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.

SARS-CoV-2 Infection in Beaver Farm, Mongolia, 2021.

We report an outbreak of COVID-19 in a beaver farm in Mongolia in 2021. Genomic characterization revealed a unique combination of mutations in the SARS-CoV-2 of the infected beavers. Based on these findings, increased surveillance of farmed beavers should be encouraged.

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.

Low Dose Gamma Irradiation of Trypanosoma evansi Parasites Identifies Molecular Changes That Occur to Repair Radiation Damage and Gene Transcripts That May Be Involved in Establishing Disease in Mice Post-Irradiation.

The protozoan parasite Trypanosoma evansi is responsible for causing surra in a variety of mammalian hosts and is spread by many vectors over a wide geographical area making it an ideal target for irradiation as a tool to study the initial events that occur during infection. Parasites irradiated at the representative doses 100Gy, 140Gy, and 200Gy were used to inoculate BALB/c mice revealing that parasites irradiated at 200Gy were unable to establish disease in all mice. Cytokine analysis of mice inoculated with 200Gy of irradiated parasites showed significantly lower levels of interleukins when compared to mice inoculated with non-irradiated and 100Gy irradiated parasites. Irradiation also differentially affected the abundance of gene transcripts in a dose-dependent trend measured at 6- and 20-hours post-irradiation with 234, 325, and 484 gene transcripts affected 6 hours post-irradiation for 100Gy-, 140Gy- and 200Gy-irradiated parasites, respectively. At 20 hours post-irradiation, 422, 381, and 457 gene transcripts were affected by irradiation at 100Gy, 140Gy, and 200Gy, respectively. A gene ontology (GO) term analysis was carried out for the three representative doses at 6 hours and 20 hours post-irradiation revealing different processes occurring at 20 hours when compared to 6 hours for 100Gy irradiation. The top ten most significant processes had a negative Z score. These processes fall in significance at 140Gy and even further at 200Gy, revealing that they were least likely to occur at 200Gy, and thus may have been responsible for infection in mice by 100Gy and 140Gy irradiated parasites. When looking at 100Gy irradiated parasites 20 hours post-irradiation processes with a positive Z score, we identified genes that were involved in multiple processes and compared their fold change values at 6 hours and 20 hours. We present these genes as possibly necessary for repair from irradiation damage at 6 hours and suggestive of being involved in the establishment of disease in mice at 20 hours post-irradiation. A potential strategy using this information to develop a whole parasite vaccine is also postulated.

Molecular characterization of African swine fever viruses from Burkina Faso, 2018.

BACKGROUND: African swine fever (ASF) is a viral hemorrhagic disease of domestic and wild swine. ASF has been endemic in Burkina Faso since 2003. In October 2018, substantial pig deaths occurred in Ouagadougou and two neighboring municipalities in central Burkina Faso. Following these mortalities, the veterinary extension services carried out investigations to begin control measures and collect samples. METHODS: We performed real-time PCR for diagnostic confirmation and molecular characterization of the virus based on the partial P72, the complete p54, the partial CD2v, and partial B602L genes. RESULTS: The field study revealed that mortalities started two weeks before our investigations. The real-time PCR results confirmed ASFV DNA in twenty samples out of sixty-two blood samples collected in four different locations. The sequencing and phylogenetic analysis showed that ASFVs causing these outbreaks belong to genotype I and serogroup 4. The study of the CVR showed 4 TRS variants, and that of the CD2v amino acid sequence revealed five variants based on the number of deleted KCPPPK motifs in the C-terminal proline-reach region of the protein. CONCLUSIONS: The existence of multiple variants in these outbreaks shows the importance of molecular characterization to understand the evolution of ASFV isolates and the link between epidemics.

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.

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 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.

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.

African swine fever virus genotype II in Mongolia, 2019.

African swine fever (ASF) is a severe haemorrhagic disease of domestic and wild pigs caused by the African swine fever virus (ASFV). In recent years, ASF has steadily spread towards new geographical areas, reaching Europe and Asia. On January 15th, 2019, Mongolia reported its first ASF outbreak to the World Organization for Animal Health (OIE), becoming, after China, the second country in the region affected by the disease. Following an event of unusual mortality in domestic pigs in Bulgan Province, a field team visited four farms and a meat market in the region to conduct an outbreak investigation and collect samples for laboratory analysis. Different organs were examined for ASF associated lesions, and total nucleic acid was extracted for real-time PCR, virus isolation and molecular characterization. The real-time PCR results confirmed ASFV DNA in 10 out of 10 samples and ASFV was isolated. Phylogenetic analysis established that ASFVs from Mongolia belong to genotype II and serogroup 8. The viruses were identical to each other, and to domestic pig isolates identified in China and Russia, based on the comparison of five genomic targets. Our results suggest a cross-border spread of ASFV, without indicating the source of infection.

African swine fever in North Sumatra and West Java provinces in 2019 and 2020, Indonesia.

African swine fever (ASF) is a highly lethal and contagious viral haemorrhagic disease of domestic and wild pigs, caused by the ASF virus (ASFV). After entering China in 2018, the disease has continued to spread through Asia. In September 2019, a team from the Indonesian Research Center for Veterinary Science, Bogor, investigated outbreaks in backyard pigs in the Dairi and Humbang Hasundutan districts of North Sumatra province. In January 2020, three pigs purchased from a pig seller in Bogor District, West Java province were also tested. Real-time PCR results confirmed ASFV DNA in sixteen out of twenty-nine samples, with nine positive samples from North Sumatra and seven from West Java. Four partial or full-length genes (i.e. p72, p54, pB602L and CD2v) and a 356-bp fragment between the I73R and I329L genes were sequenced from representative samples. Phylogenetic analysis established that the ASFV in the samples from both North Sumatra and West Java were identical, indicating a common source of infection, and that they belonged to the p72 genotype II and serogroup 8. The sequences from the Indonesian ASFVs were also identical to other genotype II ASFV from domestic pigs in Vietnam, China and Russia.

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.

Development and evaluation of a real-time PCR panel for the detection of 20 immune markers in cattle and sheep.

The establishment of a panel of immune markers is of paramount importance to understand the different transcription patterns of infectious diseases in livestock. The array of commercially available immunological assays for cattle and sheep is currently limited, due to the lack of antibodies for these species. Even though SYBR Green based real time quantitative PCR (qPCR) is the most commonly used method to study cytokine transcription in ruminants, a lack of standardization impairs its implementation in the study of different ruminant diseases. In order to obtain reliable qPCR results, several variables need to be considered: choice of reference genes for optimal normalization, variation of annealing temperature among primer sets, and assay specificity and sensitivity. In this study, we developed and validated a panel of immune markers in bovine and ovine samples using SYBR Green based qPCR in a cost-effective way with multiple primer sets optimised to amplify at a common thermal cycling temperature. Twenty primer sets were designed to quantify immune markers (IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, TNF-α, IFN-γ, IFN-α, Ki-67, NFkB-65, TLR-3, TLR-4, TLR-8 and Rig-1) in ovine and bovine templates. For optimal normalization and selection of suitable reference genes, primer sets that measure the transcription of five reference genes were also included in the panel. The amplification efficiency, linearity and specificity was validated for all target genes. Optimal amplification conditions were achieved in both ovine and bovine samples for all gene targets, with the exception of Ki67. Relative quantification studies were performed on ovine and bovine mRNA obtained from sheep peripheral blood mononuclear cells (PBMCs) stimulated with three different treatments (PMA/Ionomycin, Concanavalin A (Con A) and pokeweed mitogen (PWM)). Pokeweed and ConA efficiently induced gene transcription of most of the targeted genes, while PMA/Ionomycin showed a weaker induction. Finally, we further assessed usability of our panel by running it on bovine monocyte derived dendritic cells (MoDCs) stimulated with different vaccines. Results confirmed the induction of a specific pro-inflammatory gene transcription pattern by rabies vaccine, which resembles the one occurring during viral infection. Altogether, we validated the efficiency and usability of an extended real-time PCR panel that gives the possibility to rapidly measure a broad spectrum of ovine and bovine immune markers by using a single set of reagents and protocol thus representing a valid and cost-effective tool for research purposes.

Symptomatic and asymptomatic cases of African swine fever in Tanzania.

African swine fever (ASF) is an acute, highly contagious and deadly viral haemorrhagic disease of domestic pigs caused by African swine fever virus (ASFV). In ASF endemic countries, there are an increasing number of reports on circulating ASFV strains with different levels of virulence causing a broad range of clinical symptoms in susceptible animals. Tanzania, where ASFV is endemic since 2001, recorded several outbreaks including symptomatic and asymptomatic cases between 2015 and 2017. We collected 35 clinical samples from four outbreaks for diagnostic confirmation and sequenced the partial B646L (p72), the full E183L (p54) gene, the central variable region of the B602L gene and the intergenic region between the I73R and I329L genes to characterize molecularly the new ASFV isolates and analyse their relatedness with previously reported Tanzanian and foreign isolates. We detected ASFV in 21 samples, 15 from symptomatic and six from asymptomatic pigs. Phylogenetic analyses based on the partial p72 gene and the complete p54 (E183L) genes revealed that the ASFVs in samples from symptomatic pigs belonged to genotypes II and those in samples from asymptomatic pigs belonged to genotype IX. The CVR profiles of the p72 genotype II and genotype IX isolates differed between each other and from previously published Tanzanian sequences. The sequence analysis of the intergenic region between the I73R and I329L for the 2017 genotype II isolates showed the absence of one GGAATATATA motif in those isolates. This study showed the simultaneous circulation of two different ASFV genotypes with different levels of pathogenicity in Tanzania. Since the existence of sub-clinically infected pigs may contribute to the persistence of the virus, our findings suggest continuous surveillance and characterization of ASFV isolates in disease-endemic regions.

An HRM Assay to Differentiate Sheeppox Virus Vaccine Strains from Sheeppox Virus Field Isolates and other Capripoxvirus Species.

Sheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) affect small ruminants and cattle causing sheeppox (SPP), goatpox (GTP) and lumpy skin disease (LSD) respectively. In endemic areas, vaccination with live attenuated vaccines derived from SPPV, GTPV or LSDV provides protection from SPP and GTP. As live poxviruses may cause adverse reactions in vaccinated animals, it is imperative to develop new diagnostic tools for the differentiation of SPPV field strains from attenuated vaccine strains. Within the capripoxvirus (CaPV) homolog of the variola virus B22R gene, we identified a unique region in SPPV vaccines with two deletions of 21 and 27 nucleotides and developed a High-Resolution Melting (HRM)-based assay. The HRM assay produces four distinct melting peaks, enabling the differentiation between SPPV vaccines, SPPV field isolates, GTPV and LSDV. This HRM assay is sensitive, specific, and provides a cost-effective means for the detection and classification of CaPVs and the differentiation of SPPV vaccines from SPPV field isolates.

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.

First genetic characterization of Peste des Petits Ruminants from Niger: On the advancing front of the Asian virus lineage.

Peste des Petits Ruminants (PPR) is a serious transboundary infectious disease of small ruminants. The causal agent, PPR virus (PPRV), can be separated into four genetically distinct lineages using phylogenetic analysis. In recent decades, lineage IV of PPRV has dramatically extended its geographic distribution from Asia to the Middle East and to Africa, where it has progressively replaced other PPRV lineages. Lineages I and II are historically distributed in West Africa. Currently, lineage II appears to dominate the region, whereas the last recorded occurrence of lineage I dates back to 1994. Recent studies reported the presence of lineage IV in Nigeria, suggesting that this lineage is expanding in West Africa. In Niger, a close neighbour of Nigeria, PPRV has never been genetically characterized, despite reports of PPR incidence. In this study, pathological samples collected from sick goats were collected in 2013 during a suspected PPR outbreak in southern Niger close to the Nigerian border were compared to samples collected in a previous investigation in October 2001 in south-western Niger. These strains were characterized by sequencing and phylogenetic analysis to identify their genetic lineage. Our results show that in 2001, lineages I and II were cocirculating in south-western Niger, whereas the strain that caused the outbreak in 2013 belonged to lineage IV and is closely related to strains identified in Nigeria. These results confirm the progression of lineage IV in West Africa. The process of PPRV lineage replacement and its implications for the epidemiology and the control of the disease in this region are unclear and should be the subject of further studies in the field.

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.