ISSN 0012-4966, Doklady Biological Sciences, 2006, Vol. 406, pp. 6365. Pleiades Publishing, Inc., 2006.Original Russian Text G.G. Onishchenko, A.M. Shestopalov, V.A. Ternovoi, V.A. Evseenko, A.G. Durymanov, Yu.N. Rassadkin, Yu.V. Razumova, A.V. Zaikovskaya, S.I. Zolotykh,S.V. Netesov, L.S. Sandakhchiev, 2006, published in Doklady Akademii Nauk, 2006, Vol. 406, No. 2, pp. 278279.
The genus of A-type influenza virus that belongs toOrthomyxoviridae family is subdivided onto subtypesbased on the antigenic properties of the virion surfaceglycoproteins, hemagglutinin (HA) and neuraminidase(NA). The viruses of three hemagglutinin (HA) sub-types (H1, H2, H3) and of two neuraminidase (NA)subtypes (N1, N2) are known to circulate in humanpopulations;in birds, 15 HA subtypes (H1H15) and 9NA ones (N1N9) were identified [3, 4].
For the first time, the H5N1 serotype of influenzavirus attracted much attention during an epidemic ofhighly pathogenic poultry influenza in Hong Kong in1997, when six out of 18 patients infected with theseviruses died . To stop the outbreak of this disease, theentire poultry stock in Hong Kong has been eliminated,which stopped the epidemic and made it possible to getrid of the causative agent until 2003. In 20032004, thepathogenic H5N1 viruses repeatedly caused an unprec-edented large-scale outbreak of influenza among poul-try, which involved several countries of Southeast Asia.Fatal cases among people infected with these viruseswere also recorded. During the outbreaks in 1997 and20032004, humans were infected with influenzaviruses because of direct passing the virus from birds,and no cases of virus transmission from human tohuman were recorded, although sometimes severalmembers of the same family were infected. However,genetically changed viruses may appear, capable ofpassing from human to human, which, in turn, mayresult in a pandemic with a high death rate [5, 6].
In May and June 2005, at least a thousand of wildbirds that nested in Lake Qinghai in northwesternChina perished because of the influenza viruses of AH5N1 subtype that are closely related to the Asian vari-ants of 20032004 [7, 10]. This outbreak showed that
the highly pathogenic Asian variants of H5N1 influenzaviruses are still circulating among birds.
In addition, the outbreaks of avian influenza wererecorded in Japan in 20032004 .
Thus, an urgent task of virology is the control overthe circulation and geographical spread of H5N1 influ-enza virus.
In this study, we present the data on the first casewhen the H5N1 influenza virus that is highly patho-genic for birds and genetically related to recent (20032005) South Asian variants has been identified on theterritory of Russia. The fact that this pathogen has beenrevealed in new regions remote from countries ofSoutheast Asia testifies to the increasing hazard of anew pandemic of influenza virus.
In July 2005, large-scale loss of poultry wasrecorded in the Suzdalka village (Novosibirsk oblast).The H5N1 influenza viruses were isolated from thedead bird organs: the strain A/Turkey/Suzdalka/Nov-1/05, from turkey spleen; A/Chicken/Suzdalka/Nov-11/05 and A/Chicken/Suzdalka/Nov-12/05 strains,from chicken kidney. The viruses were isolated ongrowing chicken embryos (GCE) and identified by theconventional methods as described previously .The HA genes of the above isolates were partiallysequenced. For this purpose, viral RNA was isolatedfrom the allantoic fluid of infected GCE and cDNA wassynthetized using Uni-12 primer complementary to theterminal fragments of all eight segments of viral RNA. A region of the HA gene was amplified andsequenced using the forward H5-1 GCC ATT CCACAA CAT ACA CCC  and reverse H5-1685 AGACCA GCT ACC ATG ATT GC  primers. The result-ant nucleotide sequences were deposited in Genbank as
DQ231240, DQ231241, and DQ231242
Phylogenetic analysis of HA genes showed that thestrains that we have isolated contribute to a cluster(index of supporting = 99) that is constituted by H5N1strains isolated from birds during the outbreak of avian
Highly Pathogenic Influenza Virus H5N1 Found in Western Siberia is Genetically Related to Viruses
that Circulated in Southeast Asia in 20032005
Member of the RAMS
G. G. Onishchenko, A. M. Shestopalov, V. A. Ternovoi, V. A. Evseenko, A. G. Durymanov, Yu. N. Rassadkin, Yu. V. Razumova, A. V. Zaikovskaya,
S. I. Zolotykh,
Corresponding Member of the RAS
S. V. Netesov, and
L. S. Sandakhchiev
Received August 31, 2004
Vector State Research Center of Virology and Biotechnology, Koltsovo, Novosibirsk oblast, 630559 Russia
DOKLADY BIOLOGICAL SCIENCES
influenza on Qinghai Lake in China in 2005 , aswell as by the strains isolated in Japan in 2004  andby H5N1 strain from people and birds isolated in coun-tries of Southeast Asia during the influenza outbreak in20032004  (figure).
To determine the nucleotide sequence of the NAgene of the A/Turkey/Suzdalka/Nov-1/05 strain, theviral RNA was isolated from a homogenate of turkeyspleen. RNA isolation and cDNA synthesis were per-formed as described above for HA gene. Polymerasechain reaction and sequencing were performed usingthe calculated primers specific for the NA gene of N1subtype. The resultant nucleotide sequence of the NAgene (547 bp) was deposited in Genbank as
When compared with the other sequences published inGenbank, the fragment obtained by us proved to be100% homologous with similar fragments of the NAgenes of H5N1 strains isolated in China on QinghaiLake in 2005 (A/great black-headed gull/Qing-hai/1/2005,
. When our fragment was comparedwith the structure of the NA gene of the strain isolatedfrom a crow in Japan (2004), several individual substi-tutions were found.
Hemagglutinin amino acid sequence cleavable byproteolytic enzymes is known to be one of the mostimportant pathogenicity markers of H5N1 influenza Aviruses. Because of this cleavage, the virus becomesinfectious. In isolates that we have obtained, thissequence, ERRRKKR/GL, corresponded to that ofhighly pathogenic strains circulating in Southeast Asiacountries. In addition, the pathogenicity of the isolatedvirus was tested on chickens. For this purpose, ten six-
week-old chickens were infected by intravenous injec-tion of 0.1 ml of virus-containing allantoic fluid diluted1 : 10 by the standard procedure . All birds diedwithin 36 h after infection, which testifies to the factthat highly pathogenic avian strains (HPAI) were iso-lated.
Thus, our study has shown that the highly patho-genic H5N1 influenza viruses genetically related to theAsian variants of 20032005 were circulating on theterritory of West Siberia in summer of 2005. This testi-fies to the fact that the spread of highly pathogenicAsian variants of H5N1 viruses over the new regionsstill continues.
Until recently, it was assumed that the wide geo-graphical spread of these viruses is caused by anthropo-genic activity, transporting the infected poultry, meat,and virus-contaminated objects. However, H5N1viruses on lake Qinghai were most likely carried bymigrant birds . The outbreak of influenza amongpoultry in Novosibirsk oblast seems to be also a resultof H5N1 virus carrying by migrant birds because thisregion is on the way of spring flyby of the birds thatmigrate from Southeast Asia countries [2, 14], whereasno poultry meat export from China to Russia wererecorded. This implies that the hazard of further geo-graphic spread of these viruses by migrant birds is stillan urgent problem, because a significant proportion ofnesting birds and those flying by from the foreststeppe ofWest Siberia migrate in autumn in southern-west directionto Mediterranean, Black, and Caspian Seas .
Note that the outbreak of avian influenza on the ter-ritory of West Siberia and near-Ural region in 2005 has
A/Bar-headed Goose/Qinghai/67/2005A/Black-headed gull/Qinghai/1/2005A/Great black-headed gull/Qinghai/1/2005A/Chicken/Shantou/4231/2003A/Crow/Osaka/102/2004A/Chicken/Indonesia/BL/2003A/Chicken/Suzdalka/Nov-12/2005A/Turkey/Suzdalka/Nov-1/2005
A/Goose/Thailand/79/2004A/Viet Nam/1194/2004A/Hong Kong/156/97
A/Anas platyrhynchos/Chany Lake/9/2003A/Mallard/64650/2003
A/Tern South Africa/61
A/Duck/New York/44018-2/2000A/Mallard Duck/Pensylvania/10218/84A/Turkey/Ontario/7732/66
The phylogenetic tree of H5N1 genes of influenza A viruses. The strains isolated in this study are underlined. In phylogeneic anal-ysis, a 660-bp fragment of hemagglutinin gene was used. The phylogenetic tree has been constructed using the neighbor joiningalgorithm and MEGA2 software.