NA proteins of influenza A viruses H1N1/2009, H5N1, and H9N2 show differential effects on infection initiation, virus release, and cell-cell fusion

Abstract

Two surface glycoproteins of influenza virus, haemagglutinin (HA) and neuraminidase (NA), play opposite roles in terms of their interaction with host sialic acid receptors. HA attaches to sialic acid on host cell surface receptors to initiate virus infection while NA removes these sialic acids to facilitate release of progeny virions. This functional opposition requires a balance. To explore what might happen when NA of an influenza virus was replaced by one from another isolate or subtype, in this study, we generated three recombinant influenza A viruses in the background of A/PR/8/34 (PR8) (H1N1) and with NA genes obtained respectively from the 2009 pandemic H1N1 virus, a highly pathogenic avian H5N1 virus, and a lowly pathogenic avian H9N2 virus. These recombinant viruses, rPR8-H1N1NA, rPR8-H5N1NA, and rPR8-H9N2NA, were shown to have similar growth kinetics in cells and pathogenicity in mice. However, much more rPR8-H5N1NA and PR8-wt virions were released from chicken erythrocytes than virions of rPR8-H1N1NA and rPR8-H9N2NA after 1 h. In addition, in MDCK cells, rPR8-H5N1NA and rPR8-H9N2NA infected a higher percentage of cells, and induced cell-cell fusion faster and more extensively than PR8-wt and rPR8-H1N1NA did in the early phase of infection. In conclusion, NA replacement in this study did not affect virus replication kinetics but had different effects on infection initiation, virus release and fusion of infected cells. These phenomena might be partially due to NA proteins' different specificity to α2-3/2-6-sialylated carbohydrate chains, but the exact mechanism remains to be explored.

Figure 1. Alignment of the deduced amino acid sequences of the NA genes from the influenza virus strains A/Chicken/Jiangsu/7/2002(H9N2), A/California/04/2009 (H1N1), A/chicken/Henan/12/2004 (H5N1) and PR8 (H1N1).

Figure 2. Characterization of recombinant influenza viruses.

(A) Multiple-cycle growth curves of recombinant viruses on MDCK cells. MDCK cells were infected at a multiplicity of infection (MOI) of 0.001. The virus titers were measured by TCID₅₀. The data shown are means ± SD for triplicate wells at each time point. Statistical significance (p>0.05). (B) SDS-PAGE analysis of expression of NA proteins in recombinant viruses. Concentrated viruses were resuspended with SDS-PAGE sample loading buffer, incubated at 37°C for 30 min, and heated at 100°C for 1 min. The protein was separated by 12% SDS-PAGE, gels were stained with coomassie brilliant blue G250.

Figure 3. Pathogenicity of recombinant viruses in BALB/c mice.

Survival rates (A) and bodyweight changes (B) after challenge with the viruses. BALB/c mice were intranasally inoculated with rPR8-H5N1NA, rPR8-H9N2NA, rPR8-H1N1NA or PR8-wt virus at 1×10^6.5 EID₅₀. The survival rates and bodyweights of five mice in each group were measured daily from the date of challenge to 14 days after challenge. Values represent mean ± SD of each group of mice.

Figure 4. Virus elution in vitro.

50 µl two-fold dilutions of virus containing the HA titers of 1∶128 was incubated with 50 µl 0.5% chicken erythrocytes in microtiter plates at 4°C for 1 h. Then microtiter plates were incubated at 37°C, and the reduction of HA titers was measured periodically for 8 h.

Figure 5. Impact of different NA on initiation of influenza virus infection.

MDCK cells were infected at an MOI of 0.001 in the presence of 1 µg/ml TPCK-trypsin. After adsorption for 1 h at 37°C, the inocula were removed and the cultures were washed 3 times. The cells were incubated at 37°C for 6 h. At the indicated time, the cells were processed for immunofluorescence, and the infected cells were detected with polyclonal antisera to whole viruses. (A) Fluorescence images of the infected cells at 6 h p.i. Fluorescent photomicrographs showing the intracellular expression of virus protein in cell culture. The FITC-fluorescence signal was expressed as the infected cells. (B) Volocity Demo software analysis of the ratios of infected cells according to the Fig. 5A. *, statistical significance (p<0.05) (C) Flow-cytometric analysis of virus-infected cells at 6 h p.i. MDCK cells (2×10⁶) in suspension were incubated with PBS or anti-PR8 antibodies on ice. Then the FITC-conjugated IgG secondary antibodies were added. After washing, the cells were fixed and the number of infected cells was determined by flow cytometric analysis. *, statistical significance (p<0.05).

Figure 6. Influenza virus induced cell-cell fusion. MDCK cells were infected with the viruses at MOI of 0.1 or 0.001 in the presence of 1 µg/ml TPCK-trypsin.

After adsorption for 1 h at 37°C, the inocula were removed and the cultures were washed 3 times. The cells were incubated for the indicated times at 37°C in the maintenance media. At the indicated time, the cells were processed for indirect immunofluorescence assay, and the infected cells were detected with polyclonal antisera to whole viruses. (A) MOI at 0.1, 3 h p.i. (B) MOI at 0.1, 6 h p.i. (C) MOI at 0.001, 12 h p.i.