| Porcine circovirus (PCV) is a member of the family Circoviridae, a recently established virus family composed of small, non-enveloped viruses, with a circular, single-stranded DNA genome, it is one of the smallest animal viruses. According to the pathogenicity, antigenicity and nucleotide sequence of PCV, PCV can be divided into two types, PCV1 and PCV2. PCV1 is considered to be non-pathogenic to pigs by experimental inoculation and was circulating widely in swine population in the world. PCV2 has been recently associated with a number of disease syndromes, which have been collectively named porcine circovirus diseases (PCVD). Post-weaning multisystemic wasting syndrome (PMWS) is the most common. PMWS is distributed worldwide, and occurred in 7-15 weeks old piglets. Its clinical symptoms include weight loss, respiratory problems, diarrhea, visible mucous membranes pale, dermatitis, Lymph node swelling and kidney damage are often seen upon necropsy. Its mortality rate is high, particularly when co-infected with other pathogens.The main objectives of this study were (1) to characterize the genomic structure of PCV2 isolates prevalent in Zhejiang; (2) to establish a research platform to study TLRs pathway by using dual reporter gene assay and relative quantitative RT-PCR; and (3) to probe the effect of nucleotide fragments of PCV2 on the pathway of Toll-like receptors.1. To gain insights into the molecular epidemiological characteristics of PCV2 isolates prevalent in Zhejiang, PCV2-specific PCR method was used for mixed tissue samples, including spleen, lung and lymph nodes of diseased pigs during 2008-2009. Eighteen PCV2 isolates were then randomly selected for sequencing and sequence analysis. Out of 99 samples,47 were PCV2 positive (47.5%), and PCV2-positive rates in 2008 and 2009 remained at similar level to those during 2004-2006. Among 18 PCV2 isolates sequenced,5 belonged to subgroup 1A (27.8%),3 to subgroup 1B (16.7%), and 9 to subgroup 1C (50%). Group 1 remains to be the predominant PCV2 genotype in Zhejiang. Remarkably, subgroup 1C isolates has emerged in the region in recent years, and account for the majority at present, suggesting that this genotype probably plays a predominant role in the prevalence of PCV2. However, the pathogenic features of subgroup 1C isolates require further study.2. In order to set up a platform for research on TLR-mediated innate immune pathways, we used TLRs ligand (Poly (I:C) and ODN1826) as well as intracellular TLRs pathway inhibitor Chloroquine (Chq) as model molecules to detect NF-κB and TLRs mRNA expression levels by means of dual reporter gene assay and the relative quantitative RT-PCR. With 50μg/mL Poly (I:C) and 5μM ODN1826 stimulation, the cellular expression of NF-κB in RAW264.7 was significantly higher than unstimulated cells (238% and 113%, P＜0.01); the corresponding TLR ligands of TLR3 and TLR9 mRNA expression were also significantly higher than unstimulate cells (12.9(P<0.01) and 1.5). Therefore, the combination of dual reporter gene assay with quantitative RT-PCR may be used to examine the TLRs pathway by PCV2. We found that single strand DNA fragment of PCV2 was more potent than double strand DNA in inducing NF-κB expression.In summary, the present study revealed the molecular characteristics of PCV2 isoaltes prevalent in recent years in Zhejiang. The dual reporter assay in combination with quantitative RT-PCR could be used to examine the effect of PCV2 nucelotide fragments on expression of molecules along the TLR pathways. These works has laid the foundation for studying the relationship between amino acid mutations and PCV2 pathogenicity as well as between PCV2 infection and host immune responses.
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