Phylogenetic analysis of porcine circovirus type 2 (PCV2) between 2015 and 2018 in Henan Province, China

Background Porcine circovirus type 2 (PCV2) is the pathogen of porcine circovirus associated diseases (PCVAD) and one of the main pathogens in the global pig industry, which has brought huge economic losses to the pig industry. In recent years, there has been limited research on the prevalence of PCV2 in Henan Province. This study investigated the genotype and evolution of PCV2 in this area. Results We collected 117 clinical samples from different regions of Henan Province from 2015 to 2018. Here, we found that the PCV2 infection rate of PCV2 was 62.4%. Thirty-seven positive clinical samples were selected to amplify the complete genome of PCV2 and were sequenced. Based on the phylogenetic analysis of PCV2 ORF2 and complete genome, it was found that the 37 newly detected strains belonged to PCV2a (3 of 37), PCV2b (21 of 37) and PCV2d (13 of 37), indicating the predominant prevalence of PCV2b and PCV2d strains. In addition, we compared the amino acid sequences and found several amino acid mutation sites among different genotypes. Furthermore, the results of selective pressure analysis showed that there were 5 positive selection sites. Conclusions This study indicated the genetic diversity, molecular epidemiology and evolution of PCV2 genotypes in Henan Province during 2015–2018.


Background
Porcine circovirus (PCV) is a kind of single-stranded circular DNA virus, including PCV1, PCV2 and PCV3. The non-pathogenic PCV1 was first discovered in 1974 [1,2]. In the late 1990s, the first post-weaning multisystemic wasting syndrome (PMWS) outbreak was identified and soon PCV2 became endemic causing severe economic losses in the swine industry [3,4]. Porcine circovirus type 2 (PCV2) plays a significant role in porcine circovirus associated diseases (PCVAD) [5], previously known as PMWS [6]. Many studies have reported the co-infection of PCV2 with other swine pathogens, such as porcine reproductive and respiratory syndrome virus, porcine parvovirus, swine influenza virus, Mycoplasma pneumoniae and Salmonella spp. [7]. Recently, a novel circovirus was identified by next generation sequence (NGS) analysis from aborted fetuses of sows and named porcine circovirus type 3 (PCV3) [8]. The newly discovered virus was associated with porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, cardiac and multisystemic inflammation [8][9][10][11]. In addition, PCV3 was also detected in healthy pigs without any clinical signs [12].
PCV2 encodes at least five proteins, including two major proteins, Rep and Cap, which are encoded by open reading frame 1(ORF1) and open reading frame 2(ORF2), respectively [13,14]. ORF2 encodes the capsid, which is a unique structure protein (Cap) and main antigenic determinant of PCV2, which is also commonly used for analyzing PCV2 genetic diversity [15]. Currently, five major genotypes were distinguished, including PCV2a-e [16,17]. PCV2a was the earliest genotype. Over time, PCV2b and PCV2d are the major genotypes widely existing in the world, and PCV2c was only found in some areas [18][19][20][21][22]. PCV2e is a new genotype that has been circulating in the United States since 2015. Compared with PCV2a-PCV2d, its ORF2 sequences have 12 or 15 additional nts [17,23]. In addition, a few recombinants of different genotypes existed in nature [24]. PCV2 have a high evolutionary dynamics as single-stranded RNA viruses [3].
Although the current vaccines have decreased the economic impact of PCVAD in pig herds [25], PCV2 remains economically important in the global swine industry and still exists in pig herds. Continuous reports of newly emerging strains worldwide, but information on the genetic variation of PCV2 in the Henan province is limited. Henan Province is the hinterland of China, with well-developed transportation, as well as a large pigraising province. There are many pig farms, frequent introduction and transfer of pigs, and low biosafety level. All these will bring opportunities for the transmission, variation and recombination of pathogenic microorganisms. Therefore, to understand the current molecular epidemiology of PCV2 in central China, the objective of the present work was to investigate the epidemiological and evolutionary characteristics of PCV2 in the Henan province from 2015 to 2018.

PCV2 detection and genomic sequences
All 117 tissue samples from Henan Province during 2015-2018 were detected by PCR, of which 73 were PCV2 positive, with the infection rate as high as 62.4% (Fig. 1). 37 PCV2 genomes were selected and obtained for further analysis of PCV2 characteristics. Table 1 summarizes the strain designation, year of collection, geographic origin, genotype and GenBank accession number of these PCV2 genomes. Interestingly, 2 (HN-QY-2016 and HN-BF-2016) out of the 37 PCV2 genomes obtained in this study included 1778 nt. Apart from above two strain, the genome size of PCV2 HN-JY-2015 strain was 1766 nt and other two (HN-YY-2016 and HN-LH-2018) genome had 1768 nt, all other PCV2 genome size was 1767 nt, that is still within the size range of published PCV2 genomic sequences (1766-1768 nt). Pairwise-sequence comparisons of complete genomes revealed that the nucleotide sequence similarity between 37 PCV2 strains and 15 PCV2 reference strains varied from 93.6 to 99.9%, and the homology among 37 PCV2 strains was between 94.2 and 99.9%. In addition, the length of the ORF2 was varied from 702 to 705 nt with nucleotide homology ranged from 89.2 to 100% for ORF2. Of these, 25 sequences were 702 nt in length and 12 were 705 nt.

Phylogenetic analysis and genotype identification
In order to get a better understanding of the genetic relationship and evolution of PCV2, a phylogenetic tree based on the ORF2 gene of the 37 PCV2 strains sequenced here was constructed together with 19 reference sequences deposited in GenBank database, which  (Table 2). In comparison with the phylogenetic tree based on the ORF2 gene of PCV2, all 56 strains have the same classification according to phylogenetic tree by complete genome (Fig.  2b). From here we see that PCV2b and PCV2d become the two predominant genotypes in the Henan province from 2015 to 2018.

Molecular characterization
The

Discussion
Since PCV2 was detected in early 1990s, it has caused considerable economic loss in the swine industry and become one of the most extensively investigated viral agents of pigs globally. The genetic diversity of PCV2 is continuously increasing, and novel subtypes are still emerging [4,26]. As everyone knows, the pork industry in China contributed to nearly half of worldwide pork production in 2018. During the last 30 years, pork production in China has increased two-fold [27]. Furthermore, Henan is a major pig-raising province which located in the central part of China. To some extent, the infection and molecular epidemiology of PCV2 in Henan could represent that of China. In this study, we collected 117 tissue samples between 2015 and 2018 in Henan to investigate infection and molecular epidemiology of PCV2.
The prevalence of PCV2 among swine populations in China has considerably increasing since 2000 [28][29][30]. PCV2a was the earliest and predominant genotype in China [31]. Despite a wide variety of vaccines ranging from inactivated vaccines to baculovirus-expressed recombinant capsid protein-based vaccines have been widely used since 2009 [32,33], clinical and subclinical infections of PCV2 still existed in China. Our results show that the positive rate of PCV2 was about 62.4% in Henan. High positive rates of PCV2 have been reported in different areas of China [34][35][36]. Subsequently, a shift from PCV2a to PCV2b occurred in China, suggesting that PCV2b become the predominant genotype in China [37][38][39]. Recently, a genotype shift to PCV2d has recently occurred on a nationwide scale [4,28]. The genotype shift of PCV2 may have an impact on virulence that may related to vaccination, pathogenesis and diagnosis [40,41]. Consistent with previous results, our investigation indicates that 56.8% (21 of 37) of isolated PCV2 strains were the PCV2b genotype, 35.1% (13 of 37) belonged to the PCV2d genotype, and 8.1% (3 of 37) were classified to the PCV2a genotype. Previous studies also demonstrated that PCV2c and recombinant strains had existed in China [19,42]. However, two genotypes above weren't found in this research, which maybe because of relative small sample size and limited sampling scope (only in Henan). Interestingly, 37 PCV2 newly detected strains could be divided into four clusters (1766 nt (2.7%), 1767 nt (86.5%), 1768 nt (5.4%) and 1778 nt (5.4%)) based on genome size. Normally, genome size of PCV2 is 1767 or 1768 nt. PCV2 strains with a genome of 1766 nt have also been reported in previous investigations [37,43]. However, 1778 nt genome strain was rarely reported [18,44,45].
Furthermore, ORF2 was widely used for phylogenetic analysis and genotype identification of PCV2. The typical motifs in ORF2 can be used to identify different  [41,48]. From the results of amino acid alignment in this research, Y8F, F53I, I57V, A68N, S121T, T134 N, S169G/R and V215I, were found in PCV2d ORF2. Recent study revealed that nuclear localization signal (NLS) located in N-terminus of the PCV2 Cap could function as a cell-penetrating peptide (CPP) being capable of entering cells [49]. In addition, the aromatic side chains of tyrosine (Y) and phenylalanine (F) in NLS may insert into and anchor NLS-A on membranes. Because both F and Y include a bulky aromatic ring, Y8F may not affect cell penetration of NLS. Other amino acid mutations are possibly responsible for the immunogenicity change of the Cap protein. Further studies are required to determine the effects of these mutations on viral properties. At present, the detection rate of PCV2 is still very high in the case of widespread use of vaccines, which is related to the genetic variation of PCV2. Conversely, it is this pressure that promotes the evolution(1.2 × 10 − 3 ) of the virus, which makes PCV2 to have an evolutionary dynamics that is much closer to single-stranded RNA (ssRNA) viruses rather than ssDNA viruses [3]. In this experiment, we found five positive selection sites (30, 63, 89, 90 and 190) in PCV2 ORF2 of China under selection pressure. All positively selected sites were related to the main capsid epitope regions: Position 30 is involved the core epitope (residue 26-36) located in the nuclear localization signal region [50]; Position 63 is located in immunorelevant epitopes at residues 51-81 [41,48]. Position 89 and 90 are located in the typical motifs of different PCV2 genotypes [46,47]. Position 190 are  (51-81), B (113-134), C (161-208) and D (228-233)) and immunodominant decoy epitope E (168-180), were demarcated by the colored boxes, in bright green, blue, white, black and dark red, respectively important for determining neutralization activity [51,52]. Probably, these mutations are associated with virus escape from the host immune system.

Conclusions
In conclusion, we investigated the infection of PCV2 in Henan province from 2015 to 2018 and found that the infection rate was 62.4%. 37 new isolates of PCV2 were sequenced. Through alignment and phylogenetic tree analysis, it was found that the prevalent genotypes were mainly PCV2b and PCV2d. Surprisingly, two rare 1778 nt PCV2 strains were found. Some new amino acid mutation sites were found by ORF2 amino acid alignment. Five positive selection sites in ORF2 of PCV2 in China were found by selective pressure analysis. The results of this study will lay a foundation for understanding PCV2 infection, molecular epidemics and evolution in Henan Province.

Sample collection
A total number of 117 clinical samples (lung, lymph node, tonsil and spleen) were collected from Henan Province, in central China between 2015 and 2018. The pigs suspected to have clinical symptoms of PMWS and/or PDNS came from different farms. After collecting tissue samples, the pigs were treated innocuously. Tissue samples were suspended in sterile phosphate buffered saline (PBS) and homogenized. These homogenized samples were centrifuged at 8000 rpm for 5 min. Supernatant were aliquoted and stored at − 80°C for further analysis.

Phylogenetic analysis
The Multiple sequence alignments were carried out using Clustal W of the Megalign program (DNAStar software). To determine the genotype of the PCV2 isolates, 19 reference PCV genome sequences (Table 4) were downloaded from the GenBank database. Phylogenetic trees were constructed on the basis of ORF2 and full-length nucleotide sequence of the 37 isolates and 19 downloaded sequences with the MEGA 7.0.14 software using the neighbor-joining (NJ) method, with the Jukes- When p-value was below 0.1 in SLAC, FEL, MEME and the posterior probability was above 0.9 in FUBAR, positive selection pressure signals were considered to acceptable. Positively selected sites were confirmed when positive selection pressure signals were identified by at least two methods. "∞" represents numerical infinity cantor model as a nucleotide substitution model. The reliability of the generated trees was determined with 1000 replicates of the data set. Furthermore, the amino acid sequence of all PCV2 capsid proteins were aligned using the MegAlign software and plotted by EPSprint 3.0 [54].

Selection pressure analysis
The detection of selection pressure on the ORF2 coding sequences of 37 new isolates and 104 China PCV2 strains (Additional file 1: Table S1) was performed using Datamonkey (http://www.datamonkey.org), which based on the ratios between non-synonymous and synonymous substitution rates (dN/dS) [55]. The methods used to investigate dN/dS ratio and positive codon sites at individual codons included Single-likelihood ancestor counting (SLAC), fixed-effects likelihood (FEL), mixed effects model of evolution (MEME) and fast unconstrained Bayesian approximation (FUBAR). When p-value was below 0.1 in SLAC, FEL, MEME and the posterior probability was above 0.9 in FUBAR, positive selection pressure signals were considered to acceptable. Positively selected sites were confirmed when positive selection pressure signals were identified by at least two methods.
Additional file 1: Table S1. ORF2 sequence of PCV2 strains in China were used for the analysis of selection pressure in this study. Table S1 contains GenBank accession numbers and years of 104 PCV2 ORF2 sequences in China. These ORF2 sequences were used for selection stress analysis.