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RNA recombination between persisting pestivirus and a vaccine strain: generation of cytopathogenic virus and induction of lethal disease.

ABSTRACT: Molecular analysis of a cytopathogenic (cp) bovine viral diarrhea virus (BVDV) isolate (1741) obtained from a case of mucosal disease (MD) led to the identification of five different viral subgenomic RNAs in addition to a noncytopathogenic (noncp) strain (NCP 1741). For each of the subgenomes, a large internal deletion was found together with an inserted sequence encoding part of ribosomal protein S27a fused to an N-terminally truncated ubiquitin monomer. Surprisingly, the two cellular insertions together with flanking viral sequences encoding parts of NS3 and NS4B are >99% identical to the previously described sequence of BVDV vaccine strain RIT (P. Becher, M. Orlich, and H.-J. Thiel, J. Virol. 72:8697-8704, 1998), while the remainder of the subgenomes is derived from the genome of NCP 1741. Further analyses including molecular cloning and nucleotide sequencing of the recombination partners revealed that both homologous and nonhomologous RNA recombination contributed to the generation of the viral subgenomes. Interestingly, for another cp BVDV isolate (CP 4584) from an independent case of MD, again an insertion of a RIT-derived sequence element was detected. In contrast to CP 1741, for CP 4584 a duplication of the genomic region encoding NS3 and parts of NS4A and NS4B was found. Transfection of bovine cells with RNA transcribed from a chimeric cDNA construct showed that the RIT-derived insertion together with the CP 4584-specific duplication of viral sequences represents the genetic basis of cytopathogenicity of CP 4584. Remarkably, passages of the recovered cp virus in cell culture led to emergence of noncp BVDV and a number of viral subgenomes whose genome organization was similar to that in BVDV 1741.

SUBMITTER: Becher P

PROVIDER: S-EPMC114347 | biostudies-literature | 2001 Jul

REPOSITORIES: biostudies-literature

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RNA recombination between persisting pestivirus and a vaccine strain: generation of cytopathogenic virus and induction of lethal disease.

Becher P P Orlich M M Thiel H J HJ

Journal of virology 20010701 14

Molecular analysis of a cytopathogenic (cp) bovine viral diarrhea virus (BVDV) isolate (1741) obtained from a case of mucosal disease (MD) led to the identification of five different viral subgenomic RNAs in addition to a noncytopathogenic (noncp) strain (NCP 1741). For each of the subgenomes, a large internal deletion was found together with an inserted sequence encoding part of ribosomal protein S27a fused to an N-terminally truncated ubiquitin monomer. Surprisingly, the two cellular insertion ...[more]

PMID: 11413291

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Project description:Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth A/Puerto Rico/8/1934 (PR8) master virus. The PB1-V43I mutation was introduced to increase replication fidelity in order to design an H1N1 vaccine strain with a low error rate. The PR8-PB1-V43I virus exhibited good replication compared with that of the parent PR8 virus. In order to compare the efficiency of egg adaptation and the occurrence of gene mutations leading to antigenic alterations, we constructed 6:2 genetic reassortant viruses between the A(H1N1)pdm09 and the PR8-PB1-V43I viruses; hemagglutinin (HA) and neuraminidase (NA) were from the A(H1N1)pdm09 virus, and the other genes were from the PR8 virus. Mutations responsible for egg adaptation mutations occurred in the HA of the PB1-V43I reassortant virus during serial egg passages; however, in contrast, antigenic mutations were introduced into the HA gene of the 6:2 reassortant virus possessing the wild-type PB1. This study shows that the mutant PR8 virus possessing the PB1 polymerase with the V43I substitution may be utilized as a master virus for the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production.IMPORTANCE Vaccination represents the most effective prophylactic option against influenza. The threat of emergence of influenza pandemics necessitates the ability to generate vaccine viruses rapidly. However, as the influenza virus exhibits a high mutation rate, vaccines must be updated to ensure a good match of the HA and NA antigens between the vaccine and the circulating strain. Here, we generated a genetically stable master virus of the A/Puerto Rico/8/1934 (H1N1) backbone encoding an engineered high-fidelity viral polymerase. Importantly, following the application of the high-fidelity PR8 backbone, no mutation resulting in antigenic change was introduced into the HA gene during propagation of the A(H1N1)pdm09 candidate vaccine virus. The low error rate of the present vaccine virus should decrease the risk of generating mutant viruses with increased virulence. Therefore, our findings are expected to be useful for the development of prepandemic vaccines and live attenuated vaccines with higher safety than that of the present candidate vaccines.

Project description:BackgroundThe aim of this study was to consider the impact that multi-clone, complex infections have on a parasite population structure in a low transmission setting. In general, complexity of infection (minimum number of clones within an infection) and the overall population level diversity is expected to be minimal in low transmission settings. Additionally, the parasite population structure is predicted to be clonal, rather than sexual due to infrequent parasite inoculation and lack of recombination between genetically distinct clones. However, in this low transmission of the Peruvian Amazon, complex infections are becoming more frequent, in spite of decreasing infection prevalence. In this study, it was hypothesized that sexual recombination between distinct clonal lineages of Plasmodium falciparum parasites were altering the subpopulation structure and effectively maintaining the population-level diversity.MethodsFourteen microsatellite markers were chosen to describe the genetic diversity in 313 naturally occurring P. falciparum infections from Peruvian Amazon. The population and subpopulation structure was characterized by measuring: clusteredness, expected heterozygosity (He), allelic richness, private allelic richness, and linkage disequilibrium. Next, microsatellite haplotypes and alleles were correlated with P. falciparum merozoite surface protein 1 Block 2 (Pfmsp1-B2) to examine the presence of recombinant microsatellite haplotypes.ResultsThe parasite population structure consists of six genetically diverse subpopulations of clones, called "clusters". Clusters 1, 3, 4, and 6 have unique haplotypes that exceed 70% of the total number of clones within each cluster, while Clusters 2 and 5 have a lower proportion of unique haplotypes, but still exceed 46%. By measuring the He, allelic richness, and private allelic richness within each of the six subpopulations, relatively low levels of genetic diversity within each subpopulation (except Cluster 4) are observed. This indicated that the number of alleles, and not the combination of alleles, are limited. Next, the standard index of association (IAS) was measured, which revealed a significant decay in linkage disequilibrium (LD) associated with Cluster 6, which is indicative of independent assortment of alleles. This decay in LD is a signature of this subpopulation approaching linkage equilibrium by undergoing sexual recombination. To trace possible recombination events, the two most frequent microsatellite haplotypes observed over time (defined by either a K1 or Mad20) were selected as the progenitors and then potential recombinants were identified in within the natural population.ConclusionsContrary to conventional low transmission models, this study provides evidence of a parasite population structure that is superficially defined by a clonal backbone. Sexual recombination does occur and even arguably is responsible for maintaining the substructure of this population.

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RNA recombination between persisting pestivirus and a vaccine strain: generation of cytopathogenic virus and induction of lethal disease.

Publications

RNA recombination between persisting pestivirus and a vaccine strain: generation of cytopathogenic virus and induction of lethal disease.

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