AGRIS - 

2019

Usadov, T.R. | Kol'tsov, A.Yu. | Sukher, M.M. | Morgunov, Yu.P. | Yanzhieva, D.V. | Sal'nikov, N.I., All-Russia Research and Development Inst. of Veterinary Virology and Microbiology, Pokrov

Presented are data on the development of a test-system for detection of lumpy skin disease (LSD) virus genome in cattle by Real-Time polymerase chain reaction (PCR). This technique is based on the amplification of a fragment (113 b.p.) of LSDV 129 gene using the original oligonucleotide primers and fluorescent-labeled hybridization probe. Analytical specificity of the developed test system was evaluated by testing the strains of LSD virus, sheep pox and goat pox viruses, contagious ecthyma virus, as well as biological samples from clinically healthy animals and intact cell cultures. The analytical sensitivity of the developed test-system was determined using tenfold serial dilutions of cultural virus contained material, which was 1.0±0.15 lg TCID50/ml. To create positive amplification control to the developed test system, a fragment of LSDV 129 gene synthesized was cloned as a part of the plasmid vector pGEM-T Easy in the E. coli bacterial cells. It has been established that the range of plasmid DNA concentration, at which the amplification linearity is observed, is from 3.0х10E9 to 3.0 DNA copies/mcl. | Представлены данные по разработке тест-системы для выявления генома вируса заразного узелкового дерматита крупного рогатого скота (ЗУД КРС) методом полимеразной цепной реакции (ПЦР) в реальном времени. Методика основана на амплификации фрагмента гена LSDV 129 размером 113 пар оснований с использованием оригинальных олигонуклеотидных праймеров и флуоресцентно-меченого гибридизационного зонда. Аналитическую специфичность этой тест-системы устанавливали при исследовании панели образцов биоматериала, содержащих штаммы вируса ЗУД, оспы овец и коз, контагиозной эктимы овец, а также от клинически здоровых животных и интактные культуры клеток. Аналитическую чувствительность тест-системы определяли с помощью последовательных десятикратных разведений культурального вируссодержащего материала, она составила 1,0±0,15 lg ТЦД(50)/см3. Для создания положительного контроля амплификации к разработанной тест-системе фрагмент гена LSDV 129 клонировали в составе плазмидного вектора pGEM-TEasy в бактериальных клетках E. coli. Установили, что диапазон концентраций плазмидной ДНК, при котором соблюдается линейность амплификации, был от 3,0х10E9 до 3,0 копий ДНК/мкл.

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Central Scientific Agricultural Library - Russian Federation

2018

Upadyshev, M.T., All-Russia Selection and Technological Inst. of Horticulture and Plant Breeding, Moscow

The prevalence of viral diseases of fruit and small fruit crops in Russia varies from 32 to 80% depending on the crop. The main ways to solve the problem of sanitation of horticultural crops from viruses in Russia are the creation of breeding and nursery centers and the introduction of a modern system of planting material certification. Virus diagnosis is an important task in breeding and nursery centers. Diagnosis is carried out by testing on indicators, using serological and molecular methods, electron microscopy. The methods of ELISA and PCR are increasingly used now. Modern methods of virus diagnostics must meet the requirements of high sensitivity, specificity, reliability, suitability for mass testing of samples. Diagnostic methods need to be adapted to the biological characteristics of specific cultures and types of viruses. For successful testing, the optimal timing and type of samples, the distribution of viruses in the plant, the composition of the extractive buffer, preventing the destruction of viruses, are of great importance. The optimal period of diagnosis of viruses on fruit and small fruit crops by ELISA in open ground in Central Russia is the period from the second half of May to the end of June. The advantage of PCR is the possibility of effective diagnosis of the virus in the tissues in the rest of the plants. Improvement of the PCR method should be carried out in the direction of increasing its productivity, reducing the stages, simplifying and improving the efficiency of procedures for the release of nucleic acids from plant material. Currently, All-Russian Research Horticultural Institute for Breeding, Cultivation and Nursery is working on the sanitation and identification of plants, which are free from malicious viruses: for 2011-2017 16 thousand analysis were made and 3 thousand of virus-free plants were selected. Diagnosis of viruses on fruit and small fruit crops is one of the most important stages in the breeding and nursery centers. | Распространенность вирусных болезней в условиях России варьирует от 32 до 80% в зависимости от культуры. Основными путями решения проблемы оздоровления садовых культур от вирусов являются создание селекционно-питомниководческих центров и внедрение современной системы сертификации посадочного материала. Важная задача этих центров - диагностика вирусов. Диагностика проводится путем тестирования на индикаторах, серологическими, молекулярными методами, электронной микроскопией. Все более широкое применение находят методы иммуноферментного анализа (ИФА) и полимеразной цепной реакции (ПЦР). Современные методы диагностики вирусов должны отвечать требованиям высокой чувствительности, специфичности, достоверности, пригодности для массового тестирования образцов. Методы диагностики необходимо адаптировать к биологическим особенностям конкретных культур и видам вирусов. Для успешного тестирования большое значение имеют оптимальные сроки отбора и вид образцов, распределение вирусов по растению, состав экстрагирующего буфера, предотвращающий разрушение вирусов. Оптимальным сроком диагностики вирусов на ягодных и плодовых культурах методом ИФА в условиях открытого грунта средней полосы России является период со второй половины мая до конца июня. Преимущество ПЦР - возможность эффективной диагностики вирусов в тканях, находящихся в состоянии покоя растений. Совершенствование метода ПЦР должно проводиться в направлении повышения его производительности, сокращения этапов, упрощения и повышения эффективности процедур выделения нуклеиновых кислот из растительного материала. Во Всероссийском селекционно-технологическом институте садоводства и питомниководства проводится работа по оздоровлению и выделению свободных от вредоносных вирусов растений: за 2011-2017 гг. выполнено 16 тыс. анализов на вирусы и отобрано 3 тыс. свободных от вирусов растений. Диагностика вирусов на плодовых и ягодных культурах является одним из самых важных этапов в работе селекционно-питомниководческих центров.

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Central Scientific Agricultural Library - Russian Federation

2017

Savchenkova, I.P. | Yurov, K.P., The Ya.R. Kovalenko All-Russia Research and Development Inst. of Experimental Veterinary, Moscow

The study of cellular membrane molecules as receptors for viruses plays an important role in the antiviral agents and vaccines development. However, the molecular structure and chemical composition of receptors are not investigated in detail so far in cells as well as in viruses. The equine infectious anemia (EIA) is a viral disease of one-hoofed (horse, mule, donkey), which is characterized by recurrent fever, anemia, thrombocytopenia, and a variety of disease progression: from acute to asymptomatic latent. The information about the structure of the genome of lentivirus that induces this disease is provided. The information about equine lentivirus receptor ELR1, which is detected on the surface of horses monocytes and macrophages in vitro and identified as a protein which shared by tumor necrosis factor receptor superfamily is discussed. The ELR1 role in the penetration of virus into the cells of mammals in vitro is considered. It is shown that in vitro selection for a strain of EIAV resulted in the generation of a virus that was able to infect mammalian cells, presumably by the use of an ELR1 mediated mechanism of cell entry. Created is a laboratory model (transgenic mice) to study the pathogenesis of the virus EIA and the host immune response. Study of the receptors responsible for binding the virus to the cell membrane is an important stage for development aimed at blocking virus infection. This aim resulted in the vaccines based on modified protein shell (envs) viruses and env-receptor complexes, as well as the modified viruses based on delivery systems, targeted at specific types of cells the possibility of development. | Изучение молекул клеточной мембраны в качестве рецепторов для вирусов играет важную роль в разработке противовирусных реагентов и вакцин. Однако до настоящего времени детально не исследована молекулярная структура рецепторов и их химический состав, как у клеток, так и у вирусов. Инфекционная анемия лошадей (ИНАН) – вирусная болезнь непарнокопытных (лошадь, мул, осёл), которая характеризуется рецидивирующей лихорадкой, анемией, тромбоцитопенией и разнообразным течением: от острого до бессимптомного латентного. Представлены сведения о структуре генома лентивируса, вызывающего это заболевание. Обсуждаются данные о рецепторе вируса инфекционной анемии лошадей ELR1, который выявлен на поверхности моноцитов и макрофагов лошадей in vitro и представляет собой белок, принадлежащий к семейству рецепторов фактора некроза опухоли TNFR. Приводятся данные о его роли в проникновении вируса в клетку млекопитающих in vitro. Показано, что молекулярные знания рецепторов позволяют создавать клетки с заданными свойствами посредством генетической трансформации in vitro, которые необходимы для изучения фундаментальных вопросов. Продемонстрирована возможность создания лабораторной модели (трансгенные мыши) для изучения патогенеза вируса ИНАН и иммунного ответа организма. Таким образом, изучение рецепторов, ответственных за связывание вируса с клеточной мембраной, может служить важным этапом для разработки методов и подходов, направленных на блокирование вирусной инфекции, создания вакцин на основе модифицированных белков оболочки (envs) вирусов и комплексов env-рецепторов, а также модифицированных вирусов на основе систем доставки, ориентированных на специфические типы клеток.

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Central Scientific Agricultural Library - Russian Federation

2017

Glotov, A.G. | Koteneva, S.V. | Glotova, T.I., Siberia Federal Scientific Center of Agrobiotechnology. Institute of Experimental Veterinary of Siberia and the Far East, Novosibirsk Region

Bovine viral diarrhea is a viral disease of cattle. Bovine viral diarrhea virus (BVDV) is considered as prototype member of the Pestivirus genus. The disease is caused by two different viruses BVDV1 and BVDV2. Studies on genetic polymorphism of viruses in Russia are insufficient. The aim of this work was a phylogenetic analysis of two types of BVDV circulating in the big dairy farms in five regions of Siberia and Northern Kazakhstan based on the nucleotide sequence of highly conserved region of the genome, 5′-untranslated region (5'-UTR). Specific prevention on the disease was not carried out in these regions before. The local and imported cattle revealed the circulation of the six subtypes of BVDV1 (a, b, c, g, p, k), the presence of mixed subtypes: 1d/1n/1u and BVDV2. Predominant subtype was 1b. The number of BVDV1 was as follows: 1a, 1c, 1d/1n/1u and 1p at 3.85%; 1g – 15.38%, 1k – 7.69% and BVDV2 – 11.54%. Distribution of viruses had geographical differences. BVDV1b, BVDV1p, BVDV1g, BVDV1k and mixed subtypes 1d/1n/1u often detected in sick or persistently infected animals in farms with respiratory diseases. BVDV 1c was found in the serum of persistently infected heifer without clinical symptoms. BVDV1a and BVDV2 shed from animals with reproduction pathology, aborted fetuses and stillbirth calves. Results of the studies on the molecular epidemiology of BVDV can be used to select and to optimize control strategies and measures, and to make decisions on the vaccines usage in a particular region. | Вирусная диарея – болезнь слизистых оболочек (BVDV) – контагиозное заболевание крупного рогатого скота, вызываемое 2 прототипными членами рода Pestivirus:  BVDV1 и BVDV2. Исследование их генетического полиморфизма в России недостаточно. Целью работы был филогенетический анализ вирусов на основе секвенирования высококонсервативной области генома – 5'-нетранслируемого региона (5'-UTR), циркулирующих среди высокопродуктивного молочного скота в 5 регионах Сибири и в Северном Казахстане где на момент исследований специфическая профилактика болезни не проводилась. В результате от местного и завозного скота выделили 6 субтипов вируса первого типа (a, b, c, g, p, k), вируса второго типа, а в одном случае наличие смешанных субтипов BVDV1: 1d/1n/1u. Преобладающим субтипом являлся 1b (50% положительных проб). Количество субтипов BVDV1 было следующим: 1a, 1c, 1d/1n/1u и 1p по 3,85%; 1g – 15,38%, 1k – 7,69% и BVDV2 – 11,54%. Распространение типов и субтипов вирусов имело географические различия. BVDV1b, BVDV1p, BVDV1g, BVDV1k и смешанные субтипы 1d/1n/1u чаще выявляли у больных или персистентно инфицированных животных в хозяйствах, неблагополучных по респираторным болезням. BVDV 1c обнаружили в сыворотке крови одной персистентно инфицированной нетели без проявления клинических симптомов. Субтип 1а и тип 2 вируса установили у животных с патологией воспроизводства, у абортированных плодов и мертворожденных телят. Результаты исследования по молекулярной эпизоотологии вируса BVDV можно использовать для выбора и оптимизации стратегии контрольных мероприятий и решения вопроса о применении вакцин в конкретном регионе.

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Central Scientific Agricultural Library - Russian Federation

2016

Volkova, M.A. | Chvala, I.A. | Mudrak, N.S., Federal Centre for Animal Health, Vladimir

Egg drop syndrome-76 (EDS-76) is an infectious disease in layer hens caused by Duck adenovirus A. EDS-76 is manifested in soft-shelled or shell-less eggs and depigmentation and by a quick drop in egg production. The indirect enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) are used to detect and quantify antibodies to EDS-76 virus in chicken sera. The goal of this study was the definition of the main validation parameters ELISA method for detection of antibodies to EDS-76 virus according ISO 17025 and estimation of efficiency of using this method in the laboratory. The humoral immune responses were determined by means of ELISA and hemagglutination inhibition test (FGBI "ARRIAH"). Specificity and sensitivity of this method compared to HI is 99.36 and 98.88% accordingly. During estimation of reproducibility of the method the coefficient of variation between different lots of the analysis did not exceed 8%. The accuracy of the detection of antibodies in the ELISA was 99.25% in comparison with HI. High repeatability and reproducibility, as well as great compliance of ELISA with HI, allow using EDS -76-ELISA for serological assays with a goal of laboratory diagnosis of EDS -76, evaluation of immune status of birds and control of postvaccinal immunity. | Синдром снижения яйценоскости-76 (ССЯ-76) — инфекционная болезнь кур-несушек, вызываемая Duck adenovirus A. ССЯ-76 характеризуется размягчением, отсутствием или депигментацией скорлупы яиц и приводит к значительному снижению яичной продуктивности. В лабораторной практике для выявления антител к вирусу ССЯ-76 в сыворотках крови кур широко используют иммуноферментный анализ (ИФА) и реакцию торможения гемагглютинации (РТГА). Цель исследования — определить основные валидационные характеристики метода ИФА для выявления антител к вирусу ССЯ-76 в сыворотках крови кур в рамках подготовки к аккредитации лаборатории по ГОСТ ИСО/МЭК 17025 и оценить эффективность его применения в лабораторных условиях. Для выявления антител к вирусу ССЯ-76 в сыворотках крови кур методом РТГА и ИФА использовали коммерческие наборы производства ФГБУ "ВНИИЗЖ". Специфичность и чувствительность метода ИФА относительно РТГА составила 99,36 и 98,88%, соответственно. При оценке воспроизводимости метода коэффициент вариации между разными сериями анализа не превышал 8%. Точность выявления антител в ИФА составляла 99,25% от точности РТГА. Высокая повторяемость и воспроизводимость, а также хорошая согласованность результатов ИФА с РТГА позволяет использовать ССЯ-76-ИФА для серологических исследований с целью лабораторной диагностики ССЯ-76, оценки иммунного статуса птиц и контроля поствакцинального иммунитета.

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Central Scientific Agricultural Library - Russian Federation

2010

Panferova, A.V. | Malogolovkin, A.S. | Lunitsin, A.V. | Bobrovskaya, N.K., All-Russia Research and Development Inst. of Veterinary Virology and Microbiology, Pokrov

Genetic changes in the virus genome structure when cattle is experimentally infected with bluetongue virus (BV) 1 (BTV-1 Spain) and 8 (BTV-8 Denmark) serotypes were studied. As is shown, on the background of the absence of clinical signs the BV genome was detected at day 3, group-specific antibodies – at day 9 after infection throughput the study period (110 days). The most amount of viral RNA was present in blood samples at day 8 to 9 after infection. Serotype-specific RT-PCR discovered in blood samples taken at day 11, 13 and 15 day only BV genome, serotype 1 in a cell culture infected with the blood collected at day 29 – BV 1genome, at day 61 – BV genome, serotypes 1 and 8. A neutralization tests with a constant serum dose confirmed the results of serotype-specific RT-PCR testifying thereby to differences in reproduction times of BV of different serotypes in the course of mixed infection. Nucleotide sequencing of region 2 segment and full-size 10 segment of viral RNA showed that regions 2 and 10 of segments of genomes 1 and 2 serotypes isolated at days 29 and 61 after infection are sufficiently conservative. The percentage of homology between nucleotide sequences 2 BV-1 genome used for infection segment and BV-1 isolated at day 29 and 61 after infection was 98.8 and 98.4%, respectively, fro BV-8 – 97.9 and 98.4% of homology to the original strain BTV-8 Denmark for 2 and 10 segments. On the basis of the study carried out it is concluded about replication of BV 1 and 8 serotypes under mixed infection of cattle and absence of structural changes in segments 2 and 10 of the viral genome

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Central Scientific Agricultural Library - Russian Federation

2009

Nedachin, A. E. | Dmitrieva, R.A. | Doskina, T.V. | Dolgin, V.A. | Sanamyan, A.G. | Snegirev. D.V., Russian Academy of Medical Sciences, Moscow (Russian Federation) | Tarasov, A.V., Russian Academy of Natural Sciences, Moscow (Russian Federation) | Fedotov, Yu.A. | Lepeshin, S.A.

The paper outlines materials on how to improve a method for indication of water viral pollution in different water objects of the environment, by applying a MFM filtration module based on tangentially radial-mode water microfiltration and viral concentration on the positively charged membranes, by using the secondary stage of concentration. Experimental and full-scale studies have demonstrated that this method for virus isolation has a high effectiveness and is based on the employment of the materials made in Russia. This determines its rapid introduction into the system for the sanitary-virological monitoring of practical laboratories of various departments, which will favor the higher efficiency of epidemiological surveillance as an integral part of nonspecific prophylactic measures of virus infections of water etiology. The developed system of isolating virus from water is highly effective, based on using domestic materials and recommended for sanitary-virological control in practical laborato

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Central Scientific Agricultural Library - Russian Federation

2008

Grigorjev, V.B. | Kalnov, S.L. | Pokidyshev, A.N. | Tsibezov, V.V. | Balandina, M.V. | Gibadulin, R.A. | Verkhovskij, O.A. | Klimenko, S.M., Russian Academy of Medical Sciences, Moscow (Russian Federation). The D.I. Ivanovskij Research and Development Inst. of Virology

The transformation of normal prion protein PRP from bovine encephalopathy having alpha-spirals-in beta is based on pathogenesis of neurodegenerative zoonoses – Creutzfeldt-Jakob disease and its new variant, as well as bovine spongiform encephalopathy. Amyloidal fibrillae (scrapie-associated fibrillae SAF) being formed in the tissue are influenced by the amino acid PrP sequence, they consist of beta-Prp and have partial resistance to proteolysis. The transformation of recombinant PrP of different types of animals and human in vitro supported a possibility of interspecies transmission of prion diseases. Fibrillation of C-terminal pek-PrP-fragment of cattle in vitro was performed and presented for the first time, and the morphological peculiarities of the obtained structures are as is found peck-shown, pek-PrP of cattle in in vitro incubation in the presence of a denatured agent and pH-4.5 is polymerized into fibrillae which are similar in the structure, physical and chemical properties and high polymorphism to SAF being formed in vivo. The mechanism of self-assembling fibillae pek-PrP is shown: a) forming flexible long spiralized protofibrillae; b) forming portable rigid fibrillae due to lateral interaction of some protofibrillae; c) fragmentation of shoert rigid fibrillae followed by transformation either into short single or two badly twisted ribbon-like fibrillae having high polymorphism. It is suggested that spontaneous fragmentation of bovine PrP promotes increasing active centers of PrP conversion into a pathological isoform partially explaining easy overcoming the species barrier of PrP and its high infectivity

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Central Scientific Agricultural Library - Russian Federation

2007

Kurinnov, V.V. | Bunkova, N.I. | Balashova, E.A. | Tsybanov, S.Zh. | Zelentsov, V.A.,All-Russia Research and Development Inst. of Veterinary Virology and Microbiology, Pokrov

The role of porcine reproductive and respiratory syndrome (PRRS) virus in the etiology of massive abortions was studied at one of the pig complexes after introducing into the main herd some replacement pigs Hypur imported from Poland and seronegative for viruses of PRRS, Aujeszkij disease, transmissible gastroenteritis, Teschen disease and porcine parvoviruses. An increase in the incidence of abortions among its own and newly inseminated sows (Hypur) depended on the pregnancy stage, however, in the periods of their maximal increase, above 50%, was observed at the end of the trimester. The PRRS virus of the European variant was isolated with reference of the results of serological [EIA (PRRS-SEROTEST) and indirect immunofluorescence test (IFT)], virological (RT-PCR) and postmortem studies giving reasons to suggest it circulates among sows and boars in spite of the regular vaccination with the emulsified inactivated vaccine against PRRS (strain BD of the American variant) and porcine parvovirus infection. The infection with PRRS virus of seronegative pigs from Poland resulted in a change in its pathogenicity (hemorrhagic inflammation of navel strings in aborted fetuses at days 105-111 of pregnancy),in consequence of which the population immunity (by EIA and IFT – not above 50%) did not inhibit the transfer of the virus from a sow to sow. It is concluded, that a continuous epizootological monitoring at the pig complex, the compliance of the vaccines with the pathogenesis of diseases, the optimization of feeding and keeping conditions, as well as observing the All in, all out principle are required.

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Central Scientific Agricultural Library - Russian Federation

2007

Fevet, G. | Tencheva, D.

The infectivity of bee queens and drones with deformed wing virus (DWV), as well as its distribution within different organs and tissues were studied by the RT-PCR method, immunochemistry and electronic microscopy. DWV was detected in 67% of clinically healthy productive bee queens, 78% of drones collected at a hive entrance and 100% of drones before the hive entrance. The number of virus genome copies in RT-PCR was maximum in testes and digestive tracts of drones, lower – in glands; in queens – maximum in ovaries and the least – in heads and digestive tracts. Some attempts to identify the localization of DWV in digestive tracts and ovaries by a serological test and hybridization were not successive in spite of detection of virus RNA in RT-PCR. Adipose cells gave a strong and specific response to DWV. Therefore, the DWV infection of fatty cells in queens results in their reduced productivity. In drones DWV was detected in epithelial cells in seminal vesicles, accessory glands. The intensive replication of DWV in them influenced negatively the productivity of drones, and consequently contaminated sperm was a source of infection bee females and the next bee generations. DWV was also detected in most drones’ epithelial cells of proventriculus, mid and hind intestine. It is concluded that DWV affects certain tissues of bees’ genital and digestive tracts. Many epithelial cells are adjacent to the basal membrane which is a physiological barrier preventing from penetrating viruses into internal organs. This explains the differences in the infection running under peroral and vector infections of bees. The spread of infection in a bee family among adult bees and larvae in the nutrition process or through gland excretions is much lower.

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Central Scientific Agricultural Library - Russian Federation