Influenza A (H3N2) Outbreak, Nepal

Worldwide emergence of variant viruses has prompted a change in the 2005–2006 H3N2 influenza A vaccine strain.

. The annually updated trivalent vaccine consists of hemagglutinin (HA) surface glycoprotein components from influenza H3N2, H1N1, and B viruses.
The HA1 segment of the influenza HA protein is the most rapidly evolving gene product (3) and plays a major role in viral attachment and evasion from the adaptive immune response. Previous studies have demonstrated 5 antigenic sites on the HA1 polypeptide where antibody binding can occur (4,5). Additionally, several studies have documented specific immunodominant codons corresponding to specific amino acids of the HA protein that are directly involved in the divergence of antigenically distinct influenza viruses (6)(7)(8).
In July 2004, an outbreak of influenza A (H3N2) was detected in patients at 3 Bhutanese refugee camps in southeastern Nepal. To elucidate the molecular mechanism underlying the emergence of this H3N2 outbreak, we conducted a molecular analysis of the HA1 region of the HA protein. In this report, we describe the epidemiologic and molecular aspects of isolates obtained from this off-season influenza A (H3N2) outbreak.

Sample Collection and Antigenic Analysis
Sixty-four patients in Nepal that met US Department of Defense enrollment criteria (9) for influenzalike illness were evaluated by using onsite rapid influenza tests (Optical Immunoassay Rapid Diagnostic Tests, Thermo Electron Corp., San Jose, CA, USA) according to the manufacturer's instructions. Throat swab specimens were collected within the first 72 hours of onset of symptoms, routed through the Armed Forces Research Institute for Medical Sciences in Bangkok, Thailand, and shipped on dry ice to Brooks City Base in San Antonio, Texas, for clinical characterization and diagnosis using traditional culturing techniques and monoclonal antibody staining (10). Antigenic analysis of select isolates was performed at the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, by using the hemagglutination inhibition (HI) assay and postinfection ferret antisera (11).

Epidemiologic and Laboratory Assessment
Clinical evaluations and throat specimens were obtained from 64 patients from 3 refugee camps in southeastern Nepal ( Figure 1). Of the 64 patients, 61 were refugees from Bhutan, 1 was a foreign aid worker from Japan, and 2 were Nepalese nationals. Most of the patients were <10 years of age; 36 were male and 28 were female. None had previously been vaccinated against influenza and of the 64 specimens collected, 42 (66%) tested positive for influenza A by culture.  (Table 1).

Molecular Analysis
RT-PCR-based molecular subtyping showed that all 42 specimens were the H3N2 influenza subtype. Twenty-six of the 42 influenza A-positive samples were randomly selected for molecular characterization using direct nucleotide sequencing of the HA gene. The 26 Nepal isolates exhibited 99.8% nucleotide sequence identity and contained the Fujian-like amino acid substitutions at positions 155 (H155T) and 156 (Q156H) in the HA protein ( Table 2). Alignment of the 329-amino acid HA protein from 26 isolates obtained from this outbreak with the 2004/05 A/Wyoming/3/03 vaccine strain and previous H3N2 vaccine strains indicated 4 evident amino acid changes present in most of the isolates ( Table 2) Table 2).
The phylogeny of H3N2 HA proteins indicates a drifting of the Nepal isolates from the A/Fujian/411/03 and A/Wyoming/03/03 vaccine strains and shows that these outbreak isolates have a higher genetic homology to A/Wellington/1/04, a prototype strain selected as the 2005-2006 Southern Hemisphere H3 vaccine strain ( Figure 2). The A/Wellington/1/04 strain contains 2 of the 4 amino acid changes (S227P and S189N) observed in the Nepal isolates, but does not contain the K145N and V226I substitutions.
Three-dimensional views of influenza HA proteins highlighting amino acid changes in a representative Nepal isolate and the A/Wyoming/3/03 vaccine strains are shown in Figure 3A and B, respectively. The mutation at position 145 (shown in yellow), which is located adjacent to antibody-binding site A and within a known glycosylation site, introduces an asparagine-for-lysine substitution. This substitution results in a more accessible receptor-binding cleft located directly above residue 145 (comparing panels A and B). Located above the receptor-binding pocket is a serine-to-asparagine change (shown in green) that possibly alters the regional surface topography at position 189 within antibody-binding site B. A serine-to-proline mutation at position 227 (shown in magenta) appears to marginally affect the HA surface features. This substitution resides within antibody-binding site D, which corresponds to residues 225-228, which make up the left side of the receptor-binding pocket (14). Interestingly, this proline residue is located within a β barrel (a protein motif consisting of an antiparallel β sheet domain) and does not appreciably alter the predicted protein structure, as shown by the absence of any substantial changes in the computer-modeled, 3-dimensional structure compared with the HA1 of A/Wyoming/3/2003.

Discussion
The 4 substitutions described represent a growing lineage of influenza A (H3N2) viruses characterized since July 2004. Three amino acid changes are confined within known antibody-binding sites, i.e., the S189N change within antibody-binding site B (4,5) and the V226I and S227P changes residing in antibody-binding site D (4,5). Because of rotational restrictions, a proline substitution at position 227 (S227P) would typically give rise to considerable conformation change; however, this particular substitution is located within a β barrel motif and therefore has little effect on regional protein conformation. Cumulatively, field isolates characterized subsequent to this outbreak continue to exhibit these 4 changes, and they appear to constitute a distinct branch in the phylogeny of HA sequences when compared with H3N2 isolates from the 2003-2004 season.
The K145N mutation represents a change from a charged to uncharged amino acid R group. This change may affect protein-protein interactions since it is immediately adjacent to antibody-binding site A, where neutralizing antibodies have been shown to bind (4,5). Furthermore, since the K145N substitution is located within a glycosylation site, the charge alteration may affect glycosyl transferase activity, which results in altered glycosylation. Differences in glycosylation have been shown to contribute to antigenic variation by preventing antibody binding to antigenic sites (15). Additionally, 3-  Table 2 that were antigenically distinct from A/Wyoming/303. dimensional analysis suggests this amino acid substitution may also promote enhanced receptor binding since the asparagine R group is shorter, which may make binding requirements less stringent and the receptor cleft more accessible. The 3-dimensional depiction provides a unique regional residue perspective, demonstrating how the rapidly evolving HA surface antigens in the vaccine strain differ at the molecular level. These changes are consistent with both antigenic and genetic data.
Collectively, the clinical isolates obtained from this outbreak in Nepal cannot be considered antigenically distinct from the A/Wyoming/3/03 vaccine strain because only 4 of 9 isolates evaluated exhibited 4-fold lower titers by HI (Table 1). Furthermore, the varying reactivity noted in several isolates from this outbreak having identical HA1 sequences is suggestive that other viral antigens aside from the HA1 protein may have contributed to the antigenic variability observed in the HI panel.
With the exception of A/Nepal/1670/2004 and A/Nepal/1672/2004, all isolates evaluated by HI (Table 1) exhibited identical HA1 amino acid sequences and varying antigenicity profiles to A/Wyoming/03/2003 reference antisera. One explanation for this observation is that genetic differences in other influenza surface proteins contribute to the observed immunoreactivity. Alternative viral surface protein candidates include the neuraminidase, HA2, and M2 glycoproteins, which have been shown to exhibit antigenic properties (16)(17)(18)(19).
In this report, we describe the genetic analysis of the HA proteins from viruses obtained from an early season outbreak and compare them to current vaccine strains. Three amino acids changes (S189N, I226V, and S227P) were noted in known (4,5) antibody-binding sites ( Table  2). The fourth change (K145N), which was located within a glycosylation site, may enhance viral binding since the smaller asparagine R group is located close to the HA receptor-binding cleft ( Figure 3). Phylogenetic analyses show that the Nepal isolates make up a distinct branch in the evolution of H3N2 viruses when they are compared with vaccine and reference strains ( Figure 2). However, antigenic data appear more ambiguous, suggesting a multigenic effect that cannot solely be attributed to properties of the influenza HA (Table 1). Studies are in progress to characterize the neuraminidase, M2, and HA2 proteins to determine the molecular basis responsible for antigenicity differences observed within isolates from this outbreak.
The K145N substitution change has become a marker for an increasingly large subset of the Fujian-like viruses.  Magenta, residues 226 and 227; orange, residue 189; green, residues 155 and 156; yellow, residue 145. Hemagglutinin molecules were generated by using the respective amino acid sequences with MOLMOL (12). A/Nepal/1648/04 is available from GenBank under accession no. AY945264. this variant H3N2 virus. Our findings emphasize the importance of continued molecular surveillance for characterizing emerging influenza drift variants.