Introduction of site-specific mutations into the genome of influenza virus.

Abstract

We succeeded in rescuing infectious influenza virus by transfecting cells with RNAs derived from specific recombinant DNAs. RNA corresponding to the neuraminidase (NA) gene of influenza A/WSN/33 (WSN) virus was transcribed in vitro from plasmid DNA and, following the addition of purified influenza virus RNA polymerase complex, was transfected into MDBK cells. Superinfection with helper virus lacking the WSN NA gene resulted in the release of virus containing the WSN NA gene. We then introduced five point mutations into the WSN NA gene by cassette mutagenesis of the plasmid DNA. Sequence analysis of the rescued virus revealed that the genome contained all five mutations present in the mutated plasmid. The ability to create viruses with site-specific mutations will allow the engineering of influenza viruses with dermed biological properties.

Reverse genetics system for introduction of site-specific mutations into the double-stranded RNA genome of infectious rotavirus

“…There have been no reports on its application in other laboratories or to other Reoviridae viruses. In contrast, we demonstrated that the reverse genetics system involving the traditional T7 RNA polymerase expression system (25) developed by Palese and colleagues (18)(19)(20) could be applied to RVs with some modifications. The recombinant vaccinia virus T7 RNA polymerase-driven system allows extremely high expression of artificial viral mRNAs mimicking the natural RV mRNAs in the cytoplasm, which is favorable for the development of reverse genetics for RVs whose entire replication cycle is completed in the cytoplasm.…”

“…To generate an infectious RV transfectant that contains the previously undescribed cDNAderived gene segment, we used the helper virus-driven reverse genetics procedure, which was developed originally for influenza viruses by Palese and colleagues (18)(19)(20), with some modifications. This procedure requires a strong selection system for the transfectant virus containing the cDNA-derived gene because the majority of virus in the culture fluid is helper virus.…”

A DNA transfection system for generation of influenza A virus from eight plasmids

“…These cRNAs serve as templates for the synthesis of vRNAs. The first reverse-genetics system to be developed for influenza A virus was the RNP-transfection method (9,10). After in vitro transcription of virus-like vRNA by T3 or T7 RNA polymerase and reconstitution of viral ribonucleoprotein (vRNP) molecules, genetically altered RNP segments were introduced into eukaryotic cells by transfection.…”

Postexposure vaccination massively increases the prevalence of γ-herpesvirus-specific CD8+T cells but confers minimal survival advantage on CD4-deficient mice

“…There are some indications that postexposure boosting with HIV vaccines can improve prognosis (13), but this possibility has not been addressed for the intercurrent infections that often cause mortality in AIDS patients. (18,19) (8,12,20,21) utilized congenic (H-2 b ) embryonic fibroblasts (2214-CRL; American Type Culture Collection) seeded (10 4 ͞ well) overnight into 96-well plates. Dilutions (2-to 3-fold) of lung homogenate in DMEM ϩ 0.1% BSA were added to 16 replicate wells.…”