AP09259102 Genetic characterization of bluetongue virus isolates, established in Kazakhstan, and development of the test system for its differential detection

The project is aimed at genetic characterization of bluetongue virus genotypes circulating in Kazakhstan and at improving the effectiveness of measures to control this dangerous animal disease. Although the territory of Kazakhstan is considered bluetongue-free (the identified virus isolates are attributed to the vaccine-like topotype BTV-9w), the southern region of the country has ideal conditions for the spread of sheep catarrhal fever. The risk of introduction of non-vaccine isolates of the virus capable of causing epizootics remains. Therefore, in addition to standard serological monitoring of livestock, it is necessary to carry out differentiating detection of BTV-9w topotype and PCR-diagnostics of disease vectors. The developed domestic test system based on real-time PCR will save money on diagnostics and increase the efficiency of bluetongue control in Kazakhstan.


The RNA-containing bluetongue virus (BTV) belongs to the family Reoviridae, genus Orbivirus. The BTV genome is represented by ten linear double-stranded RNA segments. The viral genome encodes five non-structural (NS1, NS2, NS3, NS3A, NS4) and seven structural (VP1-VP7) proteins. Currently, 29 serotypes of BTV are distinguished, within which individual topotypes may be distinguished.

The vectors of the virus are blood-sucking midges of the genus Culicoides. Sheep are most susceptible to BTV. Cattle, buffalo, goats and wild ruminants may be virus carriers for long periods of time without showing clinical signs of disease. Limited testing of susceptible animals to BT by serologic assays is conducted in Kazakhstan on an ongoing basis (mainly testing imported livestock). Other research groups have also indicated the identification of bluetongue seropositive as well as PCR-positive animals in Kazakhstan. However, the serotype of the identified samples has not been determined and their genetic characterization has not been carried out.

The novelty of the project lies in the complete genetic characterization of Kazakhstani BTV isolates, testing of Kazakhstani representatives of wild ruminants and vectors of Culicoides sp. for the presence of bluetongue virus traits, as well as in the development of new primers for detection of bluetongue virus by quantitative real-time PCR and genetic characterization of BTV. Similar studies have not been conducted in Kazakhstan.

The aim

Conduct genetic characterization of bluetongue virus isolates detected in Kazakhstan and develop a domestic test system based on real-time PCR for their differential detection.

Expected Results

Limited (targeted) collection of clinical specimens from animals and insect vectors (Culicoides sp.) in bluetongue risk areas of southern Kazakhstan and their laboratory diagnosis will be conducted. Genetic characterization of bluetongue virus variants circulating in southern Kazakhstan will be carried out. A domestic test system based on real-time PCR for detection of bluetongue virus RNA and identification of topotype West serotype BTV-9 will be developed. The design of the kit and packaging of the test system will be developed, documentation for the developed test system will be formalized. Production of a trial batch of the test-system will be carried out and its quality control will be carried out.

Project manager

Мамадалиев С.М., доктор ветеринарных наук, профессор, вирусолог. Индекс Хирша: 3. ORCID: https://orcid.org/0000-0002-7767-0251. Scopus ID: 37000092700. WoS ID: N-8389-2017.

Executive team members

Zhigailov A.V. – Candidate of Biological Sciences (molecular biology). H-index: 2. ORCID: https://orcid.org/0000-0002-9646-033X. Scopus ID: 6508121286. WoS ID: N-6073-2015.

Maltseva E.R. – PhD- student (biotechnology), specialist in the field of molecular biology. H-index: 2. ORCID: http://orcid.org/0000-0001-9198-695X. Scopus ID: 57202717826. WoS ID: N-4309-2017.

Neupokoeva A.S. – Master of Biotechnology. H-index: 1. ORCID: http://orcid.org/0000-0001-7257-8037. Scopus ID: 57217703182. WoS ID: N-9341-2017.

Bisenbay A.O. – PhD-student (biotechnology). ORCID: https://orcid.org/0000-0002-7109-2534. Scopus ID: 57217425178. WoS ID: AAX-9935-2020.

Naizabaeva D.A. – Master of Biotechnology. ORCID: http://orcid.org/0000-0002-0606-4289. Scopus ID: 57218288692.WoS ID: AAY-5696-2020.

Kuatbekova S.A. – Master, specialist in veterinary medicine.

Publications of the project manager and members of the research team on the topic of the project

  1. Perfilyeva Y.V., Shapiyeva Z.Zh., Ostapchuk Y.O., Berdygulova Z.A., Bissenbay A.O., Kulemin M.V., Ismagulova G.A., Maltseva E.R., Skiba Y.A., Sayakova Z.Z., Mamadaliyev S.M., Dmitrovskiy A.M. Tick-borne pathogens and their vectors in Kazakhstan – a review. Ticks and Tick-borne diseases. 2020;11(5):101498. https://doi.org/10.1016/j.ttbdis.2020.101498. IF 2.749; Q2; Cite score 5.2; SJR 1.182; percentile 95. 
  2. Ostapchuk Y.O., Zhigailov A.V., Perfilyeva Y.V., Shumilina A.G., Yeraliyeva L.T., Nizkorodova A.S., Kuznetsova T.V., Iskakova F.A., Berdygulova Z.A., Neupokoyeva A.S., Mamadaliyev S.M., Dmitrovskiy A.M. Two case reports of neuroinvasive West Nile Virus infection in the Almaty region, Kazakhstan. IDCases. 2020;21:e00872. https://doi.org/10.1016/j.idcr.2020.e00872. Cite score 1.0; SJR 0.294; percentile 24.
  3. Bissenbay A.O., Zhigailov A.V., Maltseva E.R., Egemberdieva R.A., Skiba Y.A., Mamadaliyev S.M. Borreliosis: a Hidden Threat for Kazakhstan. Eurasian Journal of Applied Biotechnology. 2019; 2:5-27. DOI: 10.11134/btp.2.2019.2. КОКСОН РК.
  4. Остапчук Е.О., Скиба Ю.А., Мамадалиев С.М. Проблемы лабораторной диагностики клещевого боррелиоза. Вестник КазНМУ. 2019;3:58-62. КОКСОН РК.
  5. Bissenbay A.O., Zhigailov A.V., Neupokoyeva A.S., Naizabaeva D.A., Skiba Y.A., Dmitrovsky A.M., Shapiyeva Zh.Zh., Mamadaliyev S.M. West Nile fever virus: biology, epidemiology, molecular genetic characteristics and research priorities. Eurasian Journal of Applied Biotechnology. 2019;2:28-40. DOI: 10.11134/btp.2.2019.3. КОКСОН РК.
  6. Sansyzbay A.R., Erofeeva M.K., Khairullin B.M., Sandybayev N.T., Kydyrbayev Zh.K., Mamadaliyev S.M., et al. An Inactivated, Adjuvanted Whole Virion Clade 2.2 H5N1 (A/Chicken/Astana/6/05) Influenza Vaccine Is Safe and Immunogenic in a Single Dose in Humans. Clinical and Vaccine Immunology. 2013;20(8):1314-1319. https://doi.org/10.1128/CVI.00096-13. IF 3.233; Q2.
  7. Mamadaliyev S.M., Sandybayev N.T., et al. Basic results of development of a production technology and control of a pandemic influenza A/H5N1 vaccine. Influenza and Other Respiratory Viruses. 2011; 5:350-353. IF 3.288; Q2.
  8. Mamadaliyev S.M., Sandybayev N.T., et al. Development of production technology and pre-clinical testing of a pandemic influenza A/H1N1 vaccine. Influenza and Other Respiratory Viruses. 2011; 5:354-357. IF 3.288; Q2.
  9. Chervyakova O.V., Strochkov V.M., Sultankulova K.T., Sandybayev N.T., Zaitsev V.L., Mamadaliyev S.M. Molecular and genetic analysis of NS gene from high pathogenic strains of the avian influenza (H5N1) virus isolated in Kazakhstan. Gene. 2011;476(1-2):15-19. https://doi.org/10.1016/j.gene.2011.02.003. IF 2.984; Q2.
  10. Perfilyeva Yu.V., Nizkorodova A.S., Berdygulova Zh.A., Ostapchuk Ye.A., Naizabayeva D.A., Neupokoyeva A.S., Kuznetsova T.V., Shishkina T.S., Abuova G.N., Yegemberdiyeva R.A., Bissenbay A.O., Maltseva E.R., Mamadaliyev S.A., Dmitrovsky A.M.  Detection of IgG against rickettsia typhi: A population-based study in Southern Kazakhstan. Infektološki glasnik. 2019;39(4). https://doi.org/10.37797/ig.39.4.2. SJR 0.104.
  11. Мамадалиев С.М., Абдураимов Е.О., и др. Штамм “RT/RIBSP-07/16″ 16 серотип вируса катаральной лихорадки овец, для приготовления диагностических и профилактических препаратов. Патент РК № 22289. 15.02.2010. Бюл.2.
  12. Ершебулов З.Д., Мамадалиев С.М., и др. Штамм “Хуросон-07/4″ 4 серотип вируса катаральной лихорадки овец, для приготовления диагностических и профилактических препаратов. Патент РК № 22184. 15.08.2012. Бюл.8.
  13. Skiba Y., Mokrousov I., Ismagulova G., Maltseva E., et al. Molecular snapshot of Mycobacterium tuberculosis population in Kazakhstan: A country-wide study. Tuberculosis. 2015; 9. https://doi.org/10.1016/j.tube.2015.04.012. IF 6.259; Q1.
  14. Skiba Y, Mokrousov I, Nabirova D, Vyazovaya A, Maltseva E, Malakhova N, et al. Mycobacterium tuberculosis RD-Rio strain in Kazakhstan. Emerg Infect Dis. 2019 Mar; 25(3): 604–606 doi: 10.3201/eid2503.181179. IF 2.576; Q3.
  15. Ismagul A., Yang N., Maltseva E., et al. A biolistic method for high-throughput production of transgenic wheat plants with single gene insertions. BMC Plant Biology. 2018; 18:135. https://doi.org/10.1186/s12870-018-1326-1. IF 3.497; Q1.
  16. Zhigailov A.V. Alexandrova A.M., et al. Evidence that phosphorylation of the alpha-subunit of eIF2 does not essentially inhibit mRNA translation in wheat germ cell-free system. Frontiers in Plant Science. 2020, 11:936. https://doi.org/10.3389/fpls.2020.00936. IF 4.106; Q1.
  17. Nizkorodova A., Suvorova M., Zhigailov A., Iskakov B. The effect of translation promoting site (TPS) on protein expression in E. coli cells. Molecular Biotechnology (ISSN: 10736085). 2020. 62(6-7):1-9. https://doi.org/10.1007/s12033-020-00251-1. IF 1.712; Q3.
  18. Akbergenov R.Z., Zhanybekova S.S., Kryldakov R.V., Zhigailov A., et al. ARC-1, a sequence element complementary to an internal 18S rRNA segment, enhances translation efficiency in plants when present in the leader or intercistronic region of mRNAs. Nucleic Acid Research. 2004; 32(1):239-247. https://doi.org/10.1093/nar/gkh176. IF 11.501; Q1.
  19. Neupokoyeva A., Abaildayev A., et al. Association of variability in the ZNF365 gene with BC in Kazakh population. FEBS J. 2016; 283:376-376.

Achieved Results

2021 year

A limited (targeting) collection of clinical samples from animals and insect vectors (Culicoides sp.) in bluetongue risk areas of South Kazakhstan was conducted and their laboratory diagnostics was carried out

Primers for detection and genetic characterization of bluetongue virus were synthesized using computer program MEGA-X and GenBank resource, genomes of different serotypes and separate topotypes of bluetongue virus were analyzed to identify conserved parts of the genome. For detection of bluetongue virus nucleic acids, 2 primers to conserved regions of BTV segment 10 were synthesized and purified. For the detection of internal control (to the ruminant beta-actin gene), 2 primers were synthesized and purified. For genetic characterization of the bluetongue virus genome, 9 primers were synthesized and purified for BTV-9W segment 2, 6 primers each for BTV-9W segments 1 and 3, 4 primers each for BTV-9W segments 4, 5, and 6, 3 primers each for BTV segment 7, and 2 primers each for BTV segments 8, 9, and 10.

Collection of Culicoides sp. in bluetongue-risk areas of southern Kazakhstan was carried out, and their species identification was carried out. Homogenization of collected insects was carried out; RNA was isolated from homogenates. Literature sources were analyzed in relation to distribution in Kazakhstan of representatives of midge Culicoides spp. (Diptera: Ceratopogonidae). Collection of biting midges in the southern region of Kazakhstan was carried out. Coordinates were recorded for all collection points. Visual identification of collected Culicoides spp. was carried out using an insect identifier and binoculars. Insects identified to subgenus were distributed into pools by sex, collection location and systematic group. Among the collected midges, four subgenera Culisoide spp. were identified: Trithecoides, Hofmania, Oaecata and Avaritia. The formed pools of midge beetles were homogenized on an automatic homogenizer, total RNA preparations were isolated from midge homogenates using Trizol.

Whole blood and serum samples were collected from animals susceptible to BTV in risk areas of the country. Total samples were collected from 520 sheep, 27 goats, 11 cows, 27 camels and 20 Bukhara deer. Total RNA samples were isolated from the blood of animals.

Serologic analysis of animal sera by ELISA method was performed and the total prevalence was 6.15% for sheep and 3.7% for goats. No seropositive animals were found among cows, camels and deer. RT-PCR analysis of RNA from animal blood was performed. Virus nucleic acid was detected in one pool of blood flies and in three RNA samples from sheep blood.

2022 year

Genetic characterization of bluetongue virus variants circulating in southern Kazakhstan was carried out.

– Total RNA preparations were isolated from ten RT-PCR-positive samples, and reverse transcription reaction was performed using a high-yield Superscript IV revertase. From these ten samples of synthesized cDNA, amplifications were obtained using high-fidelity DNA polymerase (segments 2 and 10). 

– Genetic characterization of bluetongue virus isolates was performed using partially sequenced Seg-2 and Seg-10 followed by phylogenetic analysis. For both loci, all samples were clustered with samples of BTV-9W virus genotypes.

– Nucleotide sequences of sequenced regions of BTV segments 2 and 10 obtained from ten RT-PCR positive animals were deposited in the NCBI database and assigned numbers: OM307411 – OM307420 (segment 10) and numbers OM307421 – OM307430 (segment 2).

Genetic characterization by classical PCR of bluetongue positive samples was carried out.

– Ten PCR-positive samples (9 obtained from sheep and 1 from Culicoides sp.) were analyzed by classical OT-PCR at the Seg-2 locus using serotype-specific primers for serotype identification. All eight samples were found to belong to serotype 9.

– The samples were archived and stored at – 70°C.

Genetic characterization by PCR DNA sequencing of bluetongue-positive samples was performed. Phylogenetic analysis based on DNA sequencing results was performed.

– DNA amplification was performed on eight PCR-positive samples using Phusion high-precision polymerase at the Seg-10 and Seg-2 loci.

– Sanger sequencing of Seg-10 and Seg-2 DNA fragments was performed. Phylogenetic analysis based on the results of DNA sequencing in the MEGA-X program was carried out. It was found that all samples, serotype 9, belong to the topotype “Western”, which includes only mesogenic vaccine strains of the virus.

– Primers for genetic full genomic analysis of BTV-9W were selected. Whole-genome sequencing of the virus was performed for one RNA sample. The genome was analyzed and it was found that the virus is not reassortant: all genomic segments are genetically closest to representatives of topotype “West” of serotype 9.

2023 year

– To develop a domestic real-time PCR-based test system for detection of bluetongue virus RNA, short regions of the viral genome containing targets for RT-PCR were identified. Using primers “BTV-9W-2F” and “BTV-9W-1F”, a 542 bp DNA fragment including a Seg-2 target for RT-qPCR was amplified. Using primers “BTV-S10-F” and “BTV-S10-R”, a 746 bp DNA fragment including a Seg-10 target for RT-qPCR was amplified. Both amplifications were directly cloned into the PCR2.1-TOPO vector using the commercial TOPO™ TA Cloning™ Kit (Thermo Fisher Scientific) and competent E. coli strain DH5 cells.

– DNA clones were screened using PCR and restriction analysis techniques. Selected DNA clones containing inserts of the required sizes were isolated using a commercial GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific).

– DNA sequencing by Sanger sequencing was performed to verify the nucleotide sequence of the insert using primers M13 (forward and reverse primers).

– DNA constructs containing regions of genomic segments 2 and 10 of topotype West serotype BTV-9 under the control of the promoter and terminator of phage T7 were transformed into E. coli bacterial cells of the expression strain. Control RNAs were spiked in them and then isolated from the bacteria. RNA was isolated from BTV-9W vaccine and RT-PCR of BTV-positive and BTV-negative sheep blood samples. The sensitivity of a system of two primers and a probe for detection by the conserved locus of the Seg10 segment of BTV was tested by one-step quantitative real-time RT-PCR under different temperature conditions. Positive control dilutions of 1:10, 1:20 and 1:50 were used (Ct values of 6, 6.5 and 8, respectively). Accuracy was tested by ten points with a dilution of the control 1:20; Ct value ranged from 6.4 to 6.6.

– The composition of the reaction mixture of the test system was developed and optimized. The composition of primers and samples based on single and cross reactions was selected: “BTV-S10-qPCR-F” (5’-TGGAYAAAGCRATGTCAAA), “BTV-S10-qPCR-R” (5’-CATCATCACGAAACGCTTC), “BTV-S2-qPCR-F” (5’-ACCGTTCGGGAAATTCATG), “BTV-S2-qPCR-R” (5’-GGAATGTGTCAAGTCTATCAGC), “BTV-Seg2-qPCR-Pr” (5´-JOE-ACCGTTCGCCCAGTTGAAGAGGCA-BHQ1), “BTV-Seg10-qPCR-Pr” (5´-FAM-GCTGCATTCGCATCGTACGC-BHQ1). The optimal concentration for oligoDNA was selected. Optimization of Mg2+ ion concentration, bovine serum albumin, buffer, primer and sample concentration was carried out. Thermocycling mode of the selected test-system composition was tested.

– The sensitivity of the developed test-system was evaluated on control and field samples. To obtain positive controls, competent E. coli cells of expression strain BL21(DE3) were transformed with plasmid DNA T7-BTV9w-S10F_pET23c, T7-BTV9w-S10R_pET23c and T7-BTV9w-S2F_pET23c. The grown DNA clones were verified by PCR analysis to contain the required DNA cassettes. Expression of cloned cDNA genes in E. coli cells by induction with 1mM IPTG was performed. Residual DNA was cleaved with Turbo-DNase I, and after RNA was deproteinized and resuspended with alcohol. Total preparations were isolated from field blood samples that gave a positive response to RT-PCR for seg10 BTV as well as from the vaccine strain BTV-9W. RNA samples isolated from the blood of animals that did not show the presence of BTV RNA were used as negative controls. The dilutions of control RNA were created. The sensitivity of the test system was determined for Seg10 and Seg-2 loci. The same loci were tested on positive RNA samples isolated from vaccine and field strains of the virus detected on the territory of the country.

– Analytical specificity of the test kit on viruses affecting ungulate mammals (for testing of which the test system is supposed to be used) was evaluated: on cattle viral diarrhea virus (BVDV) types 1 and 2 (BVDV-1 and BVDV-2), sheep pox virus (SPPV), sheep contagious ecthyma virus (ORFV), sheep nodular dermatitis virus (LSDV), cattle papular stomatitis virus (BPSV), infectious rhinotracheitis virus (IBR), parainfluenza-3 virus (PI3), bovine respiratory syncytial virus (BRSV). When working with nucleic acids from the above organisms, no false positives were detected for any of the two target loci (Seg-2 BTV and Seg-10 BTV). Samples (control and field RT-PCR positive BTV samples) containing bluetongue virus strains BTV-4, BTV-14, BTV-9W were also tested. At the Seg-10 BTV locus, all samples were positive: ct for BTV-4 = 24.54±1.01; ct for BTV-14 = 23.15±1.25; ct for control strain BTV-9W = 21.28±0.95; ct for field strain BTV-9W = 22.45±0.90. At the Seg-2 BTV locus, as expected, a positive response was obtained only for bluetongue virus strains belonging to the BTV-9W topotype: ct for control strain BTV-9W = 21.06±0.98; ct for field strain BTV-9W = 22.73±0.89.

– The composition of a reagent kit for detection of RNA of all 29 known serotypes of bluetongue virus and separate detection of topotype “West” serotype 9 by triplex combined reverse transcription-polymerase chain reaction with real-time fluorescence detection (RT-PCR-RV) “BTV pan/9W plus 18S” was developed. Instructions for use of the kit were developed. Package design of the kit was carried out. A trial batch of the test kit in the amount of three units was produced, the quality of the batch was evaluated. The quality of the kit was confirmed.

– CRL-NCB-SA-SOP-045 “Conducting classical PCR for detection and further genotyping of catarrhal fever virus (Bluetongue)”, CRL-NCB-SA-SOP-052 “Procedure for collection of Culicoides midge“, CRL-NCB-SA-SOP-056 “Protocol for competitive ELISA for detection of antibodies to Bluetongue virus using the ID SCREEN Bluetongue Competition kit, ID.vet”.

Project Publications

  1. Zhigailov A.V., Perfilyeva Y.V., Maltseva ER., Ostapchuk Y.O., Cherusheva A.S., Naizabayeva D.A., Nizkorodova A.S., Berdygulova Zh.A., Mashzhan A.S., Bissenbay A.O., Kuatbekova S.A., Koshemetov Zh.K., Abdolla N., Skiba Y.A., Mamadaliyev S.M. Identification and characterization of bluetongue virus in Culicoides spp. and clinically healthy livestock in southeastern Kazakhstan // Comparative Immunology, Microbiology and Infectious Diseases. – 2022. – Vol. 90–91. – P. 101895. https://doi.org/10.1016/j.cimid.2022.101895 (Scopus – 86%; Web of Science – Q2; Impact Factor 2.729; CiteScore 3.7).
  2. Жигайлов А.В., Остапчук Е.О., Перфильева Ю.В., Абдолла Н., Мальцева Э.Р., Найзабаева Д.А., Куатбекова С., Машжан А., Низкородова А.С., Бердыгулова Ж.А., Скиба Ю.А., Мамадалиев С.М. Анализ рисков распространения катаральной лихорадки овец в Казахстане // Вестник КарУ. Серия биология. Медицина. География. – 2022. – Т. 2. – С. 71-81. doi: https://doi.org/10.31489/2022BMG1/71-81. (журнал рекомендован ККСОН МОН РК, импакт-фактор 0,028 за 2019 г. по КазБЦ).
  3. Zhigailov A.V., Perfilyeva Y.V., Ostapchuk Y.O., Kulemin M.V., Ivanova K.R., Abdolla N., Kan S.A., Maltseva E.R., Berdygulova Zh.A., Naizabayeva D.A., Skiba Y.A., Mamadaliyev S.M. Molecular detection and characterization of bovine viral diarrhea virus type 2 and bluetongue virus 9 in forest flies (Hippobosca equina) collected from livestock in southern Kazakhstan // Veterinary Parasitology: Regional Studies and Reports. – 2023. – Vol.45. – P. 100932. https://doi.org/10.1016/j.vprsr.2023.100932 (Scopus – 70%; Web of Science – Q2; Impact Factor 2.52; CiteScore 1.51; SJR 0.45).
  4. Иванова К., Найзабаева К., Куатбекова С., Низкородова А., Жигайлов А., Мамадалиев С.М. Клонирование участков геномных сегментов 2 и 10 вируса блютанга 9 серотипа (BTV-9) для разработки ПЦР тест-системы // Материалы международной научно-практической конференции, посвященной 65-летию НИИ Проблем биологической безопасности МЗ РК «Биотехнология и биологическая безопасность: достижения и перспективы развития». Алматы, 6-8 сентября, 2023. – С. 106. – Устный доклад (Иванова К.).