Molecular & antigenic evolution & geographical spread of HPAI H5N1 in W. Africa

canagica · Jul 16, 2007

Molecular and antigenic evolution and geographical spread of H5N1 highly pathogenic avian influenza viruses in western Africa [subscription required]
[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=17622635&dopt=Abstract]

Abstract:
In Africa, highly pathogenic avian influenza H5N1 virus was first detected in northern Nigeria and later also in other regions of the country. Since then, seven other African countries have reported H5N1 infections. This study reports a comparison of full-length genomic sequences of H5N1 isolates from seven chicken farms in Nigeria and chicken and hooded vultures in Burkina Faso with earlier H5N1 outbreaks worldwide. In addition, the antigenicity of Nigerian H5N1 isolates was compared with earlier strains. All African strains clustered within three sublineages denominated A (south-west Nigeria, Niger), B (south-west Nigeria, Egypt, Djibouti) and C (northern Nigeria, Burkina Faso, Sudan, C?te d'Ivoire), with distinct nucleotide and amino acid signatures and distinct geographical distributions within Africa. Probable non-African ancestors within the west Asian/Russian/European lineage distinct from the south-east Asian lineages were identified for each sublineage. All reported human cases in Africa were caused by sublineage B. Substitution rates were calculated on the basis of sequences from 11 strains from a single farm in south-west Nigeria. As H5N1 emerged essentially at the same time in the north and south-west of Nigeria, the substitution rates confirmed that the virus probably did not spread from the north to the south, given the observed sequence diversity, but that it entered the country via three independent introductions. The strains from Burkina Faso seemed to originate from northern Nigeria. At least two of the sublineages also circulated in Europe in 2006 as seen in Germany, further suggesting that the sublineages had already emerged outside of Africa and seemed to have followed the east African/west Asian and Black Sea/Mediterranean flyways of migratory birds.

HenryN · Jul 16, 2007

Re: Molecular & antigenic evolution & geographical spread of HPAI H5N1 in W. Africa

Sublineages of HPAI H5N1 in Africa on the basis of full genome sequencing
Detailed phylogenetic analysis of each of the eight gene segments and the full-length genome revealed three African sublineages with a distinct nucleotide and amino acid signature. Although two of the three sublineages, B and C, emerged from a common node, their high Kimura distance and their distinct geographical distribution within Africa suggest three rather than two distinct sublineages. Sublineage A seems to have emerged from Astrakhan 2005 and was originally found in the BA farm in south-western Nigeria from where it spread more recently to several other farms in the same part of the country and to Niger. This sublineage might be even more closely related to swans from Poland and Germany and a buzzard from Denmark (0.3 % diversity in the HA gene; Dr Ian Brown, personal communication). In Africa, sublineage B was found in a single farm in the south-west of Nigeria and is the only sublineage reported from Egypt and Djibouti. Its ancestor is less clear, as the different genes have mixed characteristics from European, Russian and Mongolian strains. Interestingly, all reported human cases in Africa (Egypt and Djibouti) have been caused by viruses of this sublineage. The African sublineage C was found in northern Nigeria (A/chicken/Nigeria/641/2006) and all strains reported from the Sudan and C?te d'Ivoire belong to this sublineage. It is closely related to A/chicken/Kurgan/3/2005, but we cannot exclude a similar or higher homology with unpublished sequences from strains circulating elsewhere. Sublineages B and C seem to have emerged from a more recent common ancestor than the Qinghai Lake 2005 strains and it cannot be excluded that this common ancestor was circulating in Africa. 

In the new strains reported here, no evidence of reassortment was found, in contrast to A/chicken/Nigeria/1047-62/2006 where HA, NP, NS and PB1 gene segments clustered with sublineage C strains and NA, M, PA and PB2 clustered with sublineage A strains (Salzberg et al., 2007

). Human cases reported so far in Africa (Egypt and Djibouti) have all been caused by sublineage B, but it would be dangerous to infer a lower infection potential of sublineages A and C strains for humans. 
Substitution rates
HPAI H5N1 substitution rates in Africa were high, reaching on average 2.6[FONT=arial,helvetica]x[/FONT]10?3 substitutions per site per year. This is well within the range of substitution rates of avian influenza viruses in general and H5N1 in particular, as described by Chen & Holmes (2006)

using the same calculation method. During the 1994 H5N2 outbreak in Mexico, higher substitution rates (0.88[FONT=arial,helvetica]x[/FONT]10?2 to 2.8[FONT=arial,helvetica]x[/FONT]10?2 substitutions per site per year) were reported (Garcia et al., 1997

) but flocks were partially vaccinated. 
To date, estimates of mutation rates from a single farm are rare. Under the assumption of a single introductory event into the SO farm, a direct ancestral relationship between strains isolated subsequently in the farm and a 5 week evolution in the farm (15 January to 20 March 2006), the substitution rate of the virus for each gene segment including 11 HA sequences was calculated. The rate of 4.58[FONT=arial,helvetica]x[/FONT]10?2 substitutions per site per year (for HA, Table 1

) was higher than the substitution rate calculated within Africa using the same calculation method (1.60[FONT=arial,helvetica]x[/FONT]10?2 substitutions per site per year; Table 1

). This suggests that HPAI H5N1 mutated faster within a farm with a virtually unlimited supply and high density of susceptible (unvaccinated) animals than on its way from Qinghai to Africa. 
Based on the HA substitution rate in the SO farm (sublineage B), it would have taken a minimum of 18 weeks for the south-west Nigerian strains (sublineages A and B) to evolve from a common ancestor and 13?14 weeks for each of the south-western strains to develop from the northern strain (A/chicken/Nigeria/641/2006, sublineage C). As the three outbreaks occurred within a maximum of 8 weeks, the strains probably did not spread from one farm to another. In particular, as H5N1 emerged essentially at the same time in the north (10 January 2006) and in the SO farm (15 January 2006), these two sublineages probably did not develop within Nigeria. Thus, these data and the fact that the closest pre-Nigerian relatives of each of the sublineages were geographically separated further support the independent introduction of each of the three H5N1 sublineages in Nigeria as suggested previously (Ducatez et al., 2006a

), even if the B and C sublineages emerged from a common ancestor. The same analysis for the sublineage A strains from the south-west revealed that the 5 months between the outbreaks was in agreement with the genetic distances between strains, as calculated with Kimura parameters. The HA substitution rate in the SO farm was also compatible with a single introduction of a sublineage C strain into Burkina Faso. Unexpectedly, but as already observed by Chen & Holmes (2006)

, the change of host (chicken to hooded vulture) did not seem to influence the mutation rate of the virus. 
Nucleotide and amino acid substitutions and biological consequences
As previously discussed for Burkina Faso strains (Ducatez et al., 2007

), west African H5N1 strains retained the amino acid pattern associated with preferential binding to
$agr.gif$
2,3-linked sialic acid (mostly present in avian species): 91Y, 130GVSS134, 149W, 151I, 179H, 186E, 190LY191 and 220NGQSGR225 (Ha et al., 2001

; Shinya et al., 2004

). Mutations towards increased binding to human receptors (to $agr.gif$
2,6-linked sialic acid) did not seem to have occurred in west African H5N1 strains. In contrast to south-east Asian lineages, PB2 segments of west African strains have a lysine instead of a glutamic acid in position 627, lysine corresponding to a more pathogenic H5N1 phenotype with accelerated viral replication, a reduced host defence and higher mortality in mice (Fouchier et al., 2004

; Hatta et al., 2001

), as well as increased virulence of H7N7 in human (Chen et al., 2006a

). Chen et al. (2006a)

studied H5N1 viruses in waterfowl in western China in 2005 and observed lysine residues in position 627 for most but not all of the virus isolates. Among the non-south-east Asian strains, some Russian strains, including A/Cygnus olor/Astrakhan/Ast05-2-4/2005, A/duck/Kurgan/08/2005 and A/chicken/Tula/Russia/Oct-5/2005, had a glutamic acid at PB2 position 627, thus also co-existing with the lysine as found in western China. Strains from Russia and western China seem to represent intermediates between the south-east Asian strains with only glutamic acid and the African strains with only lysine. 
None of the west African H5N1 strains showed a signatory amino acid pattern associated with resistance to oseltamivir or to amantadine. 274H was indeed present in all Nigerian and Burkina Faso NA sequences, whilst 274Y has been observed in oseltamivir-resistant patients in Vietnam (de Jong et al., 2005

). Moreover, all west African strains had a 27V/I, 30A, 31S and 34G pattern in M2 and none of the amantadine-resistance markers (27V, 30A, 31S and 34D) (Scholtissek et al., 1998

). 
Unlike the suggestion by Chen et al. (2006b)

, 99I and 268N in HA as well as 111R in NA are not always associated with wild birds, as shown by new sequences from domestic poultry in Africa, Europe and Russia. All Nigerian and Burkina Faso H5N1 strains from chickens had these three amino acids, whilst A/hooded vulture/Burkina Faso/1/2006 had 268Y instead of the predicted 268N. 
In all of the available NS gene sequences from Africa, Europe and Russia, a 563G<IMG alt="->" src="http://vir.sgmjournals.org.proxy.library.vcu.edu/math/rarr.gif" border=0>C substitution was observed, which may correspond to a change in RNA conformation as described by Gultyaev et al. (2007)

for the recent H5N1 strains. Thus, the NS gene of the African HPAI H5N1 2006 viruses would be likely to show a hairpin rather than a pseudoknot conformation towards the 3' extremity, which may play a role in mRNA splicing regulation (Gultyaev et al., 2007

). 
Selection pressure
In Africa, Europe and Russia, most H5N1 genes are under purifying selection with 0.10< $ohgr.gif$
<0.43 (Table 1

), suggesting that a non-synonymous mutation has only 10?43 % as much chance as a synonymous mutation of being fixed in the population. Only the PB1-F2 gene seems to be under positive natural selection ( $ohgr.gif$
=8.476, data not shown), but, as discussed by Holmes et al. (2006)

, this is probably due to the overlap of the PB1 and PB1-F2 ORFs (a shift of 1 nt compared with PB1 ORF) and therefore represents an artefact. In south-east Asian 2002?2005 genotype Z H5N1 strains, M2 was also under positive natural selection, which was not the case in the 26 African strains analysed here or in the available strains from western Asia, Russia and Europe (data not shown). No positively selected codons were detected here in HA, unlike observations in south-eastern strains (Smith et al., 2006

). 
Our data indicate that the geographical origin of west African HPAI H5N1 was not south-east Asia but the central Asian/European region. The three sublineages A, B and C already existed prior to the arrival of HPAI H5N1 in Africa. They have continued to circulate and to evolve both in Africa and in Europe, e.g. in Germany. Although the relative roles of bird migration and the poultry trade in the spread of recent H5N1 remain a matter for debate, the present study contributes to a better understanding of the evolution and geographical spread of HPAI H5N1 in Africa. H5N1 virus has been reported from 14 of the 31 Federal Nigerian States and has caused at least five outbreaks in poultry and one outbreak in wild birds in Burkina Faso. It is difficult to see how the virus can be contained without depopulation combined with surveillance and large-scale vaccination. Despite the high mortality caused by H5N1, surveillance is complicated by uncharacteristic organ lesions at necropsy and multiple viral and bacterial co-infections (Ducatez et al., 2006b

; Igbokwe et al., 1996

; Owoade et al., 2006

, 2004a

, b

), many of which are associated with significant levels of mortality. Reservations of the developed countries towards vaccination against H5N1 delay crucial decisions and prevent rapid action in Africa. Socio-economic and political hurdles further compromise control measures, whilst the virus continues to spread and threatens to become endemic.

HenryN · Jul 16, 2007

Re: Molecular & antigenic evolution & geographical spread of HPAI H5N1 in W. Africa

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AM262524?AM262578 and AM502998?AM503074.

HenryN · Jul 16, 2007

Re: Molecular & antigenic evolution & geographical spread of HPAI H5N1 in W. Africa

Many if the HA sequences discussed in the paper are in the phylogenetic tree at

http://precedings.nature.com/documents/459/version/1

which also includes the initial sequences from Ghana.

Molecular & antigenic evolution & geographical spread of HPAI H5N1 in W. Africa

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HenryN

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