A proposed update of African swine fever virus (genotype II) subgenotyping based on the central variable region (CVR) of Russian isolates.

African swine fever virus (ASFV) isolates are grouped and tracked through analysis of their central variable region (CVR) sequences. In this study, sequences of 70 ASFV isolates collected from different regions of Russia between 2018 and 2022 were analyzed. The analysis based on the CVR sequences indicated that the isolates belonged to three distinct groups. Group 1 shared 100% sequence identity to the isolate Georgia 2007/1. Group 5 had a C > A single-nucleotide polymorphism (SNP) at position 601, while group 13 is new and unique to the Far East of Russia, with five isolates from the Amur, Khabarovsk, and Primorsky regions. These findings demonstrate a new approach to phylogenomics and cladistics of ASFV isolates within genotype II on the basis of the CVR.

Subclinical infection caused by a recombinant vaccine-like strain poses high risks of lumpy skin disease virus transmission.

Lumpy skin disease (LSD) is a transboundary viral infection, affecting cattle with characteristic manifestations involving multiple body systems. A distinctive characteristic of lumpy skin disease is the subclinical disease manifestation wherein animals have viremia and shed the virus through nasal and ocular discharges, while exhibiting no nodules but enlarged lymph nodes that are easily oversighted by inexperienced vets. Further research on the role of subclinically ill animals in the transmission of LSD virus (LSDV) can contribute to the development of more effective tools to control the disease worldwide. Thus, this study aims to determine the potential role of subclinical infection in virus transmission in a non-vector-borne manner. To achieve this, we inoculated animals with the recombinant vaccine-like strain (RVLS) Udmurtiya/2019 to cause clinical and subclinical LSDV infection. After the disease manifestation, we relocated the subclinically ill animals to a new clean facility followed by the introduction of another five animals to determine the role of RVLS-induced subclinical infection in the virus transmission via direct/indirect contact. After the introduction of the naïve animals to the relocated subclinically ill ones in a shared airspace, two introduced animals contracted the virus (clinically and subclinically), showing symptoms of fever, viremia, and seroconversion in one animal, while three other introduced animals remained healthy and PCR-negative until the end of the study. In general, the findings of this study suggest the importance of considering LSDV subclinical infection as a high-risk condition in disease management and outbreak investigations.

Metagenomic profiling of viral and microbial communities from the pox lesions of lumpy skin disease virus and sheeppox virus-infected hosts.

Introduction: It has been recognized that capripoxvirus infections have a strong cutaneous tropism with the manifestation of skin lesions in the form of nodules and scabs in the respective hosts, followed by necrosis and sloughing off. Considering that the skin microbiota is a complex community of commensal bacteria, fungi and viruses that are influenced by infections leading to pathological states, there is no evidence on how the skin microbiome is affected during capripoxvirus pathogenesis. Methods: In this study, shotgun metagenomic sequencing was used to investigate the microbiome in pox lesions from hosts infected with lumpy skin disease virus and sheep pox virus. Results: The analysis revealed a high degree of variability in bacterial community structures across affected skin samples, indicating the importance of specific commensal microorganisms colonizing individual hosts. The most common and abundant bacteria found in scab samples were Fusobacterium necrophorum, Streptococcus dysgalactiae, Helcococcus ovis and Trueperella pyogenes, irrespective of host. Bacterial reads belonging to the genera Moraxella, Mannheimia, Corynebacterium, Staphylococcus and Micrococcus were identified. Discussion: This study is the first to investigate capripox virus-associated changes in the skin microbiome using whole-genome metagenomic profiling. The findings will provide a basis for further investigation into capripoxvirus pathogenesis. In addition, this study highlights the challenge of selecting an optimal bioinformatics approach for the analysis of metagenomic data in clinical and veterinary practice. For example, direct classification of reads using a kmer-based algorithm resulted in a significant number of systematic false positives, which may be attributed to the peculiarities of the algorithm and database selection. On the contrary, the process of de novo assembly requires a large number of target reads from the symbiotic microbial community. In this work, the obtained sequencing data were processed by three different approaches, including direct classification of reads based on k-mers, mapping of reads to a marker gene database, and de novo assembly and binning of metagenomic contigs. The advantages and disadvantages of these techniques and their practicality in veterinary settings are discussed in relation to the results obtained.

Genome sequence characterization of the unique recombinant vaccine-like lumpy skin disease virus strain Kurgan/2018.

In 2018, the molecular epidemiology of lumpy skin disease in Russia was characterized by a surge in novel recombinant vaccine-like strains causing outbreaks along the southern border, spreading in an easterly direction. Currently, five distinct novel recombinant vaccine-like lineages have been described, designated as clusters 2.1 to 2.5. Based on the complete genome sequence analysis of the causative lumpy skin disease virus (Kurgan/Russia/2018), obtained from an eponymous outbreak, the genome was shown to be composed of a Neethling vaccine strain virus as the dominant parental strain and KSGPO vaccine virus as its minor parental strain. These features are similar to those of Saratov/Russia/2017 and Tyumen/Russia/2018, representing clusters 2.1 and 2.4, respectively. However, Kurgan/Russia/2018 has 16 statistically significant recombination events unique to this sequence, contributing to the phylogenetic clustering of Kurgan/Russia/2018 in yet another cluster designed cluster 2.6, based on analysis involving the complete genome sequences.

Phylogenetic Characterization of Rabies Virus Field Isolates Collected from Animals in European Russian Regions in 2009-2022.

Rabies is a fatal disease of mammals that poses a high zoonotic risk to humans as well. The distribution of rabies is mainly driven by host animal migration and human-mediated dispersion. To contribute to the global understanding of the rabies virus (RABV) molecular epidemiology, 94 RABV field isolates collected from animals in 13 European Russian regions were phylogenetically characterized using the nearly full-size N gene nucleotide sequences. According to phylogenetic inferences, all isolates belonged to one of the two established phylogenetic groups, either group C (n = 54) or group D (n = 40), which are part of the clade Cosmopolitan of RABVs. Some representatives of group C collected from regions located far apart from each other had a remarkably high level of nucleotide identity. The possibility of the contribution of local bat species to the distribution of RABVs was discussed. Interestingly, over the years, the fraction of group D isolates has been constantly decreasing compared with that of group C isolates. The phylogenetic insights generated herein might have an important contribution to the control and surveillance of animal rabies epidemiology in the region.

Novel Protocol for the Preparation of Porcine Bone Marrow Primary Cell Culture for African Swine Fever Virus Isolation.

Isolation of African swine fever virus (ASFV) is a critical step towards the identification, titration, characterization, and even modification of the virus. Therefore, it is important to identify a suitable cell line that supports the efficient replication of ASFV for these purposes. This should be achieved even when starting with a low virus load, as in the case of isolating the virus from field samples. This article presents a detailed protocol on the preparation of porcine bone marrow primary (PBMP) cell culture, which has a high sensitivity towards ASFV, resulting in high viral yields with a minimal risk of bacterial contamination.

Development and application of a real-time polymerase chain reaction assay to detect lumpy skin disease virus belonging to the Kenyan sheep and goat pox group.

Lumpy skin disease (LSD) outbreaks in Southeast and South Asia are attributed to different lineages of LSD virus (LSDV). Variants belonging to the novel recombinant cluster 2.5 circulate in China and Thailand, while a Kenyan sheep and goat pox (KSGP) strain from cluster 1.1 circulates in India, Pakistan, and Bangladesh. The clusters representing these circulating strains are vastly different. However, if their distribution encroaches into each other's ranges, it will be impossible to differentiate between them due to the lack of suitable molecular tools. Thus, fit-for-purpose molecular tools are in demand to effectively and timeously diagnose and investigate the epidemiology of LSDVs in a region. These could significantly contribute to the phylogenetic delineation of LSDVs and the development of preventive measures against transboundary spillovers. This work aimed to develop a real-time polymerase chain reaction assay targeting open reading frame LW032, capable of specifically detecting KSGP-related isolates and recombinant LSDV strains containing the KSGP backbone. The analytical specificity was proven against the widest possible panel of recombinant vaccine-like LSDV strains known to date. The amplification efficiency was 91.08%, and the assay repeatability had a cycle threshold variation of 0.56-1.1 over five repetitions across three runs. This KSGP-specific assay is reliable and fast and is recommended for use in LSDV epidemiological studies where the accurate detection of KSGP genetic signatures is a priority, particularly in regions where KSGP-like and other lineages are circulating.

Comparison of Gross Pathology between Classical and Recombinant Lumpy Skin Disease Viruses.

The pathology caused by three different isolates of lumpy skin disease virus, classical field cluster 1.2 strain Dagestan/2015, recombinant vaccine-like cluster 2.1 strain Saratov/2017, and cluster 2.2 strain Udmurtiya/2019, in cattle was compared from experimental infections. The infection of cattle was performed using intravenous administration of 2 mL of 105 TCID50/mL of each specific LSDV. Both classical and recombinant forms of LSDV cause pathological changes in the skin and lymph nodes, as well as the trachea and lungs. Due to circulatory disorders in the affected organs, multiple areas of tissue necrosis were observed, which, with the resurgence of secondary microflora, led to the development of purulent inflammation. Observed pathological changes caused by the recombinant vaccine-like strain Udmurtiya/2019 were characterized by a more pronounced manifestation of the pathoanatomical picture compared to the classical field strains Dagestan/2015 and Saratov/2017. Interestingly, Dagestan/2015 and Udmurtiya/2019 caused damage to the lymph nodes, characterized by serous inflammation and focal purulent lymphadenitis caused by purulent microflora. "Saratov/2017" did not cause pathology in the lymph nodes. All LSDVs were virulent and caused pathology, which was not distinguishable between viruses. This data set will serve as the experimentally validated basis for the comparative examination of novel LSDV strains in gross pathology.

Post-genomic era in agriculture and veterinary science: successful and proposed application of genetic targeting technologies.

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.

A Guide to Molecular Characterization of Genotype II African Swine Fever Virus: Essential and Alternative Genome Markers.

African swine fever is a contagious viral disease that has been spreading through Europe and Asia since its initial report from Georgia in 2007. Due to the large genome size of the causative agent, the African swine fever virus (ASFV), the molecular epidemiology, and virus evolution are analyzed by employing different markers. Most of these markers originate from single nucleotide polymorphisms or disparities in the copy number of tandem repeat sequences observed during the comparisons of full genome sequences produced from ASFVs isolated during different outbreaks. Therefore, consistent complete genome sequencing and comparative analysis of the sequence data are important to add innovative genomic markers that contribute to the delineation of ASFV phylogeny and molecular epidemiology during active circulation in the field. In this study, the molecular markers currently employed to assess the genotype II ASFVs circulating in Europe and Asia have been outlined. The application of each of these markers to differentiate between ASFVs from related outbreaks is described to implement a guideline to their suitability for analyzing new outbreaks. These markers do not signify the complete repertoire of genomic differences between ASFVs, but will be beneficial when analyzing the first outbreaks in a new region or a large number of samples. Furthermore, new markers must be determined via complete genome sequence analyses for enabling in-depth insights into the molecular epidemiology of ASFV.

Experimentally controlled study indicates that the naturally occurring recombinant vaccine-like lumpy skin disease strain Udmurtiya/2019, detected during freezing winter in northern latitudes, is transmitted via indirect contact.

Lumpy skin disease (LSD) caused by LSD virus (LSDV), is a member of the poxvirus genus Capripoxvirus. It is classified as a notifiable disease by the World Organization for Animal Health (WOAH) based on its potential for rapid spread and global economic impact. Due to these characteristics, the mode of LSDV transmission has prompted intensive research efforts. Previous experimental studies using the virulent vaccine-derived recombinant LSDV strain Saratov/2017, demonstrated that this strain has the capacity for transmission in a vector-proof environment. This study demonstrated that a second novel recombinant vaccine-derived LSDV strain Udmurtiya/2019, can infect bulls in contact with diseased animals, in the absence of insect vectors. Bulls were housed in an insect proof animal biosafety level 3 facility, where half the animals were inoculated intravenously with the recombinant LSDV (Udmurtiya/2019), whilst the remaining five animals were mock-inoculated but kept in contact with the inoculated group. Both the infected / inoculated group (IN) and uninfected / incontact group (IC), were monitored for 41 days with continuous registration of body temperature, observations for clinical signs and collection of blood samples and nasal swabs for testing of LSDV presence using real-time PCR. Results indicated that cohabitation of animals from both groups was sufficient to transmit the virus from the IN to the IC-group, with the onset of clinical signs including pyrexia (~41°C) and classical LSD nodular skin lesions starting at 10 dpi for the IN group and 16 dpi for the IC-group. Additionally, the presence of LSDV genomes as well as anti-LSDV antibodies were detected in swabs, blood and serum samples from animals belonging to both groups. These results provides additional evidence of LSDV transmission in a controlled environment without direct contact between diseased and healthy animals, yet in the absence of vectors. Based on these observations, the question concerning a hypothetical relation between mutations in the virus genome and its mode of transmission gains more importance and requires additional investigations with direct comparisons between classical and novel recombinant LSDV strains.

Genetic characterization and epidemiological analysis of the first lumpy skin disease virus outbreak in Mongolia, 2021.

Novel lumpy skin disease virus (LSDV) strains of recombinant origin are on the rise in South East Asia following the first emergence in 2017, and published evidence demonstrates that such genetic lineages currently dominate the circulation. Mongolia reported first LSD outbreaks in 2021 in a north-eastern region sharing borders with Russia and China. For each of 59 reported LSDV outbreaks, the number of susceptible animals ranged from 8 to 8600 with a median of 572, while the number of infected animals ranged from one to 355 with a median of 14. Phylogenetic inferences revealed a close relationship of LSDV Mongolia/2021 with recombinant vaccine-like LSDV strains from Russia, China, Taiwan, Thailand and Vietnam. These findings support the published data that the circulating strain of LSDV belongs to the dominant recombinant lineage recently established in the region.

Capripoxviruses, leporipoxviruses, and orthopoxviruses: Occurrences of recombination.

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.

Genetic characterization of sheep pox virus strains from outbreaks in Central Russia in 2018-2019.

This study investigates the phylogenomic relatedness between sheep pox viruses (SPPVs) circulating in Central Russia in 2018-2019 with the NISKHI vaccine strain used in the country, based on their complete genome sequences. The sheep pox outbreaks occurred 1 year apart in the adjacent regions of Tula and Moscow. Full genome sequences were generated by sequencing DNA directly obtained from Trizol-extracted scabs, using the DNBSEQ-400 platform (MGI Tech, China). Phylogenetic analysis indicated that the SPPV isolates from Russia clusters with previously published sequences from Srinagar in the Kashmir province of India in 2000 (SPPV-Srinagar strain) as well as SPPV A strain from Kazakhstan in 2000. The aforementioned cluster belonged to a sister clade containing the NISKHI vaccine strain, thus indicating that the recent outbreaks were not genetically linked to the widely used vaccine.

Genetic Characterization of the Central Variable Region in African Swine Fever Virus Isolates in the Russian Federation from 2013 to 2017.

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.

Phylogenetic analysis of lumpy skin disease virus isolates in Russia in 2019-2021.

Lumpy skin disease continues to pose a threat to countries in the East and Asia-Pacific regions. Although only occasional LSDV outbreaks have been reported recently in Russia, these have been mainly restricted to the Far East region of the country. An increase in the number of outbreaks in South East Asia has been attributed to recombinant vaccine-like LSDV strains. In this scenario, it is epidemiologically important to perform phylogenetic analysis to track the distribution of LSDV worldwide at the genetic level to understand routes of migration and molecular evolution patterns. In this study, we investigated the RPO30 and GPCR gene regions of LSDV isolates associated with outbreaks in 2019-2021 in Siberia and the Far East region of Russia. The inferred phylogeny confirms the recombinant origin of these sequenced isolates. Based on sequences of these selected loci, the isolates from 2019 differed from isolates detected in Russia in the past and from isolates from Asian countries, while the isolates from 2020 and 2021 exhibited a high degree of similarity to the Asian isolates. These findings indicate that recombinant LSDV strains continue to persist and additionally point to the establishment of a specific lineage of recombinant isolates in the region. Full genome sequencing is still needed to gain more information about how the circulating isolates are related to each other.

Whole-genome sequencing of African swine fever virus from wild boars in the Kaliningrad region reveals unique and distinguishing genomic mutations.

Introduction: Since the first report of outbreaks of African swine fever (ASF) in Georgia in 2007, the disease has expanded into Europe, Russia, and Asia, spreading rapidly via contact with infected animals including domestic pigs and wild boars. The vast expansion of this Genotype II African swine fever virus (ASFV) across wide-ranging territories and hosts inevitably led to the acquisition of novel mutations. These mutations could be used to track the molecular epidemiology of ASFV, provided that they are unique to strains restricted within a certain area. Whilst whole-genome sequencing remains the gold standard for examining evolutionary changes, sequencing of a single locus with significant variation and resolution power could be used as a rapid and cost-effective alternative to characterize multiple isolates from a single or related outbreak. Material and methods: ASFVs obtained during active ASF outbreaks in the Russian region of Kaliningrad between 2017 and 2019 were examined. Since all of the viruses belonged to Genotype II and no clear differentiation based on central variable region (CVR) sequencing was observed, the whole-genome sequences of nine ASFV isolates from this region were determined. To obtain insights into the molecular evolution of these isolates, their sequences were compared to isolates from Europe, Asia, and Africa. Results: Phylogenetic analysis based on the whole-genome sequences clustered the new isolates as a sister lineage to isolates from Poland and Germany. This suggests a possible shared origin followed by the addition of novel mutations restricted to isolates from this region. This status as a sister lineage was mirrored when analyzing polymorphisms in MGF-505-5R and MGF-110-7L, whilst a polymorphism unique to sequences from Kaliningrad was identified at locus K145R. This newly identified mutation was able to distinguish the isolates obtained from Kaliningrad with sequences of Genotype II ASFVs available on GenBank. Discussion: The findings of this study suggest that ASFVs circulating in Kaliningrad have recently obtained this mutation providing an additional marker to the mutations previously described.

Comparative Analysis of Full Genome Sequences of African Swine Fever Virus Isolates Taken from Wild Boars in Russia in 2019.

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.

A Long-Term Study of the Biological Properties of ASF Virus Isolates Originating from Various Regions of the Russian Federation in 2013-2018.

Biological properties of the African swine fever (ASF) virus isolates originating from various regions of the Russian Federation (2013-2018) were studied in a series of experimental infections. Comparative analysis allowed us to establish the differences in the key characteristics of the infection, such us the duration of the incubation periods, disease, and the onset of death. The incubation period averaged 4.1 days, varying from 1 to 13 days. An average duration of the disease was 6.3 days and varied from 0 to 18 days. Overall case fatality was 94.5%, and antibodies were detected only in 19.3% of the animals. The biological properties of isolates Odintsovo 02/14 and Lipetsk 12/16 were significantly different from others. For this two, the presence of antibodies to the virus was detected in 71.4% and 75% of animals respectively and mortality levels were of 87.5% and 50%.