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Titolo:
RESCUE OF AN INFLUENZA-A VIRUS WILD-TYPE PB2 GENE AND A MUTANT DERIVATIVE BEARING A SITE-SPECIFIC TEMPERATURE-SENSITIVE AND ATTENUATING MUTATION
Autore:
SUBBARAO EK; KAWAOKA Y; MURPHY BR;
Indirizzi:
NIAID,INFECT DIS LAB,RESP VIRUSES SECT BETHESDA MD 20892 ST JUDE CHILDRENS HOSP,DEPT VIROL & MOLEC BIOL MEMPHIS TN 38101
Titolo Testata:
Journal of virology
fascicolo: 12, volume: 67, anno: 1993,
pagine: 7223 - 7228
SICI:
0022-538X(1993)67:12<7223:ROAIVW>2.0.ZU;2-O
Fonte:
ISI
Lingua:
ENG
Soggetto:
REASSORTANT VIRUSES; SQUIRREL-MONKEYS; SEQUENCE CHANGES; POLYMERASE; NEURAMINIDASE; H2N2; RNA;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
19
Recensione:
Indirizzi per estratti:
Citazione:
E.K. Subbarao et al., "RESCUE OF AN INFLUENZA-A VIRUS WILD-TYPE PB2 GENE AND A MUTANT DERIVATIVE BEARING A SITE-SPECIFIC TEMPERATURE-SENSITIVE AND ATTENUATING MUTATION", Journal of virology, 67(12), 1993, pp. 7223-7228

Abstract

Live attenuated influenza A virus vaccines are currently produced by the transfer of attenuating genes from a donor virus to new epidemic variants of influenza A virus, with the selection of reassortant viruses that possess the protective antigens (i.e., the two surface glycoproteins) of the epidemic virus and the attenuating genes from the donor virus. The previously studied attenuated donor viruses were produced by conventional methods such as passage of virus at low temperature or chemical mutagenesis. The present paper describes a new strategy for the generation of a donor virus bearing an attenuating, non-surface-glycoprotein gene. This strategy involves the introduction of attenuatingmutations into the cDNA copy of the PB2 polymerase gene by site-directed mutagenesis, transfection of in vitro RNA transcripts of PB2 cDNA,and recovery of the transfected PB2 gene into an infectious virus. Anavian-human influenza A virus PB2 single-gene reassortant virus (withan avian influenza A virus PB2 gene) that replicates efficiently in avian tissue but poorly in mammalian cells was used as a helper virus to rescue a transfected synthetic RNA derived from a human influenza A virus PB2 gene. The desired human influenza A virus mutant PB2 transfectant was favored in this situation because the avian influenza A virus PB2 gene restricts viral replication in mammalian cells in culture, the system used for rescue, thereby providing strong selection for thevirus bearing the human influenza A virus PB2 gene. We validated the feasibility of this approach by rescuing the PB2 gene of the wild-typeinfluenza A/Ann Arbor/6/60 virus and a mutant derivative that had a single amino acid substitution introduced at position 265 by site-directed mutagenesis. Previously, this amino acid substitution had been shown to specify both a temperature-sensitive (ts) and an attenuation (att) phenotype. The rescued mutant 265 PB2 transfectant virus exhibited the ts and att phenotypes, which confirms that these phenotypes were specified by this single amino acid substitution. The transfectant virus was immunogenic and protected hamsters from subsequent challenge with wild-type virus. The cDNA copy of this influenza A/Ann Arbor/6/60 virus mutant 265 PB2 gene will be used as a substrate for the introduction of additional attenuating mutations by site-directed mutagenesis.

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Documento generato il 21/09/20 alle ore 02:50:44