Mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype

Allende, Rossana; Kutish, G. F.; Laegreid, William W.; Lu, Zhiqiang; Lewis, Terry L.; Rock, Daniel L.; Friesen, James D.; Galeota, J. A.; Doster, Alan R.; Osorio, Fernando A.

doi:10.1007/s007050070115

Cited by 116 publications

(105 citation statements)

References 23 publications

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“…Both PRRSV CC-01 and the RespPRRS vaccine virus were derived from VR-2332 through cell culture passage and, for the ORFs sequenced, shared 100% amino acid homology. Reversion of the amino acid residue at position 151 of ORF 5 has been reported for the RespPRRS vaccine virus and VR-2332 (2,3,40,54,59). Cumulatively, these results lead us to speculate that the glycine residue at position 151 of ORF 5 may be a marker for cell culture adaptation.…”

Section: Discussionmentioning

confidence: 92%

Evolution of Porcine Reproductive and Respiratory Syndrome Virus during Sequential Passages in Pigs

Chang¹,

Yoon²,

Zimmerman³

et al. 2002

J Virol

112

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Porcine reproductive and respiratory syndrome (PRRS) viruses are recognized as possessing a high degree of genetic and antigenic variability. Viral diversity has led to questions regarding the association of virus mutation and persistent infection in the host and has raised concerns vis-à-vis protective immunity, the ability of diagnostic assays to detect novel variants, and the possible emergence of virulent strains. The purpose of this study was to describe ongoing changes in PRRS virus during replication in pigs under experimental conditions. Animals were inoculated with a plaque-cloned virus derived from VR-2332, the North American PRRS virus prototype. Three independent lines of in vivo replication were maintained for 367 days by pig-to-pig passage of virus at 60-day intervals. A total of 315 plaque-cloned viruses were recovered from 21 pigs over the 367-day observation period and compared to the original plaque-cloned virus by virus neutralization assay, monoclonal antibody analysis, and sequencing of open reading frames (ORFs) 1b (replicase), 5 (major envelope protein), and 7 (nucleocapsid) of the genome. Variants were detected by day 7 postinoculation, and multiple variants were present concurrently in every pig sampled over the observation period. Sequence analysis showed ORFs 1b and 7 to be highly conserved. In contrast, sequencing of ORF 5 disclosed 48 nucleotide variants which corresponded to 22 amino acid variants. Although no epitopic changes were detected under the conditions of this experiment, PRRS virus was shown to evolve continuously in infected pigs, with different genes of the viral genome undergoing various degrees of change.

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Section: Discussionmentioning

confidence: 92%

Evolution of Porcine Reproductive and Respiratory Syndrome Virus during Sequential Passages in Pigs

Chang¹,

Yoon²,

Zimmerman³

et al. 2002

J Virol

112

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“…In total, 11 nucleotide differences were identified (Table 2) when the DNA sequence of the full-length cDNA clone was compared to previously published full-length sequences of North American isolates (GenBank accession numbers AF046869, AF066183, AF159149, AF176348, and PRU87392) (2,3,26,38,41). Three of these differences were silent mutations that were introduced intentionally to generate the BstZ17I and HpaI restriction sites (Tables 1 and 2).…”

Section: Resultsmentioning

confidence: 99%

“…1) was sequenced in total (GenBank accession number AY150564). This sequence was compared to a "consensus" sequence of previously published full-length sequences of North American isolates (GenBank accession numbers PRU87392, AF066183, AF176348, AF046869, and AF159149) (2,3,26,38,41). In total, 11 nucleotide differences were observed, as shown in the table.…”

Section: Resultsmentioning

confidence: 99%

“…VR-2332 was chosen because it is the prototypical North American-type isolate, is well characterized, and has been shown to be highly virulent for sows and piglets (5,10,13). In addition, VR-2332 forms the basis of an attenuated live vaccine (3,22), the complete genome sequences of both VR-2332 and the vaccine virus have been published (3,26,41), and both VR-2332 and the vaccine virus are freely available. Given the availability of complete genome sequences for the vaccine and VR-2332 strains, the infectious clone of VR-2332 should be immediately applicable for the identification of genetic attenuation and virulence determinants in the PRRSV genome, with a view to the development of second-generation, genetically engineered vaccines.…”

mentioning

confidence: 99%

“…After 2 h of incubation, the cells were washed twice, and fresh medium was added (time 0). The virus titers of the supernatants were determined by immunoperoxidase monolayer assay on Marc-145 cells at 0, 3,16,20,24,48, and 72 h postinfection.…”

mentioning

confidence: 99%

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Generation of an Infectious Clone of VR-2332, a Highly Virulent North American-Type Isolate of Porcine Reproductive and Respiratory Syndrome Virus

Nielsen

Liu

Nielsen³

et al. 2003

J Virol

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A full-length cDNA clone of the prototypical North American porcine reproductive and respiratory syndrome virus (PRRSV) isolate VR-2332 was assembled in the plasmid vector pOK 12 . To rescue infectious virus, capped RNA was transcribed in vitro from the pOK 12 clone and transfected into BHK-21C cells. The supernatant from transfected monolayers were serially passaged on Marc-145 cells and porcine pulmonary alveolar macrophages. Infectious PRRSV was recovered on Marc-145 cells as well as porcine pulmonary macrophages; thus, the cloned virus exhibited the same cell tropism as the parental VR-2332 strain. However, the cloned virus was clearly distinguishable from the parental VR-2332 strain by an engineered marker, a BstZ17I restriction site. The full-length cDNA clone had 11 nucleotide changes, 2 of which affected coding, compared to the parental VR-2332 strain. Additionally, the transcribed RNA had an extra G at the 5 end. To examine whether these changes influenced viral replication, we examined the growth kinetics of the cloned virus in vitro. In Marc-145 cells, the growth kinetics of the cloned virus reflected those of the parental isolate, even though the titers of the cloned virus were consistently slightly lower. In experimentally infected 5.5-week-old pigs, the cloned virus produced blue discoloration of the ears, a classical clinical symptom of PRRSV. Also, the seroconversion kinetics of pigs infected with the cloned virus and VR-2332 were very similar. Hence, virus derived from the full-length cDNA clone appeared to recapitulate the biological properties of the highly virulent parental VR-2332 strain. This is the first report of an infectious cDNA clone based on American-type PRRSV. The availability of this cDNA clone will allow examination of the molecular mechanisms behind PRRSV virulence and attenuation, which might in turn allow the production of second-generation, genetically engineered PRRSV vaccines.

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The Genetic Diversity of European Type PRRSV Is Similar to That of the North American Type but Is Geographically Skewed within Europe

Forsberg

Storgaard²,

Nielsen³

et al. 2002

Virology

132

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Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently emerged pathogen. Two PRRSV genotypes exist, North American and European, which are only 55-70% identical at the nucleotide level. Previous studies have shown high nucleotide diversity in the North American genotype and low nucleotide diversity in the European genotype. Here, we analyzed the ORF5 and ORF7 genes for a large number of new European type PRRSV isolates in conjunction with existing database sequences. This new analysis showed that contrary to previous assumptions, genetic diversity is at least as high in the European genotype as in the North American genotype. Furthermore, we showed that genetic diversity of European type PRRSV has a marked geographical pattern, with exceptionally high genetic diversity among Italian sequences. The geographical pattern of diversity in relation to the epidemiology of PRRSV in Europe is discussed. Discrepancies between ORF5- and ORF7-based genealogies were observed, and further analysis of the data set confirmed the presence of recombination. We were therefore able to report the first observation of recombination in wild-type isolates of European genotype PRRSV.

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Mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype

Cited by 116 publications

References 23 publications

Evolution of Porcine Reproductive and Respiratory Syndrome Virus during Sequential Passages in Pigs

Evolution of Porcine Reproductive and Respiratory Syndrome Virus during Sequential Passages in Pigs

Generation of an Infectious Clone of VR-2332, a Highly Virulent North American-Type Isolate of Porcine Reproductive and Respiratory Syndrome Virus

The Genetic Diversity of European Type PRRSV Is Similar to That of the North American Type but Is Geographically Skewed within Europe

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