A global phylogenetic analysis in order to determine the host species and geography dependent features present in the evolution of avian H9N2 influenza hemagglutinin

Global nucleotide phylogenetic tree analysis

A complete phylogenetic analysis of the nucleotide dataset was carried out. Computational limits on memory and processor speed make it impossible to carry out a complete analysis on the protein dataset (especially as it contains almost twice as many sequences). A condensed view of the principal clades from the global nucleotide tree can be seen in Fig. 1. One noticeable absence from the global nucleotide tree is the quail sequences. These were omitted during the editing of the sequences to remove truncated sequences where the ends of the sequences were missing. The virus can be broken into three main clusters labelled A, B and the Main Chinese Clade. (The complete tree can be found in Fig. S1.) There is also a small clade (labelled C) that splits from the root of the tree between clades A and B. This clade contains Chinese sequences that were isolated from chickens between 1999 and 2002. This topology agrees with those from the existing literature except that there is some disagreement in the identification of the lineages (Li et al., 2003; Peiris et al., 2001; Perk et al., 2006; Yu et al., 2008). Ji used a lineage and sub-lineage nomenclature that identified four lineages and 2 sub-lineages corresponding to Clade A (lineages 9.1, 9.2 and 9.3), Clade B (sub-lineage 9.4.1) and the main Chinese Clade (sub-lineage 9.4.2). Huang and co-workers used a lineage naming system based on the prototypes Y439, TY WS 66, G1 and Y280 (Huang et al., 2010). Y439, and TY WS 66 (Turkey, Wisconsin 1966) are both in clade A. G1 is in clade B and Y280 is in the main Chinese clade in China 1 1994–2010. The large study by Dong et al. (2011) identified HK/G1/97, BJ/1/94, HK/289/78, HK/AF157/92, KR/96323/96, DE/113/95 and WI/1/66 as prototype sequences for the different HA lineages. The G1 lineage and WI/1/66 lineages are well established and correspond to Clade B and Clade A respectively. BJ/1/94 is in the same clade as Y280, HK/289/78, HK/AF157/92 and DE/113/95 are not in the current dataset because of truncations, but there are sub-clades for Hong Kong duck sequences and Korean chicken sequences that correspond to HK/289/78 and KR/96323/96 respectively. This suggests that it might be possible to divide Clade A into further sub-clades but given the limited number of sequences sampled there is insufficient evidence to be able to carry this out at present.

Within the Main Chinese clade there are a series of nested sub-clades labelled China 1–9 where the annotations follow the correct date order, with the exception of clade 8. In clade 8 there is a single sequence from a chicken in the Shandong region collected in 1999, which is a probable outlier. All of the other sequences within the clade are from 2003 onwards, which would be consistent with the splitting dates for the other sub-clades. It is possible that this is an example of convergent evolution between recent Chinese sequences and an earlier branching of the phylogenetic tree, but a single sequence is insufficient evidence to corroborate this. An alternative explanation is that this results from sequencing error but this is also unlikely given the large number of bases that would have to be incorrectly identified (for an alignment between the Shandong sequence, G1, G9/Y280, Wisconsin 1966 sequences see Fig. S2). It is possible that this could be a database error, but again this seems unlikely given the provenance and tracking systems within GenBank. In the absence of database error and a clear evolutionary connection between this lone sequence and the rest of the clade does provide evidence for the inadequate sampling of sequences. This nested structure of the Chinese sequences had previously been reported by Song, Han & Chen (2011).

The expanded tree (Fig. S1) shows that the geographical sampling has not been systematic and that it has been carried out in a sporadic and haphazard manner. There are breaks in the dates of sequence annotations in some of the clades that are homogeneous for location. Date gaps in annotations at specific locations are likely to be a result of inadequate sampling rather than reliable evidence for the loss and subsequent migratory return of the clade. The focus on China because of outbreaks of H9N2 within flocks of domestic birds has resulted in a very dense phylogenetic tree for the Chinese sequences, resulting in an artificially Chinese focused distribution to the phylogenetic trees. There is only a single sequence from South America this is the result of a sampling effect rather than reflecting the actual H9N2 distribution.

Clade A (Fig. 2) corresponds to wild bird infections that are distributed world-wide and include examples from North America, South America, Europe and the Far East. This is an important clade because it also contains the original sequence of the hemagglutinin from the H9N2 subtype that was found in a turkey in Wisconsin in 1966. This clade also contains a number of recent sequences from Korea the US and Europe and so this clade remains extant. The topology of this clade disagrees with that from Kim and co-workers who constructed a tree for regions 1-1104 using DNA Star (Kim et al., 2006). The complete sequence for HA is over 1,700 bases, and the method used here is Maximum Likelihood, whereas DNA Star uses the less reliable UPGMA tree generation algorithm within ClustalW. In their tree the recent Korean sequences were placed outside clade A and beyond the Y280 sequences as a distinct clade. The bootstrap values are high for this region of the tree (>99%) and it has geographical consistency with the rest of the clade, and so the topology presented in the current paper is most likely to be correct.

Clade B (Fig. 3) contains mostly Middle Eastern sequences although there are a few Chinese sequences that form a sub-clade. This clade corresponds to the extended G1 lineage used by Fusaro et al. (2011) and Monne et al. (2013). Iran and Israel are the two countries most strongly represented in this clade. There are also a number of sequences from the Arabian peninsular and a large grouping from the Indian sub-continent.

The sub-lineage structure of Clade B shows that the sequences have evolved substantially from the G1 prototype. The G1 prototype sub-clade became extinct in Iran in 2004 and in Israel in 2007. This was replaced by a new sub-lineage (labelled 725 sub-clade in Fig. 3) that appears to have originated in chicken flocks in Iran in 1998, although it is only found in a large number of Iranian samples after 2004. This sub-lineage is similar to that previously identified by Fusaro et al. (2011) and Monne et al. (2013) (labelled cluster C in their papers). There is a sub-clade from the Indian subcontinent that originated in the Punjab/Haryana region in 2003. There is a linking clade than originated in Pakistan in 2004 before spreading back to Iran in 2009. The final sub-clade seems to have originated in the Arabian Peninsular in 2006 and correspond to cluster B from the studies of Fusaro et al. (2011) and Monne et al. (2013). This sub-lineage then spread to Israel and most recently to Egypt. This is in good agreement with the previous studies on the origin of the Egyptian virus (Abdel-Moneim, Afifi & El-Kady, 2012). The phylogenetic tree shows that after the initial Egyptian outbreak in 2010 there has been a marked diversification in the sequences.

Clade C (Fig. 4) is a Chinese clade that has annotated dates between 1999 and 2001. In the phylogenetic analysis this clade falls between Clade A and Clade B. As there are only a small number of sequences within the clade there is no discernable pattern to the distribution of the sequences within the Chinese regions, although it appears to have originated in Guangdong in 1999. As there are no more sequences after those from 2002 this clade can be considered extinct.

The first Chinese clade contains the G9/Y280 lineage (Fig. 5). After the banning of live quail from poultry markets the G1 lineage disappeared from Chinese poultry leaving only the G9/Y280 lineage (Choi et al., 2004). In the study of Li et al. (2005) four lineages were specified G1, TY/WS/66, Y439 and Beijing/1/94, but no sequences from G1 or Y439 were found in their sample. Cong and co-workers (2007) identified two more lineages within this clade based on antigenic studies and nucleotide phylogenetic trees. These are represented by prototype sequences from Shanghai 1998 and a swine genotype. These lineages only cover a small number of the possible sub-clades within this first Chinese Clade (Fig. 5). As no sequences have been sampled from this clade since 2010 it is possible that the G7/Y280 lineage is now extinct, but this hypothesis cannot be confirmed without a longer period of absence of viruses from this clade.

This study shows a series of new clades that had not been previously identified and that originate in 1997 in Beijing. This is also the origin of the clade labelled China 2. All of the subsequent nested Chinese clades are related to this Beijing sequence. Some sub-clades such as China 4, China 5 and China 6 also appear to have become extinct and so there is good evidence for successive selective sweeps through the viral population.

As discussed previously the individual unusual sequence from 1999 in China 8 sub-clade is difficult to explain. It might suggest that there is considerable sequence diversity within these nested clades, but because of the low number of individuals carrying a particular sequence it might take a long-time for a sequence to become fixed sufficiently within the population to be found by sporadic sampling. If this is true then all of the clades probably have a much earlier origin and there is a long period before first detection. This causes some concern for tracking the evolution of potential pandemic strains, as they might be circulating quite widely before they are detected for the first time.

Clade analysis of the geographical subsets

From the global phylogenetic tree three interesting geographical subsets were identified; the tree for Korea because it is homogeneous to clade A, and the trees from Iran and Israel as they show successive waves of sequence evolution in clade B. The main Chinese clade has many interesting features but these are difficult if not impossible to untangle and coherently explain because of the limitations of sampling within the data, where there are only small numbers from some regions and then large numbers from others.

Both the protein and nucleotide phylogenetic trees for Korea are shown in Fig. 6. There is a fair agreement between the two trees but once again this emphasizes the problems of sampling and the possible effects this can have on phylogenetic reconstruction. From the nucleotide tree it is tempting to define a clade that contains only wild birds and that suddenly developed in 2005, but this clade is not present in the protein tree, and so it cannot be definitively assigned. There are also some differences in the topology surrounding the swine flu case (marked with a red diamond). Previous studies have investigated the effect of vaccination, which initially suppressed the number of cases that were seen in 2008 before an increase in the number of cases in 2009 (Park et al., 2011). From the trees presented here the period following the introduction of vaccination does correspond to a period of diversification. All of the Korean sequences are from clade A, which is the longest circulating lineage and previously it had not shown a wide diversity of sequences. Vaccination is likely to have had an impact on hemagglutinin evolution, which can be seen in the 2009–2010 clade (Lee & Song, 2013).

The protein and nucleotide phylogenetic trees for Israel and Iran are shown in Fig. 7. In the Iranian tree Clade 1 are duck sequences from Clade A. This is why this clade has the deepest origin within the tree, but it does not contain the earliest sequences. This shows that wild birds can introduce other lineages to a geographical region where another lineage currently dominates.

Clades 2 and 5 in the Iranian tree are the oldest clade from the G1-like lineage in these regions. The G1 lineage appears to have originated in Hong Kong in 1997. The initial G1 clade (Clade 2) becomes extinct in 2003 in the nucleotide tree and in 2005 in the protein tree indicating a selective sweep. This event coincides with the loss of sister Clades 1 and 2 in the Israeli protein tree and Clade 1 in the nucleotide tree. These clades were assigned to cluster A by Fusaro et al. (2011) and Monne et al. (2013). The second G1-like clade (clade 5, labelled sub-clade 725 in the global phylogenetic tree, Fusaro/Monne Cluster D) continued to be found in Iran until 2009. This has no equivalent in the Israeli trees, which seem to have inherited a G1-like lineage which originated in the Indian sub-continent and then circulated in the Arabian peninsular (see the global phylogenetic tree, Fig. S1 and Fig. 3, Fusaro and Monne cluster B) before being found in Israel in 2006/2007 (clade 3 in the nucleotide and protein trees) and in Iran in 2009 (clade 4 in the protein and nucleotide trees). A recent paper has shown that there have in fact been several introductions of H9N2 to Israel from Jordan, and the Arabian Peninsula (Davidson et al., 2014). This newly introduced G1-like sub-lineage seems to have had a selective advantage over the existing viral G1-like sub-lineages but this can only be confirmed by future sampling. Subsequently this new sub-lineage has also spread to Egypt from Israel.

Clade analysis of the host specific subsets

The interesting host species subsets are those for ducks, quail and swine. Ducks are important as a possible carrier of the virus between geographical regions and in acting as a reservoir species. Quail have also been associated with acting as a host to allow re-assortment and viral evolution. Finally swine are important because of the over-lapping glycosylation site specificity of swine and human viruses, which suggests that they may act as an intermediate for transmission to humans (Guo et al., 2005).

There are problems in interpreting the quail trees because of the absence of the Shantou sequences from the nucleotide tree because of partial sequencing (Fig. 8). Shantou was the main geographical location for the outbreak of H9N2 in quail between 2000 and 2005 (Xu et al., 2007a; Xu et al., 2007b). Live quail were banned from Chinese wet markets after a study had shown the link to the virus. In the literature it was found that by 2003 this had resulted in the G1-like lineage disappearing from China but the data here show that it was still present in quail in the Shantou region until 2005 (Choi et al., 2004). The trees show that quail are hosts for all of the main lineages clade A, clade B (G1-like lineage) and the main Chinese clade. This would support the theory that quail have been the host species responsible for the diversification of the H9N2 sub-type, especially given that the original sequence of G-like lineage that is the prototype sequence for clade B was originally found in a Hong Kong quail (Hossain, Hickman & Perez, 2008). This also fits with the epochal model of viral evolution proposed by Wagner (2008). There are periods of neutral drift, which are punctuated by selection events. Here the selection event is the formation of a new lineage within a different host species after a long period of neutral drift within clade A.

Like quail ducks provide a host for the clade A and main Chinese clade viral lineages (Fig. 9). There is only a single example of a clade B sequence in ducks, and so they might not be very effective carriers of this virus or this might be explained by their geographical exposure to that lineage (Perez et al., 2003). Ducks are of concern as a host species as they can contribute to the spread of this lineage over a wider geographical region. In the global phylogenetic tree sequences from ducks are often found clustered with those from quail showing that there is frequent transfer between the two hosts.

Most of the swine cases have been within the main Chinese clade of sequences (Fig. 10). There is a single sequence from Korea in 2004 that belongs to clade A. This is important in guiding how we monitor disease outbreaks because it shows that the most geographically widely distributed clade can also produce infection in pigs. The swine flu epidemic of H1N1 originated in Mexico whereas China had been the main focus of surveillance, because of the frequency and previous circulation of the virus. This is also true of H9N2 monitoring which is focused on Southeast Asia, but shows that more widespread monitoring is important in the early detection of pandemics.

There is no obvious geographical or temporal pattern in infection in pigs and there are multiple transmissions between birds and pigs that produce a tree with many different sub-clades usually with only a small number of members. This is consistent with there not being a widespread circulation of the virus within pigs, which would give a larger homogeneous cluster of swine viruses from different times and locations due to pig to pig transmission. Previously five amino acids changes had been identified as being swine specific, but none of these were conserved within the swine sequences or even within a single swine clade (Xu et al., 2004). There are a few cases of the S145N mutation that has been identified to change the antibody epitope but this again is sporadic (Ping et al., 2008).

Identifying the amino acid changes responsible for clade formation

The X-ray crystal structures are available for the H9 hemagglutinin and so it is possible to map the amino acid changes responsible for differentiating between the different clades onto the structure (Ha et al., 2001; Ha et al., 2002). The region between amino acids 128 and 275 makes up the receptor subdomain. This domain is responsible for binding to the cellular membrane as part of viral invasion. The stem domain is made up of the first 60 amino acids of the N-terminus and the final 275 amino acids in the C-terminus of which the last 221 are cleaved by proteolysis of the precursor protein at a conserved arginine to produce a second peptide chain. In between these domains is the remains of a catalytic domain—the vestigial enzyme domain (Ha et al., 2002). The amino acids that are specific to the four most distinct clades are given in Table 1.

The presence of four key amino acids has been shown to be essential for droplet transmission of the virus H183, A189, E190 and L226 that correspond to residues H191, A197, E198, and L234 in the H9 numbering (Sorrell et al., 2009).

There are 17 amino acids that distinguish between Clade A and the consensus sequence, only three of these are in the receptor domain and so the majority are in the stem domains. Three mutations are in the enzymatic domain of which the most significant of which is the replacement of a serine at residue 127 with an asparagine as this is on the boundary of the domain and this creates another potential glycosylation site. The receptor domain changes are Q164H, R180E and T206A. Of these the replacement of the basic arginine group by an acidic glutamic acid is the most interesting, because of the change in polarity, none of the amino acid changes affected either the glycosylation sites or altered the residues that were identified as key to viral droplet transmission. Only T8A had been previously identified as an important change when it was shown to be involved in host specificity (Perez et al., 2003).

There are 26 amino acid changes that distinguish Clade B. Eleven of these changes can be found in the receptor subdomain. Many of the changes are between leucine, isoleucine and valine. These are conservative changes that preserve the hydrophobicity of the amino acid but change the steric interactions. Another significant proportion of the changes is substitutions to threonine from serine or valine. Three of the serine to threonine changes occur in the receptor domain and this might reflect an altered binding affinity for a larger binding partner. Position A168 had been identified previously as under positive selection (Fusaro et al., 2011). This is supported by the mutation to leucine in this clade. Of the key amino acid changes required for droplet infection only the N191H mutation is clade specific.

The amino acid changes responsible for differentiating between clades gives some support to previous studies that have tried to identify residues that are under selection (Fusaro et al., 2011). However most of the clade specific changes have not been previously identified in the literature on the evolution of antigenicity (Kaverin et al., 2004; Skehel & Wiley, 2000). The amino acids responsible for glycosylation are conserved throughout the phylogenetic trees, although some of the amino acid changes introduce new asparagine residues, which could be new sites for glycosylation (Guo et al., 2000; Zhang et al., 2004). There are a very large number of sequences with the L234 substitution required for droplet infection but these are not specific to any of the major clades and this shows that the mutation has arisen multiple times.