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.

The Live Attenuated Vaccine Strain "ARRIAH" Completely Protects Goats from a Virulent Lineage IV Field Strain of Peste Des Petits Ruminants Virus.

Peste des petits ruminants (PPR) is a transboundary viral disease that affects small ruminants, such as goats and sheep, in Africa, the Middle East, and Asia, causing substantial damage to livelihoods and disrupting livestock trade. Although Russia is PPR virus (PPRV)-free, controlling PPRV in neighboring countries is the top national priority. Recent PPR outbreaks in Mongolia and other countries in the Middle East caused by a lineage IV virus represent a risk of transboundary emergence in neighboring countries, including China, Kazakhstan, and Russia. In the present study, we assessed the potency and safety of the ARRIAH live attenuated PPRV vaccine (lineage II) in Zaannen and Nubian goat breeds by challenging them with a virulent lineage IV Mongolia/2021 isolate. For comparison, two commercial vaccines of Nigeria75/1 strain were used. The ARRIAH-vaccinated animals showed an increase in body temperature of 1-1.5 °C above the physiological norm, similar to the animals vaccinated with Nigeria75/1 vaccines. In all vaccinated groups, the average rectal temperature never exceeded 39.4-39.7 °C throughout the infection period, and no clinical signs of the disease were observed, demonstrating vaccine efficacy and safety in the current experimental setting. However, the control group (mock vaccinated) challenged with Mongolia/2021 PPRV exhibited moderate-to-severe clinical signs. Overall, the findings of the present study demonstrate that the ARRIAH vaccine strain has a promising protective phenotype compared with Nigeria75/1 vaccines, suggesting its potential as an effective alternative for curbing and controlling PPR in affected countries. Although the ARRIAH vaccine against PPR is not currently endorsed by the World Organization for Animal Health due to its incomplete safety and potency profile, this study is the first step to provide experimentally validated data on the ARRIAH vaccine.

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.

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.

A Recombinant Vaccine-like Strain of Lumpy Skin Disease Virus Causes Low-Level Infection of Cattle through Virus-Inoculated Feed.

Since 1989, lumpy skin disease of cattle (LSD) has spread out of Africa via the Middle East northwards and eastwards into Russia, the Far East and South-East Asia. It is now threatening to become a worldwide pandemic, with Australia possibly next in its path. One of the research gaps on the disease concerns its main mode of transmission, most likely via flying insect vectors such as biting flies or mosquitoes. Direct or indirect contact transmission is possible, but appears to be an inefficient route, although there is evidence to support the direct contact route for the newly detected recombinant strains first isolated in Russia. In this study, we used experimental bulls and fed them via virus-inoculated feed to evaluate the indirect contact route. To provide deeper insights, we ran two parallel experiments using the same design to discover differences that involved classical field strain Dagestan/2015 LSDV and recombinant vaccine-like Saratov/2017. Following the attempted indirect contact transmission of the virus from the inoculated feed via the alimentary canal, all bulls in the Dagestan/2015 group remained healthy and did not seroconvert by the end of the experiment, whereas for those in the Saratov/2017 recombinant virus group, of the five bulls fed on virus-inoculated feed, three remained clinically healthy, while two displayed evidence of a mild infection. These results provide support for recombinant virus transmission via the alimentary canal. In addition, of particular note, the negative control in-contact bull in this group exhibited a biphasic fever at days 10 and 20, developed lesions from day 13 onwards, and seroconverted by day 31. Two explanations are feasible here: one is the in-contact animal was somehow able to feed on some of the virus-inoculated bread left over from adjacent animals, but in the case here of the individual troughs being used, that was not likely; the other is the virus was transmitted from the virus-fed animals via an airborne route. Across the infected animals, the virus was detectable in blood from days 18 to 29 and in nasal discharge from days 20 to 42. Post-mortem and histological examinations were also indicative of LSDV infection, supporting further evidence for rapid, in F transmission of this virus. This is the first report of recombinant LSDV strain transmitting via the alimentary mode.

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.

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.

Estimating evolutionary changes between highly passaged and original parental lumpy skin disease virus strains.

Research into the phylogenetic relationships of lumpy skin disease virus (LSDV) strains was long overlooked, partially due to its original restricted distribution to sub-Saharan Africa. However, recent incursions into northern latitudes, and a rapid spread causing major economic losses worldwide, have intensified additional research on the disease and the causative virus. This study delineates the phylogeny of LSDV in the context of full genome sequences of strains recovered in the field, as well as strains highly passaged in cell culture. We sequenced the oldest known field strain to date (isolate LSDV/Haden/RSA/1954 [South Africa] recovered from an outbreak in 1954), a recent field isolate (LSDV/280-KZN/RSA/2018 [South Africa] sequenced directly from blood during an outbreak in 2018) and strain LSDV/Russia/Dagestan-75 (a high-passaged cell culture strain derived from the field strain, LSDV/Russia/Dagestan/2015 [Russia]). Sequence analysis placed the field strain LSDV/Haden/RSA/1954 in the same cluster (cluster 1.1) with attenuated Neethling-type commercial vaccine viruses, with eight SNP differences, discrediting the previously held hypothesis that cluster 1.1 vaccine strains were derived from cluster 1.2 field viruses via the process of attenuation between them. In contrast, the recent LSDV/280-KZN/RSA/2018 isolate grouped with other recent field isolates in cluster 1.2, providing evidence that cluster 1.1 strains were displaced by cluster 1.2 strains in South Africa. Based on the field isolates between 1954 and 2018, the substitution rate of 7.4 × 10-6 substitutions/site/year was established, with mutations occurring in either synonymous sites or intergenic regions. This is the first evolutionary metric recorded for LSDV. Comparing the genome sequences of high-passage strains of LSDV showed that propagation in vitro without animal host selective pressure generates mainly non-synonymous SNPs in virus-replication genes. These results improve our understanding of LSDV evolution and demonstrate that the population dynamics of circulating isolates is not constant, with LSDV associated with different genetic clusters dominating the landscape during specific periods in time.

Performance of the currently available DIVA real-time PCR assays in classical and recombinant lumpy skin disease viruses.

The use of live homologous vaccines to protect against lumpy skin disease virus (LSDV) infection requires the use of molecular tools to differentiate between infected and vaccinated animals (DIVA). In this study, the commercial real-time PCR assays; ID Gene™ LSD DIVA Triplex kit and Bio-T kit® LSD - DIVA, as well as published assays targeting the GPCR gene (Journal of Virological Methods, 249, 48-57) and ORF008 and ORF126 (Sel'skokhozyaistvennaya Biologiya, 54, 347-358) were evaluated. These assays correctly identified classical field isolates (European lineage) and vaccine (Neethling vaccine). In contrast, when tested using vaccine-like recombinant viruses, the commercial and published assays were not able to correctly identify recombinant isolates. At the same time, the recombinant viruses were detected as either field and/or vaccine, or not detected at all depending on the assay. The different gene sequences present in recombinant viruses cause these DIVA assays to incorrectly assign recombinant viruses as either a field or vaccine virus. This observation has implications for using these assays and for identification of LSDV vaccine.

A lumpy skin disease virus which underwent a recombination event demonstrates more aggressive growth in primary cells and cattle than the classical field isolate.

Genomic changes by recombination have been recently observed in lumpy skin disease viruses circulating in Russia. The first characterized naturally occurring recombinant lumpy skin disease virus Saratov/2017 occurred through recombination between a live attenuated virus vaccine and the Southern African lumpy skin disease virus. Understanding if recombination can increase or decrease virulence of viruses through changes in different gene regions is required to improve the understanding of capripoxvirus biology. In this study, the in vitro and in vivo growth of the recombinant Saratov/2017 and the classical field isolate Dagestan/2015 was compared. Primary lamb kidney and lamb testis cells as well as the goat ovarian cell line were used to assess virus replication. In the goat ovarian cell line, Saratov/2017 and Dagestan/2015 induced comparable cytopathic activity and virus titres. In contrast, in primary lamb kidney and lamb testis cells, Saratov/2017 grew more aggressively causing more massive rounding up of cells, detachment and agglomeration compared to Dagestan/20152015. Growth curves of Saratov/2017 and Dagestan/2015 were assessed in primary lamb testis cells using different multiplicities of infection (MOI), with Saratov/2017 demonstrating faster replication at the different MOI and time points evaluated post-infection. In cattle, Saratov/2017 demonstrated more pronounced skin reactions when titrated by skin inoculation of serially diluted virus. In both primary cells and cattle, the titre of Saratov/2017 was significantly higher compared to Dagestan/2015 (p ≤ .05). These results demonstrate recombinant Saratov/2017 exhibits more aggressive replication properties.

Full-length genome characterization of a novel recombinant vaccine-like lumpy skin disease virus strain detected during the climatic winter in Russia, 2019.

An uncharacteristic outbreak of lumpy skin disease was reported in the Republic of Udmurtiya, Russia, during the climatic winter of March 2019. The causative lumpy skin disease virus (LSDV_Udmurtiya_Russia_2019) was shown to be a recombinant composed of a live attenuated Neethling-type vaccine strain as the dominant parental strain and a Kenyan KSGP/NI-2490-like virus as its minor parental strain, with 24 statistically significant recombination events that are not identical to those in LSDV Saratov/2017, in which 27 events were identified.