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).

RNA was extracted from 48-hour shell vial cultures (10) by using the MagnaPure LX (Roche Molecular, Mannheim, Germany) and RNA Isolation Kit II (Roche Molecular) according to the manufacturer's protocols. For reverse transcription-polymerase chain reaction (RT-PCR) amplification, 5 μL RNA was added to a 50-μL master mixture containing 1× reaction buffer, 1.6 mmol/L MgSO₄, 1× enzyme mixture, and 400 nmol/L primers (H3-F7, 5´-ACT-ATC-ATT-GCT-TTG-AGC-3´ and H3R-1184, 5´-ATG-GCT-GCT-TGA-GTG-CTT-3´) by using the SuperScript III One-Step RT-PCR System (Invitrogen, Carlsbad, CA, USA). PCR thermocycling consisted of an RT step at 50°C for 30 min, hot start activation at 95°C for 3 min, followed by 40 amplification cycles of 95°C for 30 s, 52°C for 15 s, and 68°C for 1 min, with a final extension cycle at 68°C for 7 min. All PCR products were visualized after electrophoresis in 2% precast gels stained with ethidium bromide (Invitrogen) under UV illumination. PCR products were purified by using the PCR Purification Kit (Qiagen Inc., Valencia, CA, USA). The HA1 amplicon (1177 bp) was sequenced by using the H3-F7 and H3R-1184 PCR primers (described above) and 2 additional internal oligonucleotides, H3R-466 (5´-GGT-GCA-ACC-AAT-TCA-ATC-3´) and H3F-282 (5´-CAG-CAA-CTG-TTA-CCC-3´).

Unincorporated fluorescent nucleotides were removed by using a Dye Ex 96-well plate kit (Qiagen) according to the manufacturer's recommendations. Nucleotide sequencing was performed by using the Big Dye Terminator v3.1 Kit and analyzed by using an ABI 3100 Genetic Analyzer (both from Applied Biosystems, Foster City, CA, USA) according to the manufacturer's specifications. Multiple sequence alignments, protein translation, and phylogenetic analysis were performed with the DNAStar (DNAStar Inc., Madison, WI, USA) software package. Three-dimensional HA protein structures were generated by using MOLMOL (12) and the Swiss-PDB Viewer programs (13). HA nucleotide sequences for all 26 Nepal isolates depicted in the phylogenetic analysis are available from GenBank under accession nos. AY945263–AY945288.

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.

HI was performed by using postinfection ferret antisera with reference antigens that included the 2004–2005 H3N2 vaccine seed strain (A/Wyoming/03/2003) and the 2005–2006 Southern Hemisphere H3N2 vaccine strain (A/Wellington/1/2004). When compared with A/Wyoming/03/2003, 4 of 9 Nepal isolates showed 4-fold lower titers (1:320 versus 1:1,280 HI units) than that allowed for homologous titer of the reference antisera. This indicated that these 4 isolates were antigenically distinct. Six of 9 Nepal isolates were antigenically distinct when compared with the A/Wellington/1/2004 strain and showed a 4-fold (1:160 versus 1:640) reduction in titer to ferret antisera (Table 1).

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). All 4 amino acid changes observed within most of these outbreak isolates are present within A/California/7/04, a variant strain selected as the H3N2 vaccine strain for the 2005–2006 influenza season.

Of the 26 Nepal strains examined, 24 exhibited a novel lysine-to-asparagine substitution at position 145 in the HA protein (K145N). This substitution is noteworthy because most strains characterized in 2003–2004, including the Fujian/411/2002 vaccine strain, contained a lysine (K) at this position. Prior to this outbreak, the US Department of Defense had only observed K145N substitutions in 6 strains obtained from Ramstein, Germany, (data not shown) in June 2004. Additionally, all 26 Nepal sequences exhibited a serine-to-asparagine substitution at position 189 (S189N) that had also been observed in the 6 isolates from Germany, as well as in a few isolates from Asia characterized at the end of the 2003–2004 influenza season.

Two other substitution mutations in the HA1 hemagglutinin, i.e., valine to isoleucine at position 226 (V226I) and serine to proline at position 227 (S227P), were also observed in 24 (92%) and 26 of 26 of the Nepal isolates, respectively. Both substitutions differ from most influenza A H3N2 field isolates collected in 2003–2004, including the Fujian and Wyoming vaccine strain for 2004–2005 (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.