Background Since the first description of lumpy skin disease virus (LSDV) in Africa in the 1920’s, it has brazenly spread beyond Africa into the Middle East, Europe and most recently Asia. In 2017 the first atypical LSDV recombinant strain was reported in Russia, composed of both a live-attenuated Neethling vaccine strain and Kenyan vaccine strain. An increase in LSDV research enabled a public release of numerous full genome sequences of unique recombinant LSDV strains from Kazakhstan, Russia, China and Vietnam. Prior to the recombinant strain first described in China in 2019, every new recombinant strain was genetically unique and each of these recombinants clustered in a monophyletic lineage. In this work, we provide the complete genome sequences of two novel recombinant strains of LSDV from Russia and attempt to gain more insight into genomic composition of all the recombinant strains currently available. This analysis will provide new insight into the global molecular epidemiology of LSDV. Results By sequencing and analyzing two novel recombinant strains Khabarovsk/2020 and Tomsk/2020, this study investigates the differences and similarities of all five the available recombinant LSDV lineages from different countries based on the SNPs inherited from the aforementioned parental strains. A total of seven recombinant strains: LSDV/Russia/Saratov/2017, LSDV/Russia/Udmurtya/2019, LSDV/KZ-Kostanay/Kazakhstan/2018, LSDV/Russia/Tyumen/2019, LSDV/GD01/China/2020 Khabarovsk/2020 and Tomsk/2020 were examined. It was observed that strains isolated prior to 2020 were composed of unique combinations of open reading frames, whilst from 2020 onwards all circulating strains in Russia and South-Eastern Asia belonged to a single lineage radiating out in the region. The first representative of this lineage is LSDV/GD01/China/2020. Interestingly, the other four unique recombinant strains as well as the newly established lineage, exhibit consistent patterns of targeted selection pointing to regions constantly selected for during the recombination-driven processes. Conclusion This study highlights the inexplicable emergence of novel recombinant strains to be unique introductions of sibling viruses, with the most recent recombinant lineage establishing as the dominant strain across the south eastern Asian countries as evidenced by full genome sequence data. Overall, these findings indicate that LSDVs are subjected to accelerated evolutionary changes due to recombination in the face of homologous live attenuated vaccines as well as the slow genetic drift commonly observed in capripoxviruses curculatign in the field with hardly any genetic changes over decades.

Objective The resurgence of lumpy skin disease virus isolates of different genotypic natures abolishes the accuracy of assays that target either vaccine or field strain genome. The aim of the present study was to develop a universal real-time PCR assay using TaqMan chemistry to cover field, vaccine, and recombinant strains of lumpy skin disease virus isolates. Results The PCR assay was designed based on a LSDV044 target region that offers a unique identification locus to facilitate the sensitive and specific detection of all isolates known to date. The efficiency of amplification, determined over five orders of magnitude, was 93%, with the standard deviation remaining in the range of 0.11–0.23. Evaluation of the assay repeatability on three different days revealed that the inter-run variability ranged from 0.83 to 1.22 over five repetitions across three runs. This new screening assay is proposed as a fast, efficient, and sensitive tool that can be employed in the basic or applied surveillance studies regardless of the genotype. Moreover, the assay can be used for the routine laboratory testing of animal samples during eradication programs for lumpy skin disease.

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Poxviruses are double-stranded DNA viruses with several members displaying restricted host ranges. They are genetically stable with low nucleotide mutation rates compared to other viruses, due to the poxviral high-fidelity DNA polymerase. Despite the low accumulation of mutations per replication cycle, poxvirus genomes can recombine with each other to generate genetically rearranged viruses through recombination, a process directly associated with replication and the aforementioned DNA polymerase. Orthopoxvirus replication is intimately tethered to high frequencies of homologous recombination between co-infecting viruses, duplicated sequences of the same virus, and plasmid DNA transfected into poxvirus-infected cells. Unfortunately, the effect of these genomic alterations on the cellular context for all poxviruses across the family Poxviridae remains elusive. However, emerging sequence data on currently circulating and archived poxviruses, such as the genera orthopoxviruses and capripoxviruses, display a wide degree of divergence. This genetic variability cannot be explained by clonality or genetic drift alone, but are probably a result of significant genomic alterations, such as homologous recombination, gene loss and gain, or gene duplications as the major selection forces acting on viral progeny. The objective of this review is to cross-sectionally overview the currently available findings on natural and laboratory observations of recombination in orthopoxviruses, capripoxviruses, and leporipoxviruses, as well as the possible mechanisms involved. Overall, the reviewed available evidence allows us to conclude that the current state of knowledge is limited in terms of the relevance of genetic variations across even a genus of poxviruses as well as fundamental features governing and precipitating intrinsic gene flow and recombination events.

Gene editing tools have become an indispensable part of research into the fundamental aspects of cell biology. With a vast body of literature having been generated based on next generation sequencing technologies, keeping track of this ever-growing body of information remains challenging. This necessitates the translation of genomic data into tangible applications. In order to address this objective, the generated Next Generation Sequencing (NGS) data forms the basis for targeted genome editing strategies, employing known enzymes of various cellular machinery, in generating organisms with specifically selected phenotypes. This review focuses primarily on CRISPR/Cas9 technology in the context of its advantages over Zinc finger proteins (ZNF) and Transcription activator-like effector nucleases (TALEN) and meganucleases mutagenesis strategies, for use in agricultural and veterinary applications. This review will describe the application of CRISPR/Cas9 in creating modified organisms with custom-made properties, without the undesired non-targeted effects associated with virus vector vaccines and bioactive molecules produced in bacterial systems. Examples of the successful and unsuccessful applications of this technology to plants, animals and microorganisms are provided, as well as an in-depth look into possible future trends and applications in vaccine development, disease resistance and enhanced phenotypic traits will be discussed.

Оптимизация параметров культивирования вируса респираторно-синцитиальной инфекции крупного рогатого скота штамма «Вологда/2019» РЕЗЮМЕ В настоящее время в изучении респираторно-синцитиальной инфекции крупного рогатого скота есть множество спорных вопросов. В связи с этим актуальным является изучение биологических свойств вируса, оптимизация методов его культивирования и подбор наиболее технологичных приемов конструирования средств диагностики и профилактики данного заболевания. Целью настоящей работы являлись выбор чувствительных клеточных систем и оптимизация параметров культивирования в подобранных культурах клеток. В опытах использовали штамм «Вологда/2019» вируса респираторносинцитиальной инфекции крупного рогатого скота, выделенный из биологического материала, полученного от теленка с признаками респираторной патологии. Штамм адаптирован к перевиваемой культуре клеток слизистой носовых перегородок крупного рогатого скота (BT) и депонирован в Государственную коллекцию штаммов микроорганизмов ФГБУ «ВНИИЗЖ». Установлено, что перевиваемые линии клеток трахеи эмбриона крупного рогатого скота (FBT) и почки теленка (RBT) являются наиболее чувствительными клеточными системами для репродукции респираторно-синцитиального вируса крупного рогатого скота штамма «Вологда/2019», в данных культурах клеток отмечалось максимальное накопление вируса. Цитопатическая активность вируса в культуре клеток FBT на 4-5 сут культивирования составила от 4,78 ± 0,18 до 5,50 ± 0,16 lg ТЦД 50 /см 3 , а в клеточной системе RBT -от 4,00 ± 0,23 до 4,75 ± 0,20 lg ТЦД 50 /см 3 . Определено, что при множественности заражения культур клеток FBT и RBT вирусом в 0,1 lg ТЦД 50 /кл, использовании в составе поддерживающей питательной среды 2% глютамина, а также 2% сыворотки крови лошади либо крупного рогатого скота удается получить вирусный материал с высокой цитопатической активностью.Ключевые слова: респираторно-синцитиальный вирус крупного рогатого скота, штамм «Вологда/2019», культивирование, цитопатическая активность, титр вируса.Благодарность: Работа выполнена за счет средств ФГБУ «ВНИИЗЖ» в рамках тематики научно-исследовательских работ «Ветеринарное благополучие».

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