Showing papers on "Avian Influenza A Virus published in 1993"

A single amino acid in the PB2 gene of influenza A virus is a determinant of host range.

Abstract: The single gene reassortant virus that derives its PB2 gene from the avian influenza A/Mallard/NY/78 virus and remaining genes from the human influenza A/Los Angeles/2/87 virus exhibits a host range restriction (hr) phenotype characterized by efficient replication in avian tissue and failure to produce plaques in mammalian Madin-Darby canine kidney cells. The hr phenotype is associated with restriction of viral replication in the respiratory tract of squirrel monkeys and humans. To identify the genetic basis of the hr phenotype, we isolated four phenotypic hr mutant viruses that acquired the ability to replicate efficiently in mammalian tissue. Segregational analysis indicated that the loss of the hr phenotype was due to a mutation in the PB2 gene itself. The nucleotide sequences of the PB2 gene of each of the four hr mutants revealed that a single amino acid substitution at position 627 (Glu-->Lys) was responsible for the restoration of the ability of the PB2 single gene reassortant to replicate in Madin-Darby canine kidney cells. Interestingly, the amino acid at position 627 in every avian influenza A virus PB2 protein analyzed to date is glutamic acid, and in every human influenza A virus PB2 protein, it is lysine. Thus, the amino acid at residue 627 of PB2 is an important determinant of host range of influenza A viruses.

Rescue of an influenza A virus wild-type PB2 gene and a mutant derivative bearing a site-specific temperature-sensitive and attenuating mutation.

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 attenuating mutations 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. An avian-human influenza A virus PB2 single-gene reassortant virus (with an 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 the virus bearing the human influenza A virus PB2 gene. We validated the feasibility of this approach by rescuing the PB2 gene of the wild-type influenza 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.

Analysis of influenza A virus nucleoproteins for the assessment of molecular genetic mechanisms leading to new phylogenetic virus lineages.

Abstract: The nucleoprotein (NP) gene of influenza A viruses is decisive for separating two large individually evolving reservoirs in birds and humans. A phylogenetic analysis of the NP gene revealed that all mammalian influenza viruses originated — directly or indirectly — from an avian ancestor. The stable introduction of an avian influenza A virus into a mammalian species seems to be a relatively rare event, the latest one occurred in 1979 when such an avian virus was introduced into pigs in Northern Europe which gave rise to a new lineage. At least two concomitant events are required for such a new and stable introduction: (1) The new species has to become infected, and (2) a mutation in the polymerase complex has to establish a labile variant, which is prone to provide a large number of different variants, from which some can adapt rapidly to the new host (or to any unusual environments). Since such mutator mutations might be advantageous only during stress periods, variants with a less error prone polymerase might emerge again after adaptation. Examples for such fluctuations in terms of mutational and evolutionary rates are discussed in this brief review.

Importance of conserved amino acids at the cleavage site of the haemagglutinin of a virulent avian influenza A virus

Abstract: The virulence of avian influenza A viruses depends on the cleavability of the haemagglutinin (HA) by an intracellular protease at multiple basic amino acids. Although previous studies have demonstrated the importance of these amino acids for processing by the cellular protease, with emphasis on conserved residues near the cleavage site, the minimal requirements for cleavage remain unknown. By expressing site-specific mutants of the HA of a virulent avian influenza virus, A/turkey/Ireland/1378/85 (H5N8), in the simian virus 40 system and testing for their cleavability by an endogenous protease in CV-1 cells, and their fusion activity in a polykaryon formation assay, we were able to show that glycine at the amino terminus of HA2 is not essential for cleavage and that maximal cleavage requires at least five basic residues at the cleavage site, when carbohydrate is nearby. Moreover, we confirmed, that a conserved proline upstream of the cleavage site is not essential for HA cleavage or fusion activity, and that lysine replacement of the carboxyl-terminal arginine of HA1 abolishes cleavability. These findings should help identify the proteases responsible for intracellular cleavage of the HA of virulent avian influenza viruses.