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strains between human and animal inuenza viruses, as well asemergence of local outbreaks of human morbidity caused byuncommon variants of inuenza viruses.
In view of the above facts, it seems obvious that it is necessaryto carry out widespread study of circulating viruses, their molecu-lar biological properties, and phylogenetic links in Western Siberia.
2. Materials and methods
Work with inuenza A/H1N1, A/H1N1pdm, and A/H3N2 wascarried out in BSL-2 virological laboratories. All studies of inuenzaA/H5N1 viruses were conducted in BSL-3 lab. The use of clinicalsamples (nasopharyngeal swabs, autopsy material, blood sera)
Corresponding author at: Novosibirsk State University, Pirogov St., 2, Novosi-birsk 630090, Russia. Tel.: +7 383 3367540; fax: +7 383 3367540.
Infection, Genetics and Evolution 20 (2013) 177187
Contents lists availab
.e lsE-mail addresses: firstname.lastname@example.org, email@example.com (T. Ilyicheva).structure and relatively high (for Siberia) population density.Therefore, there is high probability of emergence of reassortant
epidemiologic monitoring of inuenza in humWestern Siberia in 20082012.1567-1348/$ - see front matter 2013 Published by Elsevier B.V.http://dx.doi.org/10.1016/j.meegid.2013.08.025leculartion ofout in nesting periods, and vast station where even more amountof birds stop during their migration and nest in boreal coniferousforests and Arctic prairie. Territorial relations of Western Siberianbirds that are established during seasonal migration are extremelywide since this region is an intersection point of bird migrationows wintering in different regions of the world: Europe, Africa,Middle East, Central Asia, Hindustan, and South East Asia. Further-more, the South of Western Siberia is a region with extended infra-
subtype, previously having isolated only in the Southernhemisphere (Marchenko et al., 2012). Additionally, we showed thatseveral Western Siberian lakes are of key importance in highlypathogenic inuenza A/H5N1 epizootology. Therefore research ofcirculating inuenza virus strains and herd immunity in WesternSiberia is of great importance, since it enables to detect nontypicalvirus variants that are likely to emerge in this region.
The objective of this work is to analyze results of mo1. Introduction
Western Siberia is of great imporology of inuenza. The South of thisvalleys, ObIrtysh interuves) is replie in migration paths of many birdnesting area for great amount of bnected with water. Forest steppe oboth huge incubator where millin ecology and epidemi-(the Ob and Irtysh riverth rivers and lakes thats. Thus, this territory isecies ecologically con-tern Siberia representsnestlings are brought
We have studied epizootology and ecology of avian inuenzaviruses since 2002. We showed large variety of inuenza A virusesamong different wild bird species (Marchenko et al., 2012;Sharshov et al., 2010; Sivay et al., 2013, 2012). It was revealed thatduring 10 years inuenza A/H3N8 and A/H4N6 virus subtypesprevail among birds (Marchenko et al., 2012; Sivay et al., 2012).Genome reassortation was detected in a number of inuenzaviruses (Marchenko et al., 2012; Sivay et al., 2013). Interestingly,we isolated in Western Siberia reassortant inuenza H15N4 virusMolecular epidemiologic monitoringWestern Siberia
2013 Published by Elsevier B.V.Monitoring of inuenza viruses in Weste
T. Ilyicheva a,b,, I. Sobolev a,b, I. Susloparov b, O. KursA. Shestopalov b
aNovosibirsk State University, Pirogov St., 2, Novosibirsk 630090, Russiab State Research Center of Virology and Biotechnology Vector, Koltsovo, Novosibirsk 63
a r t i c l e i n f o
Article history:Received 16 July 2013Received in revised form 23 August 2013Accepted 27 August 2013Available online 5 September 2013
Keywords:Human anti-H5 antibodiesInuenza virus
a b s t r a c t
Western Siberia is of greatarea for great amount of bduring seasonal migrationows wintering in differenSouth East Asia. ReassortanWestern Siberia with highcirculated viruses, their moimmunity to inuenza viru
journal homepage: wwwSiberia in 20082012
ya a,b, A. Durymanov a,b, K. Sharshov a,b,
ortance in ecology and epidemiology of inuenza. This territory is nestingpecies. Territorial relations of Western Siberian birds that are establishedextremely wide since this region is an intersection point of bird migrationions of the world: Europe, Africa, Middle East, Central Asia, Hindustan, anduenza viruses that can cause outbreak among population may emerge inbability. Thus, it is extremely important to carry out widespread study ofular biological properties, phylogenetic links in this region, as well as herderotypes with epidemic potential.le at ScienceDirect
s and Evolution
evier .com/locate /meegid
was approved by Ethics Committee IRB 00001360 (protocol #2 d.d.20.05.2008).
Nasopharyngeal swabs and autopsy material were collected inhospitals of Novosibirsk, Tomsk, Omsk, Krasnoyarsk, Barnaul, andin hospitals from several towns of Novosibirsk region: Dovolnoe,Zdvinsk, Karasuk (230, 280, and 400 km to the SouthWest ofNovosibirsk, respectively), and Koltsovo (the nearest suburb). In to-tal 1132 samples were collected. Ten percent of patients were vac-cinated. Google map (Appendix A) demonstrates collection sites.
Virus was isolated from nasopharyngeal swabs collected frompatients with provisional diagnosis of inuenza or from autopsymaterial (10% homogenate in Hanks solution). Homogenate ortransport medium containing clinical material was centrifuged at400g for 10 min and inoculated into culture plates with a mono-layer MDCK cells. Virus reproduction was checked visually on thebase of cytopathic effect in the hemagglutination of goose and hu-man erythrocytes. All nasopharyngeal swabs and isolated inuenzavirus strains were tested in PCR.
Blood sera were collected in towns Dovolnoe, Zdvinsk, Karasuk,Kogalym, Novoagansk, KhantyMansiysk, and Rostov-on-Don. Intotal 2282 blood sera samples were collected.
Sera from participants were tested for antibodies to H5 virus bymicroneutralization assay (MN) and hemagglutinin inhibition test(HI). In test work we used A/Commongull/Chany/06 (HPAI H5N1)(clade 2.2) virus isolated in Western Siberia from dead Common
used as positive control sera for the assays. Human antisera were
erythrocytes as described in (Rowe et al., 1999). Before study alltested sera were treated with RDE (Denka Seiken, Tokyo, Japan)that destroys non-specic inhibitors. After that twofold sera dilu-tions were mixed with four HAU of inactivated by b-propiolactonevirus (Rowe et al., 1999) and incubated for 60 min at room temper-ature. Then equal amount of one percent erythrocytes suspensionwas added in each well of the plate. Sera were considered positiveif the antibody titer was equal to or greater than 40.
2.3. Microneutralization test
Sera were analyzed in microneutralization assay as described in(Rowe et al., 1999). Twofold sera dilutions previously heated for30 min at 56 C were mixed with 100 TCID50/100 ll of virus, keptfor 60 min at 37 C in 5% CO2 atmosphere. After that we added1.5 104 MDCK cells and incubated for 1820 h. Then cells werexed; presence of virus antigen in cells was detected using ELISA.Mice anti-NP monoclonal antibodies (CDC) were used as the rstantibodies, goat antimouse IgG conjugated by horseradish peroxi-dase (Sigma) as the second ones. Sera were considered positive ifthe antibody titer was equal to or greater than 80.
InuenzavirusRNA isolationwascarriedoutwith thekitPROME-GA SV Total RNA Isolation System (Promega Corporation, Madison,WI, USA) in compliancewith themanufacturers recommendations.
To type and subtype inuenza virus strains we used a set of re-agents for the detection of inuenza A and inuenza B virus RNA in
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178 T. Ilyicheva et al. / Infection, Genetics and Evolution 20 (2013) 177187tested at a starting dilution of 1:10.
2.2. Hemagglutination-inhibition test
Presence of antibodies to inuenza A/H5N1 in blood sera wasdetected according to WHO recommendations in HI test with horse
Primers for amplication of HA gene (segment 4)SW-HA-F1 TGTAASW-HA-F351 TGTAASW-HA-F736 TGTAASW-HA-R943 CAGGSW-HA-R1204 CAGGSW-HA-R1340 CAGGSW-HA-R1541 CAGGSW-HA-R1778 CAGGPrimers for amplication of NA gene (segment 6)SW-NA-F0 TGTAASW-NA-F318 TGTAASW-NA-F536 TGTAASW-NA-F941 TGTAASW-NA-R740 CAGGSW-NA-R1063 CAGGSW-NA-R1346 CAGGSW-NA-R1452 CAGGPrimers for amplication of HA gene (segment 4)HA_F_1BM13 TGTAAHA_R_589M13 CAGGHA_F_453BM13 TGTAAHA_R_975M13 CAGGgull European and Asian subspecies of Larus canus (Sharshovet al., 2010).
Sera were also tested for antibodies to human inuenza Aviruses of the H1 and H3 subtypes for control purposes (data notshown). Ferret antisera raised against homologous viruses wereclinical materials using PCR with hybridization-uorescence detec-tion AmpliSens Inuenza virus A/B-FL and the kit for typing (sub-type identication of H1N1 and H3N2) of inuenza A virusesAmpliSens Inuenza virus A-type-FL made by Central researchinstitute of epidemiology of the Ministry of health of Russia (Mos-cow, Russia).
To produce cDNA from inuenza virus RNA matrix reversetranscription was carried out with the kit Fermentas RevertAid(Fermentas International Inc., Vilnius, Lithuania).
To amplify certain gene segments encoding inuenza virussurface glycoproteins PCR with gene-specic primers was used(Ghedin et al., 2005). We used the following primer sequences:
nuenza A/H1N1pdm09CGACGGCCAGTATACGACTAGCAAAAGCAGGGGCGACGGCCAGTACRTGTTACCCWGGRGATTTCACGACGGCCAGTAGRATGRACTATTACTGGACCAGCTATGACCGAAAKGGGAGRCTGGTGTTTACAGCTATGACCTCTTTACCYACTRCTGTGAACAGCTATGACCTTCTKCATTRTAWGTCCAAACAGCTATGACCTCATAAGTYCCATTTYTGACAGCTATGACCGTGTCAGTAGAAACAAGGGTGTTTnuenza A/H1N1pdm09CGACGGCCAGTAGCAAAAGCAGGAGTCGACGGCCAGTTACACAAAAGACAAYAGCCGACGGCCAGTGGTCAGCAAGCGCATGYCATG ACGACGGCCAGTTAGGATACATCTGCAGTGGCAGCTATGACCGGRCCATCGGTCATTATGCAGCTATGACCCATATYTGTATGAAAACCCAGCTATGACCGCTGCTYCCRCTAGTCCAGATCAGCTATGACCAGTAGAAACAAGGAGnuenza A/H3N2CGACGGCCAGTAGCARAAGCAGGGGACAGCTATGACCTTGTTTGGCATRGTCACGTTCCGACGGCCAGTTTCRAYTGGRCTGGRGTCRCCAGCTATGACCTTTTGAAADGGYTTGTCATTGG
ics aAmplication was carried out with DNA Engine Dyad PeltierThermal Cycler (Bio-Rad, USA). In order to extract gene segmentsobtained by PCR which were coding NA and HA of studied inu-enza A/H3N2 virus strains a set of reagents QIAquick Gel ExtractionKit (Qiagen GmbH, Hilden) was used.
Sequence analysis was conducted with BigDye terminator cyclesequencing ready reaction kit (Applied BioSystems, USA). Ampli-cation passed in accordance with the manufacturers recommenda-tions. To purify the product the BigDye XTerminator puricationkit (Applied BioSystems, USA) was used. Automatic sequenator310 Genetic Analyzer (Applied BioSystems, USA) was used for theanalysis of products.
Analysis of nucleotide sequences was carried out with the pro-gram package SeqMan (Lasergene) and specic Inuenza Virus Re-source (Bao et al., 2008) and BLAST (National Center forBiotechnology Information, U.S. National Library of Medicine). Tocarry out multiple sequence alignment and nucleotide sequencetranslation as well as to analyze obtained amino-acid sequenceswe used BioEdit sequence alignment editor.
Phylogenetic trees based on adjusted nucleotide sequenceswerebuilt with the program MEGA5 using the neighbor-joining method(model: maximum composite likelihood) (Saitou and Nei, 1987). Tovalue certainty bootstrap test (1000 replications) was used.
In summer, 2005 highly pathogenic inuenza A/H5N1 virus wasisolated and described for the rst time in Russia (Onishchenkoet al., 2006). It seems that the virus was carried by migrating birdsduring spring migration from South East Asia. The rst outbreakamong poultry was registered in the South of Western Siberia.After that the virus spread up to CaspianBlack Sea region and fur-ther to the South of West Europe. During 20052007 in the Southof Western Siberia there were numerous HPAI outbreaks caused by
HA_F_872M13 TGTAAHA_R_1425M13 CAGGHA_F_1300M13 TGTAAHA_R_1778BM13 CAGGPrimers for amplication of NA gene (segment 6)NA_F_1M13 TGTAANA_R_560M13 CAGGNA_F_415M13 TGTAANA_R_984M13 CAGGNA_F_880BM13 TGTAANA_R_1465BM13 CAGG
T. Ilyicheva et al. / Infection, GenetA/H5N1 virus serotype among poultry and wild birds.Since August, 2005 we have studied clinical samples and au-
topsy material from people that were suspected to have an illnesscaused by avian inuenza A/H5N1 virus. We analyzed 289 samplesusing PCR, presence of virus was veried by inoculation in embry-onated chicken eggs and MDCK cell culture (at least three pas-sages). We did not detect A/H5N1 virus in samples.
We made permanent serological monitoring among populationin adverse regions to detect antibodies to A/H5N1 virus in humanblood sera. From August, 2005 to December, 2007 we collected1115 samples. From 2007 to April, 2009 we collected 1167 bloodsera samples in several regions differed by level of risk of avianinuenza A/H5N1 contamination. Among them 33 samples werecollected in August, 2005 from residents of Suzdalka (Novosibirskregion) where HPAI A/H5N1 was isolated for the rst time in Rus-sia. All samples were obtained from farmers whose poultry died.We collected 1880 samples in different localities of Novosibirskregion where outbreaks among birds caused by A/H5N1 wereregistered. We also collected 350 samples from people inKhantyMansi Autonomous Area (KMAA). We did not detect avianinuenza A/H5N1 viruses. However, a lot of residents of that areaare hunters; thus, they could belong to risk group. Finally, we col-lected 51 samples from poultry farm personnel in Rostov-on-Donwhere avian inuenza outbreak of A/H5N1 occurred (see Table 1).
Sera from all age groups older than 18 were analyzed. All serawere tested in HI and MN assays. There were no sera positive toA/H5N1 before 2008. We detected antibodies to A/H5N1 virus in2008 in blood sera of residents from KMAA, and in 2009 fromKMAA and Novosibirsk region. The results are shown in Table.
As shown in Table in 31 (2.66%) samples we detected antibodiesto HPAI A/H5N1 serotype in HI test, and in 8 (0.69%) samples anti-bodies to A/H5N1 were detected with the use of HI and MN assays.Since it is unknown who contacted with A/H5 virus serotype andwhen, it is difcult to suppose why neutralizing antibodies are ab-sent on low level in sera. It is also possible that we sometimes gotfalse-positives results in HI test. But even in that case part of serawas positive both in HI test and MN assay. In accordance withWHO recommendations (Katz et al., 1999), this fact is sufcientto prove the presence of antibodies to inuenza virus serotype A/H5 in human sera. So far, this is the rst and the only case of detec-tion of antibodies to A/H5N1 virus serotype in human sera in Rus-sia. Since 2009 among 4266 samples we have not detected anyserum positive to H5.