More than 1,250 samples of influenza A virus from New York State and New Zealand have been sequenced and published as part of the IGSP. In the IGSP standard sequencing pipeline all eight virus genomic segments of each isolate are reconstructed from sequenced overlapping PCR products (or amplicons) and consensus assembly of the sequence reads. No cloning is involved. For 0.5% of the isolates sequenced, irresolvable base calls at multiple sites in genes indicated virus sequence heterogeneity, and full genomes could not be assembled. One of these isolates was collected in New Zealand in 2004, and its assembly led to a large number of unresolved ambiguities: A/Canterbury/200/2004 (abbreviated NZ094). We cloned each segment from this isolate to evaluate the extent of the genetic diversity present. An average of six clones per segment was sequenced by the Sanger method (Table ).
Phylogenetic analysis of NZ094 clones revealed that at least two distinct lineages of A/H3N2 were present in this isolate: one closely related to viruses cocirculating in New Zealand during 2004 and a second lineage that clustered with A/H3N2 viruses that became dominant in the following (2005) influenza season in the southern hemisphere (Fig. ). Notably, one of the nine clones of the M segment was closely related to 2005 viruses that carry the S31N mutation in the M2 ion channel protein and which confers resistance to adamantane drugs (18
) (Fig. ). The adamantanes (amantadine and rimantadine) normally block the M2 ion channel thus preventing the fusion of the virus and host-cell membranes. A single nonsynonymous point mutation (G to A at nucleotide 92 of the M2 open reading frame, leading to S31N) within the transmembrane region of this protein succeeds in producing a resistant virus.
FIG. 1. Maximum-likelihood phylogenetic trees for M (a), PB1 (b), PA (c), and NP (d) segment clones from sample NZ094 compared to a representative sample of viruses circulating in the years 2004 and 2005. Clones from sample NZ094 are shown in red and italics, (more ...)
Analysis of the other seven genome segments confirmed the presence of these two A/H3N2 lineages (Fig. and ). Specifically, a minority of the M, polymerase basic protein 1 (PB1), polymerase (PA), and nucleoprotein (NP) segment clones were more closely related to later 2005 viruses rather than to 2004 viruses (Fig. ). In contrast, all clones for the other segments, HA, PB2, NA, and nonstructural protein (NS) either fell within the genetic diversity sampled in 2004 (HA and PB2) or possessed insufficient phylogenetic signal to clearly resolve evolutionary history (NA and NS) (Fig. ). It is important to note that although two viral lineages must obviously be present in all segments, these are only clearly distinguishable in the PB1, PA, NP, and M segments.
FIG. 2. Maximum-likelihood phylogenetic trees for the HA (a), PB2 (b), NA (c), and NS (d) segment clones from sample NZ094 compared to a representative sample of viruses circulating in the years 2004 and 2005. Clones from sample NZ094 are shown in red and italics, (more ...)
This phenotypically important intrahost genetic diversity was confirmed through deep sequencing of specific regions of the NA, M, and HA1 domain of the HA derived from patient NZ094. Specific primers were used to amplify 250- to 400-nt regions of these segments, and amplicons were subjected to pyrosequencing on the GS-FLX (Roche/454). In both HA1 and NA, deep sequencing revealed no mutations characteristic of adamantane-resistant A/H3N2 (positions 193 and 225 for HA1 and position 93 for NA [18
]) (Fig. ). In marked contrast, a comparison with two positions in the M segment, one that corresponds to a residue in the M1 protein (K174R) and the other to the residue associated with drug resistance in M2 (S31N), shows the extent of the mixed variants (Fig. ).
FIG. 3. Color-coded representation of protein residues for translated pyrosequencing reads of the HA1, M, and NA segments of isolate NZ094. This representation shows the alignment of specific residues that differ from the N-lineage isolates A/Canterbury/20/2005 (more ...)
This complex pattern of genetic diversity indicates that patient NZ094 was carrying two phylogenetically distinct lineages of influenza A virus. That individual segments have such different evolutionary histories is strongly suggestive of mixed infection with already distinct lineages rather than generation de novo within this patient. That one lineage contains viruses that are adamantane resistant while the other does not further indicates that this patient was coinfected with the “parental” (i.e., prereassortant) strains of an adamantane-resistant lineage that later came to dominate global influenza virus diversity following a major reassortment event in early 2005, denoted the N lineage (18
). A detailed analysis of each clone for any of the 17 specific residues that were shown to be characteristic of the N lineage (18
) further confirmed the presence of two separate lineages (Table ).
Amino acid replacements characterizing the adamantane-resistant N-lineage that were found in cloned segments from patient NZ094
The presence of cocirculating influenza A virus variants within one individual, with potentially important consequences for viral emergence, was also apparent in the case of a patient from New York State. Sample A/NewYork/537/1998 (abbreviated WW537) was collected during the 1997-1998 northern hemisphere influenza season. Phylogenetic analysis of eight fully sequenced HA clones (Table ) revealed that these fell into two lineages separated by viruses sampled in other patients (Fig. ), indicating that the genetic diversity within this patient was also generated through mixed infection rather than de novo. Notably, clones corresponding to these two lineages differed at multiple amino acid residues in the HA1 domain, suggesting that they are also antigenically distinct (Fig. ). Focusing solely on the HA1 domain (residues 16 to 350, from the first methionine), 14 amino acid changes distinguish the two viral lineages, all falling at potential antibody binding sites. By comparing the HA1 domain of the clones to the A/H3N2 strain used in the vaccine that season, A/Wuhan/359/1995, it is apparent that four of the clones (Cr08, C036, C006, and Cr07; lineage 1) consistently differ at 11 residues, again all representing antigenic residues. Indeed, these four clones are A/Sydney/05/97-like in appearance, such that they represent a drift variant of the A/Wuhan/359/1995 strain that spread rapidly in 1997 and to which the vaccine administered throughout North America during that year provided inadequate protection (1
). A/Sydney/05/97 was chosen for the vaccine in the following year. This is the first reported case of a single patient being simultaneously infected with two cocirculating and antigenically distinct variants.
FIG. 4. Maximum-likelihood phylogenetic tree for HA segment clones from sample WW537 in comparison with those circulating during the same time period (1997-1998 northern hemisphere season) in New York State. Clones from sample WW537 are shown in red and italics. (more ...)
FIG. 5. Alignment of nonconserved HA1 residues of the HA across all eight clones from sample WW537. Numbering of the residues is given both from the first methionine of the coding sequence (CDS) and from the first residue of the HA1 domain (HA1 numbering). Gray (more ...)
A final dramatic example of mixed infection was seen in another A/H3N2 patient from New York State—A/New York/347/1999 (WW347)—who was found to harbor both influenza A and influenza B viruses. Although this isolate was originally typed as influenza A virus (GenBank accession numbers AAZ74562 to AAZ74572), a complete influenza B virus genome was also recovered (GenBank accession numbers CY037367 to CY037374). No material remained from the original specimen, a primary swab collected in 1999. However, a portion of the harvest from the original pRhMK cell culture inoculated in 1999 was available for analysis. We therefore quantitated this sample alongside the subsequent, passaged cell culture isolate by real time-PCR. Diluted viral RNA from recently circulating 2006 influenza A/H1N1 and B virus strains, originally isolated in New York State, were used as positive amplification controls (Table ). In New York State, this was the only case found of mixed influenza A and influenza B virus infection out of the more than 500 isolates analyzed in the IGSP. After reviewing the more recent Wadsworth Center test records, however, two additional mixed influenza A/B virus coinfections were noted, and the relevant samples were retrieved for further analysis. Influenza A and B viral copy numbers were determined in both the original primary specimen and the first-passage isolate for both of these samples, using real-time RT-PCR as described above. The quantitation results confirm the presence of the two viruses in both of the primary swabs, as well as in the first passaged isolate in one of them (Table ). No influenza B virus was detected in the other isolate, most likely due to the relatively low level of influenza B virus in the sample and consequent overgrowth of influenza A virus.
Influenza A and B viral RNA copy numbers from isolate WW347, as determined using quantitative real-time RT-PCR