Submitted to: Electronic Publication
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/28/2004
Publication Date: 9/1/2004
Citation: Grebennikova, T.V., Zaberezhny, A.D., Musienko, M.I., Mengeling, W.L., Lager, K.M., Aliper, T.I., Nepoklonov, E.A. 2004. Genomic characterization and library of infectious genomes of the attenuated North American strain of porcine reproductive and respiratory syndrome virus (in Russian). Voprosy Virusologli (Rus). 3:56-63. Interpretive Summary: Porcine reproductive and respiratory syndrome (PRRS) is a disease of swine caused by the PRRS virus (PRRSV). PRRS is the number one infectious disease problem of the US swine industry. Although there are now several PRRSV vaccines available for use by pork producers, the vaccines have not solved all of the PRRS control and prevention problems. In order to improve PRRSV vaccines, a better understanding of how the virus causes disease in pigs is needed. This study reports the genetic relationship of 3 different passages of the same PRRSV and how these genetic differences may relate to the capability of each passage of virus to cause disease in a pig. The potential for a virus to cause disease is known as virulence. Differences were found in the virulence of the 3 passages of PRRSV and they are referred to as virulent, intermediate, and avirulent. Avirulent virus was derived by continually passing the virulent virus in cell culture to cause mutations that would decrease the virulence of the virus. Intermediate virus was derived by passage of avirulent virus in a pig, the pig passage allowed the avirulent virus to regain some virulence through additional mutations. There were genetic differences found among the 3 virus passages. The differences between virulent and avirulent virus consisted of 50 changes and a small deletion of genetic material. The differences between avirulent and intermediate virus consisted of 8 changes of which 3 of these mutations were changes back to the original virulent virus. Changes in some regions of the virus' genetic material, especially those mutations seemingly associated with changing the avirulent virus to an intermediate virulent virus, may be involved in the control of PRRSV replication and virulence. This information may be used to develop more effective vaccines.
Technical Abstract: Three different passages of the NADC-8 strain of porcine reproductive and respiratory syndrome virus (PRRSV) were used to investigate the relationship between genotypic and phenotypic properties. Differences were found in the virulence of the 3 passages referred to as virulent, intermediate, and avirulent. Avirulent virus was derived by attenuation of virulent virus in cell culture and intermediate virus was derived by passage of avirulent virus in a pig. Nucleotide sequence differences between virulent and avirulent virus consisted of 50 nucleotide changes and a 3-nucleotide deletion, and between avirulent and intermediate virus consisted of 8 nucleotide changes resulting in 6 amino acid changes. Three of these amino acid changes were direct reversions to virulent virus. Genetic changes, especially those seemingly associated with attenuation followed by some degree of reversion to virulence, in ORF 1a, ORF1b, and ORF 6 regions of the genome may be involved in the control of PRRSV replication and virulence. A full-length genomic cDNA clone of the avirulent NADC-8 PRRSV strain (passage 251) was assembled in the plasmid vector pACYC177. A plasmid library was constructed and screened by restriction analysis. Thirty-two clones had the appropriate digestion pattern and 8 of these were randomly selected for transcription and transfection studies. The capped polyadenylated full-length positive sense genomic RNA was obtained in vitro and used for transfection of BHK-21 and MARC-145 cells. Supernatants from both transfected cell monolayers were serially passaged on MARC-145 cells. Progeny viruses were revealed by specific fluorescence microscopy and they induced cytopathic effect in MARC-145 cells. Three of the 8 selected recombinant viruses had growth characteristics similar to the parental strain of PRRSV. Four recombinant viruses had reduced growth characteristics and the 8th selected recombinant did not replicate. This model will be used for functional analysis of the attenuation and virulence determinants in the PRRSV genome.