Porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 7 (Nsp7) plays an important role in the induction of host humoral immune response and could serve as an ideal antigen for serological genotyping assay for PRRSV based on the significant difference in immunoreactivities of North American (NA) and European (EU) PRRSV Nsp7. In this study, Nsp7 of NA and EU PRRSVwas separately expressed and purified using prokaryotic expression system. The purified recombinant Nsp7 proteins reacted with serum antibodies against corresponding genotype PRRSV in Western blotting. However, nonspecific reaction of whole recombinant Nsp7 with antibodies against another genotype PRRSV was observed, indicating that whole NA PRRSV Nsp7 and EU PRRSV Nsp7 have similar antigenic epitopes and recombinant proteins could not be used for genotyping of antibodies against PRRSV. Based on the analysis of similar antigenic epitopes at the hydrophilic region of NA PRRSV Nsp7 and EU PRRSV Nsp7 by bioinformatics assessment, partial Nsp7 gene region deleted sequences encoding similar antigenic epitopes was constructed by fusion PCR. The recombinant truncated Nsp7 (NA-deltaNsp7 and EU-deltaNsp7, about 43 kDa) was expressed and the molecular weight was about 43 kDa. The results of Western blotting showed that NA-deltaNSP7 and EU-deltaNSP7 could be specifically recognized by positive serum to NA or EU PRRSV individually and nonspecific reaction was eliminated. This study provided a basis for further development of serological genotyping assay for North American and European genotype PRRSV infection.

In order to understand porcine circovirus type 2 （PCV2） infection in Chinese pig population and analyze the relationship between the PCV2 infection and pig age,a total of 2 905 serum samples were detected for PCV2 by PCR method,which were collected from 71 small to medium scale farms,22 slaughter houses and 62 backyard farms in 28 provinces of mainland China in 2009. The results showed that 429 positive serum samples were found,and the aver- age positive rate was 14. 8~. The positive farm rate of slaughter houses, scale farms and backyard farms were 86.4%, 52.1% and 58.1%, respectively, and the infection rate of 48.6 % positive scale farms was less than 10%. To analyze the correlation between the PCV2 infection and pig age, the pigs of scale farms were divided into five age groups. The results showed that PCV2 infection was not detected in 2-4 week group, but in all other groups. The positive rate of 11-14 weeks group and 15-26 weeks group was higher than 5-7 weeks group and 8-10 weeks group. This work investigated the PCV2 infection in most Chinese provinces and explored the relationship between the PCV2 infection and pig age,and it provided valuable data for controlling the possible risk of PCV2 infection for the Chinese pig industry.

ABSTRACT Here, we report a novel porcine circovirus type 2a (PCV2a) strain with 11 nucleotides (nt) inserted in the origin of genome replication (Ori). This is the first report of a PCV2a strain with nucleotide insertion in Ori. Our study will help further epidemiological studies and extend our knowledge of evolutionary characteristics of PCV2.

The aim of this study was to investigate the prevalence of porcine circovirus 2(PCV2) and Torque teno sus virus(TTSuV) co-infection in China,1898 swine serum samples were collected from 29 provinces in China in 2009 and detected for TTSuV1,TTSuV2 and PCV2 by PCR.1 103 samples were TTSuV positive(58%),435 samples were PCV2 positive(23%).Among the positive samples,275(14%) samples were both positive for PCV2 and TTSuV,including 249(13%) infected with both TTSuV1 and PCV2,200(10%) infected with both TTSuV2 and PCV2,and 174 were triple infection.These results suggested that co-infection of TTSuV and PCV2 was highly prevalent in China.Distribution analyses of positive pig herds demonstrated that there were significant difference in different regions,and the breed pattern might not be a factor of aggravation in the co-infection.

Porcine circovirus type 2 (PCV2) is continuously evolving through point mutation and genome recombination. In the present study, genetic affiliations of 40 PCV2 strains were determined by amplification, sequencing and phylogenetic analyses during the nationwide molecular epidemiology investigation from 2009 to 2010 in mainland China. The results revealed that PCV2b was the predominant genotype in mainland China from 2009 to 2010 and PCV2b-1C within PCV-2b genotype was an emerging predominant subtype. More interestingly, five strains (09HaiN-1, 09HaiN-2, 10AH, 10GX and 10QH) were classified into a novel cluster along with the two main PCV2 genotypes PCV2a and PCV2b. Further analyses revealed that this novel cluster arose from recombination between PCV2a and PCV2b stains within the ORF2 gene. Moreover, BLAST search on NCBI website revealed that PCV2 strains of the novel cluster also emerged in Thailand, Indonesia and Laos, indicating that the novel cluster of PCV2 has also been circulating in some other Asian countries. This study is the first time to perform comprehensive analyses to demonstrate a cluster of PCV2 strains derived from the same type of inter-genotypic recombination pattern. Our findings provide valuable information on PCV2 evolution.

Porcine circovirus diseases (PCVD) are globally emerging diseases that have huge economic impacts on swine industry. Porcine circovirus type 2 (PCV2) is considered to be the essential primary causative agent of PCVD. In the present study, recombination analyses of PCV2 identified two possible recombination events with high confidence using recombination detection program, phylogenetic analysis and base-by-base comparison. These recombination events occurred between strains 09CQ (HQ395024) and ZhuJi2003 (AY579893), giving rise to two recombinants 09GS (HQ395028) and HN0907 (GU938303). Phylogenetic analyses of the parental strains at full length level suggest that natural recombination happened between PCV2b strains. Interestingly, recombination of the two parental strains yielded two recombinants through different recombination patterns with crossover regions mainly located in ORF1 and ORF2, respectively. These results demonstrate that recombination between PCV2b strains can occur both in non-structural protein coding region and structural protein coding region. Our study not only indicates that PCV2b strains can undergo recombination through a variety of patterns, but also suggests that recombination contributes to the genetic diversity of PCV2.

Postweaning multisystemic wasting syndrome of swine(PMWS)is associated with porcine circovirus type 2(PCV2).Simple and reliable diagnostic methods are needed to detect antibodies against PCV2 for monitoring early PCV2 infection.An indirect enzyme linked immunosorbent assay(ELISA) method based on purified recombinant capsid protein(Cap) was established to detect IgM antibodies against PCV2.The purified PCV2 Cap protein was used to coat 96 well plate as the antigen,and the following factors affected the experiments were optimized.The results demonstrated that optimal concentration of Cap protein for coating was 1.25 μg/mL,the optimal dilution of serum sample was 1∶100.The cutoff value was 0.35.The results showed no cross-reactivity with positive serum of other five porcine virus.The CV of intra-assay and interassay rangedfrom 2% to 6%.The indirect ELISA was used to detect 100 field serum samples,compared with control IgG/IgM kit,the diagnostic sensitivity,specificity,and accuracy of PCV2 IgM were 90.3%,92.8% and 92%.The result showed that the indirect ELISA was not only sensitive and specific,but also suitable for large scale epidemiological investigation of early PCV2 infection.

A high-mortality swine disease, the highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS), reappeared in some regions of China in 2009. To explore the possible mechanisms underlying the emergence of HP-PRRSV and more fully understand the extent of the genetic diversity of this virus in China, the complete genome of 14 isolates from 10 provinces in China from 2009 were analyzed. Full-length genome sequencing analysis showed that the 14 isolates were closely related to HP-PRRSV, with 98.0–98.9% nucleotide similarity, although 2 of the 14 strains exhibited a new, discontinuous 29-amino acid deletion in the Nsp2 gene. Furthermore, amino acid analysis of the GP5 protein indicated that the 14 isolates had a concurrent mutation in a decoy epitope and different mutations in glycosylation sites. Additionally, the antigenic drift in GP3 and a 1-nucleotide deletion in both the 5′-UTR and 3′-UTR, which are found in almost all highly pathogenic Chinese PRRSV isolates, were examined in all 14 isolates. The phylogenetic analysis showed that the 14 strains belonged to the North American genotype and were clustered in a subgroup with other HP-PRRSV isolates that have been found in China since 2006. However, compared with other Chinese HP-PRRSV isolates collected in 2006–2008, the phylogenetic tree showed that the 14 isolates had a closer relationship with each other. These results indicated that HP-PRRSV remained an extensive pandemic, affecting swine farms in China in 2009 and revealed new genetic diversity.