Neuraminidase and hemagglutinin matching patterns of a highly pathogenic avian and two pandemic H1N1 influenza A viruses

Abstract

Background: Influenza A virus displays strong reassortment characteristics, which enable it to achieve adaptation in human infection. Surveying the reassortment and virulence of novel viruses is important in the prevention and control of an influenza pandemic. Meanwhile, studying the mechanism of reassortment may accelerate the development of anti-influenza strategies.

Methodology/principal findings: The hemagglutinin (HA) and neuraminidase (NA) matching patterns of two pandemic H1N1 viruses (the 1918 and current 2009 strains) and a highly pathogenic avian influenza A virus (H5N1) were studied using a pseudotyped particle (pp) system. Our data showed that four of the six chimeric HA/NA combinations could produce infectious pps, and that some of the chimeric pps had greater infectivity than did their ancestors, raising the possibility of reassortment among these viruses. The NA of H5N1 (A/Anhui/1/2005) could hardly reassort with the HAs of the two H1N1 viruses. Many biological characteristics of HA and NA, including infectivity, hemagglutinating ability, and NA activity, are dependent on their matching pattern.

Conclusions/significance: Our data suggest the existence of an interaction between HA and NA, and the HA NA matching pattern is critical for valid viral reassortment.

Figure 1. Infectivity assay of all combinations.

Infectivity of normalized pps with various HA/NA combinations; infectivity is presented as the Mean± SD percentage of infected cells from 3 repeats. All pps were grouped by NA; 2009 H1N1, 1918 H1N1, and H5N1 (A/Anhui/1/2005) are abbreviated as 09, 1918, and AH, respectively.

Figure 2. HA/NA expression analysis.

A. Western blot of HA. HA of each pp combination was blotted with purified 300ng wild viruses as positive control (H5N1: A/Vietnam/1194/2005; H1N1: A/Carifornia/7/2009). B. Immunocellular staining of HA and NA on pp producer 293T cells. HA (bottom) and NA (upper). Mock, normal 293T cell lysate.

Figure 3. Hemagglination assay of various pps.

All pps were two-fold diluted serially in 96-well plate. The hemagglutinating ability was expression as the mean HA titer (log₂ HA units/50 µl) of each pp, n = 3.

Figure 4. NA activity in the various pps, NA activity is presented as the Mean± SD from 3 repeats.

A. NA activity of 09N1 combined with 09H1, 1918H1, and AH H1. B. NA activity of 1918N1 combined with 1918H1, 09H1, and AH H1. C. NA activity of AH N1 combined with AH H1, 09H1, and 1918H1.

Figure 5. Primary sequence alignment of the HAs and NAs.

The H1N1 1918 strain was used as a standard. Those residues that are identical in the 1918 strain are shown by “.” The signal peptide is indicated in bold. The aas marked in red represent the cleavage site of the HA precursor, linking the functional HA1 and HA2 domains, while those marked in blue indicate the binding sites for the sialic acid receptor. The gray boxes indicate potential glycosylation sites, as predicted from the sequence, while the green box indicates conserved active site residues from the influenza virus NA.