ISSN 0012-4966, Doklady Biological Sciences, 2007, Vol. 414, pp. 226–230. © Pleiades Publishing, Ltd., 2007.Original Russian Text © V.A. Evseenko, A.V. Zaykovskaya, V.A. Ternovoi, A.G. Durimanov, S.I. Zolotykh, Yu.N. Rassadkin, A.S. Lipatov, R.G. Webster, A.M. Shestopalov,S.V. Netesov, I.G. Drosdov, G.G. Onishchenko, 2007, published in Doklady Akademii Nauk, 2007, Vol. 414, No. 4, pp. 561–565.

226

INTRODUCTION

Highly pathogenic avian influenza (HPAI) type Aviruses with the H5 subtype of hemagglutinin (HA)were first isolated form a hen in 1959 in Scotland.Later, avian influenza viruses of this subtype were iso-lated in South Africa in 1961; in the North Americancontinent in 1975, 1981, and 1983; and in England andScotland in 1991 [1]. The H5 subtype varieties of HPAIvirus that are the source of the HA gene of currently cir-culating H5N1 HPAI virus were first isolated from agoose in 1996 and from a human that died in 1997 inHong Kong [2, 3]. By 2006, the H5N1 HPAI viruseswere already widespread and became endemic inSoutheast Asia. These viruses affected mostly poultry;however, humans were sometimes also infected. Thepatients infected with H5N1 HPAI virus have severeforms of primary virus pneumonia accompanied byfever, and the disease was fatal for 70% of infectedpatients [4]. Beginning from early 2003, the H5N1-sub-type viruses have spreading throughout Southeast Asia.In 2005–2006, the geographical area of the highlypathogenic H5N1 viruses extended significantly: epi-zootics among wild birds and poultry were recorded inMongolia, Kazakhstan, Russia, Ukraine, the EU andMediterranean countries, the Middle East, and Africa[5]. In 2006, cases of human disease with high mortal-ity were recorded in China, Indonesia, Turkey, Azer-baijan, some African countries, and Iraq [5]. The virusexpansion over long distances was promoted by asymp-tomatic infection in the wild migrant birds and resis-tance of some individuals in the wild and domesticduck populations [6].

Wild migrant birds with asymptomatic chronicinfection migrated mostly along the Western Siberianflyway where they either nested on the lakes and riversof the Barabo-Kulundinskaya plain or rested at thisplace before flying to the north of Russia [7]. Domesticwater birds are traditionally brought for feeding on thesame lakes where wild birds nest; therefore, poultryinfection was most probable here.

An epizootic of the H5N1 HPAI in Western Siberiawas first recorded in mid-July of 2005 around theSuzdalka village of Dovolenskii raion, Novosibirskoblast (NSO). The village is on the shore of a lakewhere domestic water birds are kept in summer. Closecontact between waterfowl and domestic ducks andgeese and afterwards between the latter and hens andturkeys, which are the most sensitive animals [8],finally resulted in cross-infection and high mortalityamong poultry.

Later, devastating loss of domestic poultry occurredin other settlements of Novosibirsk and Omsk oblasts;afterwards, avian influenza caused by H5N1 virus wasestablished to be the cause of poultry mortality. It hasalso been found that in all settlements where the poultryloss was observed the poultry was in contact with wildbirds. Because of this, it was suggested that wild birdsplayed the decisive role in the distribution of the H5N1HPAI virus in Russia [9, 10]. Since wild birds winter invarious geographically remote regions of Asia, thequestion arose about the degree of similarity betweenthe Southern Asian and Siberian virus isolates, as wellas the degree of heterogeneity and pathogenicity of thelatter for birds and mammals. Note that, during theperiod of avian influenza virus epizootic, we isolated62 varieties of the H5N1 HPAI virus.

The subject of our study was representatives of iso-lates of various groups from the remotest settlements,as well as isolates obtained in different time from vari-ous avian species.

The goal of this study was to evaluate the diversityof isolates of the HPAI H5N1 virus, the causative agentof the 2005 epizootic in Southern Siberia.

Diversity of Highly Pathogenic Avian Influenza H5N1 Viruses That Caused Epizootic in Western Siberia in 2005

V. A. Evseenko

a

, A. V. Zaykovskaya

a

, V. A. Ternovoi

a

, A. G. Durimanov

a

, S. I. Zolotykh

a

, Yu. N. Rassadkin

a

, A. S. Lipatov

b

, R. G. Webster

b

, A. M. Shestopalov

a

,

Corresponding Member of the RAS

S. V. Netesov

a

, I. G. Drosdov, and G. G. Onishchenko

c

Received April 5, 2006

DOI:

10.1134/S0012496607030179

a

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

b

St. Jude Children’s Research Hospital, 332 North Lauderdale St., Memphis, TN 38105-2794, United States

c

Federal Service for Surveillance in the Sphere of Consumer Rights and Human Well-being

GENERAL BIOLOGY

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DIVERSITY OF HIGHLY PATHOGENIC AVIAN INFLUENZA H5N1 VIRUSES 227

MATERIALS AND METHODS

H5N1 HPAI viruses were isolated in the vicinities ofNovosibirsk oblast settlements (Suzdalka, Dovol’noye,Krasnozerskoe, Chany) either from lungs or from cloa-cal lavage fluid of dead and ill birds, respectively. Sam-ples from Omsk oblast (Omsk and Cherlak raions) werealso examined. Virus isolation and their serologic andgenetic analyses were performed according to [9]. Thenucleotide sequences determined were deposited inGenBank under accession numbers DQ676838,DQ231240, and DQ676840. The virus pathogenicityfor mice and ferrets was determined by the methodsused in [14].

RESULTS AND DISCUSSION

The results of the antigenic analysis of the isolatesare shown in Table 1. On the basis of these data, all thestrains studied were divided into three groups. Theantigens of viruses A/mallard/Chany/222/05 andA/Coot/Chany/204/05 displayed no affinity to the seraobtained against viruses circulating in Southeast Asia,and the pathogenicity biological tests showed that theseviruses belonged to nonpathogenic strains of the H5serotype circulating in Western Siberia and the Far East[11] (the negative results of biological tests and moleculargenetic analysis are not shown). The isolates under-lined in Table 1 displayed cross-reactivity with thesera against H5N1 HPAI viruses from Southeast Asiancountries. The subjects of our subsequent study wereA/turkey/Suzdalka/Nov-01/05 and A/chicken/Dovol-noye/03/05 isolates with different antigenic propertiesand A/goose/Krasnoozerska/627/05 isolate obtained inautumn 2005.

Phylogenetic analysis of hemagglutinin genes fromisolates of the first two groups showed that theybelonged to the same cluster. The nucleotide sequencesof hemagglutinin genes from the HPAI H5N1 virus pre-viously isolated in central China (Lake Quinghai) andMongolia [12] were also classified with this cluster. Itwas more reasonable to assign the A/goose/Krasnooz-erska/627/05 strain to a separate branch (Fig. 1).

Analysis of the hemagglutinin amino acid sequenceshowed that all highly pathogenic viruses studies con-tained the incision site PQGERRRKKR/GL inherent inthe H5N1 HPAI virus [4].

Our results suggest that several phylogeneticallyrelated but not identical viruses were brought into theRussian Federation and different phenotypic propertiesof these viruses should be studied.

According to the results of antigenic and phylogeneticanalyses, the strains A/turkey/Suzdalka/Nov-01/05,A/chicken/Dovolnoye/03/05, and A/Goose/Krasnoozer-skoye/627/05 are members of three different groups;their pathogenicity for animals has been studied onchickens, mice, and ferrets. The first clinical signs ofdisease and the start of chicken mortality wereobserved on the second day after infection. The IVPIindex for the viruses A/turkey/Suzdalka/Nov-01/05 andA/goose/Krasnoozerska/627/05 was 3.0, which is themaximum value.

The onset of mouse death was recorded on the thirdday and depended on the virus dose. Autopsy showedlung fever, enlarged spleen with hemorrhages, edema-tous and strongly enlarged liver, an enlarged kidneywith an indistinguishable boundary between the coreand medullar layers. Table 2 shows virus distribution inorgans and both lethal and infection doses of the patho-

Table 1.

Antigenic analysis of the isolates obtained

Isolate

Monoclonal ABs Sera

CP

58

CP4

6

CP2

4

CP

406/

7

HK

03-

3

VN

04

Ck/

Hid

alg/

95

Gs/

HK

/99

HK

/156

/97

HK

/213

/03

VN

/120

3/04

Prac

hinb

rr/0

4

Tk/

Suzd

alka

/05

Tk/Suzdalka/Nov-01/05 <100 <100 100 300 <100 <100 80 160 10 160 160 20 20

Ck/Suzdalka/2/05 <100 <100 <100 200 <100 <100 160 320 <10 80 40 10 ND

Gs/Suzdalka/6/05 100 100 200 100 100 <100 160 320 10 80 80 20 ND

Ck/Omsk/108/05 <100 100 100 400 100 <100 80 160 10 160 80 20 ND

Mallard/Omsk/105/05 <100 <100 <100 400 <100 <100 160 160 10 160 80 20 ND

Chicken/Dovolnoye/5/05 <100 400 100 1600 400 <100 10 10 10 640 320 10 <10

Mallard/Chany/222/05 <100 <100 <100 <100 <100 <100 640 <10 <10 <10 <10 <10 <10

Coot/Chany/204/05 <100 <100 <100 <100 <100 <100 640 <10 <10 <10 <10 <10 ND

Gs/Krasnoozerk/627/05 <100 <100 <100 200 <100 <100 160 160 <10 80 80 10 80

Note: Dilutions of sera and monoclonal antibodies that inhibit 4GAE virus agglutination are indicated. Isolate designations are abbrevi-ated. The monoclonal antibodies used have been previously described in [15]. ND, no data.

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gen. Thus, A/goose/Krasnoozerska/627/05 virus washighly pathogenic and infectious for mice. Unlike otherstrains studied, this strain seems to be extremely infec-tious also in other mammals, because cell membranesin mice and other mammals bear similar influenza virusreceptors [13]. Further detailed analysis of the molecu-lar mechanisms underlying the influenza virus pathoge-nicity for other mammals made it possible to extrapo-late our results to other animals and humans.

Note that the isolate A/turkey/Suzdalka/Nov-01/05was low-infectious and low-pathogenic for mice. Theisolate A/chicken/Dovolnoye/03/05 was not infectiousfor mice at all.

The effects of viruses A/goose/krasnoozerska/627/05and A/turkey/Suzdalka/Nov-01/05, which are the mostpathogenic for mice, were also studied in experimentson ferrets. They were intranasally infected with 6 LG

33

46

6625

17

880

9573

100

96

99

5084

100

78

9897

100

15

29

94482026

2

7

10

21

92

0.005

100

A/chicken/Crimea/01/2005

A/swan/Italy/179/06

A/turkey/Suzdalka/Nov-01/05

A/chicken/Dovolnoye/03/05

A/cygnus olor/Astrakhan/Ast05-2-2/2005

A/whooper swan/Mongolia/6/05

A/chicken/Kurgan/3/2005

A/chicken/Nigeria/641/2006

A/grebe/Novosibirsk/29/2005

A/Bar-headed Goose/Qinghai/5/05

A/goose/Krasnoozerska/627/05

Viruses isolated in Japan and Korea in 2003–2004

A/chicken/Guang xi/12/2004

A/chicken/Indonesia/2A/2003

A/chicken/Indonesia/4/2004

A/Hong Kong/213/03

A/Thailand/16/2004

A/chicken/Indonesia/7/2003

A/chicken/Vietnam/NCVD10/2005

A/Hanoi/30408/2005

A/goose/Cambodia/28/2004

A/Viet Nam/1203/2004

A/Cambodia/JP52a/2005

A/chicken/Cambodia/022LC3b/2005

A/chicken/Malaysia/5858/2004

A/Hatay/2004

A/Prachinburi/6231/2004A/chicken/Bangkok/Thailand/CU-3/04

A/chicken/Laos/7191/2004

A/chicken/Thailand/Kamphaengphet/3-02/05

A/chicken/Hubei/327/2004

Viruses isolated in Southeast Asia before 2004

Fig. 1.

The phylogenetic tree of the genes encoding HA1 subunits of Asian genotype H5 hemagglutinin. The viruses causing epi-zootics in Russia and phylogenetically related isolates are indicated in braces. The values at the bases of branches are bootstrapindices.

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EID

50

(the 50% infectious dose for growing chickenembryos). Beginning from the first day after infection,the body temperature was measured daily, the generalstate was recorded, and nasopharyngeal lavages weremade. Diseased ferrets displayed anorexia, hyperther-mia, tremor, coughing, sneezing, and short-term diar-rhea; their feces changed color from white to light-yel-low, which suggests liver damage. Figure 2 shows thechanges in the amount of virus in the nasopharyngeallavages. The A/turkey/Suzdalka/Nov-01/05 virus wasdetected from the first to sixth day, the titers changingfrom 2.4 to 0.7 Lg EID

50

. Discharge of theA/goose/Krasnoozerska/627/05 virus was recordedfrom the first to the eleventh day; the titer ranged from6 to 1.75 Lg EID

50

. These results show a high level of theisolate replication; the A/goose/Krasnoozerska/627/05

virus was discharged in significant amounts with thesputum.

Thus, a series of phylogenetically related but notphenotypically identical isolates were obtained duringepizootics of the H5N1 HPAI virus in Siberia in sum-mer 2005. These isolates showed a significant serolog-ical cross-reactivity with the viruses circulating inSoutheast Asia. At the same time, our study suggestssignificant differences between the pathogenic proper-ties of the isolates studied.

Our results show clearly that the isolates highlypathogenic for mammals are already present amongavian influenza viruses brought to Russia. This sug-gests that the potential of H5 influenza viruses is suffi-cient for their evolutionary drift towards formation of avirus epidemic for people, which is only a matter oftime and probability.

The results obtained also suggest that during recur-rent epizootics and epidemics caused by the influenzavirus in Russia, samples should be taken in several tensof epizootic localities to be studied individually. Other-wise, we may overlook the moment when the viruspathogenic for humans is formed in wild bird or animalpopulations and, therefore it, will be difficult to preventhuman disease.

This study was supported by the Reserve Founda-tion of Governor of the Novosibirsk oblast, project Bio-industry Initiative Gap no. RUB2-20440-NO-06.

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Table 2.

Virus virulence for mice

Virus EID

50

/ml MID

50

MLD

50

Organ

lung spleen brain liver kidney

A/Gs/Krasnoozerska/627/05 9.2 2.2 2.3 6.1 1.6 5.2 1.6 2.6

A/Tk/Suzdalka/Nov-01/05 9.3 5.3 6.3 4.1 < 2.3 <1 <1

A/VN/1204* 9.8 2.3 3.8 6.9 3.4 2.2 <1 <1

A/Ck/Indonesia/05* 9.3 5.3 >7 4.3 <1 <1 <1 <1

A/rh/Dovolnoye/05 8.7 IS IS IS IS IS IS IS

Note: Data are indicated in Lg EID

50

. Reference data marked with asterisk are from [14]. 100 mg of each organ was analyzed. IS, insensitive.

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1

Log

EID

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/

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Days

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A/VN/1204

A/Ck/Indonesia/05

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Fig. 2.

Virus titers in nasopharyngeal lavage fluid. The timedependence of virus titers in lavage fluid of ferrets is plot-ted. The data on virus strains (the dashed lines) are takenform [14]. Each virus was studied in two animals; the aver-age values are shown.

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