Genetic Variation Of Porcine Reproductive And Respiratory Syndrome Virus Isolates From China Based On NSP2 Gene And Imuno-Enhancing Effect Of GM-CSF On The Viral GP3/GP5 Protein

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus, belonging to Arteriviridae. PRRSV was firstly isolated in the last century, and then it has kept varying and caused great economic losses in pig farms. In 2000, the atypical PRRSV appeared in America and the immunized herds still encountered with serious mortality. PRRSV firstly emergeed in China in 1996, and then rapidly spread out to all the provinces. In 2006, there was a harrowing outbreak of PRRSV in the Mid-East China, which was characterized by durative fever (>41.0℃) and high mortality in herds of each age. The pathogen was identified as a new variant of PRRSV, with a unique hallmark in NSP2 gene (with two discontinues deletion of 30aa). In order to reveal the variation rule of PRRSV genetics and the contribution of NSP2 to PRRSV virulence, thirty-eight PRRSV isolates were isolated from different farms in the southeast of China during 2004-2009, and the NSP2 of the isolates were sequenced partly and analyzed. Then 4 representative isolates were selected to challenge piglets and the relationship between the NSP2 variation and virulence change was analyzed. Meanwhile, the recombinant adenovirus co-expressing the porcine granulocyte/macrophage colony stimulating factor (GM-CSF) and PRRSV GP3/GP5 was constructed and the immunogenicity was detected in mice and piglets.1. Isolation and identification of porcine reproduction and respiratory syndrome virusBase on the clinical epidemiology of the pig disease, we collected the lung samples from pathogenic pig farm in Jiangsu, Shanghai, Shandong, Anhui, Zhejiang and Guangxi provinces. After homogenization, freeze-thawing and then filtrated with filter membrane, the supernatants were collected and inoculated into Macr-145 cells or PAM for virus isolation. Thirty-eight PRRSV isolates were isolated and identified by RT-PCR and IFA. Among them, thity-two were isolated directly with Marc-145 cells and 6 were recovered with first PAM and then passaged in Marc-145. The 38 PRRSV isolates had different characters for in vitro culture and graw up with different titers of 10-3.75-10-6.33TCID50/ml. These virus isolates could be used to study on the disese in the future.2. Genetic variation of porcine reproduction and respiratory syndrome virus based on NSP2 during the period of 2004-2009Base on NSP2 sequence of PRRSV, two pairs of primers were designed for Nest-PCR to amplify the high variation region of NSP2. Totally 5 fragments with different length,1215,1275,1287,1373 and 1374bp were obtained from the 38 isolates. The homology of the isolates base on the nucleotide sequence was 72.7%-99.6% and the derivation of amino sequence was 63.7%-98.3%. According to the polygenic analysis results, the 38 PRRSV isolates could be classified into two subgenomic groups:NSP2-subtypeⅠand NSP2-subtypeⅡ. Of the 38 isolates,28 were sorted into NSP2-subtypeⅠsharing with 1 and 29 amino acids deletion, and the homology of NSP2-subtypeⅠwas 92.9%-99.2%. The isolates in NSP2-subtypeⅡhad 1 or no amino acid deletion with 76.5%-99.6% genic homology. Moreover, five different deletion patterns were identified in NSP2 in these 38 isolates. The most popular isolates were the virus with 30aa deletion. Two new deletion patterns were identified in these 38 isolates, which isolated since 2008. It indicated that the NSP2 of the PRRSV isolates in China had high variety and it was need to monitor on the variation of NSP2 in the future.3. The relationship between the NSP2 variation and the virulence of porcine reproduction and respiratory syndrome virusBased on the NSP2 sequence of the 38 PRRSV iolates, four different isolates named as YX0907, BB0907, SY0909 and NT0801 were selected. Among them, YX0907, BB0907 and SY0909 isolates were ranged to NSP2-subtypeⅠwith 30aa deletion and NT0801 isolate to NSP2-subtypeⅡwithout any amino acids deletion. The homology of these 4 isolates base on GP5 nucleotide sequence was 98.0%-99.8% and the derivation of amino sequence was 97.5%-99.5%. Twenty-three 45-day old piglets free of PRRSV, PCV2 and HPs were selected and randomly separated into 5 groups. The first 4 groups were challenged individully with the 4 PRRSV isolates by injection of 2ml of 2×104.32TCID50/ml. Three pigs in the 5th group were served as control. After challeng, the rectal temperatures of the pigs were recorded every day. The sera were collected at 7,14 and 21 days post challenge (dpc) for detection of the viremia. The dead and sacrificed pigs at 21 dpc were autopsied and the gross lesions were valued. The results showed that BB0907 isolate had highest virulence. It caused apparent fever and clinical disease with great pathological changes. And 3 pigs out of 5 were dead after infection. Comparing with BB0907, YX0907 isolate had less virulent. The pigs in this group also showed apparent clinical syndrome and tissue gross lesion, and 2 pigs were dead after inoculation. But NT0801 isolate has relatively lower pathogenic ability and caused the pigs had 1-2 days fever with slight clinical signs. SY0909 isolates has lowest virulence. Only some pigs in this group only experienced 1-2 days fever without clinical sign. Meanwhile, the pigs in the control group were quite healthy throughout the trail. These results revealed that the level of the virulence of these four isolates is BB0907> YX0907> NT0801> SY0909, indicating that the isolates with 30aa deletion have different pathogenic ability and the 30aa deletion has no relationship with the virulence of isolate.4. Construction and identification of the recombinant adenovirus co-expressing pig GM-CSF and GP3 and GP5 of porcine reproduction and respiratory syndrome virusPRRSV GP3 and GP5 were amplified from high pathogenic isolate SY0608 and pig GM-CSF was amplified from stimulated PBMC by RT-PCR, respectively. And then they were inserted into the different sites of the adenovirus shuttle vector one by one. Between GM-CSF and GP3, the gene of 2A protein from FMDV was used as a linker, and "AAGCTT" as a linker between GP3 and GP5. The recombinant plasmid was identified by restriction enzyme digestion and then sequenced. The linearized positive shuttle plasmid was electro-transformated into BJ5183 and recombined with the adenovirus backbone vector, and then recombined plasmid linearized with PacⅠ. After that, the recombinant adenovirus, named as pAd-GF35, were transfected into 293A cells. At 10 days post incubation, the recombinant adenovirus came up with CPE. Then, the expressions of these target proteins were identified by IFA and western blot, the bioactivity of the GM-CSF was confirmed by MTT assay and colony formation assay. It indicated that a recombinant adenovirus, rAd-GF35, co-expressing the GM-CSF-GP3-GP5 fusion protein was successfully constructed and it could be used to deteced the immunogenicity in the future.5. Immunogenicity of recombinant adenovirus co-expressing GM-CSF and GP3-GP5 of porcine reproduction and respiratory syndrome virus in miceSixty BALB/c mice were randomly separated into 4 groups with15 in each. The mice in the first two groups were ranged as control, empty control with PBS and scramble control with wtAd, respectively. Group3 were immunized with rAd-GP35 (expressing GP3-GP5 fusion protein) and group 4 with rAd-GF35 (expressing the GM-CSF-GP3-GP5 fusion protein). After 3 weeks, the all groups were boosted vaccined except those mice were slaughtered. And PRRSV-specific antibody, lymphocyte proliferation index, IFN-γand IL-4 were detected to evaluate the immunization at different time after vaccination. The results showed that the antibody against PRRSV became detectable in group3 and group4 at 21 days post immunization (dpi), and reached to the peak at 42dpi. At 42dpi, the neutralizing antibody (NA) titers in some mice from rAd-GF35 group could reach to 1:128 and the average level was 1:80. It was significantly higher than that in rAd-GP35 group with the level of 1:32. The lymphocytes proliferation index in rAd-GF35 group was significant higher than that of the rAd-GP35 group. The secretion of IFN-γand IL-4 of the lymphocytes from the mice immunized with rAd-GF35 is 175pg/ml and 87.3pg/ml and which are significant higher than that with rAd-GP35 (117.3 pg/ml and 46.5pg/ml). These results indicated that the recombinant adenovirus rAd-GF35 could induce distinctly higher humoral and cell immune response than rAd-GP35. GM-CSF could enhance the immune response induced by GP3/GP5.6. Protective efficacy of recombinant adenovirus co-expressing GM-CSF and GP3-GP5 of porcine reproduction and respiratory syndrome virusFifiteen 2-week old commericall pigs free of PRRSV, PCV2 and HPS were randomly separated into 3 groups with 5 in each. The first two groups were immunized with the recombinant adenovirus rAd-GP35 and rAd-GF35 (5×10.0TCID50/ml), respectively, and the last one as control, inoculated with wtAd. After 3 weeks, the all groups were boosted vaccined. PRRSV specific antibody, lymphocyte proliferation index, IFN-γand IL-4 were detected at different time after vaccination. And at 42 days post immunized (dpi), all the animals were challenged with high pathogenic PRRSV isolate SY0608 (2×104.0 TCID50/ml). And then the rectal temperature, virema, gross tissue lesion and microscope lesion were used to judgment the protective efficacy. The results showed that at 21dpi, rAd-GP35 and rAd-GF35 immunized pigs developed significant higher level of PRRSV-specific antibodies compared with the control. The highest NA titer in rAd-GF35group was 1:10 and the average level was 1:8. But the average NA titer in group rAd-GP35 was less than 1:4. The lymphocytes proliferation index of rAd-GF35 group was significantly higher than rAd-GP35 group. The contents of IFN-γand IL-4 in sera were 276.2pg/ml and 312.7pg/ml in rAd-GF35 immunized pigs, which are significantly higher than that immunized with rAd-GP35 (117.3 pg/ml and 46.5pg/ml). The pigs immunized with rAd-GF35 showed less clinical signs with integrated score 26.2, while the score in rAd-GP35 group is 44 and that in wtAd is 82.2. The pigs in rAd-GF35 group developed lower level of viremia (1.5×103.0 TCID50), which was significant with other two groups (3.3×103.0 and 104.0TCID50). Meanwhile, the piglets in control groups experienced the most serious tissue lesion. All results indicated rAd-GF35 could provide partly a protection against PRRSVchalleng in piglets.In summary, based on the NSP2 sequences of PRRSV isoaltes, it proved that there were a lot of PRRSV variants with various kinds of NSP2 deletion in China, which enriched our current epidemiologic knowledge of PRRSV. The results of challenge experiment revealed that there was no relationship between the 30 aa deletion of NSP2 gene and the virulence of PRRSV. The recombinant adenoviruses co-expressing porcine GM-CSF and PRRSV GP3/GP5 could induce PRRSV-specific immune response in mice and pigs. GM-CSF could enhance the immune response induced by GP3-GP5 and increased the protective effect to PRRSV challenge. This study provides new strategies for developing new efficient PRRSV vaccine.