In this study, we report on the full genome phylogenetic analysis of four ASFV isolates obtained from wild boars in Russia. These samples originated from two eastern and two western regions of Russia in 2019. Phylogenetic analysis indicated that the isolates were assigned to genotype II and grouped according to their geographical origins. The two eastern isolates shared 99.99% sequence identity with isolates from China, Poland, Belgium, and Moldova, whereas the western isolates had 99.98% sequence identity with isolates from Lithuania and the original Georgia 2007 isolate. Based on the full genome phylogenies, we identified three single locus targets, MGF-360-10L, MGF-505-9R, and I267L, that yielded the same resolving power as the full genomes. The ease of alignment and a high level of variation make these targets a suitable selection as additional molecular markers in future ASFV phylogenetic practices.

African swine fever virus (ASFV), classified as genotype II, was introduced into Georgia in 2007, and from there, it spread quickly and extensively across the Caucasus to Russia, Europe and Asia. The molecular epidemiology and evolution of these isolates are predominantly investigated by means of phylogenetic analysis based on complete genome sequences. Since this is a costly and time-consuming endeavor, short genomic regions containing informative polymorphisms are pursued and utilized instead. In this study, sequences of the central variable region (CVR) located within the B602L gene were determined for 55 ASFV isolates submitted from 526 active African swine fever (ASF) outbreaks occurring in 23 different regions across the Russian Federation (RF) between 2013 and 2017. The new sequences were compared to previously published data available from Genbank, representing isolates from Europe and Asia. The sequences clustered into six distinct groups. Isolates from Estonia clustered into groups 3 and 4, whilst sequences from the RF were divided into the remaining four groups. Two of these groups (5 and 6) exclusively contained isolates from the RF, while group 2 included isolates from Russia as well as Chechnya, Georgia, Armenia, Azerbaijan and Ukraine. In contrast, group 1 was the largest, containing sequences from the RF, Europe and Asia, and was represented by the sequence from the first isolate in Georgia in 2007. Based on these results, it is recommended that the CVR sequences contain significant informative polymorphisms to be used as a marker for investigating the epidemiology and spread of genotype II ASFVs circulating in the RF, Europe and Asia.

The lack of data on the whole-genome analysis of genotype II African swine fever virus (ASFV) isolates significantly hinders our understanding of its molecular evolution, and as a result, the range of single nucleotide polymorphisms (SNPs) necessary to describe a more accurate and complete scheme of its circulation. In this regard, this study aimed to identify unique SNPs, conduct phylogenetic analysis, and determine the level of homology of isolates obtained in the period from 2019 to 2022 in the central and eastern regions of Russia. Twenty-one whole-genome sequences of genotype II ASFV isolates were assembled, analyzed, and submitted to GenBank. The isolates in eastern Russia form two clades, “Amur 2022” and “Asia”. Within the latter clade, five subclusters can be distinguished, each characterized by a unique set of SNPs and indels. The isolates from the central regions of Russia (2019; 2021) form the “Center of Russia” clade, with two subclusters, “Bryansk 2021” and “Center of Russia 2021” (bootstrap confidence index = 99). The presence of the previously unique genetic variant ASFV for the Kaliningrad region in the wild boar population of the Khabarovsk region (eastern Russia; 2021) has also been confirmed.

Для успешного искоренения африканской чумы свиней, построения эффективных программ надзора и контроля за болезнью, поиска перспективных геномных маркеров для создания профилактических препаратов, реализации стратегии по дифференциации инфицированных и вакцинированных животных, а также кластеризации изолятов необходимо продолжать изучение эпизоотической ситуации и молекулярно-биологических свойств современных изолятов вируса африканской чумы свиней, выделенных на приграничных территориях Российской Федерации. Ретроспективный анализ некоторых эпизоотологических данных в отношении эпизоотии африканской чумы свиней и сравнительный генетический анализ изолятов, циркулирующих на территории Дальневосточного федерального округа, позволили предположить пути заноса и распространения возбудителя, а также определить сезонность регистрации инфекции в Приморском крае. Отмечена циркуляция двух субгенотипов вируса африканской чумы свиней (IGR-I и IGR-II), определенных по интергенному региону I73R/I329L, на территории изучаемого края в течение первых месяцев неблагополучия по заболеванию. Исследования по изучению биологических свойств изолята вируса африканской чумы свиней ASFV/Primorsky 19/WB-6723, выделенного из трубчатой кости от павшего дикого кабана на территории Приморского края, позволили охарактеризовать его как высоковирулентный, способный вызывать от сверхострой до подострой формы течения инфекции с гибелью до 100% зараженных животных. Инкубационный период и длительность течения болезни у экспериментально инфицированных свиней составили от 4 до 6 и от 3 до 5 дней после заражения соответственно. Геном вируса африканской чумы свиней при использовании метода полимеразной цепной реакции в реальном времени детектировали в пробах крови, полученной от зараженных животных на 5-8-й день после инфицирования. Специфические антитела в образцах сыворотки крови обнаружены не были. Подтверждена необходимость проведения дальнейшего изучения молекулярно-биологических свойств современных изолятов вируса африканской чумы свиней. Во избежание продолжения эпизоотии и ухудшения сложившейся ситуации требуется корректировка применяемых подходов к осуществлению эпизоотического надзора и контроля...

Introduction. Up-to-date data and full characterization of circulating ASFV isolates play a crucial role in virus eradication and control in endemic regions and countries. The aim of the study was to evaluate and characterize the molecular and biological properties of the ASFV isolate ASF/Tatarstan 20/WB-12276, conduct phylogenetic analysis, and compare the results with isolates circulating in Europe and Asia. Materials and methods. For bioassay, eight heads of the Large White pigs weighing 1520 kg/head were used. Detection of specific anti-ASFV antibodies by ELISA and immunoperoxidase method. Detection of ASFV genome was performed by qPCR. Isolation of ASF/Tatarstan 20/WB-12276 and determination of titer were performed in pig spleen cell culture. Sequencing was carried out by the Sanger method. Results. The virus was characterized as highly virulent and capable of causing acute to subacute forms of ASF. Phylogenetic analysis revealed substitutions in the genome of the ASF/Tatarstan 20/WB-12276 isolate (IGR/I73R-I329L and I267L markers) that supported the clustering of the studied variant with isolates prevalent in most of Europe and Asia. Conclusion. For the first time, the molecular and biological properties of the ASF/Tatarstan 20/WB-12276 virus isolate taken from a wild boar shot on the territory of the Republic of Tatarstan were studied and analyzed.

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.