In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses

Abstract

Influenza A viruses cause recurrent outbreaks at 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 11 June 2009 the World Health Organization 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 viruses. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; hereafter referred to as 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 non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human 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 that these compounds could function as a first line of defence against the recently declared S-OIV pandemic.

Figure 1. Pathologic examination of the lungs of infected cynomolgus macaques

Representative pathologic images of CA04- (macaque #1, a-d), KUTK-4- (macaque #7, e-g), and mock- (h) infected lungs on day 3 pi. One or two sections per lung lobe were examined; representative findings are shown to depict the distribution of lesions in the sections (shown as cross sections placed next to illustrations of each lung lobe), with or without viral antigen, as follows: brown, severe lung lesion containing moderate to many viral antigen-positive cells; pink, mild lung lesions containing a few viral antigen-positive cells; blue, lung lesions with alveolar wall thickening, with remaining air spaces unaffected.

Figure 1. Pathologic examination of the lungs of infected cynomolgus macaques

Representative pathologic images of CA04- (macaque #1, a-d), KUTK-4- (macaque #7, e-g), and mock- (h) infected lungs on day 3 pi. One or two sections per lung lobe were examined; representative findings are shown to depict the distribution of lesions in the sections (shown as cross sections placed next to illustrations of each lung lobe), with or without viral antigen, as follows: brown, severe lung lesion containing moderate to many viral antigen-positive cells; pink, mild lung lesions containing a few viral antigen-positive cells; blue, lung lesions with alveolar wall thickening, with remaining air spaces unaffected.

Figure 2. CA04 sensitivity to antiviral compounds in mice

Mice were intranasally inoculated with 10⁴ PFU (50 μl) of CA04, KUTK-4, or A/Kawasaki/UTK-23/08 (H1N1). At 1 h pi, mice were administered oseltamivir phosphate, zanamivir, CS-8958, T-705, or distilled water and PBS (control). Three mice per group were euthanized on days 3 and 6 pi and the virus titres in lungs were determined by plaque assays in MDCK cells. *, p<0.05, **, p<0.01.

Figure 3. Neutralization activities in human sera against viruses

Human sera of donor groups #1 (collected in 1999) and #2 (collected in April and May of 2009) were subjected to neutralization assays with CA04 and KUTK-4. Since the sera of donor group #1 were collected in 1999, little neutralization activity was expected against KUTK-4, which was isolated in 2009.