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Molecular Bases of Different Virulence between Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus and Its Attenuated Derivative

Author: JiangYiFeng
Tutor: TongGuangZhi
School: Chinese Academy of Agricultural Sciences
Course: Preventive Veterinary Medicine
Keywords: PRRSV Virulence difference ORF1a ORF1b ORF2-7
CLC: S852.65
Type: PhD thesis
Year: 2012
Downloads: 171
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Abstract


Porcine Reproductive and Respiratory Syndrome (PRRS) caused by PRRS virus (PRRSV) is one of the most important diseases destructing the swine industry worldwide and is characterized by the productive failure and stillbirth in sows, and respiratory problems in piglet. In China, the first report about the prevalence of PRRSV is in1996. In2006a pandemic of highly pathogenic PRRS (HP-PRRS) occurred and resulted in significant economic loss to swine industry. So far the mechanism for the virulence increasement of HP-PRRSV is not clear. To elucidate the mechanism involved the virulence enhancement from molecular level may be immensely contributed to the elimination and control PRRS.To investigate the molecular mechanisms of the different virulence between highly pathogenic porcine reproductive and respiratory virus (HP-PRRSV) HuN4-F5strain and its attenuated vaccine strain HuN4-F112, we generated six full length infectious cDNA clones with interchange of ORF1a, ORFlb, or ORF2-7region, between the two viruses, named rHuN4-F5-ORF1a, rHuN4-F5-ORF1b, rHuN4-F5-ORF2-7(genetic backbone of virulent HP-PRRSV, HuN4-F5with ORFla, ORF1b or ORF2-7from attenuated HP-PRRSV, HuN4-F112) and rHuN4-F112-ORF1a, rHun4-F4112-ORF1b, rHun4-F112-ORF2-7(genetic backbone of HuN4-F112with ORFla, ORFlb or ORF2-7from HuN4-F5), respectively. The growth kinetics showed that rHuN4-F5-ORFla had significantly higher titer than its parental virus rHuN4-F5, and rHuN4-F112-ORF1a had significantly lower titer than its parental virus rHuN4-F112, while other chimeric viruses had similar growth curve with their parental viruses on MARC-145cell. When PRRSV-negative piglets of30-day old were infected with these chimeric viruses, all piglets remained normal as groug of rHuN4-F112infected pigs with measurements of body temperature and clinical signs.The piglets were infected with rHuN4-F5-ORFla, rHuN4-F5-ORFlb and their parental virus rHuN4-F5, and the sera from each pig were collected. The virus titer in sera from pigs infected with the chimeric viruses was significantly lower than that from parental virus at3,5,7and14dpi. But pigs infected with rHuN4-F5-ORF2-7showed significantly lower titer than that of rHuN4-F5at7and14dpi. There was no significantly difference among pigs infected with chimeric viruses except rHuN4-F112-ORFla at7dpi. The antibodies in response to rHuN4-F5-ORF1b, rHuN4-F5-ORF2-7and rHuN4-F5were quickly produced as early as7dpi, which is faster than infected with the other chimeric viruses and the parental virus rHuN4-F112.The level of IL-1in response to rHuN4-F5was significantly increased and all the chimeric viruses were not significantly chenged at a range from3d pi to28dpi. The levels of IL-6in response to rHuN4-F5infection were much higher than that infected with chimeric viruses or rHuN4-F112at3,5,7,14,21and28dpi. While the infection with rHuN4-F5-ORF2-7led to significantly higher level of IL-6than that infected with the other chimeric viruses or rHuN4-F112at both3and5dpi.When it was infected with rHuN4-F5-ORFlb, rHuN4-F5-ORF2-7and rHuN4-F5, IL-10was produced significantly. At3,5,7and14dpi, higher levels of IL-10were induced by the infection of rHuN4-F5-ORF2-7or rHuN4-F5than that induced by the other virus. And at3,5,7and14dpi the infection of rHuN4-F5-ORFlb triggered much higher amount of IL-10than that infected by the other viruses except rHuN4-F5-ORF2-7and rHuN4-F5. The rescued viruses rHuN4-F5-ORFlb and rHuN4-F5-ORF2-7with a common backbone of rHuN4-F5stimulated higher amount of IL-10than that rHuN4-F5-ORFla and rHuN4-F112-ORFla with a backbone of rHuN4-F112. Actually, there was no constant dogma for the influence of backbone on the excitement of IL-10, which was indicated by the fact that the infection of both rHuN4-F5-ORF1a and rHuN4-F112-ORFla stimulated IL-10with similar level.The infection of all the viruses also resulted in the secretion of1FN-γ with a moderate level. And amoug them rHuN4-F5showed the strongest competency, while the remainings showed similar ability.On the other hand recombinant viruses rHuN4-F112-nsp9, rHuN4-F112-nsp10, rHuN4-F112-nsp11(genetic backbone of rHuN4-F112with nsp9, nsp10and nsp11from rHuN4-F5) were rescued to reveal the role of three important protein in ORFlb(nsp9,nsp10and nsp11) in virulence of PRRSV. Compared to the growth characteristics of the recombinant viruses with their parental viruses by muti-step growth kinetics, it was found that rHuN4-F112-nsp9and rHuN4-F112-nsp10had higher titers than rHuN4-F112-nsp11and rHuN4-F112at early stage(12-24h) of infection. Further study of the pathogenesis of the recombinant viruses in PRRSV-negative piglets indicated that the three recombinant viruses with nsp9, nsp10or nsp11from virulent PRRSV exhibited low virulence for piglets, like their parental HuN4-F112.Taken together, in this study we concluded that:the regions encoding for Non-structure proteins were important for the virus infection, especially in the production of viremia and the induction of IL-6and IL-10. ORFla plays a very important role in virus adaptation on MARC-145cell, viremia production, antibody production, IL-6and IL-10induction. ORFlb was involved in the production of IL-6and IL-10. Maybe Nsp9and nsp10were associated with the virus growth on MARC-145at the early time during the infection. But nsp9, nsp10or nspll was not individually related to the pathogenecity and replication of HP-PRRSV. Therefore, this study set up a foundation for further elucidation of the diversity between HP-PRRSV and avirulent PRRSV in genetic level.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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