Influenza A (H15N4) Virus Isolation in Western Siberia, Russia

Abstract

The rarely identified influenza A viruses of the H15 hemagglutinin subtype have been isolated exclusively in Australia. Here we report the isolation of an H15N4 influenza A virus (A/teal/Chany/7119/2008) in Western Siberia, Russia. Phylogenetic analysis demonstrated that the internal genes of the A/teal/Chany/7119/2008 strain belong to the Eurasian clade and that the H15 and N4 genes were introduced into the gene pool of circulating endemic avian influenza viruses through reassortment events.

TEXT

Influenza A viruses are antigenically and genetically diverse and undergo continuous evolution. Influenza A viruses of 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes have been identified based on antigenic properties of the 2 surface glycoproteins, while RNAs encoding potential 17 HA and 10 NA subtypes have been detected based on sequence information alone (1–4). Influenza viruses of the 16 HA and 9 NA subtypes can be found in waterfowls and shorebirds and can be divided into two major phylogenetic clades, one in the Americas and the other in Eurasia, with the latter containing a distinct Australian subclade (5, 6). Avian influenza viruses (AIV) of the H15 subtype are rarely identified (7, 8), and only 6 strains are available in the GenBank database. All 6 strains have been isolated in Australia (6, 7, 9).

Western Siberia is an area under 4 major avian flyways binding bird populations from Europe, Africa, Asia, Oceania, and North America (including the Alaska Peninsula). This convergence of flyways creates an environment in which influenza viruses from different lineages and hosts can reassort, a process common to influenza viruses of wild birds (10). During surveillance for AIV in Western Siberia in May through September of 2008, 1,445 cloacal swab samples were collected from healthy wild birds (11). Virus was isolated by injection of the original sample into the allantoic cavity of 10-day-old, embryonating, specific-pathogen-free (SPF) chicken eggs (12). Allantoic fluid was screened by performance of HA and hemagglutinin inhibition (HI) assays with antisera to H1 to H15 subtypes and 0.5% chicken red blood cells (12). A total of 25 AIVs were isolated (isolation rate, 1.73%). The majority of AIVs were detected in the Anatidae (80%) and Laridae (12%) families. AIVs of the H1 (n = 1), H3 (n = 14), H4 (n = 4), and H15 (n = 1) subtypes and N2 (n = 1), N4 (n = 1), N6 (n = 4), and N8 (n = 11) subtypes were isolated. Five AIVs were not subtyped. The influenza virus A/teal/Chany/7119/2008 was isolated from a cloacal swab obtained from a healthy common teal (Anas crecca; Anatidae family). This wild-bird species is a major natural reservoir of viruses of different HA and NA subtypes (7, 13, 14). The virus had HA activity and reacted with antiserum to the H15 HA subtype.

Influenza virus detection was confirmed by performance of real-time PCR assays using TaqMan probes (available upon request) targeting the viral M gene (11). Viral RNA was isolated from virus-containing allantoic fluid by using the SV Total RNA isolation system (Promega, Madison, WI). Virus was subtyped by using universal primers for the HA and NA genes (15). The results of the sequence analysis of HA and NA genes and the subsequent BLAST search revealed that A/teal/Chany/7119/2008 virus belonged to the H15N4 subtype. The virus replicated efficiently in eggs (10^6.3 50% egg infective doses [EID₅₀]/ml) and Madin Darby canine kidney (MDCK) cells (10^7.0 50% tissue culture infective doses [TCID₅₀]/ml).

To determine the pathogenicity of A/teal/Chany/7119/2008 virus, 6 SPF White Leghorn chickens (SRC VB “Vector,” Russia) were inoculated intravenously with 0.1 ml of virus-containing allantoic fluid (10^5.3 EID₅₀/ml). None of the chickens showed any clinical signs of disease, and none died during the 14-day postinoculation (p.i.) observation period (intravenous pathogenicity index = 0) (12). Sera were harvested from the inoculated chickens on day 21 p.i., and HI assay results showed that all inoculated birds had seroconverted (HI titers ranged from 1:640 to 1:1,280).

To assess the pathogenicity of A/teal/Chany/7119/2008 (H15N4) virus in mice, we inoculated intranasally 9-week-old BALB/c mice (SRC VB “Vector,” Russia) with 50 μl of 10¹ to 10⁶ EID₅₀/ml virus in phosphate-buffered saline (PBS) and monitored weight loss and clinical scores for 20 days. Mice did not lose weight, show signs of disease, or have detectable levels of anti-HA antibodies. These results suggest that A/teal/Chany/7119/2008 (H15N4) virus is nonpathogenic for chickens and does not replicate in the lungs of mice.

The results of antigenic analysis with 2 polyclonal antisera revealed that A/teal/Chany/7119/2008 (H15N4) virus is antigenically distinct from the reference A/shearwater/Australia/2376/1979 (H15N6) strain. Although postinfection antiserum raised against A/shearwater/Australia/2376/1979 reacted well with A/teal/Chany/7119/2008 in an HI assay (HI titer of 640, compared to a homologous titer of 1,280), the reverse did not hold, and postinfection antiserum against A/teal/Chany/7119/2008 (homologous HI titer of 1,280) did not react with A/shearwater/Australia/2376/1979.

To study the genetic relatedness of A/teal/Chany/7119/2008 (H15N4) to other viruses, we conducted sequence analysis of the full viral genome, followed by phylogenetic analysis of individual gene segments (10). Comparing the HA amino acid sequences of A/teal/Chany/7119/2008 (H15N4) with those of H15 viruses in the GenBank database revealed the following 16 unique amino acid substitutions: 35T, 37S, 46R, 53F, 71D, 93S, 112T, 125V, 147R, 160K, 173I, 180E, 184Q, 199R, 256L, and 327T (H15 numbering of the mature protein here and throughout the text). Two amino acid substitutions (147R and 199R) were in the potential A and D antigenic sites (based on the three-dimensional [3D] structure of the H3 HA subtype) (16–18) (Fig. 1). Three amino acid changes (125V, 180E, and 184Q) were in the hypothetical receptor-binding site (130 loop and 190 helix). The main characteristic feature of previously characterized H15 genes, a specific insertion in the HA1 subunit at positions 253 to 262 (8), was identified in the HA of A/teal/Chany/7119/2008 virus, although it had a unique single amino acid change at residue 256 (P256L) within this region. The Russian virus possessed Q217 and G219 residues (Q226 and G228, H3 numbering) at the HA receptor-binding pocket, suggesting that its binding preference is for sialic acid α-2,3 receptors. We identified 4 potential HA glycosylation sites based on the existence of NXS and NXT sequences. The lack of a multibasic motif at HA's cleavage site confirmed our pathogenicity data indicating that this virus was a low-pathogenic AIV. Overall, sequence analysis results showed that the HA gene of A/teal/Chany/7119/2008 virus was distinct from that of other H15 subtype viruses.

Fig 1.

HA1 protein sequence alignment of H15 influenza viruses. The 7 predicted HA1 proteins of H15 influenza viruses were aligned and compared to those of avian influenza viruses of the H3 and H7 subtypes. The GenBank accession numbers are as follows: A/teal/Chany/7119/2008 (H15N4), GenBank accession number CY098540, this study; A/Australian shelduck/Western Australia/1762/1979 (H15N9), accession number CY077616; A/shearwater/Australia/2576/79 (H15N9), accession number GU052260; A/Australian shelduck/Western Australia/1756/1983 (H15N2), accession number CY006032; A/duck/Australia/341/1983 (H15N8), accession number AB295613; A/wedge-tailed shearwater/Western Australia/2327/1983 (H15N9), accession number CY006034; A/sooty tern/Western Australia/2190/1983 (H15N9), accession number CY006033. The HA sequences of avian H3 A/duck/Memphis/928/1974 (H3N8) (accession number M73772) and H7 A/Anas crecca/Spain/1460/2008 (H7N9) (accession number HQ244407) influenza are included for reference purposes. Antigenic sites A (green), B (violet), C (orange), and D (blue) were designated on the basis of the H3 HA subtype (32). The residues forming the receptor-binding domain are indicated by a diamond (224). An 8-amino-acid insertion (positions 253 to 262) in the H15 HA of A/teal/Chany/7119/2008 virus is identical to that of H15 A/duck/Australia/341/1983. Underlined residues represent possible N-glycosylation sites. The amino acid numbering of the mature H15 HA protein is based on H3 numbering (upper line) and H15 numbering (lower line).

Analysis of antiviral susceptibility of A/teal/Chany/7119/2008 was based on the presence of mutations encoding amino acid substitutions at key residues that confer resistance to M2-ion channel blockers and NA inhibitors. Resistance markers were not detected in the transmembrane region of M2 protein or the catalytic and framework NA residues, suggesting that the virus is susceptible to both classes of drugs.

The results of the HA phylogenetic analysis (Fig. 2A) confirmed that A/teal/Chany/7119/2008 belongs to the H15 subtype, although it was placed at a basal position with respect to the other 6 H15 viruses. These results revealed a previously unrecognized level of genetic diversity between H15 strains and the possible formation of a separate sublineage of H15 subtype AIVs in Russia. The phylogenetic tree of the NA gene shows that A/teal/Chany/7119/2008 belongs to the N4 subtype, which has not been previously isolated in Western Siberia (Fig. 2B). The results of phylogenetic analysis showed that internal genes (PB1, PB2, PA, NP, MP, and NS) of A/teal/Chany/7119/2008 belong to the Eurasian clade (Fig. 3). The additional analysis of the nucleotide and amino acid identities of the internal genes of A/teal/Chany/7119/2008 virus and its closely related strains confirmed this observation (data not shown). This composition of internal genes was identified previously in Western Siberia in influenza viruses of H8N8, H3N6, and H3N8 subtypes (Fig. 3), suggesting that viruses of Eurasian lineage persist in this region. Overall, phylogenetic analysis results revealed that A/teal/Chany/7119/2008 (H15N4) is a reassortant with novel HA and NA genes and endemically circulating internal genes.

Fig 2.

Phylogenetic trees for the HA and NA genes of A/teal/Chany/7119/2008 (H15N4) influenza virus. To identify specific influenza virus populations, we analyzed all full-length, publicly available sequence data from avian hosts of all geographic regions (n = 18,000 gene segments). For hemagglutinin (HA) and neuraminidase (NA), full-length sequence data were downloaded and aligned by using the NCBI Influenza Virus Resource. Final data sets were restricted to coding regions read in the first reading frame of each gene. Sequences with insertions or deletions resulting in frame shifts or amino acid insertions were excluded from the analyses. Here and in Fig. 3, provisional phylogenetic inference was performed by using Mega v5.3 software and neighbor-joining methods (500 replicates). The number at each branch point indicates a bootstrap value of at least 75% in the bootstrap interior branch test. Duplicate sequences and those with 100% similarity that were isolated from the same region and during the same year were excluded from the analyses while ensuring that representative virus sequences of each monophyletic clade, as determined from the phylogenetic analysis described above, were included in all analyses. The A/teal/Chany/7119/2008 (H15N4) virus is marked with a red circle.

Fig 3.

Phylogenetic trees for the PB2, PB1, PA, NP, MP, and NS genes of A/teal/Chany/7119/2008 (H15N4) influenza virus. For each gene segment (including polymerase basic 2 [PB2], polymerase basic 1 [PB1], polymerase acidic [PA], nucleoprotein [NP], membrane [M], and nonstructural [NS]), full-length sequence data were downloaded and aligned by using the NCBI Influenza Virus Resource. Final data sets were restricted to coding regions read in the first reading frame of each gene. Sequences with insertions or deletions resulting in frame shifts or amino acid insertions were excluded from the analyses. The trees were rooted with the A/duck/Ukraine/1/63 (H3N8) virus (GenBank accession numbers CY005819, CY005818, CY005817, CY00515, CY005814, and CY005816). The A/teal/Chany/7119/2008 (H15N4) virus is marked with a closed circle.

Influenza viruses of the H15 subtype may have less diversity than viruses of the other HA subtypes, more likely because of the geographic separation of the Australian continent (1, 6, 7) and then being introduced into Russia by the migration of bird species between the continents (9–11). The crossing of major avian pathways and climate features suggest that the territory of Western Siberia may play an important role in influenza viral persistence, evolution, and long-distance distribution (13, 14). Overall, our results show the necessity for continuous surveillance and novel methods for diagnostics and identification of AIVs.

Nucleotide sequence accession numbers.

The genome sequences of A/teal/Chany/7119/2008 (H15N4) have been deposited in GenBank under accession numbers CY098537 through CY098544.