3
ISSN 0012-4966, Doklady Biological Sciences, 2006, Vol. 406, pp. 63–65. © 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. 278–279. 63 The genus of A-type influenza virus that belongs to Orthomyxoviridae family is subdivided onto subtypes based on the antigenic properties of the virion surface glycoproteins, 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 human populations;in birds, 15 HA subtypes (H1–H15) and 9 NA ones (N1–N9) were identified [3, 4]. For the first time, the H5N1 serotype of influenza virus attracted much attention during an epidemic of highly pathogenic poultry influenza in Hong Kong in 1997, when six out of 18 patients infected with these viruses died [5]. To stop the outbreak of this disease, the entire poultry stock in Hong Kong has been eliminated, which stopped the epidemic and made it possible to get rid of the causative agent until 2003. In 2003–2004, the pathogenic 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 viruses were also recorded. During the outbreaks in 1997 and 2003–2004, humans were infected with influenza viruses because of direct passing the virus from birds, and no cases of virus transmission from human to human were recorded, although sometimes several members of the same family were infected. However, genetically changed viruses may appear, capable of passing from human to human, which, in turn, may result in a pandemic with a high death rate [5, 6]. In May and June 2005, at least a thousand of wild birds that nested in Lake Qinghai in northwestern China perished because of the influenza viruses of A H5N1 subtype that are closely related to the Asian vari- ants of 2003–2004 [7, 10]. This outbreak showed that the highly pathogenic Asian variants of H5N1 influenza viruses are still circulating among birds. In addition, the outbreaks of avian influenza were recorded in Japan in 2003–2004 [8]. Thus, an urgent task of virology is the control over the circulation and geographical spread of H5N1 influ- enza virus. In this study, we present the data on the first case when the H5N1 influenza virus that is highly patho- genic for birds and genetically related to recent (2003– 2005) South Asian variants has been identified on the territory of Russia. The fact that this pathogen has been revealed in new regions remote from countries of Southeast Asia testifies to the increasing hazard of a new pandemic of influenza virus. In July 2005, large-scale loss of poultry was recorded in the Suzdalka village (Novosibirsk oblast). The H5N1 influenza viruses were isolated from the dead 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 on growing chicken embryos (GCE) and identified by the conventional methods as described previously [11]. The HA genes of the above isolates were partially sequenced. For this purpose, viral RNA was isolated from the allantoic fluid of infected GCE and cDNA was synthetized using Uni-12 primer complementary to the terminal fragments of all eight segments of viral RNA [11]. A region of the HA gene was amplified and sequenced using the forward H5-1 GCC ATT CCA CAA CAT ACA CCC [12] and reverse H5-1685 AGA CCA GCT ACC ATG ATT GC [13] primers. The result- ant nucleotide sequences were deposited in Genbank as DQ231240, DQ231241, and DQ231242. Phylogenetic analysis of HA genes showed that the strains that we have isolated contribute to a cluster (index of supporting = 99) that is constituted by H5N1 strains 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 2003–2005 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 Academician L. S. Sandakhchiev Received August 31, 2004 DOI: 10.1134/S0012496606010170 Vector State Research Center of Virology and Biotechnology, Kol’tsovo, Novosibirsk oblast, 630559 Russia GENERAL BIOLOGY

Highly pathogenic influenza virus H5N1 found in Western Siberia is genetically related to viruses that circulated in Southeast Asia in 2003–2005

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Page 1: Highly pathogenic influenza virus H5N1 found in Western Siberia is genetically related to viruses that circulated in Southeast Asia in 2003–2005

ISSN 0012-4966, Doklady Biological Sciences, 2006, Vol. 406, pp. 63–65. © 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. 278–279.

63

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 (H1–H15) and 9NA ones (N1–N9) 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 [5]. 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 2003–2004, 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 and2003–2004, 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 2003–2004 [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 2003–2004 [8].

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 (2003–2005) 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 [11].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[11]. A region of the HA gene was amplified andsequenced using the forward H5-1 GCC ATT CCACAA CAT ACA CCC [12] and reverse H5-1685 AGACCA GCT ACC ATG ATT GC [13] primers. The result-ant nucleotide sequences were deposited in Genbank as

DQ231240, DQ231241, and DQ231242

.Phylogenetic analysis of HA genes showed that the

strains 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 2003–2005

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

Academician

L. S. Sandakhchiev

Received August 31, 2004

DOI:

10.1134/S0012496606010170

Vector State Research Center of Virology and Biotechnology, Kol’tsovo, Novosibirsk oblast, 630559 Russia

GENERALBIOLOGY

Page 2: Highly pathogenic influenza virus H5N1 found in Western Siberia is genetically related to viruses that circulated in Southeast Asia in 2003–2005

64

DOKLADY BIOLOGICAL SCIENCES

Vol. 406

2006

ONISHCHENKO

et al

.

influenza on Qinghai Lake in China in 2005 [10], aswell as by the strains isolated in Japan in 2004 [8] andby H5N1 strain from people and birds isolated in coun-tries of Southeast Asia during the influenza outbreak in2003–2004 [6] (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

DQ231243

.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,

etc.)

[7]. 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 [11]. 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 2003–2005 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 [7]. 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 forest–steppe ofWest Siberia migrate in autumn in southern-west directionto Mediterranean, Black, and Caspian Seas [1].

Note that the outbreak of avian influenza on the ter-ritory of West Siberia and near-Ural region in 2005 has

0.02

8868

47

33

49

75 7075

9993

64

929846

5571

5697

9794

10068

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/Duck/Anyang/AVL-1/2001

A/Environment/Hong Kong/437-6/99

A/Anas platyrhynchos/Chany Lake/9/2003A/Mallard/64650/2003

A/Tern South Africa/61

35

A/Chicken/Suzdalka/Nov-11/2005

A/Chicken/Hong Kong/220/97

A/Goose/Guang Dong/1/96

A/Goose/Hong Kong/385.3/2000A/Duck/Potsdam/1402-6/86A/Chicken/Italy/312/97

A/Mallard/Netherlands/3/99

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.

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HIGHLY PATHOGENIC INFLUENZA VIRUS H5N1 FOUND IN WESTERN SIBERIA 65

already resulted in a material damage of several millionrubles (data on August 15), which is still increasing.Because of this, the monitoring for circulation of avianinfluenza virus is required to alarm early the authoritiesand be ready to the outbreaks. It is also extremelyimportant to estimate the potential pathogenicity of thecirculating viruses for people and poultry to performadequate prophylactic control.

ACKNOWLEDGMENTSThe authors are grateful to collaborators who have

helped in this study, especially to G.F. Lazikova,V.A. Gergert, V.N. Mikheev, O.P. Krashennikov, andDr. Robert G. Webster.

This study was supported by the Reserve Founda-tion of the Governor of Novosibirsk oblast and by aCRDF (project no. RUX2-20411-NO-2004).

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