Development And Application Of Infectious Molecular Clones Based On Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus And Its Attenuated Derivative

Porcine reproductive and respiratory syndrome (PRRS) has become one of the most common and economically significant infectious diseases in theswine industry worldwide. It is characterized by mild to severe reproductive failure in sows and gilts and respiratory problems in piglets. Since May 2006, atypical PRRS (so-called porcine high fever syndrome (PHFS) in china) was pandemic in china. Several studies have confirmed that the causative agent of this outbreak was highly pathogenic PRRSV (HP-PRRSV), several HP-PRRSV strains were isolated and their reverse genetic systems were developed by different lab. Many questions of PRRSV, such as, the mechanism of the PRRSV virlence determents are still unclear. So it is viry crucial to development reverse genetic systems of PRRSV. In this study, reverse genetic systems of HP-PRRSV HuN4 and vaccine strain HuN4-F112 were developted, and a marker vaccine was developed based on the HuN4-F112 reverse genetic systems.A total of six fragments, covering the complete PRRSV HuN4 strain genome, were PCR amplified. All PCR-amplified fragments were gel purified, digested with the proper enzymes, and cloned into the pG1SK vector. The bacteriophage SP6 RNA polymerase promoter was engineered into 5'end of HuN4 genome, SwaI enzyme was engineered downstream of the poly (A) tail for the further linearization of the full-length cDNA. The A at position 14680 was mutated to G by PCR to create a restriction enzyme site MluI as the genetic marker. The completely assembled full-length cDNA clone was confirmed by sequence and enzyme digestion. Capped RNA was transcribed in vitro from a full-length cDNA clone of the viral genome and transfected into BHK-21C cells. The supernatant from transfected monolayers were serially passaged on Marc-145 cells. The virus rescued from this newly assembled cDNA clone was identified by IFA and the detection of the genetic marker. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. The availability of a full-length cDNA clone of PRRSV HuN4 strain lays a new ground for further investigation of PRRSV virulence and development of new potent vaccine.In order to develop a reverse genetic system for the research of PRRSV, a full-length cDNA clone was constructed in this study. A total of six fragments, covering the complete PRRSV HuN4-F112 strain genome, were PCR amplified. All PCR-amplified fragments were gel purified, digested with the proper enzymes, and cloned into the pG2SK vector. The bacteriophage SP6 RNA polymerase promoter was engineered into 5′end of HuN4-F112 genome, NotIenzyme was engineered downstream of the poly (A) tail for the further linearization of the full-length cDNA. The A at position 14680 was mutated to G by PCR to create a restriction enzyme site MluI as the genetic marker. Capped RNA was transcribed in vitro from a full-length cDNA clone of the viral genome and transfected into BHK-21C cells. The supernatant from transfected monolayers were serially passaged on Marc-145 cells. The virus rescued from this newly assembled cDNA clone were indistinguishable from the parental virus. The availability of genome sequence information and infectious cDNA clone of HuN4-F112 lays a new ground for further investigation of PRRSV virulence and development of new potent vaccine.Nsp2 protein of PRRSV is an immunogenic protein capable of eliciting specific antibody production during viral infections.The Nsp2 protein was found to contain a large number of linear B-cell epitopes in both the European type and North American type PRRSV strains. Based on the reverse genetic system of PRRSV vaccine strain HuN4-F112, a B-cell epitope of the S2 glycoprotein of murine hepatitis virus (MHV) was inserted in-frame to replace a linear B-cell epitope of nsp2(430-454aa). Recombinant viruses properly expressing the introduced MHV epitope were successfully generated, demonstrating that the B-cell epitopes not only is dispensable for virus replication but also can be replaced by foreign sequences. Our results provide proof of the concept that DIVA PRRSV vaccines can potentially be developed by replace of individual"marker"immunodominant epitopes.