A naturally occurring deletion in its NS gene contributes to the attenuation of an H5N1 swine influenza virus in chickens

Abstract

In 2001 and 2003, we isolated two H5N1 viruses, A/swine/Fujian/1/01 (SW/FJ/01) and A/swine/Fujian/1/03 (SW/FJ/03), from pigs in Fujian Province, southern China. Genetically, these two viruses are similar, although the NS gene of the SW/FJ/03 virus has a 15-nucleotide deletion at coding positions 612 to 626. The SW/FJ/01 virus is highly lethal for chickens, whereas the SW/FJ/03 virus is nonpathogenic for chickens when administrated intravenously or intranasally. To understand the molecular basis for the difference in virulence, we used reverse genetics to create a series of single-gene recombinants of both viruses. We found that a recombinant virus containing the mutated NS gene from the SW/FJ/03 virus in the SW/FJ/01 virus background was completely attenuated in chickens. We also found that viruses expressing the mutant NS1 protein of SW/FJ/03 did not antagonize the induction of interferon (IFN) protein. Conversely, only the recombinant virus containing the wild-type SW/FJ/01 NS gene in the SW/FJ/03 background was lethal in chickens and antagonized IFN protein levels. Further, we proved that the NS1 genes of the two viruses differ in their stabilities in the host cells and in their abilities to interact with the chicken cleavage and polyadenylation specificity factor. These results indicate that the deletion of amino acids 191 to 195 of the NS1 protein is critical for the attenuation of the SW/FJ/03 virus in chickens and that this deletion affects the ability of the virus to antagonize IFN induction in host cells.

FIG. 1.

Weight changes in mice inoculated with H5N1 swine influenza viruses and a duck influenza virus. Groups of five mice were inoculated i.n. with 10⁶ EID₅₀ (in 50 μl) of DK/ZJ/00 (▴), SW/FJ/01(•), or SW/FJ/03 (⧫) and weighed daily for 14 days.

FIG. 2.

Induction of IFN-α/β in CEFs infected with different influenza viruses. (A) CEFs were infected with the indicated influenza viruses, and the UV-treated supernatants were used to examine IFN-mediated inhibition of VSV-GFP replication in CEFs as described in Materials and Methods. The cells were visualized by fluorescence microscopy at 12 and 24 h p.i. (B) RT-PCR analysis of IFN-α/β- and chicken β-actin-specific mRNAs from CEFs infected with R-SW/FJ/03 (lane 1), SW/FJ/01-03NS (lane 2), R-SW/FJ/01 (lane 3), or SW/FJ/03-01NS (lane 4) or from mock-infected CEFs (lane 5). Lane 6, marker.

FIG. 3.

Virus protein levels determined by Western blotting. Lysates of mock-infected cells (lane 1) or cells infected with R-SW/FJ/03 (lane 2), SW/FJ/01-03NS (lane 3), R-SW/FJ/01 (lane 4), or SW/FJ/03-01NS (lane 5) were incubated with rabbit anti-NS1 antiserum, a mouse monoclonal antibody to the viral M1 protein, or β-actin antiserum.

FIG. 4.

Stability of NS1 proteins and their interaction with chicken CPSF. (A) Analysis of NS1 expression by pulse-chase radioactive labeling and immunoprecipitation. The SW/FJ/01 and SW/FJ/03 NS1 proteins were expressed and labeled as described in Materials and Methods. The cells were lysed after being pulse labeled or at different chase time points, and cell lysates were immunoprecipitated with antibodies against NS1, followed by SDS-PAGE analysis. Lane 1, control (empty plasmid DNA vector-transfected cells); lanes 2 to 4, pBlue-NS1-01-transfected samples; lanes 5 to 7, pBlue-NS1-03-transfected samples. Lanes 2 and 5, samples prepared after being pulse labeled; lanes 3 and 6, samples prepared after a 4-h chase; lanes 4 and 7, samples prepared after a 7-h chase. The arrow indicates the NS1 protein band. (B) Analysis of CPSF binding. HA-tagged NS1 constructs were expressed by in vitro translation. Flag-tagged CPSF was expressed in transfected 293T cells. The cell lysate was mixed with the NS1 proteins and subjected to coimmunoprecipitation (IP) using a monoclonal anti-Flag antibody coupled to protein A-Sepharose. The precipitated proteins were analyzed for the HA-tagged NS1 protein and Flag-CPSF by Western blot analysis using HA tag- and Flag-specific monoclonal antibodies, respectively. Lane 1, SW/FJ/01 NS1; lane 2, SW/ FJ/03 NS1.