{Parvoviridae} , subfamily \emph{Parvovirinae} , which includes
human bocavirus (HBoV), porcine bocavirus (PBoV), canine minute virus
(CMV), bovine bocavirus (BPV), gorillas bocavirus (GBoV), and California
sea lion bocavirus (CslBoV) (Lau et al., 2008; Li et al., 2011). In
2009, porcine boca-like virus (PBo-likeV) was first reported from lymph
nodes of pigs with post-weaning multi-systemic wasting syndrome (PMWS)
in Sweden (Blomstrom et al., 2009). PBo-likeV was subsequently
discovered in China in 2010 and named as porcine bocavirus (PBoV) or
PBoV1 (Zhai et al., 2010). Since then, PBoV has been identified in
Europe, Asia, North America and Africa (Zhou et al., 2014).

PBoV is a non-enveloped single-stranded DNA virus which consists of
three open reading frames (ORFs), ORF1, ORF2, and ORF3 (Arthur et al.,
2009). ORF1 encodes a nonstructural protein 1 (NS1), ORF2 encodes viral
caspid proteins 1 and 2 (VP1/2), and \emph{VP1} consists of the
entire\emph{VP2} sequence and an additional N-terminal region (Sun et
al., 2009), ORF3 encodes nuclear phosphoprotein 1(NP1). Based on
the\emph{VP} 1 and \emph{VP} 2 sequences, PBoV has been classified into
different clades, which includes PBoV1, PBoV2, PBoV3, PBoV4, PBoV5,
PBoV3C, PBoV-6V and PBoV-7V. And PBoV was proposed to be classified into
three different groups, PBoV G1, PBoV G2, and PBoV G3 (Zhou et al.,
2014).

Co-infection of PBoV with other porcine viruses has also been reported,
such as porcine epidemic diarrhea virus (PEDV), porcine circovirus type
2 (PCV2), pseudorabies virus (PRV), porcine reproductive and respiratory
syndrome virus (PRRSV), porcine torque teno virus (PTTV) and classic
swine fever virus (CSFV) (Blomstrom et al., 2010; McMenamy et al., 2013;
Zhang et al., 2014; Huang et al., 2014; Zhou et al., 2014; Luo et al.,
2015). PBoV was detected from livers of healthy pigs with prevalence of
6\% in Brazil, and the co-infection of PBoV with torque teno sus virus
1(TTSuV1), TTSuVk2 and porcine parvovirus (PPV) was also found in these
healthy pigs (Silva et al., 2020). In addition, PBoV has a significantly
higher infection rate in diseased pigs than in healthy pigs, and the
co-infection rate of PEDV and PBoV was higher in samples of diarrheal
pigs than that of healthy pigs, suggesting that the PBoV might play an
important role in causing diarrhea in piglets (Zhai et al., 2010).

To date, PBoVs have been reported in pigs in 20 provinces or regions in
China with the prevalence between 7.3\% and 64\% (Wang et al., 2014;
Zhang et al., 2015; Zhou et al., 2018). However, little is known about
the presence of PBoV in central China. Here, we aimed to investigate the
occurrence of PBoV and characterized two PBoV strains from diarrheal
pigs in central China.

\textbf{2 MATERIALS AND METHODS}

\textbf{2.1 Sample collection and DNA preparation}

From 2016 to 2019, a total of 281 samples including 236~intestinal
tissue samples and 45 fecal samples were collected from diarrheal
piglets in 37 different pig farms in 18 cities of central China. Each
sample was collected from different pigs, and the origin and the number
of samples were summarized in Table 1.

Samples were diluted 1:10 with phosphate-buffered saline (PBS, pH7.4),
and the suspensions were vortexed and centrifuged at 12 000 \selectlanguage{ngerman}× g for 5
min at 4°C to collect the clarified supernatants. The viral DNA was
extracted using an UNlQ-10 Column Virus Genomic DNA Isolation Kit
(Sangon Biotech Shanghai Co, Ltd., China) according to the
manufacturer's instructions, and stored at -80°C.

\textbf{2.2. Construction of standard plasmids for quantitative PCR}

Genomic sequences of PBoVs were downloaded from the GenBank and aligned
using the MegAlign program of DNAStar software (version 7.1, DNASTAR
Inc., Madison, USA). The highly conserved sequences within the\emph{NS1}
gene were determined from an alignment of nine PBoV1/2 genomic sequences
(GenBank accession number HM053693, HM053694, HQ291309, KF025392,
KF025393, KF206155, KF206157, KF206161 and KM402139), and a pair of
primers PBoV1/2-F/R was designed using the Primer Premier (version 6.0,
USA) software based on the reference sequence (HM053693) (Table S1). The
specific primers for PBoV3/4/5 were found from an alignment of nine
sequences of PBoV3/4/5 (GenBank accession number JF429834, JF713714,
KC473563, JF713715, JN681175, JF429836, KF206158, JN621325 and
JN831651), and a pair of primers PBoV3/4/5-F/R targeting the \emph{VP} 1
gene of PBoV3/4/5 was designed based on the reference sequence
(JF429834) (Table S1).

PCR amplifications with primers PBoV1/2-F/R and PBoV3/4/5-F/R were
performed using the PCT-200 Peltier thermal cycler (MJ Research, USA).
The positive PCR products were purified using the Gel Extraction Mini
Kit (Watson Biotechnologies. INC) according to the manufacturer's
instructions, and cloned into pMD18-T vector (Takara, Dalian, China) to
produce the recombinant plasmids, which were designated as pMD18-327 and
pMD18-209 respectively. Plasmids were transformed into \emph{Escherichia
coli} DH-5\selectlanguage{greek}α \selectlanguage{english}competent cells (Takara) for sequencing, and their
concentrations and purities were quantified by ultraviolet absorbance at
260 nm and 280 nm using a Nano-100 micro-spectrophotometer (Thermo
Fisher Scientific, Waltham, MA, USA). The copy numbers of plasmids were
determined and 10-fold serial diluted in TE buffer (10mM Tris--HCl, 1mM
EDTA).

\textbf{2.3 SYBR Green I-based quantitative PCR assays}

The quantitative PCR standard curves of detecting PBoV1/2 and PBoV3/4/5
were generated with a known copy number (from 1\selectlanguage{ngerman}×10\textsuperscript{9} to
1×10\textsuperscript{0} copies/\selectlanguage{greek}μ\selectlanguage{english}L) of the recombinant plasmids pMD18-327
and pMD18-209, and performed in triplicate, respectively. The parameters
of the SYBR Green I-based quantitative PCR (qPCR) reactions were
optimized, and the sensitivity, specificity and reproducibility were
evaluated.

The qPCR was performed using the Rotor Gene 2000 real-time thermal
cycler (Corbett Research, Australia). qPCR protocol was executed in a
20-\selectlanguage{greek}μ\selectlanguage{english}L volume containing 3 \selectlanguage{greek}μ\selectlanguage{english}L DNA of samples, 10 \selectlanguage{greek}μ\selectlanguage{english}L of 2\selectlanguage{ngerman}×SYBR Premix
Ex\emph{Taq} I, 0.5 \selectlanguage{greek}μ\selectlanguage{english}L (25 \selectlanguage{greek}μ\selectlanguage{english}mol/L) of each primer and 6 \selectlanguage{greek}μ\selectlanguage{english}L
distilled~water. The amplification conditions were 95\selectlanguage{ngerman}°C for 3 min as
initial denaturation followed by 39 cycles of 95°C for 30 s, 58°C for 25
s and 72°C for 30 s.

\textbf{2.4 Complete genomic sequence amplification}

Another three pairs of specific primers (Q1F/Q1R, Q2F/Q2R and Q3F/Q3R)
were designed according to the reference sequence (HM053693) and used to
amplify three overlapping fragments covering the entire PBoV genome
(Table S1). For the two primer sets (Q1F/Q1R and Q2F/Q2R), PCR was
carried out in 25 \selectlanguage{greek}μ\selectlanguage{english}L of the reaction mixture: 12.5 \selectlanguage{greek}μ\selectlanguage{english}L of 2Phanta Max
Buffer, 2 \selectlanguage{greek}μ\selectlanguage{english}L of sample DNA, 1 \selectlanguage{greek}μ\selectlanguage{english}L of Phanta Max Super, 0.5 \selectlanguage{greek}μ\selectlanguage{english}L of dNTP
Mix, 0.5 \selectlanguage{greek}μ\selectlanguage{english}L of each primer (50 \selectlanguage{greek}μ\selectlanguage{english}M), and 8 \selectlanguage{greek}μ\selectlanguage{english}L of ddH\textsubscript{2}O.
The PCR parameters were as follows: pre-denaturation at 94\selectlanguage{ngerman}°C for 3 min;
followed by 35 cycles of denaturation at 98°C for 10 s, annealing at
55°C for 15 s, extension at 72°C for 2 min, and then followed by a final
extension step at 72°C for 10 min. For the Q3F/Q3R primer sets, the PCR
reaction mixture consisted of 12.5 \selectlanguage{greek}μ\selectlanguage{english}L of 2Es Taq Master Mix (Dye), 3 \selectlanguage{greek}μ\selectlanguage{english}L
of sample DNA, 0.5 \selectlanguage{greek}μ\selectlanguage{english}L of each primer (50 \selectlanguage{greek}μ\selectlanguage{english}M) and ddH\textsubscript{2}O
to a total volume of 25 \selectlanguage{greek}μ\selectlanguage{english}L. The reaction was performed by preheating for
5 min at 95\selectlanguage{ngerman}°C, followed by 35 cycles of 95°C for 45 s, 60°C for 45 s,
and 72°C for 2 min, and a final extension step for 10 min at 72°C. The
purified products were inserted into the pMD18-T Vector (Takara) for
sequencing, and all sequencing reactions were performed in duplicate.

\textbf{2.5 Sequence alignment and phylogenetic analyses}

Genomic sequences of PBoV strains obtained in this study were screened
for sequence similarities using BLAST
(http://www.ncbi.nlm.nig.gov/BLAST) against the database in GenBank, and
aligned altogether with 35 reference strains (Table 2) using the
MegAlign program of the DNAstar software (version 7.1, DNASTAR Inc.,
USA). Phylogenetic analysis was performed using the minimum-evolution
method in the Molecular Evolutionary Genetics Analysis (MEGA) software
(version 6.0) with Kimura distances, and a bootstrap of 1,000
replicates.

Seven recombination detection methods (RDP, GENECONV, BOOTSCAN, MaxChi,
Chimaera, SiScan and 3Seq) embedded in Recombination detection program
(RDP 4.39) were used to detected recombinant events. Only those
recombination events supported by more than four programs were deemed to
avoid dependence on a single detection method as described previously
(Zhai et al., 2010), and the window size was set to 20 bp, the highest
acceptable P value was 0.01.

\textbf{3 RESULTS AND DISCUSSION}

SYBR Green I-based qPCR assays were developed to detect PBoV1/2 and
PBoV3/4/5 in clinical samples from diarrheal piglets in 18 cities of
central China. The standard curve of PBoV1/2 was y = -3.6726x + 41.425,
with R\textsuperscript{2} values (square of the correlation coefficient)
of 0.99, and the detection limit was 16.6 copies/\selectlanguage{greek}µ\selectlanguage{english}L. The standard curve
of PBoV 3/4/5 was determined to be y = -3.3379x + 39.137, with
R\textsuperscript{2} values of 0.99, and the minimum detection limit was
33 copies/\selectlanguage{greek}µ\selectlanguage{english}L. For the evaluation of specificity, PBoV1/2 was detected
with a specific melting peak that the melting temperatures (Tm) was 88\selectlanguage{ngerman}°C
and PBoV3/4/5 with the Tm of 85°C. However, transmissible
gastroenteritis virus (TGEV), PEDV, PRRSV and PRV had no melting peaks.
The reproducibility of the assays was determined in triplicate using
inter- and intra-assay comparisons. The values of the intra-assay
standard deviation (SD) and co-efficient of variation (CV) ranged from
0.010 to 0.042 and 0.076\% to 0.678\%, respectively. The values of the
inter-assay SD and CV ranged from 0.112 to 0.539 and 0.768 \% to
2.304\%, respectively. These results showed that the qPCR assays for
PBoV1/2 and PBoV3/4/5 detection were really good to use with highly
sensitive, specific, and reproducibility.

According to the qPCR detection results of 281 clinical samples, 148
samples (148/281, 52.67\%) were positive for PBoV1/2, 117 samples
(117/281, 41.63\%) were positive for PBoV3/4/5, 55 samples (55/281,
19.57\%) were positive for both PBoV1/2 and PBoV3/4/5, and PBoVs were
detected on 32 pig farms in 17 cities except Pingdingshan (Table 1),
with 86.89\% (32/37) of pig farms harboring PBoVs, indicating that PBoV
was currently circulating in swine herds in central China. Overall, the
prevalence of PBoV was 74.73\% in central China (210/281), and it was
much higher than that of previous reports (Zhang et al. 2015; Zhou et
al. 2018b) which showed that PBoV mainly distributed in the east and
south coastal areas of China. The results suggested that PBoV has
broadly distributed among swine farms in diarrheal piglets in China.

In this study, PEDV and PCV2 were also detected as described qPCR
previously (Han et al., 2019; Zhao et al., 2019). The infection rate of
PEDV was 76.87\% (216/281), and the co-infection rate of PBoV and PEDV
was 49.11\% (138/281). PCV2 was found in all the positive samples of
PBoV. The high co-infection rate of PCV2 and PBoV in this study
demonstrated that the higher PBoV prevalence in samples from pigs with
PWMS, which were similar to results reported previously (Blomstrom et
al., 2010), suggested that PBoV might play a role in the development of
PMWS. In addition, the co-infection of PBoV with other pathogens was
common, such as PRRSV, CSFV (Blomstrom et al., 2010; Meng, 2012;
McMenamy et al., 2013; Luo et al., 2015). Pfankuche et al. (Pfankuche et
al., 2016) reported that PBoV should be considered as a pathogen that
triggers encephalomyelitis. Zhang et al. (Zhang et al., 2015) found a
higher incidence of PBoV in diarrheic pigs (73.95\%) when compared with
healthy pigs (47.83\%). However, until now, there has no definite
clinical disease associated to PBoV.

Complete genome sequences of two PBoV strains CH/HNZM and PBoV-TY from
Zhumadian in 2016 and Taiyuan in 2017 respectively were sequenced in
this study. The alignment analyses showed that two PBoV strains shared
94.8\% genomic nucleotide identity with each other, and also had three
putative ORFs. The genomic length of PBoV strain CH/HNZM was 5173 nt,
encoding \emph{NS1} (2,112 nt), \emph{VP1} and \emph{VP2} (2,115 nt,
including 1,701 nt \emph{VP2} ) and \emph{NP1} (687 nt), respectively.
The PBoV strain PBoV-TY comprised 5156 nt in length, with 2,112
nt\emph{NS1} gene, 2118 nt \emph{VP1} /\emph{VP2} gene (1,704
nt\emph{VP2} ), and 690 nt \emph{NP1} gene. The percent of complete
genome,\emph{NS1} , \emph{NP1} , \emph{VP1} and \emph{VP2} nucleotide
identity of two PBoV strains CH/HNZM and PBoV-TY with other 35 reference
strains were shown in Table 2.

The phylogenetic analyses of genomic sequences showed these
representative PBoV strains could be divided into three distinct
clusters, PBoV1, PBoV2 and PBoV3/4/5. The two PBoV strains CH/HNZM and
PBoV-TY were clustered into the PBoV2 group, formed a large branch with
other 12 PBoV2 strains (86.8\%-95.8\% genomic nucleotide identity), but
phylogenetically distinct from PBoV1, PBoV3/4/5 or HBoV groups
(44.5\%-55.3\%) (Figure 1). Additionally, the phylogenetic trees based
on the amino acid (aa) sequences of NS1, NP1, VP1 and VP2 proteins for
all 37 bocaviruses were constructed. The results showed that PBoV1 and
PBoV2 strains were closer based on NS1 analysis (Figure S1) and were
placed in a large cluster altogether with CslBoV1 and CMV strains,
whilst PBoV3/4/5 strains were in an independent branch. PBoV1 and PBoV2
strains were in a large cluster based on the NP1 sequences (Figure S2),
whilst PBoV3/4/5 strains were situated in another branch with 7 other
bocaviruses. Based on their VP1 and VP2 sequences (Figures S3 and S4),
PBoV1 and PBoV2 formed a large cluster altogether with CslBoV1 and CMV
strains, whilst PBoV3/4/5 was in a large cluster altogether with HBoV
and BPV1. On the basis of the phylogenetic tree analysis of the NS1,
NP1, VP1 and VP2 sequences, both PBoV strains CH/HNZM and PBoV-TY
belonged to PBoV2, which was identical to the result of phylogenetic
tree analysis of the genomic nucleotide sequences. Moreover, some PBoV
strains are closely related to HBoVs, leading to the hypothesis that
HBoV could be of zoonotic origin (Malecki et al., 2011; Zhang et al.,
2013). Thus, it is vital to pay more attention to the emerging and
reemerging PBoVs of swine.

In this study, the amino acids of NS1, VP1, VP2 and NP1 of the PBoV
strains were compared and analyzed. Among the 28 PBoV reference strains
we listed here, PBoV strain ZJD (HM053694)-China is a nearly full-length
genome sequence with 5186 nt that identified for the first time in
porcine samples in China in 2010 (Cheng et al., 2010). Hence, this
strain was chosen as the comparison, and the amino acids of NS1, VP1,
VP2 and NP1 of the two PBoV strains were compared with those of the
reference strain ZJD (HM053694)-China.

ORF1 that encoding NS1 is located at the 5' end of the PBoV genome, and
is essential for DNA replication. It has been illustrated to contain
conserved motifs associated with rolling-circle replication, helicase
and ATPase activities (Lau et al., 2011). After compared the \emph{NS} 1
amino acids of the two PBoV strains with the reference strain ZJD
(HM053694)-China, PBoV strain CH/HNZM contained eight major aa variation
sites, and PBoV strain PBoV-TY contained four aa variation sites. There
were six aa variation sites between the two PBoV strains CH/HNZM and
PBoV-TY (Table S2). Moreover, a higher variation in the NS1 amino acids
of PBoVs was found in this study, which was inconsistent with PBoV
strains from pigs in the USA (Jiang et al., 2014). ORF2 codes the capsid
proteins VP1 and VP2, and a conserved ``YXGXF'' motif domain was in the
unique VP1 protein (VP1u) (Yang et al., 2012). This domain indicates a
secretory phospholipase A2 (sPLA2) activity that is critical for
parvovirus infectivity (Cheng et al., 2010). On the basis of VP1 and VP2
amino acids of the two PBoV strains and comparison with reference strain
ZJD (HM053694)-China, PBoV strain CH/HNZM contained three major aa
variation sites, and PBoV strain PBoV-TY contained two aa variation
sites in VP1. PBoV strain CH/HNZM contained four major aa variation
sites, and PBoV strain PBoV-TY contained three aa variation sites in
VP2. There were only one aa variation site between the two PBoV strains
(CH/HNZM and PBoV-TY) in both VP1 and VP2 amino acids (Table S2). ORF3
encodes the NP1 protein that is located between ORF1 and ORF2, which is
a characteristic genetic feature of the \emph{Bocavirus} genus. Although
the function of NP1 in PBoV remains unclear, it is essential for CMV DNA
replication (Sun et al., 2009). There were four aa variation sites when
comparison with reference strain ZJD (HM053694)-China, and two aa
variation sites were found between PBoV strains CH/HNZM and PBoV-TY
(Table S2).

The accumulation of point mutations is an important mechanism for the
evolution of the Parvovirinae, and recombination could increase the
generation of new genotypes of PBoVs, which improve their capacity to
adapt and spread among Suidae hosts worldwide (Lau et al., 2011; Zeng et
al., 2011). The recombination analysis revealed that one putatively
recombinant breakpoint was existed in GD18 (KJ755666) strain located in
the \emph{VP1/2} gene at position 3,458 to 4,986 nt of the genome
(Figure 2). The major parent strain was 0912/2012 (MH558677) and the
minor parent strain was 57AT-HU (KF206160), which were supported by 7
programs (RDP, Av. P-Val = 3.158 × 10\textsuperscript{-14}; GENECONV,
Av. P-Val = 5.595 × 10\textsuperscript{-10}; BOOTSCAN, Av. P-Val = 3.85
× 10\textsuperscript{-14}; MaxChi, Av. P-Val = 1.89 ×
10\textsuperscript{-12}; Chimaera, Av. P-Val = 1.988 ×
10\textsuperscript{-13}; SiScan, Av. P-Val = 9.891 ×
10\textsuperscript{-19}; 3Seq, Av. P-Val = 8.082 ×
10\textsuperscript{-14}). Although no recombination signal was found for
either PBoV strain CH/HNZM or PBoV-TY with other PBoVs, the phylogenetic
analysis of VP1 and VP2 sequences showed the PBoV strain CH/HNZM was
clustered in a small branch altogether with PBoV strain GD18
from~Guangdong~province~of~China~in~2013, while the phylogenetic
analysis of genome and NS1 sequences showed the PBoV strain PBoV-TY and
PBoV strain GD18 were clustered in a small branch. The results
demonstrated that~PBoV~strains~CH/HNZM and PBoV-TY obtained in the study
may~be~novel~members~of~the~genus~bocavirus. Besides, a high prevalence
of mixed infection with PBoV1/2 and PBoV3/4/5 was reported, which
provided the possibility of recombination in the future.

In conclusion, this is the first study to demonstrate the prevalence of
PBoV in pig farms in central China. Complete genomic sequences of two
PBoV2 field strains were characterized, phylogenetic and recombination
analyses were conducted. The present study indicates that PBoVs are
prevalent in central China, and the co-infection with PEDV or PCV2 is
very common. The results of this study provide valuable information for
the further analysis of epidemiology and biological characteristics of
PBoV.

\textbf{Nucleotide sequence accession numbers}

The two complete genome sequences of PBoV strains CH/HNZM and PBoV-TY
have been submitted to the GenBank, and accession numbers are KX017193
and MH454686, respectively.

\textbf{ACKNOWLEDGEMENTS}

This work was supported by the National Natural Science Foundation of
China {[}grant number 31802218{]};Young Teacher Foundation of Henan
Province {[}grant number 2019GGJS051{]};
Zhongyuan~high~level~talents~special~support~plan {[}grant
number~204200510015{]} and Henan Agricultural University Technology
Innovation Fund {[}grant number KJCX2018C02{]}.

\textbf{CONFLICT OF INTEREST}

The authors declare that they have no conflict of interest.

\textbf{DATA AVAILABILITY STATEMENT}

The data that support the findings of this study are openly available in
this manuscript and the Supporting Information attached.

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\textbf{Figure legends}

Fig.1 Phylogenetic analysis based on the complete genome sequences of
two PBoV strains CH/HNZM and PBoV-TY together with 35 reference strains.
The tree was generated by the neighbor-joining method in MEGA7 software,
and bootstrap values were indicated for each node from 1, 000
replicates. The two PBoV strains~from the present~study~are~marked~with
black solid circles, and the GenBank accession numbers are shown in
parentheses.

Fig.2 Determination of potential recombination events by RDP4.39
programs (RDP, GENECONV, BOOTSCAN, MaxChi, Chimaera, SiScan and 3Seq)
(\emph{P} \textless{} 0.01). One putatively recombinant breakpoint was
existed in GD18 (KJ755666) strain that in the \emph{VP1/2} gene and
located at position 3,458 to 4,986 nt of complete genome. The analysis
was performed with an RDP distance model with window size of 20.

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