Because proinflammatory cytokines are markedly elevated during H5N1 influenza virus infection, the "cytokine storm" is hypothesized to be the main cause of mortality. Here, we demonstrate that mice ...deficient in the hallmark inflammatory cytokines TNF-α, IL-6, or CC chemokine ligand 2 succumb to infection with A/Vietnam/1203/04 (H5N1) virus, as do wild-type mice treated with glucocorticoids for suppression of cytokines. Because cytokine inhibition does not protect against death, therapies that target the virus rather than cytokines may be preferable.
We have developed an eight-plasmid DNA transfection system for the rescue of infectious influenza A virus from cloned cDNA. In this plasmid-based expression system, viral cDNA is inserted between the ...RNA polymerase I (pol I) promoter and terminator sequences. This entire pol I transcription unit is flanked by an RNA polymerase II (pol II) promoter and a polyadenylation site. The orientation of the two transcription units allows the synthesis of negative-sense viral RNA and positive-sense mRNA from one viral cDNA template. This pol I-pol II system starts with the initiation of transcription of the two cellular RNA polymerase enzymes from their own promoters, presumably in different compartments of the nucleus. The interaction of all molecules derived from the cellular and viral transcription and translation machinery results in the generation of infectious influenza A virus. The utility of this system is proved by the recovery of the two influenza A viruses: A/WSN/33 (H1N1) and A/Teal/HK/W312/97 (H6N1). Seventy-two hours after the transfection of eight expression plasmids into cocultured 293T and MDCK cells, the virus yield in the supernatant of the transfected cells was between 2 × 105and 2 × 107infectious viruses per milliliter. We also used this eight-plasmid system for the generation of single and quadruple reassortant viruses between A/Teal/HK/W312/97 (H6N1) and A/WSN/33 (H1N1). Because the pol I-pol II system facilitates the design and recovery of both recombinant and reassortant influenza A viruses, it may also be applicable to the recovery of other RNA viruses entirely from cloned cDNA.
The spread of H5N1 avian influenza viruses (AIVs) from China to Europe has raised global concern about their potential to infect humans and cause a pandemic. In spite of their substantial threat to ...human health, remarkably little AIV whole-genome information is available. We report here a preliminary analysis of the first large-scale sequencing of AIVs, including 2196 AIV genes and 169 complete genomes. We combine this new information with public AIV data to identify new gene alleles, persistent genotypes, compensatory mutations, and a potential virulence determinant.
H5N1 influenza viruses transmitted from poultry to humans in Asia cause high mortality and pose a pandemic threat. Viral genes important for cell tropism and replication efficiency must be identified ...to elucidate and target virulence factors. We applied reverse genetics to generate H5N1 reassortants combining genes of lethal A/Vietnam/1203/04 (VN1203), a fatal human case isolate, and nonlethal A/chicken/Vietnam/C58/04 (CH58) and tested their pathogenicity in ferrets and mice. The viruses' hemagglutinins have six amino acids differences, identical cleavage sites, and avian-like alpha-(2,3)-linked receptor specificity. Surprisingly, exchanging hemagglutinin and neuraminidase genes did not alter pathogenicity, but substituting CH58 polymerase genes completely attenuated VN1203 virulence and reduced viral polymerase activity. CH58's NS gene partially attenuated VN1203 in ferrets but not in mice. Our findings suggest that for high virulence in mammalian species an avian H5N1 virus with a cleavable hemagglutinin requires adaptive changes in polymerase genes to overcome the species barrier. Thus, novel antivirals targeting polymerase proteins should be developed.
The successful replication of a viral pathogen in a host is a complex process involving many interactions. These interactions develop from the coevolution of pathogen and host and often lead to a ...species specificity of the virus that can make interspecies transmissions difficult. Nevertheless, viruses do sporadically cross species barriers into other host populations, including humans. In zoonotic infections, many of these interspecies transfer events are dead end, where transmission is confined only to the animal-to-human route but sometimes viruses adapt to enable spread from human to human. A pathogen must overcome many hurdles to replicate successfully in a foreign host. The viral pathogen must enter the host cell, replicate with the assistance of host factors, evade inhibitory host products, exit the first cell and move on to the next, and possibly leave the initial host and transmit to another. Each of these stages may require adaptive changes in the pathogen. Although the factors that influence each stage of the replication and transmission of most agents have not been resolved, the genomics of both hosts and pathogens are now at hand and we have begun to understand some of the molecular changes that enable some viruses to adapt to a new host.
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