An in-depth bioinformatic analysis of the novel recombinant lumpy skin disease virus strains: from unique patterns to established lineage.

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.

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.

An Attenuated Vaccine Virus of the Neethling Lineage Protects Cattle against the Virulent Recombinant Vaccine-like Isolate of the Lumpy Skin Disease Virus Belonging to the Currently Established Cluster 2.5.

Lumpy skin disease (LSD) is an emerging transboundary and highly infectious viral disease mainly affecting cattle. The fact that it was initially confined to Africa and then spread beyond its geographical range to other regions, including the Middle East, Turkey, Europe, the Balkans, Russia and Asia, is an indication of the underestimation and neglect of this disease. Vaccination is considered the most effective way to control the spread of LSDV, when combined with other control measures. LSD is now on the rise in Southeast Asia, where the circulating virus belongs to recombinant lineage 2.5. In this study, we evaluated the efficacy of an attenuated LSDV strain belonging to the Neethling cluster 1.1 by challenge with a virulent recombinant vaccine-like LSDV isolate "Mongolia/2021" belonging to cluster 2.5. Some of the vaccinated animals showed an increase in body temperature of 1-1.5 °C above the physiological norm, without clinical signs, local reactions, vaccine-induced viremia or generalization, demonstrating the efficacy and safety of the vaccine strain against a recombinant strain. Furthermore, all the vaccinated animals showed strong immune responses, indicating a high level of immunogenicity. However, the control group challenged with "Mongolia/2021" LSD showed moderate to severe clinical signs seen in an outbreak, with high levels of virus shedding in blood samples and nasal swabs. Overall, the results of the present study demonstrate that the attenuated LSDV Neethling strain vaccine has a promising protective phenotype against the circulating strains, suggesting its potential as an effective tool for the containment and control of LSD in affected countries from Southeast Asia.

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.

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.

Molecular characterization of a novel recombinant lumpy skin disease virus isolated during an outbreak in Tyumen, Russia, in 2019.

Lumpy skin disease virus causes a debilitating pox disease of domesticated cattle and water buffalos. In the last decade, LSDV has spread from Africa into the Middle East, Europe and most recently Asia. As of 2017, atypical outbreaks caused by novel LSDV strains were reported in Russia, followed by China and Vietnam between 2018 and 2020. In this work, we describe another unique recombinant LSDV strain recovered from Tyumen, Russia in 2019. Typing of the virus using currently available qPCR protocols produced inconclusive results and subsequently the complete genome of the isolate was determined. The consensus genome contained statistically significant signals of possible recombination events between parental strains KSGPO-240/Kenya/1958 and the live attenuated vaccine LW/1958. The novel strain carries 25 unique breakpoints different from the known recombinant strains. Additionally, the findings reiterate the importance of complete genome sequencing when analysing outbreak samples caused in particular by mosaic LSDV, in contrast to only performing specified qPCRs.

Overwintering of recombinant lumpy skin disease virus in northern latitudes, Russia.

Lumpy skin disease is an emerging transboundary infection demonstrating a great range expansion worldwide recently. With many knowledge gaps, there is a lack of understanding how lumpy skin disease virus (LSDV), including naturally occurring vaccine-like LSDV, is capable of surviving under different climatic conditions. In this study, we describe a recombinant vaccine-like LSDV from an outbreak in Saratov region of Russia in 2019, where the first recombinant Saratov/2017 was documented. Although the two isolates were two years apart, Saratov/2019 seems to be clonally derived from Saratov/2017 with accrual of mutations characteristic of circulating under selective conditions. The obtained findings demonstrate the persistence of LSDV during winter and successful overwintering in in cold climate, necessitating an objective need for deeper research into LSDV biology.

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.

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.