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 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 NS1 (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 ntNS1 gene, 2118 nt VP1 /VP2 gene (1,704 ntVP2 ), and 690 nt NP1 gene. The percent of complete genome,NS1 , NP1 , VP1 and 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 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 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 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 × 10-14; 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 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.