ЭПИЗООТОЛОГИЯВспышка заболевания, вызванная вирусом геморрагической болезни кроликов 2-го генотипа на территории РФ РЕЗЮМЕ Актуальность. Вирус геморрагической болезни кроликов 2-го типа (ВГБК-2) (rabbit hemorrhagic disease virus (RHDV GI2, RHDV-2)) впервые был обнаружен в 2010 году во Франции и быстро распространился по европейским странам. В отличие от штаммов первой геногруппы, RHDV GI2 поражает и кроликов моложе двух месяцев, а также различные виды зайцев. Кроме того, RHDV GI2 вызывает гибель кроликов, вакцинированных вакцинами, изготовленными на основании вирусов геморрагической болезни 1-го генотипа. Летом 2019 года в приусадебном хозяйстве в Тульской области произошла вспышка заболевания кроликов, характеризующаяся внезапной гибелью кроликов разных возрастных групп. Целью нашей работы было обнаружение и получение молекулярно-генетической характеристики возбудителя.Методы. Образцы патологического материала от павших животных были исследованы в ИФА с целью выявления антигена ВГБК и в ОТ-ПЦР с использованием пары праймеров, амплифицирующих фрагмент длинной 510 нуклеотидов, включающийучастки генов VP1 и VP60. В дальнейшем проводили секвенирование по Сэнгеру на геномном анализаторе AB3130, компьютерный анализ полученных последовательностей и построение филогенетических дендрограмм.Результаты. Лабораторные исследования образцов печени павших животных в ИФА и ОТ-ПЦР показали наличие антигена и РНК вируса геморрагической болезни кроликов. Анализ нуклеотидной последовательности участка генома длиной 470 пар оснований, содержащего 5-концевой участок гена VP1 и 3-концевой участок гена VP60, показал, что выявленный вирус принадлежит к ВГБК 2-го генотипа. Наиболее близкими к нему оказались последовательности вирусов из США, Франции и Польши, выделенные в период 2016-2020 годов. В аминокислотной последовательности белка VP60 выявленный вирус содержит замену А46Т (аланин на треонин в позиции 46), что может влиять на конформацию капсидного белка, и как следствие, антигенную структуру вируса. Outbreak of the disease caused by the rabbit hemorrhagic disease virus 2 in the Russian Federation ABSTRACTRelevance. Rabbit haemorrhagic disease virus type 2 (RHDV-2, GI2,) was first detected in 2010 in France and quickly spread across еuropean countries. Unlike strains of the first genogroup, RHDV GI2 also affects rabbits under 2 months old, as well as various types of hares. In addition, RHDV GI2 causes the death of rabbits vaccinated with vaccines based on type 1 hemorrhagic disease viruses. The rabbit hemorrhagic disease virus 2 (RHDV-2) was detected during an outbreak of the disease, characterized by the sudden death of rabbits of different age groups, in the summer of 2019 in a private farm in the Tula region, Russia. The purpose of this study was to detect and obtain the molecular genetic characteristics of the pathogen.
Viral hemorrhagic disease of rabbits is an acute, highly contagious disease characterized by the phenomena of hemorrhagic diathesis in all organs, especially in the lungs and liver. The causative agent of viral haemorrhagic disease of rabbits is a virus of haemorrhagic disease of rabbits, belonging to the family Caliciviridae, genus Lagovirus. Currently, there are 4 genogroups of lagoviruses, two pathogenic: GI1 (GI1a-GI1d) and GI2, and two non-pathogenic: GI3 and GI4. The greatest danger to rabbits in the Russian Federation is posed by viruses of the GI1 genotype. The virulence of these viruses for rabbits is extremely high, the incubation period is 48-72 hours. Clinically, the disease is almost not manifested. Mortality can reach 100%. For the prevention of HBV in the Russian Federation, inactivated tissue vaccines are used, which are a suspension of the liver of rabbits infected with virulent strains of the rabbit hemorrhagic disease virus. Currently, in veterinary practice, subunit recombinant vaccines based on virus proteins obtained in the baculovirus gene expression system are increasingly used. The authors obtained the recombinant VP60 virus of rabbit hemorrhagic disease of the GI1 genotype in the baculovirus gene expression system and studied its antigenic and immunogenic activity for rabbits. It was found that the recombinant capsid protein VP60 of the hemorrhagic disease virus, administered to rabbits at a dose of 50 pg, causes the synthesis of specific antibodies in animals, detected by enzyme immunoassay and in the hemagglutination inhibition reaction, and protects 80% of animals during control infection with the virulent strain «Voronezh-87» at a dose of 1000 LD 50. These data indicate the possibility of using this protein as a specific component of a subunit vaccine against rabbit hemorrhagic disease caused by strains of the GI1 gene group.
Introduction. Rabbit hemorrhagic disease is an acute highly contagious infection associated with two genotypes of pathogenic Lagovirus. Antibodies to major capsid protein (Vp60) are protective. The aim of the work ‒ is an evaluation of antigenic and immunogenic activity of virus-like particles (VLPs) based on recombinant major capsid proteins of both genotypes of rabbit hemorrhagic disease virus (RHDV) (recVP60-GI1 and recVP60-GI2). Materials and methods. Baculovirus-expressed VLPs were evaluated using electron microscopy and administered to clinically healthy 1.53 month old rabbits in a dose of 50 g. Rabbits were challenged with 103 LD50 of virulent strains Voronezhsky-87 and Tula 21 days post immunization. Serum samples were tested for the presence of RHDV-specific antibodies. Results. VLPs with hemagglutination activity forming VLP 3040 nm in size were obtained in Hi-5 cell culture. Specific antibody titers in rabbits measured by ELISA were 1 : 200 to 1 : 800 on 21th day post immunization with VLPs. Immunogenic activity of recVP60-GI1 VLPs was 90 and 40%, while it was 30 and 100% for recVP60-GI2 VLPs after the challenge with RHDV genotypes 1 and 2 respectively. The immunogenicity of two VLPs in mixture reached 100%. Discussion. VLPs possess hemagglutinating, antigenic and immunogenic activity, suggesting their use as components in substances designed for RHDV specific prophylaxis in rabbits. Results of the control challenge experiment demonstrated the need to include the antigens from both RHDV genotypes in the vaccine. Conclusion. Recombinant proteins recVP60-GI1 and recVP60-GI2 form VLPs that possess hemagglutinating an antigenic activity, and provide 90100% level of protection for animals challenged with RHDV GI1 and GI2 virulent strains.
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