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Rescue of recombinant Thogoto virus from cloned cDNA.

ABSTRACT: Thogoto virus (THOV) is a tick-transmitted orthomyxovirus with a genome consisting of six negative-stranded RNA segments. To rescue a recombinant THOV, the viral structural proteins were produced from expression plasmids by means of a vaccinia virus expressing the T7 RNA polymerase. Genomic virus RNAs (vRNAs) were generated from plasmids under the control of the RNA polymerase I promoter. Using this system, we could efficiently recover recombinant THOV following transfection of 12 plasmids into 293T cells. To verify the recombinant nature of the rescued virus, specific genetic tags were introduced into two vRNA segments. The availability of this efficient reverse genetics system will allow us to address hitherto-unanswered questions regarding the biology of THOV by manipulating viral genes in the context of infectious virus.

SUBMITTER: Wagner E

PROVIDER: S-EPMC114495 | biostudies-literature | 2001 Oct

REPOSITORIES: biostudies-literature

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Rescue of recombinant Thogoto virus from cloned cDNA.

Wagner E E Engelhardt O G OG Gruber S S Haller O O Kochs G G

Journal of virology 20011001 19

Thogoto virus (THOV) is a tick-transmitted orthomyxovirus with a genome consisting of six negative-stranded RNA segments. To rescue a recombinant THOV, the viral structural proteins were produced from expression plasmids by means of a vaccinia virus expressing the T7 RNA polymerase. Genomic virus RNAs (vRNAs) were generated from plasmids under the control of the RNA polymerase I promoter. Using this system, we could efficiently recover recombinant THOV following transfection of 12 plasmids into ...[more]

PMID: 11533190

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Project description:We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of the Birnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segment A encodes a 106-kDa precursor protein which is cleaved to yield mature VP2, nonstructural protease, and VP3 proteins, whereas segment B encodes the RNA-dependent RNA polymerase VP1. Plus-sense RNA transcripts of both segments were prepared by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of chinook salmon embryo (CHSE) cells with combined transcripts of segments A and B generated infectious IPNV particles 10 days posttransfection. Furthermore, a transfectant virus containing a genetically tagged sequence was generated to confirm the feasibility of this system. The presence and specificity of the recovered virus were ascertained by immunofluorescence staining of infected CHSE cells with rabbit anti-IPNV serum and by nucleotide sequence analysis. In addition, 3'-terminal sequence analysis of RNA from the recovered virus showed that extraneous nucleotides synthesized at the 3' end during in vitro transcription were precisely trimmed or excluded during replication, and hence these were not incorporated into the genome. An attempt was made to determine if RNA-dependent RNA polymerase of IPNV and infectious bursal disease virus (IBDV), another birnavirus, can support virus rescue in heterologous combinations. Thus, CHSE cells were transfected with transcripts derived from IPNV segment A and IBDV segment B and Vero cells were transfected with transcripts derived from IBDV segment A and IPNV segment B. In either case, no infectious IPNV or IBDV particles were generated even after a third passage in cell culture, suggesting that viral RNA-dependent RNA polymerase is species specific. However, the reverse genetics system for IPNV that we developed will greatly facilitate studies of viral replication and pathogenesis and the design of a new generation of live attenuated vaccines.

Project description:Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A- or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system. Segment-reassorted virus containing the A segment of the classical attenuated isolate (CEF94) and the B segment of the very virulent isolate (D6948) is not released until 15 h after an in vitro infection. This indicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection. Next to segment reassortants, we generated and analyzed mosaic IBDVs (mIBDVs). In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by the corresponding region of D6948. Analysis of these mIBDV isolates showed that tropism for non-B-lymphoid cells was exclusively determined by the viral capsid protein VP2. However, the very virulent phenotype was not solely determined by this protein, since mosaic virus containing VP2 of vvIBDV induced neither morbidity nor mortality in young chickens.

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Rescue of recombinant Thogoto virus from cloned cDNA.

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Rescue of recombinant Thogoto virus from cloned cDNA.

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