оСоБенноСти реПЛикаЦии ВируСа аФриканСкоЙ ЧумЫ СВинеЙ В ПриСутСтВии рекомБинантнЫх БеЛкоВ CD2v, pX69R и pE248R ФГБУ «ВНИИЗЖ» федеральное государственное бюджетное учреждение «Федеральный центр охраны здоровья животных», 600901, Владимирская область, г. Владимир, микрорайон Юрьевец, Россия Введение. Африканская чума свиней (АЧС) является особо опасной геморрагической болезнью свиней, которую вызывает крупный ДНК-содержащий вирус семейсмтва Asfaviridae). Поскольку нет эффективных и безопасных вакцин против АЧС, актуально изучение функций белков вируса путём анализа особенностей репликации вируса АЧС в присутствии рекомбинантных белков in vitro. Цель-изучить функции и степень влияния CD2v, pE248R и pX69R на скорость и уровень репродукции вируса АЧС in vitro для разработки подходов к созданию вакцины против АЧС. материал и методы. Использовали вирус АЧС изолят Krasnodar 07/17 и штамм АЧС/ВНИИЗЖ/CV-1. Гены X69R, EP402R и E248R клонировали в векторе pJET1.2/blunt в клетках Е. coli JM-109. Локализацию рекомбинантных белков в клетках CV-1 изучали в реакции прямой иммунофлюоресценции с использованием ФИТЦ-конъюгата поликлональных антител. Уровень репродукции вируса АЧС оценивали в реакции гемадсорбции и в полимеразной цепной реакции в режиме реального времени. результаты. Сконструированы экспрессирующие рекомбинантные плазмиды pCI-neo/E248R, pCI-neo/EP402R и pCI-neo/Х69R. Определена локализация и подтверждена специфичность полученных белков CD2v, pE248R и pX69R, для которых установлено, что они повышают уровень накопления вируса АЧС на 3-5-е сутки эксперимента на ~1,2-1,5 lgГАдЕ 50 /см 3 по сравнению с отрицательным контролем. обсуждение. В результате анализа установлена важная роль белков CD2v, pX69R и pE248R в репродукции вируса, поскольку они влияют на её уровень. Функция белка pX69R неизвестна, однако в проведённых экспериментах определено его влияние на репродукцию вируса АЧС, проявившееся в увеличении уровня его накопления. Заключение. Данная методология позволяет изучить характер влияния белков с неизвестной функцией на репликацию вируса АЧС.
Introduction. The causative agent of African swine fever (Asfarviridae: Asfivirus: African swine fever virus) (ASF) is a double-stranded DNA virus of 175–215 nm. To date, 24 of its genotypes are known. Clustering of ASF genotype II isolates is carried out by examining a limited number of selected genome markers. Despite the relatively high rate of mutations in the genome of this infectious agent compared to other DNA viruses, the number of known genome molecular markers for genotype II isolates is still insufficient for detailed subclustering. The aims of this work were the comparative analysis of ASFV/Zabaykali/WB-5314/2020 virus isolate and determination of additional molecular markers which can be used for clustering of viral genotype II sequences. Material and methods. ASF virus isolate ASFV/Zabaykali/WB-5314/2020 was used to extract genomic DNA (gDNA). Sequencing libraries were constructed using the Nextera XT DNA library prepare kit (Illumina, USA) using the methodology of the next generation sequencing (NGS). Results. The genome length was 189,380 bp, and the number of open reading frames (ORFs) was 189. In comparison with the genome of reference isolate Georgia 2007/1, 33 single nucleotide polymorphisms (SNPs) were identified, of which 13 were localized in the intergenic region, 10 resulted to the changes in the amino acid sequences of the encoded proteins, and 10 affected the ORF of ASF virus genes. Discussion. When analyzing intergenic regions, the ASFV/Zabaykali/WB-5314/2020 isolate is grouped separately from a number of isolates from Poland and three isolates from People’s Republic of China (PRC), since it does not harbor additional tandem repeat sequence (TRS). At the same time, the construction of a phylogenetic tree based on DP60R gene sequencing relates ASFV/Zabaykali/WB-5314/2020 to isolates from PRC and Poland. Moreover, phylogenetic analysis of full-genome sequences confirmed previous studies on the grouping of viruses of genotype II, and as for the studied isolate, it was grouped with the variants from China. Conclusion. A new variable region was identified, the DP60R gene, clustering for which gave a result similar to the analysis of full-length genomes. Probably, further study of the distribution of ASF virus isolates by groups based on the analysis of this gene sequences will reveal its significance for studying the evolution of the virus and its spread.
The paper describes the results of testing of biomaterial from domestic pigs and wild boars by real-time PCR used for African swine fever virus genome detection, carried out in the FGBI “Federal Centre for Animal Health” (Vladimir). In 2017 8,500 samples from 44 subjects of the Russian Federation were tested within the framework of the state laboratory monitoring. African swine fever virus genome was detected in 504 samples. In 2017 ASF outbreaks were registered in the Urals and Siberian Federal Districts of the RF for the first time. The conducted research and persistent ASF infection in the territory of the RF have demonstrated the need for further surveillance in the populations of susceptible animals. Development, organization and implementation of the program for ASF spread surveillance in wild fauna remains a high priority. It is necessary to create and implement sampling schedules with uniform sampling of biomaterial and submission of the collected samples to the research laboratories for timely ASF outbreak containment at the regional level.
Introduction. Prevention and control of African swine fever (ASF) transmission on the territory of the Russian Federation requires monitoring based on testing of samples from pigs and wild boars. Specific anti-ASFV antibodies are rarely detected in samples during routine serological diagnostics. Although, ASF isolates with weakened virulence were confirmed in Russia and neighboring countries.The aim of this work was to determine the possibility of using alternative samples for ASF diagnosis and evaluate the effectiveness of the diagnostic methods used on the territory of Russia.Materials and methods. Biological materials obtained from experimentally infected animals and samples collected in the “field” conditions were used in this study.Results. Complex testing (RT-PCR and ELISA) is a more effective approach to diagnose chronic and asymptomatic forms of ASF compared to the separate use of these techniques. The possibility and efficiency of using alternative samples in diagnostics are demonstrated. It was confirmed that IPT method overcomes ELISA by high diagnostic sensitivity and detection of antibodies on earlier stages in extended range of samples. Anti-ASFV antibodies were detected in domestic and wild pigs in five regions of Russia. Samples from infected pigs that are negative in RT-PCR can be positive for anti-ASFV antibodies. The detection of antibodies in samples from shot wild boars (negative or uncertain in RT-PCR test) suggests the existence of animals surviving ASF infection.Conclusion. The data obtained suggest a revision of the ASF surveillance strategy, by introducing complex diagnostic methods aimed at detection of both the virus genome and anti-ASFV antibodies simultaneously.
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