| Porcine circovirus type 2(PCV2),which is known as an important pathogen of porcine circovirus associated disease(PCVAD), cause emaciation, low growth rate and other clinicalsymptoms in weaned pig. Moreover PCV2 can cause immunosuppression, which result in coinfection with other pathogens.since PCV2 was reported for the first time in China in 2000, it quicklyspreads to many provinces andthen cause the outbreak of post-weaning multisystemic wastingsyndrome(PMWS), which result in a large economical losses in pig industry in China.however, the PCV2 is rather difficult to culture in vitro and its replication in cell culture get a low output. Thus, it is urgent need of a viral strainthat could well replication in PK-15 cell line.The purpose of thisstudy is to isolate PCV2 strains in clinical tissue samples. First we detect the tissuesamples by polymerase chain reaction(PCR) directly.Then we inoculate the positive samples in PK-15 cells and passage three times blindly.Influeoresence Assay is performed to detect cell cultureof the third viral passage. At the same time, the whole genomes sequencing is necessary to investigate the genetic variation of the isolated viral strain.The results of this study showed:(1)Five PCV2 strains were successfully isolated from the tissue samples, which were named 311-JS01,312-JS02, FJ-JS03,119-GD01 and YJ-GD02 respectively;(2)These sequencingshowed that the sizes of three isolated strainswere 1766bp(311-JS01,312-JS02 and FJ-JS03) and the remaining two(119-GD01 and YJ-GD02)were 1767bp;(3)Phylogenetic tree analysis found that the four of the isolates(311-JS01,312-JS02,FJ-JS03 and 119-GD01) belong PCV2b-1C, while YJ-GD02 belong PCV2b-1A/1B. These five isolatesshared 95.7%-99.9%homology with each other and 95%-99.0% with other PCV2 strains from home and abroad in Gen Bank;(4)Determineda culture method of PCV2 isolates: after inoculation 24 h, treat PK-15 cell with 300mmol/L of D-glucosamine for 25 min and then continue culturing for 72 h. With the successful isolation of these 5strains, the research in biological characteristics, pathogenesis,vaccine development of PCV2 became easier.Challenge PCV2sero-negative commercial pigs with 119-GD01 strain through intranasal inoculation and neck intramuscular injection. The pigs in challenge group were immunized with keyhole limpet hemocyanin(KLH) on the 3th day before challenge and the 4th, 7th day after challenge respectively, while intraperitoneal injection of thioglycollic broth were giving on the 3th day before challenge and the 4th, 7th,11 th,18th day after challenge.Additionally,set up the negative control group without challenge. All the experimental pigs were slaughtered on the 28 thday.The results showed:(1)The challenge group show a fever last for 2 weeks;(2)Viremia appeared in the challenge groups at 7DPI and disappeared at 25 DPI, while serologic sero-conversion occurred at 18DPI;(3)By quantitative polymerase chain reaction(q-PCR), a high load viral DNA was detected in inguinal lymph nodes, tonsils,spleen and lungs, especially in the inguinal lymph nodes;(4)Histopathological changes including lymphocyte depletion and granulomatous inflammation were observed by HE staining. Meanwhile IHC showed that strong PCV2 positive signals were detected in inguinal lymph nodes, tonsils and spleen which meant that abundant PCV2 antigen were accumulated in those organ.According to the standards including persistent fever, viremia, high viral load titer in organ and positive reaction of IHC, 119-GD01 strain had infected the commercial pigs. This meant a PCV2b-infection model in pig was established which could be used for the research on PCV2 b pathogenesis and efficacy assessments of new vaccine. |
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