В статье представлены результаты сравнения методов выделения при проведении SNP генотипирования крупного рогатого скота (КРС). Успешное проведение генотипирования зависит от получения ДНК из образцов в достаточном количестве и качестве для проведения молекулярных реакций. В работе применялись три метода экстракции нуклеиновых кислот. Методика выделения с использованием коммерческого набора PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific, USA) показала наибольшую степень выхода и чистоты нуклеиновых кислот. При выделении ДНК из волосяных луковиц КРС набором PureLink Genomic DNA Mini Kit средняя концентрация ДНК в образцах составила 151,78 нг/мкл, извлечение ДНК комплектом «ДНК-сорб-В» составила 21,51 нг/мкл и фенол-хлороформный метод соответствовал 175,08 нг/мкл. Экстрагированные ДНК животных в дальнейшем использовали для проведения генотипирования SNP методом. Большинство оцененных методов экстракции позволили амплифицировать значительное количество ДНК. Результаты генотипирования в образцах выделенных с применением PureLink Genomic DNA Mini Kit по сравнению с остальными образцами показали 100 % амплификацию продуктов. Из методов, протестированных в этом исследовании, экстракция данным набором была наиболее эффективной с точки зрения дальнейшего обнаружения ДНК. амплифицировать значительное количество ДНК. Результаты генотипирования в образцах выделенных с применением PureLink Genomic DNA Mini Kit по сравнению с остальными образцами показали 100 % амплификацию продуктов. Из методов, протестированных в этом исследовании, экстракция данным набором была наиболее эффективной с точки зрения дальнейшего обнаружения ДНК

Вспышки птичьего гриппа среди диких и домашних птиц все больше являются частым явлением во всем мире. За последние 15 лет вспышки гриппа птиц, в частности вызванные вирусами H5, происходящими из линии A/Goose/Guangdong/1/1996, возникшей в Юго-Восточной Азии в 1996 г., все чаще происходили в Европе и Азии. В период с 2005 по 2020 год в мире было выявлено не менее десяти глобальных вспышек ВПГ H5, что привело к массовой гибели домашних и диких птиц. В последние годы применение молекулярных методов для обнаружения вирусных нуклеиновых кислот стало важным инструментом для выявления вируса гриппа птиц и идентификации подтипов гемагглютинина (HA) и нейраминидазы (NA). К числу таких методов относится ОТ-ПЦР. Обратная транскриптазная - полимеразная цепная реакция – реакция синтеза ДНК-копии (кДНК) на матрице РНК с использованием специального фермента – обратной транскриптазы, также называемой ревертазой. Затем кДНК амплифицируется с помощью полимеразной цепной реакции (ПЦР). В данной статье представлены результаты типирования вируса гриппа птиц методом ПЦР. В 2020 году в одном из частных подворий на территории Алматинской области был отмечен падеж домашней птицы, с симптомами похожими на вирус птичьего гриппа. В результате проведенных лабораторных исследований был типирован вирус гриппа птиц подтипа H5 методом ОТ-ПЦР.

The clostridial bacteria emerged from dead saiga in 2010 - 2013 were identified on the basis of 16S rRNA gene as perfringens species and by the presence of plc gene, encoding only alpha- toxin and were classified as type A. The genetic variation of Clostridium perfringens alpha- toxin strains emerged from saiga was characterized in 2010 - 2013. N- and C- domains of Clostridium perfringens alpha- toxin strain structure, emerged from the saiga in 2010 - 2012 showed a very high conservatism. The structure of Clostridium perfringens alpha- toxin strain, 2013 revealed previously unknown mutations - 4 amino acid substitutions: Ala13Thr (threonine -> alanine), Val47Ile (valine, isoleucine ->), Ala202Asp (alanine -> aspartic acid), Thr205Ala (threonine -> Alanine) in the N- domain of alpha- toxin and 1 replacement of Ser363Pro (serine -> proline) in the C- domain. We may assume that this mutation affects the reduction of alpha- toxin toxicity and as a result, causes Clostridium perfringens pathogenicity reduction. The degree of identity in the N- terminal catalytic domain of alpha- toxin reaches 98.4% and in C domain responsible for the binding to membranes the identity makes 99.2%. Plc gene sequence of the strains Clostridium / Saigas / 2010 / ZKO / KZ, Clostridium / Saigas / 2011 / ZKO / KZ, Clostridium / Saigas / 2012 / Kostanay / KZ and Clostridium / Saigas / 2013 / Kostanay / KZ are deposited in the GenBankdatbase under the numbers KP143658, KP143659, KP143660, KP143661, respectively.

Brucellosis is an urgent infectious disease of livestock and wild animals and the commonest human zoonosis.Diagnosis of brucellosis is rather complicated and it has to be obligatorily confirmed by laboratory testing.Direct bacteriological and molecular methods and indirect serological tests are used for brucellosis diagnostics.The choice of the diagnostic tools depends on the overall epidemiological situation in the region and the objectives of the study: validation of the diagnosis, screening (monitoring), cross-sectional studies or confirmation of brucellosis-free status of the region.The review describes current bacteriological, serological and molecular methods, routinely used for the diagnosis of brucellosis in humans and animals.The perspectives of brucellosis diagnostics are also discussed.

The diagnostic oligonucleotide microarray for subtyping of human and animal influenza A viruses (IAVs) was developed. We proposed a simple method of the fluorescent labeling of genomic segments of all known IAVs subtypes, the composition of the hybridization buffer, as well as the software of the data processing. 48 IAVs strains of different subtypes were analyzed using our microarray. All of them were identified, while 45 of 48 strains were unambiguously subtyped.

Influenza is one of the most widespread respiratory viral diseases, infecting humans, horses, pigs, poultry and some other animal populations. Influenza A viruses (IAV) are classified into subtypes on the basis of the surface hemagglutinin (H1 to H16) and neuraminidase (N1 to N9) glycoproteins. The correct determination of IAV subtype is necessary for clinical and epidemiological studies. In this article we propose an oligonucleotide microarray for subtyping of IAV using universal one-step multisegment RT-PCR fluorescent labeling of viral gene segments. It showed to be an advanced approach for fast detection and identification of IAV.

Influenza reassortant viruses A/SPb/HK/09(H1N1), A/Astana/HK/2009 (H5N1), A/Otar/HK/2010(H3N8), and A/Perth/ HK/2011(H3N2), carrying surface antigens of different subtypes, were constructed on the basis of new potential unified donor strain A/HK/1/68/162/35(H3N2). The virulence and reproduction activity of the obtained reassortants were tested. The safety of the candidate live and inactivated influenza vaccines produced from the reassortant viruses was demonstrated. The study demonstrates that A/HK/1/68/162/35 can be used as a unified donor for attenuated and high-yield vaccine reassortants.

AIM:Development of technological stages of preparation of experimental influenza whole-virion inactivated adsorbed vaccine based on recombinant influenza virus strains NIBRG-14 and A/Astana/RG/6:2/2009.MATERIALS AND METHODS:2 recombinant vaccines influenza strains were used in the study--NIBRG-14 and A/Astana/RG/6:2/2009. Purification of native virus-containing allantoic fluid was performed by ion-exchange chromatography. The virus was inactivated by formaldehyde. Merthiolate at concentration of 0.1 mg/ml was added to the vaccine as a preserving substance. Aluminium hydroxide was used as an adjuvant. Harmlessness and immunogenicity (HI) of the constructed preparation are determining.RESULTS:Virus-containing materials from recombinant strains with biological activity of 8.5 - 9.0 lg EID50/cm3 and hemagglutination activity of 1:256 - 1:1024 in chicken embryos were obtained. Optimal inactivation regimen of non-purified suspensions by formaldehyde was established and combined scheme of purification and concentration of influenza virus was selected that provide harmlessness and immunogenicity of experimental samples of inactivated vaccines against highly pathogenic influenza A/H5N1 in experiments in mice.CONCLUSION:The data obtained on quality parameters of intermediate products and final vaccine give evidence on their compliance with normative parameters for whole-virion influenza purified vaccine.

Microarray technology is one of the most challenging methods of influenza A virus subtyping, which is based on the antigenic properties of viral surface glycoproteins – hemagglutinin and neuraminidase. On the example of biochip for detection of influenza A/H5N1 virus we showed the possibility of using multisegment RTPCR method for amplification of fluorescently labeled cDNA of all possible influenza A virus subtypes with a single pair of primers in influenza diagnostic microarrays.

ABSTRACT Here we present the genomic sequence of horsepox virus (HSPV) isolate MNR-76, an orthopoxvirus (OPV) isolated in 1976 from diseased Mongolian horses. The 212-kbp genome contained 7.5-kbp inverted terminal repeats and lacked extensive terminal tandem repetition. HSPV contained 236 open reading frames (ORFs) with similarity to those in other OPVs, with those in the central 100-kbp region most conserved relative to other OPVs. Phylogenetic analysis of the conserved region indicated that HSPV is closely related to sequenced isolates of vaccinia virus (VACV) and rabbitpox virus, clearly grouping together these VACV-like viruses. Fifty-four HSPV ORFs likely represented fragments of 25 orthologous OPV genes, including in the central region the only known fragmented form of an OPV ribonucleotide reductase large subunit gene. In terminal genomic regions, HSPV lacked full-length homologues of genes variably fragmented in other VACV-like viruses but was unique in fragmentation of the homologue of VACV strain Copenhagen B6R, a gene intact in other known VACV-like viruses. Notably, HSPV contained in terminal genomic regions 17 kbp of OPV-like sequence absent in known VACV-like viruses, including fragments of genes intact in other OPVs and approximately 1.4 kb of sequence present only in cowpox virus (CPXV). HSPV also contained seven full-length genes fragmented or missing in other VACV-like viruses, including intact homologues of the CPXV strain GRI-90 D2L/I4R CrmB and D13L CD30-like tumor necrosis factor receptors, D3L/I3R and C1L ankyrin repeat proteins, B19R kelch-like protein, D7L BTB/POZ domain protein, and B22R variola virus B22R-like protein. These results indicated that HSPV contains unique genomic features likely contributing to a unique virulence/host range phenotype. They also indicated that while closely related to known VACV-like viruses, HSPV contains additional, potentially ancestral sequences absent in other VACV-like viruses.

ABSTRACT Sheeppox virus (SPPV) and goatpox virus (GTPV), members of the Capripoxvirus genus of the Poxviridae , are etiologic agents of important diseases of sheep and goats in northern and central Africa, southwest and central Asia, and the Indian subcontinent. Here we report the genomic sequence and comparative analysis of five SPPV and GTPV isolates, including three pathogenic field isolates and two attenuated vaccine viruses. SPPV and GTPV genomes are approximately 150 kbp and are strikingly similar to each other, exhibiting 96% nucleotide identity over their entire length. Wild-type genomes share at least 147 putative genes, including conserved poxvirus replicative and structural genes and genes likely involved in virulence and host range. SPPV and GTPV genomes are very similar to that of lumpy skin disease virus (LSDV), sharing 97% nucleotide identity. All SPPV and GTPV genes are present in LSDV. Notably in both SPPV and GTPV genomes, nine LSDV genes with likely virulence and host range functions are disrupted, including a gene unique to LSDV (LSDV132) and genes similar to those coding for interleukin-1 receptor, myxoma virus M003.2 and M004.1 genes (two copies each), and vaccinia virus F11L, N2L, and K7L genes. The absence of these genes in SPPV and GTPV suggests a significant role for them in the bovine host range. SPPV and GTPV genomes contain specific nucleotide differences, suggesting they are phylogenetically distinct. Relatively few genomic changes in SPPV and GTPV vaccine viruses account for viral attenuation, because they contain 71 and 7 genomic changes compared to their respective field strains. Notable genetic changes include mutation or disruption of genes with predicted functions involving virulence and host range, including two ankyrin repeat proteins in SPPV and three kelch-like proteins in GTPV. These comparative genomic data indicate the close genetic relationship among capripoxviruses, and they suggest that SPPV and GTPV are distinct and likely derived from an LSDV-like ancestor.

Camelpox virus (CMLV), a member of the Orthopoxvirus genus in the Poxviridae, is the etiologic agent of a disease of camels. Here we report the CMLV genomic sequence with analysis. The 205,719-bp CMLV genome contains 211 putative genes and consists of a central region bound by identical inverted terminal repeats of approximately 7 kb. A high degree of similarity in gene order, gene content, and amino acid composition in the region located between CMLV017 and CMLV184 (average 96% amino acid identity to vaccinia virus (VACV)) indicates a close structural and functional relationship between CMLV and other known orthopoxviruses (OPVs). Notably, CMLV contains a unique region of approximately 3 kb, which encodes three ORFs (CMLV185, CMLV186, CMLV187) absent in other OPVs. These ORFs are most similar to B22R homologues found in other chordopoxvirus genera. Among OPVs, CMLV is the most closely related to variola virus (VARV), sharing all genes involved in basic replicative functions and the majority of genes involved in other host-related functions. Differences between CMLV and VARV include deletion and disruption of a large number of genes. Twenty-seven CMLV ORFs are absent in VARV, including seven full-length homologues of NMDA-like receptor, phospholipase D, Schlafen, MT-4 virulence, kelch, VACV C8L, and cowpox (CPXV) B21R proteins. Thirty-eight CMLV ORFs, some of which are fragments of larger genes, differ in size from corresponding VARV ORFs by more than 10% (amino acids). Genome structure and phylogenetic analysis of DNA sequences for all ORFs indicate that CMLV is clearly distinct from VARV and VACV and, as it has been suggested for VARV, it may have originated from a CPXV virus-like ancestor.