Submitted to: Journal of Virology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/18/2001
Publication Date: N/A
Citation: Interpretive Summary: Here, using a novel in vivo marker rescue strategy, a previously unidentified swine virulence determinant located on the left variable region of the ASFV genome was identified. Further experiments using both forward and reverse genetic techniques identified members the ASFV multi-gene families MGF360 and MGF 530 to have significant roles in swine virulence, independent of swine macrophage cell growth. These findings have assigned significant roles for these unique virus genes in ASFV host interactions.
Technical Abstract: Previously we have shown the ASFV NL gene deletion mutant E70DNL to be attenuated in pigs. Recent observations that NL gene deletion mutants of two additional pathogenic ASFV isolates, Malawi Lil-20/1 and Pr4, remained highly virulent in swine suggested that these isolates encoded additional virulence determinant(s) that was absent from E70. To map this putative virulence determinant, in vivo marker rescue experiments were performed b inoculating swine with infection/transfection lysates containing E70 NL deletion mutant virus (E70 DNL) and cosmid DNA clones from the Malawi NL gene deletion mutant (MalDNL). A cosmid clone representing the left hand 38 kb region of the MalDNL genome was capable of restoring full virulence to E70DNL. Southern blot analysis of recovered virulent viruses confirmed that they were recombinant E70DNL genomes containing a 23-28kb DNA fragment of the Malawi genome. Additional in vivo marker rescue experiments identified a 20kb fragment, encoding members of multi-gene family (MGF) 360 and 530, to be capable of fully restoring virulence to E70DNL. Comparative nucleotide sequence analysis of the left variable region of the E70deltaNL and Malawi Lil-20/1 genomes identified an 8kb deletion in the E70DNL isolate which resulted in the deletion and or truncation of 3 MGF 360 genes and four MGF530 genes. A recombinant MalDNL deletion mutant lacking three members of each MGF gene family was constructed and evaluated for virulence in swine. The mutant virus replicated normally in macrophage cell culture but was avirulent in swine. These results indicate that a region within the left variable region of the ASFV genome represents a previously unrecognized virulence determinant for domestic swine.