Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006 [Virology Journal]

[Giuseppe]

http://hygimia69.blogspot.com/2008/10/undiagnosed-fatalities-south-africa-ex_07.html


Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006 [Virology Journal]

Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006

Istvan Kiss, Peter Gyarmati, Siamak Zohari, Karin Wilbe Ramsay, Giorgi Metreveli, Elisabeth Weiss, Maria Brytting, Marielle Stivers, Sofia Lindstrom, Ake Lundkvist, Kirill Nemirov, Peter Thoren, Mikael Berg, Gyorgy Czifra and Sandor Belak

Virology Journal 2008, 5:113
doi:10.1186/1743-422X-5-113
Published: 6 October 2008

Abstract (provisional)

Background
The analysis of the nonstructural (NS) gene of the highly pathogenic (HP) H5N1 avian influenza viruses (AIV) isolated in Sweden early 2006 indicated the co-circulation of two sub-lineages of these viruses at that time. In order to complete the information on their genetic features and relation to other HP H5N1 AIVs the seven additional genes of twelve Swedish isolates were amplified in full length, sequenced, and characterized.

Results
The presence of two sub-lineages of HP H5N1 AIVs in Sweden in 2006 was further confirmed by the phylogenetic analysis of approximately the 95% of the genome of twelve isolates that were selected on the base of differences in geographic location, timing and animal species of origin. Ten of the analyzed viruses belonged to sub-clade 2.2.2. and grouped together with German and Danish isolates, while two 2.2.1. sub-clade viruses formed a cluster with isolates of Egyptian, Italian, Slovenian, and Nigerian origin. The revealed amino acid differences between the two sub-groups of Swedish viruses affected the predicted antigenicity of the surface glycoproteins, haemagglutinin and neuraminidase, rather than the nucleoprotein, polymerase basic protein 2, and polymerase acidic protein, the main targets of the cellular immune responses. The distinctive characteristics between members of the two subgroups were identified and described.

Conclusion
The comprehensive genetic characterization of HP H5N1 AIVs isolated in Sweden during the spring of 2006 is reported. Our data support previous findings on the coincidental spread of multiple sub-lineage H5N1 HPAIVs via migrating aquatic birds to large distance from their origin. The detection of 2.2.1. sub-clade viruses in Sweden adds further data regarding their spread in the North of Europe in 2006. The close genetic relationship of Swedish isolates sub-clade 2.2.2. to the contemporary German and Danish isolates supports the proposition of the introduction and spread of a single variant of 2.2.2. sub-clade H5N1 avian influenza viruses in the Baltic region. The presented findings underline the importance of whole genome analysis.
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<cite cite="http://www.virologyj.com/content/5/1/113">Abstract | Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006</cite>

 

[Giuseppe]

Re: Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006 [Virology Journal]

Excerpt:

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Polymerase genes
A single amino acid substitution, from glutamic acid (E) to Lysine (K) in position 627 in PB2 is a determinant of mammalian host range [13, 14].

Most avian isolates have E in this position.

The substitution to K in this position converts a nonlethal H5N1 influenza A virus isolated from a human to a lethal virus in mice [13].

Among the H5N1 HPAIV sequences we investigated a larger proportion of those originating from 1998-2005 had PB2-E627 than more recent isolates.

The 2.2.2.-like Swedish viruses along with the most closely related Danish and German isolates encoded K at this site while the two sub-clade 2.2.1.-like Swedish isolates (A/tufted duck/Sweden/599/06 and A/herring gull/Sweden/1116/06) possessed E at position 627.

The mutations D701N and S714R in PB2 contribute to virulence by enhancing polymerase activity [15]. All Swedish isolates had D and S at position 701 and 714, respectively.

PB1-F2 has been identified as a proapoptotic mitochondrial protein expressed from a second open reading frame of the PB1 gene [16] and it has been shown to contribute to viral pathogenesis in mice [17]. Aspargine in position 66 in PB1-F2 has been demonstrated to play a key role in the pathogenicity of H5N1 viruses [18] and its presence was determined in all
Swedish viruses. Furthermore, Swedish 2.2.1. subclade viruses had a K26Q substitution compared to 2.2.2. subclade viruses. Isolate A/tufted duck/Sweden/599/06 solely contained a T323I and a H562P, while A/herring gull/Sweden/1116/06 a V719M substitution, respectively.
The H5N1 viral polymerase activity is enhanced by the presence of PB2 701N and 714R, PB1 13P, PA 615N, further, NP 319K and 678N [15]. Among the Swedish isolates the presence of PB1 13P was determined.
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[Giuseppe]

Re: Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006 [Virology Journal]

(...)
Surface glycoprotein genes
The HA sequences of isolates A/Mute swan/Sweden/827/06, A/Canada goose/Sweden/978/06, and A/peregrine/Denmark/6632/06 proved to be identical.

The amino acid sequence flanking the cleavage site of the HA gene was PQGERRRKKRGLF alike all other 2.2. viruses with the exception of some French isolates that had the PQGERKRKKR/G sequence [11].

The identified amino acid markers of H5N1 influenza viruses isolated at Qinghai and Poyang Lakes from migratory birds (HA-I99, HA-N268, and NA-R110) were present in all Swedish isolates as well [11].

No ?sub-clade?-specific amino acid changes were identified in the HA among the two subgroups of Swedish isolates.

All the Swedish isolates had the 238Q and 240G (numbered from the H5 start codon) which indicates preferred receptor specificity for the avian alpha-(2,3) linkage to galactose [19, 20].

All HA sequences contained 6 N-linked potential glycosylation sites, as analysed with NetNglyc server (threshold: 0.5) at the following positions: 27, 39, 181, 302, 500, 559; none of them is adjacent to the cleavage site.

Furthermore, the substitutions S145L and A172T, which are associated with viral adaptation to poultry [21] were not determined in association with the Swedish H5N1 viruses.

The amino acid substitutions R178I and I248V in HA that were found in the domestic birds of the Danish isolates [22] were not present in any of the Swedish viruses, nor the V73I substitution that was found in the Danish swan isolates.

However, the D387N substitution found in the German and most of the Danish isolates was also present in the Swedish isolates.

The H5N1 virus isolated from a mink (A/Sweden/mink/2006/V907) was examined in order to reveal any possible adaptation towards mammals.

As a result, a unique E513G substitution was found in the HA gene but no substitutions that could be regarded as host-related were found, which is consistent with previous findings, i.e. that a single passage in mammals is not necessarily associated with changes in receptor-binding sites [9].

As in the other 2.2. viruses, NA-R110 was present in the Swedish isolates, and a 20 amino acid deletion was also found at positions 49-68 similarly to the majority of the recent H5N1 strains [22]. The N228S substitution was present only in A/Herring gull/1116/06 Swedish 2.2.1. virus (alike with several other member of the sub-clade) and not in A/Tufted duck/Sweden/599/06 isolate. These two isolates differed further in amino acid residues 414 and 434 by bearing N/K and S/G corresponding to A/Herring gull/1116/06 and A/Tufted duck/Sweden/599/06 viruses, respectively.

Interestingly, while the Danish and German isolates shared unique amino acids in the NA (G336D), PB1 (K531R) and NS2 (G63E) proteins the swedish isolates were not homogenous in this regard: although NA-G336D was a characteristic of the Swedish viruses too, two isolates retained the PB1-531K, and NS2-63G.

Reported substitutions in NA, inducing oseltamivir resistance [9], were not found in the Swedish isolates.
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[Giuseppe]

Re: Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006 [Virology Journal]

(...)
Concerning the M2 gene, all Swedish viruses contained the L26-
V27-A30-S31-G34 amino acid pattern, thus, no adamantan drug resistant variant was revealed [9].

Substitutions S64A and E66A that were present in the M2 genes of H5N1 AIV isolates from Hong Kong [11] did not appear in Swedish viruses.

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