Molecular characterization of porcine circovirus 2 isolated from diseased pigs co-infected with porcine reproductive and respiratory syndrome virus

Postweaning multisystemic wasting syndrome (PMWS), characterized by growth retardation, paleness of the skin, dyspnea, and increased mortality rates [1, 2], was first described in Canada in 1991, and is now widespread throughout swine production areas of the world [3, 4]. The genome of PCV is a single-stranded circular DNA of about 1.76 kb. ORF1 encodes the Rep proteins involved in virus replication and is highly conserved among isolates [5]. ORF2 encodes a 234 amino acid (aa) Cap protein, which is the main structural protein and also the major antigen inducing neutralizing immune responses [6]. The ORF3 protein is involved in PCV2-induced apoptosis by the caspase-8 and caspase-3 pathways [7]. However, healthy pigs experimentally inoculated with PCV2 developed only mild clinical symptoms [8, 9], suggesting that other concomitant factors may be needed for the development of typical clinical PMWS [10, 11]. Experimental studies on co-infection with PRRSV and PCV2 resulted in the microscopic lesions associated with PMWS and/or porcine dermatitis and nephropathy syndrome (PDNS), and lead to the development of severe disease [12].

In may 2008, severe disease, known as ''high fever'' occurred in several pig farms in shanghai, leading to a 57% death rate. PRRSV was detected in all the diseased piglets, genome sequence blast showed the strain belongs to genotype 2, 99.4% homologous to the PRRS virus strain JXA1 isolated in China, which has been proved to cause porcine high fever disease with high morbidity and mortality[13]. There was no Porcine Parvovirus (PPV) detected in all the samples, but we detected PCV2 from all the PRRS infected piglets (Figure 1). To investigate the genetic relationship of this newly identified Shanghai PCV2 isolate with existing viruses isolated from other parts of the world, we sequenced the complete genome of the sh0901 strain and carried out phylogenetic and polymorphic analyses.

In the present study, the diseased piglets were infected with PRRSV and PCV2 viruses, detected by PCR and RT-PCR amplification, therefore, the levels of exposure to the infectious agents were theoretically identical for all animals. The severe lesions and clinical symptoms indicated that PRRSV could be a predisposing factor to the high mortality, as previously suggested [14]. Several hypothesis have been suggested to explain this epidemiological situation, PRRSV may interfere with PCV2 clearance, favor the persistence of the virus. However, the nature of the interaction between PRRSV and PCV2 has not been elucidated to date. The ORF2 protein has been considered as a major immunogenic capsid protein, able to stimulate a protective response in pigs. Five immunoreactive epitopes in ORF2 were identified by Mahe' et al using Pepscan analysis[15], these included residues 25-43, 65-87, 113-147,157-183 and 193-207. Larochelle et al (2002) also identified three major regions of aa heterogeneity among ORF2 sequences at residues 59-80, 121-136 and 180-191, and two of these regions corresponded to two of the immunoreactive epitopes demonstrated by Mahe' et al [16]. In this study, we identified six highly polymorphic regions, 26-50, 57-83, 90-120, 134-154, 169-191 and 206-215, by polymorphic analysis, overlapped with corresponded immunoreactive epitopes, thus demonstrated that polymorphic analysis could be applied to deduce the immunoreactive epitopes of viruses. Our analysis show that ORF1 is highly conserved with only four polymorphic sites, and the amino acids at each of the sites belong to the same groups regarding chemical structure, which indicate that these mutations have no big influence on the activity of the Rep protein. Polymorphic sites were clustered in the capsid protein, and overlapped with the immunoreactive epitopes, So ORF2 may play a major role in the varied pathogenicity of PCV2 isolates.