Evolution and ecology of influenza A viruses.

RNA viruses show high mutation frequencies partly because of a lack of the proofreading enzymes that assure fidelity of DNA replication. This high mutation frequency is coupled with high rates of replication reflected in rates of RNA genome evolution which can be more than a millionfold greater than the rates of the DNA chromosome evolution of their hosts. There are some disease implications for the DNA-based biosphere of this rapidly evolving RNA biosphere.

https://science.sciencemag.org/content/sci/215/4540/1577.full.pdf

Rapid evolution of RNA genomes

Defective interfering virus (DI) particles represent a major controlling element of virus replication. They are constantly generated at low levels by infectious virus and only amplify to interfering levels when the parent helper vims is abundant. This autointerference phenomenon, as it was called when first discovered, is achieved by rearrangements and deletions of the standard virus genome such that the resulting “incomplete form” of the virus can preferentially replicate.

Origin and replication of defective interfering particles.

We have recovered infectious Sendai virus (SeV) from full-length cDNA (FL-3) by transfecting this cDNA and pGEM plasmids expressing the nucleocapsid protein (NP), phosphoprotein and large proteins into cells infected with a vaccinia virus which expresses T7 RNA polymerase. These cells were then injected into chicken eggs, in which SeV grows to very high titers. FL-3 was marked with a BglII site in the leader region and an NsiI site (ATGCAT) in the 5' nontranslated region of the NP gene, creating a new, out-of-frame, 5' proximal AUG. All the virus stocks generated eventually removed this impediment to NP expression, by either point mutation or recombination between FL-3 and pGEM-NP. The recovery system was found to be highly recombinogenic. Even in the absence of selective pressure, one in 20 of the recombinant SeV generated had exchanged the NP gene of FL-3 with that of pGEM-NP. When a fifth plasmid containing a new genomic 3' end without the presumably deleterious BglII site was included as another target for recombination, the new genomic 3' end was found in the recombinant SeV in 12 out of 12 recoveries. Using this approach, a novel copy-back nondefective virus was generated which interferes with wild-type virus replication.

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A highly recombinogenic system for the recovery of infectious Sendai paramyxovirus from cDNA: generation of a novel copy-back nondefective interfering virus.

Methods are described which allow direct quantitation and sequence analysis of base substitution levels at predetermined single nucleotide positions in cloned pools of an RNA virus genome or in its RNA transcripts in vitro. Base substitution frequencies for vesicular stomatitis virus (VSV) at one highly conserved site examined were reproducible and extremely high, averaging between 10(-4) and 4 X 10(-4) substitutions per base incorporated at this single site. If polymerase error frequencies averaged as high at all other sites in the 11-kilobase VSV genome, then every member of a cloned VSV population would differ from most other genomes in that clone at a number of different nucleotide positions. The preservation of a consensus sequence in such variable RNA virus genomes then could only result from strong biological selection (in a single host or multihost environment) for the most fit and competitive representatives of extremely heterogeneous virus populations.

Debra W. Frielle

Papers

Persistent infection with influenza A virus: Evolution of virus mutants

Generation and amplification of temperature-sensitive mutants during serial undiluted passages of vesicular stomatitis virus

Dominance of temperature-sensitive phenotypes: I. Studies of the mechanism of inhibition of the growth of wild-type vesicular stomatitis virus

Temperature sensitive mutants of vsv interfere with the growth of wild-type virus at the level of rna synthesis1