Molecular characterization of recombinant LSDV isolates from 2022 outbreak in Indonesia through phylogenetic networks and whole-genome SNP-based analysis.

Lumpy skin disease (LSD) is a transboundary viral disease of cattle and water buffaloes caused by the LSD virus, leading to high morbidity, low mortality, and a significant economic impact. Initially endemic to Africa only, LSD has spread to the Middle East, Europe, and Asia in the past decade. The most effective control strategy for LSD is the vaccination of cattle with live-attenuated LSDV vaccines. Consequently, the emergence of two groups of LSDV strains in Asian countries, one closely related to the ancient Kenyan LSDV isolates and the second made of recombinant viruses with a backbone of Neethling-vaccine and field isolates, emphasized the need for constant molecular surveillance. This current study investigated the first outbreak of LSD in Indonesia in 2022. Molecular characterization of the isolate circulating in the country based on selected LSDV-marker genes: RPO30, GPCR, EEV glycoprotein gene, and B22R, as well as whole genome analysis using several analytical tools, indicated the Indonesia LSDV isolate as a recombinant of LSDV_Neethling_vaccine_LW_1959 and LSDV_NI-2490. The analysis clustered the Indonesia_LSDV with the previously reported LSDV recombinants circulating in East and Southeast Asia, but different from the recombinant viruses in Russia and the field isolates in South-Asian countries. Additionally, this study has demonstrated alternative accurate ways of LSDV whole genome analysis and clustering of isolates, including the recombinants, instead of whole-genome phylogenetic tree analysis. These data will strengthen our understanding of the pathogens' origin, the extent of their spread, and determination of suitable control measures required.

First molecular characterization of poxviruses in cattle, sheep, and goats in Botswana.

BACKGROUND: Poxviruses within the Capripoxvirus, Orthopoxvirus, and Parapoxvirus genera can infect livestock, with the two former having zoonotic importance. In addition, they induce similar clinical symptoms in common host species, creating a challenge for diagnosis. Although endemic in the country, poxvirus infections of small ruminants and cattle have received little attention in Botswana, with no prior use of molecular tools to diagnose and characterize the pathogens. METHODS: A high-resolution melting (HRM) assay was used to detect and differentiate poxviruses in skin biopsy and skin scab samples from four cattle, one sheep, and one goat. Molecular characterization of capripoxviruses and parapoxviruses was undertaken by sequence analysis of RPO30 and GPCR genes. RESULTS: The HRM assay revealed lumpy skin disease virus (LSDV) in three cattle samples, pseudocowpox virus (PCPV) in one cattle sample, and orf virus (ORFV) in one goat and one sheep sample. The phylogenetic analyses, based on the RPO30 and GPCR multiple sequence alignments showed that the LSDV sequences of Botswana were similar to common LSDV field isolates encountered in Africa, Asia, and Europe. The Botswana PCPV presented unique features and clustered between camel and cattle PCPV isolates. The Botswana ORFV sequence isolated from goat differed from the ORFV sequence isolated from sheep. CONCLUSIONS: This study is the first report on the genetic characterization of poxvirus diseases circulating in cattle, goats, and sheep in Botswana. It shows the importance of molecular methods to differentially diagnose poxvirus diseases of ruminants.

Molecular characterization of lumpy skin disease virus (LSDV) emerged in Bangladesh reveals unique genetic features compared to contemporary field strains.

BACKGROUND: Lumpy skin disease (LSD) is a contagious viral disease of cattle caused by lumpy skin disease virus (LSDV). LSD has recently spread in Asia following outbreaks in the Middle East and Europe. The disease emerged in Bangladesh in July 2019 in the Chattogram district, then rapidly spread throughout the entire country. We investigated six LSD outbreaks in Bangladesh to record the clinical signs and collect samples for diagnostic confirmation. Furthermore, we performed the molecular characterization of Bangladesh isolates, analyzing the full RPO30 and GPCR genes and the partial EEV glycoprotein gene. RESULTS: Clinical observations revealed common LSD clinical signs in the affected cattle. PCR and real-time PCR, showed the presence of the LSDV genome in samples from all six districts. Phylogenetic analysis and detailed inspection of multiple sequence alignments revealed that Bangladesh isolates differ from common LSDV field isolates encountered in Africa, the Middle East, and Europe, as well as newly emerged LSDV variants in Russia and China. Instead, they were closely related to LSDV KSGP-0240, LSDV NI2490, and LSDV Kenya. CONCLUSIONS: These results show the importance of continuous monitoring and characterization of circulating strains and the need to continually refine the strategies for differentiating vaccine strains from field viruses.

First detection and molecular characterisation of pseudocowpox virus in a cattle herd in Zambia.

BACKGROUND: Pseudocowpox virus (PCPV) of the genus Parapoxvirus in the family Poxviridae causes pseudocowpox in cattle worldwide and presents a zoonotic concern. Most poxviruses produce diseases of similar clinical signs in affected animals, which are impossible to differentiate clinically or by serology. It is, therefore, vital to use molecular assays to rapidly identify the causative agents of poxvirus infections. This study aimed to detect, diagnose, and characterize the causative agent of pox-like skin lesions in a cattle herd in Zambia, initially suspected to be infected with Lumpy Skin Disease virus. METHODS: We used a High-Resolution Melting (HRM) analysis assay to detect the PCPV genome and sequenced the major envelope protein (B2L gene) for comparative sequence and phylogenetic analysis. RESULTS: Our field investigations showed cattle presenting atypical skin lesions and high morbidity within the herd. The laboratory diagnosis, based on the HRM assay revealed PCPV DNA in the samples. Phylogenetic and comparative sequence analyses confirmed PCPV in the samples and revealed genomic differences between samples collected in 2017 and 2018 from the same farm. CONCLUSION: Our work is the first documented report of PCPV in Zambia. It shows the strength of molecular methods to diagnose pox-like infections in cattle and discriminate between diseases causing similar clinical signs. This rapid and accurate diagnosis improves the response time for more accurate veterinary interventions.

A gel-based PCR method to differentiate sheeppox virus field isolates from vaccine strains.

BACKGROUND: Sheeppox (SPP) and goatpox (GTP) caused by sheeppox virus (SPPV) and goatpox virus (GTPV), respectively of the genus Capripoxvirus in the family Poxviridae, are severely afflicting small ruminants' production systems in Africa and Asia. In endemic areas, SPP and GTP are controlled using vaccination with live attenuated vaccines derived from SPPV, GTPV or Lumpy skin disease virus (LSDV). Sometimes outbreaks occur following vaccination. In order to successfully control the spread of the virus, it is essential to identify whether the animals were infected by the field strain and the vaccine did not provide sufficient protection. Alternatively, in some cases the vaccine strain may cause adverse reactions in vaccinated animals or in rare occasions, re-gain virulence. Thus, diagnostic tools for differentiation of virulent strains from attenuated vaccine strains of the virus are needed. The aim of this study was to identify an appropriate diagnostic target region in the capripoxvirus genome by comparing the genomic sequences of SPPV field isolates with those of the most widely used SPP vaccine strains. RESULTS: A unique 84 base pair nucleotide deletion located between the DNA ligase gene and the VARV B22R homologue gene was found only in SPPV vaccines derived from the Romanian and Yugoslavian RM/65 strains and absent in SPPV field isolates originated from various geographical locations of Asia and Africa. In addition, we developed and evaluated a conventional PCR assay, exploiting the targeted intergenic region to differentiate SPPV vaccine virus from field isolates. The assay produced an amplicon size of 218 bp for the vaccine strains, while the SPPV field isolates resulted in a 302 bp PCR fragment. The assay showed good sensitivity and specificity, and the results were in full agreement with the sequencing data of the PCR amplicons. CONCLUSION: The developed assay is an improvement of currently existing diagnostic tools and, when combined with a capripox virus species-specific assay, will enhance SPP and GTP diagnosis and surveillance and facilitate epidemiological investigations in countries using live attenuated SPP vaccines. In addition, for laboratories with limited resources, the assay provides a simple and cost-effective alternative for sequencing.

Investigation of Marek's disease virus from chickens in central Ethiopia.

Marek's disease (MD) is a lymphoproliferative and neuropathic disease of domestic chickens and less commonly, turkeys and quails, caused by a highly contagious, cell-associated, oncogenic herpesvirus. In Ethiopia, MD is believed to be introduced with importation of exotic and crossbred to improve the poultry production and has been reported to be a potential threat to the poultry sector both in backyard and commercial farming systems. This study was aimed at isolation and molecular analysis of MD virus isolates circulating in chicken population in the central part of Ethiopia where commercial farms are populated. From September 2013 to January 2014, clinical and post-mortem examination were conducted on diseased chickens suspected of MD virus infection. Representative spleen and feather follicle samples were collected following sterile procedure, and infectious virus isolation was performed using primary chicken fibroblast cell culture. Cell culture inoculated with suspension of pathological samples developed characteristic MD virus cytopathic effect of rounding of the cells and small plaques. Further analysis of the virus was conducted by conventional PCR amplifying the ICP4 gene fragment from eleven tissue samples using MD virus specific primers. PCR products were further sequenced and analyzed. Nucleotide sequence similarity search of the local isolates resulted a high degree of sequence similarity with Gallid Herpes virus type 2 strain (Marek's disease virus type 1, JN034558). To our knowledge, the present study is the first report conducted on virus isolation and molecular characterization of MD virus isolates circulated in Ethiopia. Eleven ICP4-like gene fragment (318 bp) sequences generated in the present study were uploaded in the public database (KU842366-76). Further research on virus isolation, genetic characterization, and infection dynamics is recommended targeting chickens of all age groups reared in different agro-ecological zones under different production system.

Molecular characterization of orf virus from sheep and goats in Ethiopia, 2008-2013.

BACKGROUND: Orf is a contagious disease of sheep, goats and wild ungulates caused by orf virus (ORFV) a member of the genus Parapoxvirus, Poxviridae family. Although orf is endemic in Ethiopia, little attention has been given so far as it is not a notifiable disease by the World Organization for Animal Health. In this work, we have investigated orf outbreaks representing five different geographical locations of Ethiopia, in Amba Giorgis, Gondar zuria, Adet, Debre zeit and Adami Tulu, between 2008 and 2013. RESULTS: The viral isolation and the sequence analysis of the A32L and the B2L genes of eighteen representative isolates confirmed that sampled animals were infected by ORFVs. The phylogenetic study and the comparative analysis of the deduced amino acid profile suggests that there were two main clusters of ORFV isolates which were responsible for the investigated outbreaks. Additionally the analysis of these two genes showed limited variability to ORFVs encountered elsewhere. This is the first report on the genetic characterization of the ORFV isolates from sheep and goats in Ethiopia. CONCLUSION: The molecular characterization of Ethiopian ORFV isolates highlighted the circulation of two main clusters causing orf disease in sheep and goats. The use of laboratory based methods and a constant monitoring of Ethiopian ORFV isolates is needed to better understand the dynamic of ORFV circulating in the country and facilitate the implementation of control measures.

Genetic Characterization of Lumpy Skin Disease Viruses Circulating in Lesotho Cattle.

Lumpy skin disease is one of the fast-spreading viral diseases of cattle and buffalo that can potentially cause severe economic impact. Lesotho experienced LSD for the first time in 1947 and episodes of outbreaks occurred throughout the decades. In this study, eighteen specimens were collected from LSD-clinically diseased cattle between 2020 and 2022 from Mafeteng, Leribe, Maseru, Berea, and Mohales' Hoek districts of Lesotho. A total of 11 DNA samples were analyzed by PCR and sequencing of the extracellular enveloped virus (EEV) glycoprotein, G-protein-coupled chemokine receptor (GPCR), 30 kDa RNA polymerase subunit (RPO30), and B22R genes. All nucleotide sequences of the above-mentioned genes confirmed that the PCR amplicons of clinical samples are truly LSDV, as they were identical to respective LSDV isolates on the NCBI GenBank. Two of the elevem samples were further characterized by whole-genome sequencing. The analysis, based on both CaPV marker genes and complete genome sequences, revealed that the LSDV isolates from Lesotho cluster with the NW-like LSDVs, which includes the commonly circulating LSDV field isolates from Africa, the Middle East, the Balkans, Turkey, and Eastern Europe.

Poxvirus Infections in Dairy Farms and Transhumance Cattle Herds in Nigeria.

Lumpy Skin disease (LSD) is an economically important disease in cattle caused by the LSD virus (LSDV) of the genus Capripoxvirus, while pseudocowpox (PCP) is a widely distributed zoonotic cattle disease caused by the PCP virus (PCPV) of the genus Parapoxvirus. Though both viral pox infections are reportedly present in Nigeria, similarities in their clinical presentation and limited access to laboratories often lead to misdiagnosis in the field. This study investigated suspected LSD outbreaks in organized and transhumance cattle herds in Nigeria in 2020. A total of 42 scab/skin biopsy samples were collected from 16 outbreaks of suspected LSD in five northern States of Nigeria. The samples were analyzed using a high-resolution multiplex melting (HRM) assay to differentiate poxviruses belonging to Orthopoxvirus, Capripoxvirus, and Parapoxvirus genera. LSDV was characterized using four gene segments, namely the RNA polymerase 30 kDa subunit (RPO30), G-protein-coupled receptor (GPCR), the extracellular enveloped virus (EEV) glycoprotein and CaPV homolog of the variola virus B22R. Likewise, the partial B2L gene of PCPV was also analyzed. Nineteen samples (45.2%) were positive according to the HRM assay for LSDV, and five (11.9%) were co-infected with LSDV and PCPV. The multiple sequence alignments of the GPCR, EEV, and B22R showed 100% similarity among the Nigerian LSDV samples, unlike the RPO30 phylogeny, which showed two clusters. Some of the Nigerian LSDVs clustered within LSDV SG II were with commonly circulating LSDV field isolates in Africa, the Middle East, and Europe, while the remaining Nigerian LSDVs produced a unique sub-group. The B2L sequences of Nigerian PCPVs were 100% identical and clustered within the PCPV group containing cattle/Reindeer isolates, close to PCPVs from Zambia and Botswana. The results show the diversity of Nigerian LSDV strains. This paper also reports the first documented co-infection of LSDV and PCPV in Nigeria.

Harnessing Attenuation-Related Mutations of Viral Genomes: Development of a Serological Assay to Differentiate between Capripoxvirus-Infected and -Vaccinated Animals.

Sheeppox, goatpox, and lumpy skin disease caused by the sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively, are diseases that affect millions of ruminants and many low-income households in endemic countries, leading to great economic losses for the ruminant industry. The three viruses are members of the Capripoxvirus genus of the Poxviridae family. Live attenuated vaccines remain the only efficient means for controlling capripox diseases. However, serological tools have not been available to differentiate infected from vaccinated animals (DIVA), though crucial for proper disease surveillance, control, and eradication efforts. We analysed the sequences of variola virus B22R homologue gene for SPPV, GTPV, and LSDV and observed significant differences between field and vaccine strains in all three capripoxvirus species, resulting in the truncation and absence of the B22R protein in major vaccines within each of the viral species. We selected and expressed a protein fragment present in wildtype viruses but absent in selected vaccine strains of all three species, taking advantage of these alterations in the B22R gene. An indirect ELISA (iELISA) developed using this protein fragment was evaluated on well-characterized sera from vaccinated, naturally and experimentally infected, and negative cattle and sheep. The developed wildtype-specific capripox DIVA iELISA showed >99% sensitivity and specificity for serum collected from animals infected with the wildtype virus. To the best of our knowledge, this is the first wildtype-specific, DIVA-capable iELISA for poxvirus diseases exploiting changes in nucleotide sequence alterations in vaccine strains.

Molecular Characterization of the 2020 Outbreak of Lumpy Skin Disease in Nepal.

Lumpy skin disease (LSD) is a transboundary viral disease of cattle and buffaloes transmitted by blood-feeding vectors and causes high morbidity and low-to-moderate mortality. Since the first observation of LSD in Zambia in 1929, it has spread in cattle populations across African countries, the Middle East, Europe, and Asia. Following the recent outbreaks of LSD in South Asian countries such as India and Bangladesh, the disease was first reported in cattle farms in Nepal in June 2020. This study investigated the Nepalese LSD outbreak and confirmed that the disease spread rapidly to three neighboring districts in a month, infecting 1300 animals. Both cattle and buffaloes showed common clinical signs of LSD, with the exception that the buffaloes presented small nodular lesions without centered ulcerations. The collected samples were first tested for the presence of LSDV by real-time PCR. We further applied molecular tools, RPO30, GPCR, EEV glycoprotein gene, and B22R, for additional characterization of the LSDV isolates circulating in Nepal. Using a PCR-based Snapback assay, we confirmed that samples collected from cattle and buffaloes were positive of LSDV. Furthermore, sequence analysis (phylogenetic and multiple sequence alignments) of four selected LSDV genes revealed that the Nepal LSDVs resemble the Bangladesh and Indian isolates and the historic isolates from Kenya. We also highlight the importance of a unique B22R gene region harboring single-nucleotide insertions in LSDV Neethling and LSDV KSGPO-240 vaccine strains, enabling us to differentiate them from the Nepalese isolates and other fields isolates. This study demonstrates the importance of disease surveillance and the need to determine the source of the disease introduction, the extent of spread, modes of transmission, and the necessary control measures.

First Report of Lumpy Skin Disease in Myanmar and Molecular Analysis of the Field Virus Isolates.

Lumpy skin disease virus (LSDV) causes lumpy skin disease in cattle and buffaloes, which is associated with significant animal production and economic losses. Since the 2000s, LSDV has spread from Africa to several countries in the Middle East; Europe; and Asia; including, more recently, several south-east Asian countries. In November 2020, Myanmar reported its first LSD outbreak. This study reports on the first incursion of LSD in Myanmar and the molecular analysis of the LSDV detected. Staff from the Livestock Breeding and Veterinary Department (LBVD) of the Ministry of Agriculture, Livestock, and Irrigation collected samples from cattle with suspected LSD infection. The Food and Agriculture Organization (FAO) of the United Nations' Emergency Centre for Transboundary Animal Diseases (ECTAD) and the Joint International Atomic Energy Agency (IAEA)/FAO program's Animal Health and Production laboratory provided LSDV diagnostic support to two regional veterinary diagnostic laboratories in Myanmar. Samples from 13 cattle tested positive by real-time PCR. Selected samples underwent sequence analysis in IAEA laboratories. The results show that the Myanmar LSDV sequences clustered with LSDV isolates from Bangladesh and India, LSDV Kenya, and LSDV NI-2490. Further characterization showed that the Myanmar LSDV is 100% identical to isolates from Bangladesh and India, implying a common source of introduction. These findings inform diagnosis and development of control strategies.

Development and Optimization of Indirect ELISAs for the Detection of Anti-Capripoxvirus Antibodies in Cattle, Sheep, and Goat Sera.

Sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD) are economically significant pox diseases of ruminants, caused by sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively. SPPV and GTPV can infect both sheep and goats, while LSDV mainly affects cattle. The recent emergence of LSD in Asia and Europe and the repeated incursions of SPP in Greece, Bulgaria, and Russia highlight how these diseases can spread outside their endemic regions, stressing the urgent need to develop high-throughput serological surveillance tools. We expressed and tested two recombinant truncated proteins, the capripoxvirus homologs of the vaccinia virus C-type lectin-like protein A34 and the EEV glycoprotein A36, as antigens for an indirect ELISA (iELISA) to detect anti-capripoxvirus antibodies. Since A34 outperformed A36 by showing no cross-reactivity to anti-parapoxvirus antibodies, we optimized an A34 iELISA using two different working conditions, one for LSD in cattle and one for SPP/GTP in sheep and goats. Both displayed sound sensitivities and specificities: 98.81% and 98.72%, respectively, for the LSD iELISA, and 97.68% and 95.35%, respectively, for the SPP/GTP iELISA, and did not cross-react with anti-parapoxvirus antibodies of cattle, sheep, and goats. These assays could facilitate the implementation of capripox control programs through serosurveillance and the screening of animals for trade.

Review: Vaccines and Vaccination against Lumpy Skin Disease.

The geographical distribution of lumpy skin disease (LSD), an economically important cattle disease caused by a capripoxvirus, has reached an unprecedented extent. Vaccination is the only way to prevent the spread of the infection in endemic and newly affected regions. Yet, in the event of an outbreak, selection of the best vaccine is a major challenge for veterinary authorities and farmers. Decision makers need sound scientific information to support their decisions and subsequent actions. The available vaccine products vary in terms of quality, efficacy, safety, side effects, and price. The pros and cons of different types of live attenuated and inactivated vaccines, vaccination strategies, and associated risks are discussed. Seroconversion, which typically follows vaccination, places specific demands on the tools and methods used to evaluate the effectiveness of the LSD vaccination campaigns in the field. We aimed to give a comprehensive update on available vaccines and vaccination against LSD, to better prepare affected and at-risk countries to control LSD and ensure the safe trade of cattle.

Innate Immune Responses to Wildtype and Attenuated Sheeppox Virus Mediated Through RIG-1 Sensing in PBMC In-Vitro.

Sheeppox (SPP) is a highly contagious disease of small ruminants caused by sheeppox virus (SPPV) and predominantly occurs in Asia and Africa with significant economic losses. SPPV is genetically and immunologically closely related to goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which infect goats and cattle respectively. SPPV live attenuated vaccines (LAVs) are used for vaccination against SPP and goatpox (GTP). Mechanisms related to innate immunity elicited by SPPV are unknown. Although adaptive immunity is responsible for long-term immunity, it is the innate responses that prevent viral invasion and replication before LAVs generate specific long-term protection. We analyzed the relative expression of thirteen selected genes that included pattern recognition receptors (PRRs), Nuclear factor-κß p65 (NF-κß), and cytokines to understand better the interaction between SPPV and its host. The transcripts of targeted genes in sheep PBMC incubated with either wild type (WT) or LAV SPPV were analyzed using quantitative PCR. Among PRRs, we observed a significantly higher expression of RIG-1 in PBMC incubated with both WT and LAV, with the former producing the highest expression level. However, there was high inter-individual variability in cytokine transcripts levels among different donors, with the expression of TNFα, IL-15, and IL-10 all significantly higher in both PBMC infected with either WT or LAV compared to control PBMC. Correlation studies revealed a strong significant correlation between RIG-1 and IL-10, between TLR4, TNFα, and NF-κß, between IL-18 and IL-15, and between NF-κß and IL-10. There was also a significant negative correlation between RIG-1 and IFNγ, between TLR3 and IL-1 ß, and between TLR4 and IL-15 (P< 0.05). This study identified RIG-1 as an important PRR in the signaling pathway of innate immune activation during SPPV infection, possibly through intermediate viral dsRNA. The role of immunomodulatory molecules produced by SPPV capable of inhibiting downstream signaling activation following RIG-1 upregulation is discussed. These findings advance our knowledge of the induction of immune responses by SPPV and will help develop safer and more potent vaccines against SPP and GTP.

Use of an Alignment-Free Method for the Geographical Discrimination of GTPVs Based on the GPCR Sequences.

Goatpox virus (GTPV) belongs to the genus Capripoxvirus, together with sheeppox virus (SPPV) and lumpy skin disease virus (LSDV). GTPV primarily affects sheep, goats and some wild ruminants. Although GTPV is only present in Africa and Asia, the recent spread of LSDV in Europe and Asia shows capripoxviruses could escape their traditional geographical regions to cause severe outbreaks in new areas. Therefore, it is crucial to develop effective source tracing of capripoxvirus infections. Earlier, conventional phylogenetic methods, based on limited samples, identified three different nucleotide sequence profiles in the G-protein-coupled chemokine receptor (GPCR) gene of GTPVs. However, this method did not differentiate GTPV strains by their geographical origins. We have sequenced the GPCR gene of additional GTPVs and analyzed them with publicly available sequences, using conventional alignment-based methods and an alignment-free approach exploiting k-mer frequencies. Using the alignment-free method, we can now classify GTPVs based on their geographical origin: African GTPVs and Asian GTPVs, which further split into Western and Central Asian (WCA) GTPVs and Eastern and Southern Asian (ESA) GTPVs. This approach will help determine the source of introduction in GTPV emergence in disease-free regions and detect the importation of additional strains in disease-endemic areas.

Molecular Analysis of East African Lumpy Skin Disease Viruses Reveals a Mixed Isolate with Features of Both Vaccine and Field Isolates.

Lumpy skin disease (LSD), an economically significant disease in cattle caused by lumpy skin disease virus (LSDV), is endemic to nearly all of Africa. Since 2012, LSDV has emerged as a significant epizootic pathogen given its rapid spread into new geographical locations outside Africa, including the Middle East, Eastern Europe, and Asia. To assess the genetic diversity of LSDVs in East Africa, we sequenced and analyzed the RPO30 and GPCR genes of LSDV in twenty-two archive samples collected in Ethiopia, Kenya, and Sudan before the appearance of LSD in the Middle East and its incursion into Europe. We compared them to publicly available sequences of LSDVs from the same region and those collected elsewhere. The results showed that the East African field isolates in this study were remarkably similar to each other and to previously sequenced field isolates of LSDV for the RPO30 and GPCR genes. The only exception was LSDV Embu/B338/2011, a field virus collected in Kenya, which displayed mixed features between the LSDV Neethling vaccine and field isolates. LSDV Embu/B338/2011 had the same 12-nucleotide insertion found in LSDV Neethling and KS-1 vaccines. Further analysis of the partial EEV glycoprotein, B22R, RNA helicase, virion core protein, NTPase, and N1R/p28-like protein genes showed that LSDV Embu/B338/2011 differs from previously described LSDV variants carrying the 12-nucleotide insertion in the GPCR gene. These findings highlight the importance of the constant monitoring of genetic variation among LSDV isolates.

Molecular characterization of African Swine fever viruses in Burkina Faso, Mali, and Senegal 1989-2016: Genetic diversity of ASFV in West Africa.

African swine fever (ASF) has been endemic in sub-Saharan Africa since the 1960s. Following its introduction in Senegal, in 1957, ASF steadily progressed through West Africa, reaching Burkina Faso in 2003, and later Mali in 2016. Despite the heavy burden of disease on pig production, little information is available on the genetic diversity of Africa swine fever virus (ASFV) in Burkina Faso, Mali and Senegal. Here, we used real-time PCR ASFV to detect the ASFV genome in samples collected between 1989 and 2016, in Burkina Faso, Mali and Senegal, and conventional approaches for isolate characterization. The C-terminal end of the p72 protein gene, the full E183L gene and the central variable region (CVR) within the B602L gene in ASFV genome were sequenced and compared to publicly available sequences. ASFV genome was found in 27 samples, 19 from Burkina Faso, three from Mali and five from Senegal. The phylogenetic analyses showed that all viruses belong to genotype I, with the ASFVs from Burkina Faso and Mali grouping with genotype Ia and ASFV serogroup 4, and those from Senegal with genotype Ib and the ASFV serogroup 1. The analysis of the CVR tetrameric tandem repeat sequences (TRS) showed four TRS variants in Burkina Faso, two in Senegal and one in Mali. The three countries did not share any common TRS, and all CVRs of this study differed from previously reported CVRs in West Africa, except for Senegal. Three of the five isolates from Senegal fully matched with the CVR, p72 and p54 sequences from ASFV IC96 collected during the 1996 ASF outbreak in Ivory Coast. This study shows the spread of the same ASFV strains across countries, highlighting the importance of continuous monitoring of ASFV isolates. It also calls for an urgent need to establish a regional plan for the control and eradication of ASF in West Africa.

Molecular characterization of peste-des-petits ruminants virus from Nepal, 2005 to 2016.

Outbreaks of peste-des-petits ruminants (PPR) has been reported regularly in Nepal since 1994. Despite this, there has been limited molecular characterization of circulating virus in the country. In this study a 351 bp segment of the nucleoprotein gene of the PPR virus (PPRV) was amplified and sequenced from ten samples collected between 2005 and 2016. Phylogenetic trees were estimated from these sequences using the maximum likelihood method confirming that all of the PPRV from the samples analysed belonged to the sub-clade IV of clade I of lineage IV and that they shared a common origin with other PPRV isolates in the region.

Genetic characterization of African swine fever virus in Cameroon, 2010-2018.

African swine fever (ASF) is a highly lethal haemorrhagic disease in domestic and wild swine that has acquired great importance in sub-Saharan Africa since 1997. ASF was first reported in Cameroon in 1982 and was detected only in Southern Cameroon (South, West, East, Northwest, Southwest, Littoral, and Centre regions) until February 2010 when suspected ASF outbreaks were reported in the North and Far North regions. We investigated those outbreaks by analysing samples that were collected from sick pigs between 2010 and 2018. We confirmed 428 positive samples by ELISA and real-time PCR and molecularly characterized 48 representative isolates. All the identified virus isolates were classified as ASFV genotype I based on the partial B646L gene (C-terminal end of VP72 gene) and the full E183L gene encoding p54 protein analysis. Furthermore, analysis of the central variable region (CVR) within the B602L gene demonstrated that there were 3 different variants of ASFV genotype I, with 19, 20, and 21 tetrameric tandem repeat sequences (TRSs), that were involved in the 2010-2018 outbreaks in Cameroon. Among them, only variant A (19 TRSs) was identical to the Cam/82 isolate found in the country during the first outbreaks in 1981-1982. This study demonstrated that the three variants of ASFV isolates involved in these outbreaks were similar to those of neighbouring countries, suggesting a movement of ASFV strains across borders. Designing common control measures in affected regions and providing a compensation programme for farmers will help reduce the incidence and spread of this disease.