Dataset Information

Instability of the restriction fragment length polymorphism pattern of open reading frame 5 of porcine reproductive and respiratory syndrome virus during sequential pig-to-pig passages.

ABSTRACT: Restriction fragment length polymorphism (RFLP) analysis is one of the tools commonly used to study the molecular epidemiology of porcine reproductive and respiratory syndrome viruses (PRRSVs). As PRRSVs are genetically variable, the stability of the RFLP pattern of a PRRSV during in vivo replication was evaluated by carrying out 13 sequential pig-to-pig passages (P1 to P13) of PRRSV ATCC VR-2332 in three independent pig lines for a total of 727 days. During P1 the pigs were inoculated with a homogeneous inoculum (CC-01) prepared through a series of plaque purifications, and during P2 to P13 the pigs were inoculated with a tissue filtrate from the corresponding pig in the previous passage. Fifteen viral plaque clones were directly isolated from CC-01 and the day 7 serum of each pig of each passage, open reading frame 5 of the clones was sequenced, and the clones were compared to CC-01 to assess the mutation rates and RFLP patterns (obtained by digestion with MluI, HincII, and SacII) over time. Among the 495 viral clones recovered during the passages, 398 clones, including CC-01, had pattern 2-5-2 (MluI-HincII-SacII); however, the remaining 97 viral clones showed different patterns (2-6-2 [P2], 1-5-2 [P3], 2-5-4 [P7], and 2-1-2 [P10]). Importantly, the MluI site that was reported to be present in only one of the PRRS modified live virus vaccine strains (Ingelvac) and its parental strain (ATCC VR-2332) can disappear during in vivo replication. Furthermore, sequence homology between CC-01 and clones with pattern 2-5-2 or clones with other patterns differed by 0.05 to 1.58% and 0.5 to 1.45%, respectively, suggesting that RFLP analysis cannot accurately predict genetic relatedness between PRRSVs. Collectively, precaution should be taken when the molecular epidemiology of PRRSVs is evaluated by RFLP analysis.

SUBMITTER: Cha SH

PROVIDER: S-EPMC522335 | biostudies-literature | 2004 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Instability of the restriction fragment length polymorphism pattern of open reading frame 5 of porcine reproductive and respiratory syndrome virus during sequential pig-to-pig passages.

Cha Sang-Ho SH Chang Chih-Cheng CC Yoon Kyoung-Jin KJ

Journal of clinical microbiology 20041001 10

Restriction fragment length polymorphism (RFLP) analysis is one of the tools commonly used to study the molecular epidemiology of porcine reproductive and respiratory syndrome viruses (PRRSVs). As PRRSVs are genetically variable, the stability of the RFLP pattern of a PRRSV during in vivo replication was evaluated by carrying out 13 sequential pig-to-pig passages (P1 to P13) of PRRSV ATCC VR-2332 in three independent pig lines for a total of 727 days. During P1 the pigs were inoculated with a ho ...[more]

PMID: 15472294

Similar Datasets

Project description:UnlabelledIn a previous study, ribavirin-resistant porcine reproductive and respiratory syndrome virus (PRRSV) mutants (RVRp13 and RVRp22) were selected, and their resistance against random mutation was shown in cultured cells. In the present study, these ribavirin-resistant mutants were evaluated in terms of their genetic and phenotypic stability during three pig-to-pig passages in comparison with modified live virus (MLV) (Ingelvac PRRS MLV). Pigs challenged with RVRp22 had significantly lower (P< 0.05) viral loads in sera and tissues than pigs challenged with MLV or RVRp13 at the first passage, and the attenuated replication of RVRp22 was maintained until the third passage. Viral loads in sera and tissues dramatically increased in pigs challenged with MLV or RVRp13 during the second passage. Consistently, all five sequences associated with the attenuation of virulent PRRSV in RVRp13 and MLV quickly reverted to wild-type sequences during the passages, but two attenuation sequences were maintained in RVRp22 even after the third passage. In addition, RVRp22 showed a significantly lower (P< 0.001) mutation frequency in nsp2, which is one of the most variable regions in the PRRSV genome, than MLV. Nine unique mutations were found in open reading frames (ORFs) 1a, 2, and 6 in the RVRp22 genome based on full-length sequence comparisons with RVRp13, VR2332 (the parental virus of RVRp13 and RVRp22), and MLV. Based on these results, it was concluded that RVRp22 showed attenuated replication in pigs; further, because of the high genetic stability of RVRp22, its attenuated phenotype was stable even after three sequential passages in pigs.ImportancePRRSV is a rapidly evolving RNA virus. MLV vaccines are widely used to control PRRS; however, there have been serious concerns regarding the use of MLV as a vaccine virus due to the rapid reversion to virulence during replication in pigs. As previously reported, ribavirin is an effective antiviral drug against many RNA viruses. Ribavirin-resistant mutants reemerged by escaping lethal mutagenesis when the treatment concentration was sublethal, and those mutants were genetically more stable than parental viruses. In a previous study, two ribavirin-resistant PRRSV mutants (RVRp13 and RVRp22) were selected, and their higher genetic stability was shown in vitro Consequently, in the present study, both of the ribavirin-resistant mutants were evaluated in terms of their genetic and phenotypic stability in vivo RVRp22 was found to exhibit higher genetic and phenotypic stability than MLV, and nine unique mutations were identified in the RVRp22 genome based on a full-length sequence comparison with the RVRp13, VR2332, and MLV genomes.

Dataset Information

Instability of the restriction fragment length polymorphism pattern of open reading frame 5 of porcine reproductive and respiratory syndrome virus during sequential pig-to-pig passages.

Publications

Instability of the restriction fragment length polymorphism pattern of open reading frame 5 of porcine reproductive and respiratory syndrome virus during sequential pig-to-pig passages.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets