| In late 2006, there was an outbreak of the so called"Mystery Fever Disease Syndrome"and''high fever''disease in pig herds in China. It was happened at Jiangxi, Hunan Province at the beginning and then spread to almost the whole country covered with more than 20 provinces, and made tremendous loss to the pig industry of China. The symptom of the disease includes the high body temperatures as high as 42℃, the red body skin, the anorexia, constipation/diarrhoea for some cases and sometimes with nervous symptom, secretion with the nose especially with the eyes, absorption in gestation sows. The most sensitive stages were the growing and finishing pigs, the gestation sows. The incidence and mortality were very high. The aim of this study was to investigate the role of the PRRSV in this epidemic, including, the nucleic acid variation and the virulence analysis, and eventually uncover the"mystery"of the disease or syndrome. The paper include six parts:From the 96 herds in Jiangxi,Hunan,Henan,Hainan,Guangxi,Guangdong 6 provinces in south China, the serum, lung and spleen/lymph node 225 samples were detecteded to RT-PCR, and isolated the virus by Marc-145/ PAMS cells meanwhile. The results showed that total positive of PRRSV was 37.5% in nosopoietic pigs, PRRSV were isolated in 53 samples by Marc145 cells, PRRSV were isolated in 48 samples by PAMS cells. All of PRRSV were American type by RT-PCR, which were indicated that pigs were infected serious in''high fever''disease in pig herds.Whole-genome of isolation Jiangxi-3 were obtained by splicing of more products of RT-PCR many fragments according to the sequence of VR2332. Whole-genome analysis revealed that Jiangxi-3 isolate are highly homologous to HB-1(sh)/2002, a Chinese strain of PRRSV (94.7% amino acids identity of Nsp2). More importantly, we observed a unique molecular hallmark in the viral isolate, namely a discontinuous deletion of 30 amino acids in nonstructural protein 2 (Nsp2). Compared with HB-1(sh)/2002, 480 aa were mutated (D→E) and the position of deletion were 481 aa and 532～560 aa. Compared with JXA1 isolate 99.2%～100% homology for every reading code, what is a Chinese strain of PRRSV isolated from''high fever''disease in pig herds in 2006. Jiangxi-3 shared 94.7%, 77.8%, 77.7% nucleic acid homology for Nsp2 compared with HB-1(sh)/2002,VR2332,RespPRRS MLV. The results showed that Jiangxi-3 and JXA1 were evolved by HB-1(sh)/2002.Eight PRRSV isolated newly were isolated from nosopoietic pigs in Jiangxi, Hunan, Hainan and Guangdong provinces respectively. The sequence analysis of Nsp2, ORF3 and ORF5 showed that these isolates were observed unique molecular hallmark in these viral isolates, namely a discontinuous deletion of 30 amino acids in Nsp2. The phylogenetic tree suggested that the strains from different provinces had close relationship, which were evolved by same strain.The primer E1/E2 were designed and synthesised according to the position of deletion, what can distinguish typical PRRS and Nsp2 deletion mutation PRRSV. The best condition of primer E1/E2 were determined by direct cross method,then RT-PCR which can detect Nsp2 deletion mutation PRRSV were established. The detection results of isolates showed that isolates were Nsp2 deletion mutation PRRSV, which isolated in''high fever''disease in pig herds.The pathogenicity of PRRSV variation (Jiangxi-3,Jiangxi-4,Hunan-1,Hunan-2)were researched, which were isolated from pigs of the so called"high fever"in pig herds in China. TCID50 of the four PRRSV were detected by Reed-Muench method, The 8 healthy pigs of 40 ages in days which were negative of antibody and antigen of PCV2 and PRRSV were separated 4 groups. One of every group was challenged by 4 isolations respectively, another is sentinel pig. The pigs were isolated artificial feeding. The TCID50 of the isolations were 103.75～104.5/mL respectively. The challenged and sentinel pigs appeared the symptom, the high body temperatures, secretion with the nose especially with the eyes, the red ears, anorexia. It is noticeable that all pigs show the symptom of different level diarrhea, and sometimes with nervous symptom. The results of pathological histologic slice suggested typical interstitial pulmonitis, lymphocyte sparseness and reticulocyte hyperplasia of groin, edema of gastric and intestinal wall proper layer,virulence meningitis. PRRSV RNA had been detected. The result showed the pathogenicity of variation isolations were stronger. The TCID50 of the mutation 9th generation of Jiangxi-3 isolate were 105.25/mL by Reed-Muench. The healthy pigs of 60 days ages in days were inoculated in order to detect LD50, which were free antibody and antigen of PCV2 and PRRSV. Inactivated vaccine were maded by Jiangxi-3 F9 according to common method,the pigs of immunity groups were inoculated by intramuscularly with inactivated vaccine. The immunity groups were challenged by 200 LD50 after frist immunity 21th, 28th and after second immunity 28th.Immunity effect of inactivated vaccine were evaluated by change of body temperature, clinical symptom and regular detection of PRRSV antibody and antigen in sera. The body temperature The pigs of the immunity group increased when were chanllenged 21th, 28th after frist immunity,but fervescence of the immunity group were not obvirous when were chanllenged 28th after second immunity. The monitoring of antibody indicates that the specific antibody to PRRSV increases within the 21~28 days after the first injection, the antibody 28th after frist immunity have difference compared with 21th after frist immunity(0.01 |
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