Detection of PCV2e strains in Southeast China

Porcine circovirus 2 (PCV2) has been prevalent in swine herds in China since 2002, causing severe economic loss to the pig industry. The number of live pigs in southeast China is > 20 million. Since information on the genetic variation of PCV2 in the Fujian province is limited, the objective of the present work was to investigate the epidemiological and evolutionary characteristics of PCV2 in southeast China from 2013 to 2017. Of the 685 samples collected from 90 different swine herds from 2013 to 2017, 356 samples from 84 different swine herds were positive for PCV2. PCV2a, PCV2b, PCV2d, and PCV2e co-existed in the Fujian province, with PCV2d being the predominant circulating strain in swineherds and PCV2e being reported for the first time in China. Strikingly, PCV2-FJ-water DNA comes from contaminated river water and not infected animals. Sequence comparison among all isolates indicated that 95 isolates shared approximately 78.7%–100% nucleotide identity and 74.5%–100% amino acid identity for open reading frame 2 (ORF2). Amino acid alignment showed that the Cap protein of PCV2e differed markedly from those of PCV2a, PCV2b, PCV2c, and PCV2d. These results indicated that various PCV2 genotypes exist in China, and that PCV2 is continuously evolving, leading to rapid emergence of new variant stains.

Currently, PCV2 is the primary causative agent of swine viral disease worldwide, which has resulted in considerable economic loss since it was first identified in Canada in 1991(Harding & Clark, 1997Krakowka et al., 2000;Bolin et al., 2001). The viral genomes range from 1,766 to 1,768 nucleotides (nt) in length and contain four major identified open reading frames (ORFs) (ORF1-ORF4). ORF2 encodes the capsid protein (Cap), which is the only structural protein of PCV2 and is related to viral antigenicity; hence, the gene encoding the Cap protein is suitable as a phylogenetic and epidemiological marker (Gomes et al., 2007;Choi & Chae, 2008;Jiang et al., 2017). Currently, at least five distinct genotypes have been described in swine based on the ORF2 sequence, which have been classified as PCV2a, PCV2b, PCV2c, PCV2d, and PCV2e (Segalés et al., 2008;Harmon et al., 2015;Xiao, Halbur & Opriessnig, 2015;Davies et al., 2016;Karuppannan & Opriessnig, 2017). PCV2e is a new genetic group which was recently proposed by Davies et al. (2016). Currently, PCV2a, PCV2b, and PCV2d are found globally, whereas PCV2c has only been identified in Denmark and Brazil (Dupont et al., 2008;Franzo et al., 2015). PCV2e is a new genotype that has been circulating in the USA since 2015, the ORF2 sequences of which have 12 or 15 additional nts compared to those of PCV2a-PCV2d (Harmon et al., 2015;Davies et al., 2016).
PCV2 was first reported in China in 1999, and has become the causative agent of one of the country's most serious swine diseases (Lang et al., 2000;Shuai et al., 2007;Wang et al., 2009;Guo et al., 2012;Jiang et al., 2017). Previous studies showed that various genotypes, including PCV2a, PCV2b, and PCV2d are present in China. Among these genotypes, PCV2b and PCV2d have been predominant in China since 2004(Lang et al., 2000Shuai et al., 2007;Wang et al., 2009;Guo et al., 2012;Jiang et al., 2017). Recently, several groups showed that PCV2 isolates in pig farms had recombined owing to the co-existence of different PCV2 genotypes (Hesse, Kerrigan & Rowland, 2008;Cai et al., 2011;Huang et al., 2013;Ramos et al., 2013;Anoopraj et al., 2015). Despite continuous reports of newly emerging strains worldwide, information on the genetic variation of PCV2 in the Fujian province is limited. Therefore, to understand the current molecular epidemiology of PCV2 in Southeast China, the objective of the present work was to investigate the epidemiological and evolutionary characteristics of PCV2 in the Fujian province from 2013 to 2017.

Animal ethics
Sampling procedures were approved by the Animal Ethics Committee of the South China Agricultural University, with a reference number of SCAU-AEC-2014-10.

Clinical samples
Six hundred and eighty-five clinical samples (blood, lungs, kidneys, livers, and lymph nodes) were collected from unthrifty pigs (4-10 weeks of age) in different farms of

Polymerase chain reaction (PCR) amplification and nucleotide sequencing
Total viral DNA was extracted from tissue samples using DNA extraction kit ver.3.0 (TaKaRa, Dalian, China) following the manufacturer's protocol. ORF2 was amplified from the extracted DNA using specific primers according to previous studies (Fort et al., 2007). PCR products were purified using the gel extraction kit (Tiangen Biotech Co. Ltd., Beijing, China) and cloned into the pGEM-T Easy vector (Promega). Recombinant clones were sequenced by Sangon Biotech (Shanghai) Co. Ltd. Each fragment was independently sequenced at least thrice.

Phylogenetic analysis
Ninety-five ORF2 genes of PCV2 isolates were identified in the Fujian province during 2013-2017. Representative PCV2 sequences, including PCV2a, PCV2b, PCV2c, PCV2d, and PCV2e isolates in the GenBank, were used for sequence alignments and phylogenetic analyses. Multiplex sequencing alignments were performed using CLUSTAL X (version 1.83). The phylogenetic tree was constructed using the neighbor joining (NJ) method with MEGA 6.0, the maximum composite likelihood model, and a bootstrap confidence value of 1,000 replicates.

Recombination analysis
To detect putative recombination events in PCV2, recombinant strains were investigated on the complete genome database using a recombination detection program (RDP v.4.24) (Martin et al., 2010) as described by Ramos et al. (2013). Briefly, seven methods (RDP, GeneConv, BootScan, MaxChi, Chimera, SiScan, and 3Seq) were used for preliminary scan and only events detected by more than two methods with a highest acceptable p-value of 0.01 were considered. The recombination event was further confirmed using SimPlot program (v.3.5.1) and boot scanning analysis was performed with a 200-bp window sliding along a step size of 20 bp (Lole et al., 1999).

PCV2 detection
Of the 685 samples collected from 90 different swine herds located in Fujian province from 2013 to 2017, 356 samples from 84 different swine herds were positive for PCV2. Ninety-five positive samples were selected for further analysis of PCV2 characteristics (Table 1).

DISCUSSION
PCV2 has been prevalent in Chinese swine herds since it was first identified in China in 1999. Previous studies demonstrated that various PCV2 genotypes exist in China, including PCV2a, PCV2b, PCV2c, PCV2d, and a minor recombinant group. Surprisingly, different PCV2 genotypes or strains have co-existed in Chinese swine herds. Currently, the amount of live pigs in the Fujian province (Southeast China) is >20 million heads, although the epidemic status and genetic diversity of PCV2 is still unknown. Thus, we sought to investigate the genetic diversity and epidemiology of PCV2 in southeast China in 2013-2017. On the basis of ORF2 nucleotide sequences, the genotypes of Fujian PCV2 have been classified as PCV2a, PCV2b, PCV2d, and PCV2e (Opriessnig, Meng & Halbur, 2007;Guo et al., 2010;Guo et al., 2012;Harmon et al., 2015;Davies et al., 2016), of which, PCV2d was predominant in Fujian from 2014 to 2017. The present data indicate that the prevalence of PCV2d increased from 57.1% in 2013 to 87.5% in 2016, suggesting that PCV2d has replaced PCV2b and has now become the predominant PCV2 genotype in Fujian since 2013. Additionally, CN-FJ-CTS04 and FJ-6S-2017 strains emerged in cases of in cases of apparent vaccine failure. Overuse of vaccine has probably contributed to the variation of PCV2. PCV2e, a new PCV2 genotype, was identified in the USA in 2015, the ORF2 sequence of which contains 12 or 15 additional nt compared to other known PCV2 ORF2 sequences and shows approximately 85% identity with other known PCV2 ORF2 sequences (Harmon et al., 2015;Davies et al., 2016). In the present study, only two PCV2e sequences were detected in Fujian, suggesting that PCV2e replication is low in the swine herds. The group members encoded a capsid protein of 238 amino acids, which is markedly different from all known capsid proteins of 233 or 234 amino acids (Xiao et al., 2016). The pathogenicity of PCV2e warrants further investigation. Recently, several groups detected PCV2 DNA in water samples, human stool, beef, calf bone marrow, farm air, house flies, and mosquito (Verreault et al., 2010;Blunt et al., 2011;Garcia et al., 2012;Yang et al., 2012). In the present study, PCV2 DNA was also detected in river water, which is indicative of a potential human health hazard.
PCV2 nucleotide substitution rate has been calculated to vary between 1.2 × 10 −3 and 6.6 × 10 −3 substitutions/site/year, which is close to that of RNA viruses (Firth et al., 2009;Pérez et al., 2011). Recombination is critical for viral evolution, and recombination events between PCV2 strains could generate PCV2 of varying pathogenicity (Olvera, Cortey & Segales, 2007;Hesse, Kerrigan & Rowland, 2008;Wang et al., 2009). Both inter-genotypic and intra-genotypic recombination events of PCV2 have been recorded; inter-genotypic recombination occurs within ORF1 and ORF2, whereas intra-genotypic recombination mainly occurs within ORF2. Recently, a PCV2c genotype strain (GenBank accession no. KC823058) was reported in China, and three strains were generated from PCV2b and PCV2c recombination (Liu et al., 2016). In the present study, we detected that one natural strain (PCV2-FJ-SY) was generated by recombination between different lineages of PCV2, while CN-PCV2-FJ1145 was generated by intra-genotypic recombination. These results indicated that PCV2 is continuously evolving through genome recombination, which leads to rapid emergence of new variant stains.
In summary, PCV2a, PCV2b, PCV2d, and PCV2e co-exist in Fujian, and PCV2d-2 is the predominant strain that has been circulating in Fujian since 2013. Our results enhance the understanding of the PCV2 epidemic in Fujian, and may assist veterinary workers in establishing suitable prevention and control policies for the novel strains emerging from viral evolution.

CONCLUSIONS
The findings of this study demonstrated that PCV2 is ubiquitous in China, with PCV2d being the predominant strain circulating from 2013 to 2017. PCV2 is continuously evolving through genome recombination or new viral introductions from distant geographic regions, which leads to rapid emergence of new variant stains.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This work was supported by Major Project of the Science and Technology Program of Fujian Province, China (2014NZ0002-3), the Fujian Natural Science Foundation, Fujian Province, China (2016J01168), and the Collaborative Innovation Center of Animal Health and Food Safety Application Technology in Fujian, Fujian Vocational College of Agriculture (Dongxie 201701). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors: Major project of Science and Technology Program of Fujian Province, China: 2014NZ0002-3.