Construction Of The Infectious Clones And Chimeric Viruses Of PRRSV HuN4 And Ch-1a Strains And Their Preliminary Application

Porcine reproductive and respiratory syndrome(PRRS) is one of the most economically significant infectious diseases all over the world. It was caused by PRRS virus. The disease has many clinical manifestations but the two most prevalent are severe reproductive failure in sows and gilts and respiratory problems in pigs of all ages. Since the disease could induce cyanosis in the ears of swine, it is known as blue ear disease. In 2006, the highly pathogenic PRRS(HP-PRRS) occurred in China, characterized by high fever, high morbidity and mortality in pigs of all ages. Although PRRSV was extensive researched, the genes which determine its replication and virulence are unknown. Infectious clone is an important operation platform for PRRSV research and most infection clone of PRRSV at present are based on vitro transcription, it not only reduce the efficiency of the rescue, but also increased the complexity of the virus rescue.For efficiently to rescue PRRSV, the infection clone of PRRSV strains HuN4-F5 and CH-1a based on eukaryotic promoter were constructed. The fulllength genomes of the virus were obtained by RT-PCR and then cloned into the pOKM vector, followed by plasmid transfection and the corresponding PRRSV pOK-HuN4 and pOK-CH were rescued. The pOK-CH contributes to the storage of PRRSV CH-1a strain. On the basis of pOK-HuN4 and pOK-CH, the genes of structural and non-structural protein, and the ORF1 a and ORF1 b of pOK-HuN4 and pOK-CH were swapped to obtain six chimeric viruses including pOK-HuN4nsp-CHsp, pOK-Chnsp-HuN4 sp, pOK-HuN4-CHORF1 a, pOK-CH-HuN4ORF1 a, pOK-HuN4-CHORF1 b, pOK-CH-HuNORF1 b and all the chimeric viruses were rescued. Besides, Renilla luciferase(Rluc) was inserted between ORF7 and 3’UTR of pOK-HuN4 and the luciferase was successfully detected. It can be used for monitoring the virus replication and detecting the level of neutralizing antibodies.The infection clone of PRRSV based on eukaryotic promoter omits the steps of in vitro synthesis of genomic RNA and RNA transfection which simplifies the virus rescue process and improves the success rate of the virus rescue. The infectious clone of HuN4 and CH-1a PRRSV strains and pOK-HuN4-Rlu and six chimeric viruses lay the foundation for studying the key gene affecting PRRSV replication and its virulence, meanwhile pOK-HuN4-Rlu provide the technic support for monitoring the virus replication and detecting the level of neutralizing antibodies.