Lumpy Skin Disease Virus ORF137 Protein Inhibits Type I Interferon Production by Interacting with and Decreasing the Phosphorylation of IRF3

Lumpy skin disease (LSD) is a severe infectious, emerging transboundary disease of cattle, caused by a Pox family DNA virus. Lumpy skin disease virus (LSDV) infection is associated with a febrile response followed by emergence of widespread dermal nodules. In addition to the skin, LSDV resides in multiple internal organs and can be isolated from the blood of infected cattle. LSDV is suggested to be mechanically transmitted by biting arthropods. Live attenuated vaccines are commonly used to control disease and its spread. We have characterized the tropism, replication, and dissemination of a LSDV field isolate and of an attenuated vaccine strain usingin vitrosystems. To follow virus infection and dissemination in living cells, we have generated recombinant viruses expressing green fluorescent protein (GFP) under a synthetic viral promoter. Recombinant, GFP-expressing, LSDVs demonstrated similar replication kinetics to their corresponding parental LSDV strains in a bovine kidney cell line (MDBK). We further demonstrated that LSDV-GFP productively replicated in a bovine macrophage cell line and in primary bovine foreskin cells with no apparent differences between the field isolate and the vaccine strain. When bovine peripheral blood mononuclear cells (PBMCs) were infected with either LSDV recombinant strain, we observed specific viral driven GFP fluorescence as well as significant viral gene expression. However, infected PBMCs failed to support substantial viral DNA replication and release of infectious progeny. Subsequent analysis of the anti-viral response revealed that heat treated (HT) LSDV induced the expression of interferon- stimulated genes (ISGs) in PBMCs, but this response was suppressed by infectious viruses. Finally, we show that despite failed replication, LSDV infected PBMCs transmitted the virus to recipient co-cultured MDBK cells and produced infectious foci, suggesting a potential role of PBMCs in LSDV dissemination.HighlightsVirulent and attenuated LSDV productively replicated in bovine kidney and bovine macrophage cell lines as well as in primary fibroblasts.Adherent white blood cells were susceptible to LSDV field and attenuated vaccine infection.LSDV showed active viral transcription in PBMCs yet no significant viral genome replication or production of infectious progeny.PBMCs infected with heat-treated LSDV but not with fully infectious viruses upregulated ISGs’ RNA.PBMCs transmitted and disseminated LSDV to contacting permissive cells.

Lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus within the family Poxviridae, causes significant disease in cattle and is classified as a notifiable disease by the World Organization for Animal Health (WOAH). The virus contains a double-stranded linear DNA genome of approximately 151 kbp, encoding 156 predicted open reading frames (ORFs) for various proteins. However, only a limited number of these proteins have been characterized, with the functions of many—particularly those encoded within the inverted terminal repeat (ITR) regions—remaining largely unknown. In this study, we utilized homologous recombination to generate LSDV mutants with deletions of the LSDV 001/156 gene to investigate its role. LSDV 001/156, an uncharacterized protein located within the ITR region, was identified as a late-expressed gene product incorporated into virions and involved in viral replication. Further analysis revealed that LSDV 001/156 acts as a negative regulator of the interferon (IFN) signaling pathway. It interacts with interferon regulatory factor 3 (IRF3), disrupting its dimerization and nuclear translocation, thereby attenuating IFN production. Functional studies demonstrated that the LSDV mutant lacking the 001/156 gene exhibited reduced replication and virulence in cattle compared to the wild-type virus, likely due to enhanced IFN responses in the absence of this immune-evasive protein. In summary, our findings uncover a novel role of the LSDV 001/156 gene in modulating the host intrinsic antiviral response, shedding light on the mechanisms underlying LSDV pathogenesis. This study highlights the importance of ITR-encoded genes in immune evasion and virulence, providing new insights into LSDV biology and its interactions with the host immune system.

Lumpy skin disease virus (LSDV) causes a severe emerging and transboundary disease in cattle. Infection with LSDV leads to the development of widespread dermal nodules. In addition to the skin, LSDV resides in multiple internal organs and can be isolated from the blood of infected cattle. We have characterised the tropism, replication, and dissemination of both a field isolate of LSDV and an attenuated vaccine strain in vitro. To study virus infection and dissemination in living cells, we generated recombinant viruses that express a green fluorescent protein (GFP) under a synthetic viral promoter. The recombinant LSDVs expressing GFP displayed replication kinetics similar to their parental strains in a bovine kidney cell line. These LSDV-GFP strains also replicated effectively in a bovine macrophage cell line and primary bovine foreskin cells, showing no apparent differences between the field isolate and the vaccine strain. Bovine peripheral blood mononuclear cells (PBMCs) infected with either LSDV-GFP strain displayed specific viral-driven GFP fluorescence and significant viral gene expression. However, these infected PBMCs did not support substantial viral DNA replication or the release of infectious progeny. Further analysis of the anti-viral response revealed that heat-treated LSDV, but not infectious viruses, induced the expression of interferon-stimulated genes (ISGs) in PBMCs. Thus, although LSDV did not replicate productively in PBMCs, it evaded the anti-viral response of these cells. Finally, we demonstrated that despite the lack of productive replication, infected PBMCs effectively transmitted LSDV to recipient permissive cells in co-culture, leading to the formation of infection foci. This suggests a potential role for PBMCs in the dissemination of LSDV.

BackgroundLumpy skin disease (LSD) is an acute or subacute infectious disease caused by lumpy skin disease virus (LSDV) of genus Capripoxvirus. The outbreaks of LSD were confirmed in the Yili area of the Xinjiang autonomous region in August 2019 and the Fujian province in June 2020. We detected LSDV in our daily monitoring work, then isolated, identified and sequenced the virus, and analyzed the whole genome characteristics of the isolated strain.ResultsWhole genome sequencing revealed that the strains isolated were all LSDV and were named as LSDV XJ201901 and LSDV FJ2019. The results showed that the identity based on whole genome sequences between LSDV XJ201901 and LSDV FJ2019 was 100% and the identity based on whole genome sequences between the two isolated strains and the global LSDV strains was 97.28%-99.99%, with the strain LSDV72/PrachuapKhiriKhan/Thailand/2021 (99.99%) having the highest sequence identity. Analysis of potential recombination events revealed that a total of 18 potential recombination events were identified in strains LSDV XJ201901 and LSDV FJ2019. The two strains are a recombination of Neethling vaccine LW 1959 (GeneBank: AF409138.1) with KSGP 0240 (GeneBank: KX683219.1). It was observed that Neethling vaccine LW 1959 (11/18) and KSGP 0240 (10/18) are involved in most of the potential recombination events.ConclusionsThe virus isolate in this study was LSDV and was identified as a vaccine recombinant strain. The most likely potential parent strains of the two strains in this study are Neethling vaccine LW 1959 and KSGP 0240. The strains in this study are very similar to those isolated in East and Southeast Asia since 2019.

Lumpy skin disease (LSD) is a severe animal infectious disease caused by lumpy skin disease virus (LSDV), inducing extensive nodules on the cattle mucosa or the scarfskin. LSDV genome encodes multiple proteins to evade host innate immune response. However, the underlying molecular mechanisms are poorly understood. In this study, we found that LSDV could suppress the expression of IFN-β and interferon-stimulated genes (ISGs) in MDBK cells during the early stage of infection. Subsequently, an unbiased screen was performed to screen the LSDV genes with inhibitory effects on the type I interferon (IFN-I) production. ORF127 protein was identified as one of the strongest inhibitory effectors on the expression of IFN-β and ISGs, meanwhile, the 1-43 aa of N-terminal of ORF127 played a vital role in suppressing the expression of IFN-β. Overexpression of ORF127 could significantly promote LSDV replication through inhibiting the production of IFN-β and ISGs in MDBK cells. Mechanism study showed that ORF127 specifically interacted with TBK1 and decreased the K63-linked polyubiquitination of TBK1 which suppressed the phosphorylation of TBK1 and ultimately decreased the production of IFN-β. In addition, truncation mutation analysis indicated that the 1-43 aa of N-terminal of ORF127 protein was the key structural domain for its interaction with TBK1. In short, these results validated that ORF127 played a negative role in regulating IFN-β expression through cGAS-STING signaling pathway. Taken together, this study clarified the molecular mechanism of ORF127 gene antagonizing IFN-I-mediated antiviral, which will helpfully provide new strategies for the treatment and prevention of LSD.

Background and Aim:Lumpy skin disease (LSD) and sheep pox are economically important Capripoxvirus-induced diseases of cattle and sheep, respectively. Despite the extensive vaccination program adopted by Egyptian veterinary authorities, LSD and sheep pox are still prevalent and spread throughout the whole country. The current study was designed for molecular characterization and phylogenetic analysis of LSD virus (LSDV) and Sheep pox virus (SPPV) recovered from field cases in Egypt along with vaccinal strains to assess their genetic relatedness.Materials and Methods:Skin biopsies were collected from naturally infected cases of LSD in Ismailia (n=3 farms) and Beni-Suef (n=2 farms) Governorates and sheep pox in Beni-Suef (n=1 flock). Virus isolation was carried out on primary ovine fetal kidney and heart cell cultures. DNA was extracted from infected materials (skin lesions, infected cell cultures) as well as LSDV Neethling vaccine strain and Romanian SPPV vaccine strain. Polymerase chain reaction was performed using oligonucleotide primers targeting the entire open reading frame of G protein-coupled receptors (GPCR) gene and gene sequences were analyzed.Results:Virus isolation on primary ovine fetal kidney and heart cell culture revealed a cytopathic effect at the third passage characterized by rounding of infected cells and margination of nuclear chromatin. Comparative sequence analysis of GPCR gene revealed that Egyptian LSDV isolated from Ismailia and Beni-Suef shared 99:100% nucleotide and amino acid (AA) identities with each other. In comparison to the vaccinal strains, Egyptian LSDV isolates shared 98:99 nucleotide and AA identities with LSDV Neethling vaccine strain and 93:94% with SPPV Romanian vaccine strain. No differences at the nucleotide or AAs were observed between the SPPV vaccine and virulent strains (100% identity). Phylogenetic analyses revealed that LSDV Neethling vaccine strain is more related to field Egyptian LSDV and clustered within the LSDV group while Romanian SPPV vaccine strain clustered in a separate clade with SPPV field isolates.Conclusion:Comparative sequencing and phylogenetic analyses of the GPCR gene reveal a minimal genetic variation between LSDV field isolates from different locations and a close relationship between virulent field strains and homologous vaccines.

Lumpy skin disease is caused by lumpy skin disease virus (LSDV), which can induce cattle with high fever and extensive nodules on the mucosa or the scarfskin, seriously influencing the cattle industry development and international import and export trade. Since 2013, the disease has spread rapidly and widely throughout the Russia and Asia. In the past few decades, progress has been made in the study of LSDV. It is mainly transmitted by blood-sucking insects, and various modes of transmission with distinct seasonality. Figuring out how the virus spreads will help eradicate LSDV at its source. In the event of an outbreak, selecting the most effective vaccine to block and eliminate the threat posed by LSDV in a timely manner is the main choice for farmers and authorities. At present, a variety of vaccines for LSDV have been developed. The available vaccine products vary in quality, protection rate, safety and side effects. Early detection of LSDV can help reduce the cost of disease. In addition, because LSDV has a huge genome, it is currently also used as a vaccine carrier, forming a new complex with other viral genes through homologous recombination. The vaccine prepared based on this can have a certain preventive effect on many kinds of diseases. Clinical detection of disease including nucleic acid and antigen level. Each method varies in convenience, accuracy, cost, time and complexity of equipment. This article reviews our current understanding of the mode of transmission of LSDV and advances in vaccine types and detection methods, providing a background for further research into various aspects of LSDV in the future.

Platform establishment of the Cre-loxP recombination system for genetic manipulation of the Lumpy skin disease virus

Construction and biological characteristics analysis of a gene-deleted recombinant virus strain of LSDV-ORF151.

Lumpy skin disease (LSD) is a highly contagious viral disease in cattle caused by lumpy skin disease virus (LSDV), which belongs to the genus Capripoxvirus (CaPVs) within the family Poxviridae. Since its first outbreak in China in August 2019, LSD has spread widely across mainland China, posing significant threats to the cattle industry. This study aimed to isolate and identify a clinical strain of LSDV via Vero cells. Skin tissue samples from lump lesions were homogenized and inoculated onto cell cultures. After 7 passages, the inoculated cells exhibited typical cytopathic effects (CPEs). PCR amplification of the LSDV132 gene confirmed the presence of LSDV nucleic acid. In addition, quantitative PCR (qPCR) demonstrated a significant increase in viral copy number over time. Transmission electron microscopy (TEM) revealed typical brick-shaped viral particles. Furthermore, an indirect immunofluorescence assay (IFA) of infected Vero cells exhibiting CPEs produced a positive reaction with antiserum from cattle naturally infected with LSDV. Additionally, nucleotide similarity analysis of 123 LSDV strains revealed a high degree of similarity (98.4%-100%) among different geographic lineages. Nucleotide sequencing and recombination analysis of the LSDV011 gene from LSDV/China/HB01/2020 revealed close similarity to Asian strains and revealed a recombination event. Furthermore, similarity plot analysis confirmed two genomic exchange sites at nucleotide positions 120 and 762 within the LSDV011 gene. Recombination events between 65 Asian LSDV strains and 13 goatpox virus (GTPV) strains have raised safety concerns regarding the use of attenuated goatpox vaccines, highlighting the need for novel and safer LSDV vaccines. In summary, this study successfully isolated a clinical LSDV strain, demonstrating its evolutionary status and providing crucial insights for LSD control in the cattle industry.

Lumpy skin disease (LSD), caused by the lumpy skin disease virus (LSDV), is an emerging infectious disease in China that primarily affects cattle. LSDV and goatpox virus (GTPV) belong to the Capripoxvirus genus and exhibit high genomic homology, enabling cross-immunogenicity. Comparative genome analysis revealed that LSDV contains a unique gene, Orf132, whose function remains uncharacterized. In this study, we first confirmed that the recombinant ORF132 protein exhibits immunoreactivity against sera from LSDV-infected cattle. To investigate the biological role of Orf132, we generated an Orf132 deletion strain (LSDV-ΔOrf132). Compared with the wild-type LSDV, the replication capacity of LSDV-ΔOrf132 was reduced approximately tenfold, indicating that Orf132 is critical for viral replication. Transcriptomic analysis of infected MDBK cells revealed significant alterations in endoplasmic reticulum (ER) protein processing and unfolded protein response (UPR) pathways following Orf132 deletion. qRT-PCR validation showed marked upregulation of ER stress markers, including Grp78, Chop, and Gadd34. Subsequent apoptosis assays established that Orf132 deletion triggers CHOP-Caspase-12-mediated apoptotic pathways. This dysregulated stress response cascade culminates in premature apoptotic scenarios, possibly weakening viral replication. We also showed that adding ORF132 protein effectively inhibited ER stress in LSDV-ΔOrf132-infected cells and rescued the attenuation phenotype of LSDV-ΔOrf132. Our findings collectively revealed that Orf132 is a critical gene for LSDV replication and a negative regulator of ER stress. It plays an essential role in the virus’s life cycle, and its deletion significantly impairs viral replication while inducing ER stress-related apoptosis.

BackgroundSince 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.ResultsBy 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.ConclusionThis 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.

Lumpy skin disease virus (LSDV) infection is a major socio-economic issue that seriously threatens the global cattle-farming industry. Here, a recombinant virus LSDV-ΔTK/EGFP, expressing enhanced green fluorescent protein (EGFP), was constructed with a homologous recombination system and applied to the high-throughput screening of antiviral drugs. LSDV-ΔTK/EGFP replicates in various kidney cell lines, consistent with wild-type LSDV. The cytopathic effect, viral particle morphology, and growth performance of LSDV-ΔTK/EGFP are consistent with those of wild-type LSDV. High-throughput screening allowed to identify several molecules that inhibit LSDV-ΔTK/EGFP replication. The strong inhibitory effect of theaflavin on LSDV was identified when 100 antiviral drugs were screened in vitro. An infection time analysis showed that theaflavin plays a role in the entry of LSDV into cells and in subsequent viral replication stages. The development of this recombinant virus will contribute to the development of LSDV-directed antiviral drugs and the study of viral replication and mechanisms of action.

Vaccination with live attenuated vaccines is a key element in the prevention of lumpy skin disease. The mechanism of virus attenuation by long-term passaging in sensitive systems remains unclear. Targeted inactivation of virulence genes is the most promising way to obtain attenuated viruses. Four virulence genes in the genome of the lumpy skin disease virus (LSDV) Dermatitis nodulares/2016/Atyrau/KZ were sequentially knocked out by homologous recombination under conditions of temporary dominant selection. The recombinant LSDV Atyrau-5BJN(IL18) with a knockout of the LSDV005, LSDV008, LSDV066 and LSDV142 genes remained genetically stable for ten passages and efficiently replicated in cells of lamb testicles, saiga kidney and bovine kidney. In vivo experiments with cattle have shown that injection of the LSDV Atyrau-5BJN(IL18) at a high dose does not cause disease in animals or other deviations from the physiological norm. Immunization of cattle with the LSDV Atyrau-5BJN(IL18) induced the production of virus-neutralizing antibodies in titers of 4–5 log2. The challenge did not cause disease in immunized animals. The knockout of four virulence genes resulted in attenuation of the virulent LSDV without loss of immunogenicity. The recombinant LSDV Atyrau-5BJN(IL18) is safe for clinical use, immunogenic and protects animals from infection with the virulent LSDV.

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.