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 R2 values (square of the correlation coefficient)
of 0.99, and the detection limit was 16.6 copies/µL. The standard curve
of PBoV 3/4/5 was determined to be y = -3.3379x + 39.137, with
R2 values of 0.99, and the minimum detection limit was
33 copies/µL. For the evaluation of specificity, PBoV1/2 was detected
with a specific melting peak that the melting temperatures (Tm) was 88°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 SYBR Green I-based
qPCR assays for PBoV1/2 and PBoV3/4/5 detection were really good to use
with highly sensitive, specific, and reproducibility.
According to the SYBR Green I-based PCR 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, and
55 samples (55/281, 19.57%) were positive for both PBoV1/2 and
PBoV3/4/5. 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) in China which showed that PBoV mainly
distributed in the east and south coastal areas of China, and Jiangsu
province was the center of high PBoV occurrence frequency. However, our
results suggested that PBoV has broadly distributed among swine farms in
diarrheal piglets in central China, indicating that this virus should be
thought as a probable cause of diarrhea in pigs.
In this study, PEDV and PCV2 were also detected. 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.
Two complete genomes of PBoV strains (CH/HNZM and PBoV-TY) were acquired
and then the alignment analyses showed that they also had three putative
ORFs. The genomic length of PBoV strain CH/HNZM was 5173 nt, encodingNS1 (2,112 nt), VP1 and VP2 (2,115 nt, including
1,701 nt VP2 ) and NP1 (687 nt), respectively. The PBoV
strain PBoV-TY comprised 5156 nt in length, with 2,112 nt NS1gene, 2118 nt VP1 /VP2 gene (1,704 nt VP2 ), and 690
nt NP1 gene. The genetic relationships of two PBoV strains with
35 reference strains (Table 2) were analyzed by constructing the
phylogenetic tree based on genomic nucleotide sequences. The results
showed that the pairwise similarity of the two PBoV strains was 94.8%,
and these representative PBoV strains could be divided into three
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, and had 86.8%-95.8% nucleotide sequence
homology. However, they were phylogenetically distinct from PBoV1,
PBoV3/4/5 or HBoV groups (44.5%-55.3%) (Fig.1). Interestingly,
PBoV3/4/5 group showed a closer phylogenetic relationship with HBoV than
that with PBoV1 or PBoV2 groups, which indicated that PBoV3/4/5 group
might be a further source of HBoV. Additionally, the phylogenetic trees
based on the amino acid sequences of NS1 , NP1 , VP1and VP2 proteins for all 37 bocaviruses were constructed. The
results showed that PBoV1 and PBoV2 strains were closer based onNS1 analysis (Fig.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 (Fig.S2), whilst PBoV3/4/5
strains were situated in another branch with 7 other bocaviruses. Based
on their VP1 and VP2 amino acid sequences (Fig.S3, 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 theNS1 , NP1 , VP1 and VP2 protein sequences, the
two 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.
Capsid proteins of parvoviruses have close relationship with
immunogenicity and tissue tropism. Minor genetic changes of proteins
could alter the pathogenic potential and host range of parvoviruses
(Kapoor et al., 2012). In this study, the amino acids of NS 1,VP1, VP2 and NP1 of the PBoV strains were compared and
analyzed. Among the 28 PBoV reference strains we listed in this study,
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). We chose this strain as the
comparison, and the amino acids of NS 1, VP1, VP2 andNP1 of the two PBoV strains were compared with the reference
strain ZJD (HM053694)-China.
ORF1 that encoding NS 1 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 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.S1).
Moreover, a higher variation in the NS 1 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 proteinsVP1 and VP2 , and a conserved “YXGXF” motif domain was in
the unique VP1 protein (VP1 u) (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) both
in VP 1 and VP 2 amino acids (Table.S1). ORF3 encodes theNP1 protein that is located between ORF1 and ORF2, which is a
characteristic genetic feature of the 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.S1).
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 VP1/2 gene at position 3,458 to 4,986 nt of the genome
(Fig.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 × 10-14; GENECONV, Av. P-Val =
5.595 × 10-10; BOOTSCAN, Av. P-Val = 3.85 ×
10-14; MaxChi, Av. P-Val = 1.89 ×
10-12; Chimaera, Av. P-Val = 1.988 ×
10-13; SiScan, Av. P-Val = 9.891 ×
10-19; 3Seq, Av. P-Val = 8.082 ×
10-14).
Although
no recombination signal was found for either PBoV strain CH/HNZM or
PBoV-TY with other PBoVs, the PBoV strain PBoV-TY was clustered in a
small branch altogether with PBoV strain GD18
from Guangdong province of China in 2013, with 94.2% genomic nucleotide
identity. The results demonstrated that PBoV strain CH/HNZM or PBoV-TY
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. Two complete genome sequences of
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.