We describe a new reverse-genetics system that allows one to efficiently generate inf luenza A viruses entirely from cloned cDNAs.

Influenza A viruses cause recurrent outbreaks of local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On June 11, 2009, the WHO declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses1. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) appear to be mild; however, more than 50% of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To better assess the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in nonhuman primates, causes more severe pathologic lesions in the lungs of infected mice, ferrets, and nonhuman primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting these compounds as a first line of defence against the recently declared S-OIV pandemic.

Ebola virus causes hemorrhagic fever in humans and nonhuman primates, resulting in mortality rates of up to 90%. Studies of this virus have been hampered by its extraordinary pathogenicity, which requires biosafety level 4 containment. To circumvent this problem, we developed a novel complementation system for functional analysis of Ebola virus glycoproteins. It relies on a recombinant vesicular stomatitis virus (VSV) that contains the green f luorescent protein gene instead of the receptor-binding G protein gene (VSV⌬G*). Herein we show that Ebola Reston virus glycoprotein (ResGP) is efficiently incorporated into VSV particles. This recombinant VSV with integrated ResGP (VSV⌬G*-ResGP) infected primate cells more efficiently than any of the other mammalian or avian cells examined, in a manner consistent with the host range tropism of Ebola virus, whereas VSV⌬G* complemented with VSV G protein (VSV⌬G*-G) efficiently infected the majority of the cells tested. We also tested the utility of this system for investigating the cellular receptors for Ebola virus. Chemical modification of cells to alter their surface proteins markedly reduced their susceptibility to VSV⌬G*-ResGP but not to VSV⌬G*-G. These findings suggest that cell surface glycoproteins with N-linked oligosaccharide chains contribute to the entry of Ebola viruses, presumably acting as a specific receptor and͞or cofactor for virus entry. Thus, our VSV system should be useful for investigating the functions of glycoproteins from highly pathogenic viruses or those incapable of being cultured in vitro.