Capripoxvirus Research Articles

Species-specific inhibition of antiviral protein kinase R by capripoxviruses and vaccinia virus.

Double-stranded RNA-activated protein kinase R (PKR) is an important and rapidly evolving antiviral kinase. Most poxviruses contain two distinct PKR inhibitors, called E3 and K3 in vaccinia virus (VACV), the prototypic orthopoxvirus. E3 prevents PKR homodimerization by binding double-stranded RNA, while K3 acts as a pseudosubstrate inhibitor by binding directly to activated PKR and thereby inhibiting interaction with its substrate eIF2α. In our study here, we analyzed E3 and K3 orthologs from the phylogenetically distinct capripoxviruses (CaPVs), which include lumpy skin disease virus, sheeppox virus, and goatpox virus. Whereas the sheeppox virus E3 ortholog did not substantially inhibit PKR, all three CaPV K3 orthologs showed species-specific inhibition of PKR, with strong inhibition of sheep, goat, and human PKR but only weak inhibition of cow and mouse PKR. In contrast, VACV K3 strongly inhibited cow and mouse PKR but not sheep, goat, or human PKR. Infection of cell lines from the respective species with engineered VACV strains that contained different K3 orthologs showed a good correlation of PKR inhibition with virus replication and eIF2α phosphorylation. Our results show that K3 orthologs can have dramatically different effects on PKR of different species and indicate that effective PKR inhibition by K3 orthologs is crucial for virus replication.

Clinico-biochemical and Electrocardiographic Changes in Cattle Naturally Infected with Lumpy Skin Disease

Lumpy skin disease (LSD) is an economically important viral disease of cattle caused by Capripox virus. The disease is characterized clinically by fever, nodules on the skin, mucous membranes and internal organs, emaciation, enlarged lymph nodes, edema of the skin and deaths in some cases. The aim of this study is to investigate the clinico-biochemical picture of LSD infected cattle with special reference to the cardiac abnormalities. This study was carried out on LSD infected cattle showing typical clinical signs of the disease in Kaliobia governorate (𝑛 = 50; and aged between 2 to 5 years old). Clinical and electrocardiographic examination (ECG) were performed firstly, then skin nodules were aseptically collected from infected cattle for molecular detection of LSDV using PCR, on the other hand, serum samples were collected for biochemical analysis. The results of the current study revealed that, all examined tissue samples showed successful amplification of the viral attachment protein gene of LSDV with presence of positive bands at the target size (192 bp). Furthermore, troponin I (cTnI), urea, creatinine ALT, AST, ALP and potassium were significantly increased in LSD infected cattle compared to the control ones. In contrast, serum calcium, iron and sodium concentrations were significantly decreased. The ECG of LSD infected cattle showed abnormal P waves, shortening of P and T-waves amplitudes and decrease in RT interval compared to healthy cattle. The cardiac alterations in case of LSD infection could be related to the elevation of cTnI because of direct effect of the virus on the heart beside electrolyte imbalance, acidosis and hypocalcaemia.

Species-specific inhibition of capripoxvirus replication by host antiviral protein kinase R.

The role of interferon (IFN)‐induced protein kinase R (PKR) in capripoxvirus (CaPV)‐infected cells remains unknown. In this study, we show that CaPV infection triggered PKR and eukaryotic translation initiation factor 2 alpha (eIF2α) protein phosphorylation in a dose‐dependent manner, and that this leads to decreased CaPV replication. Overexpression of PKR compromised viral gene expression and inhibited sheeppox virus (SPPV) replication. Downregulation of PKR with siRNAs significantly decreased eIF2α phosphorylation and reduced the mRNA level of IFN‐β, which increased virus replication. In luciferase assays, species‐different CaPVs K3L proteins inhibited sheep PKR (sPKR): goatpox virus K3L strongly inhibited sPKR and goat PKR (gPKR), but SPPV K3L only partially inhibited gPKR. These results are the first to show that SPPV infection induces phosphorylation of eIF2α through PKR activation, which then results in restriction of CaPV replication. Furthermore, our data show that CaPV K3L inhibits PKR in a species‐specific manner. The results presented are consistent with the hypothesis that different levels of PKR inhibition by K3L orthologs from various viruses could potentially contribute to the host range function of K3L.

A Review on Sheeppox and Goatpox: Insight of Epidemiology, Diagnosis, Treatment and Control Measures in Ethiopia

Sheeppox and goatpox are caused by a genus of Capripox virus causing a severe problem and great economic loss in Ethiopia and also in sheep and goat rearing countries in many parts of the world. The objective of this review is to give insight on epidemiology, diagnosis, treatment and control measures of sheeppox and goatpox disease. Generally, the disease is less commonly seen in indigenous breeds in area where it is endemic as compared to exotic breeds. Mostly the disease is transmitted by direct contact. After it enters, goatpox virus replicates locally in the tissues. In endemic areas the morbidity rate reaches 70-90% whereas the mortality rate is up to 5-10% and approaches 100% in newly imported animals. In Ethiopia, the disease is distributed in all parts of the country and in endemic areas it is economically important due to lose of production, loss of weight, decreased milk yield, damage to hide and skin, cause abortion and expose to other diseases, while also being a direct cause of death.

Validation of a diagnostic tool for the diagnosis of lumpy skin disease.

Lumpy skin disease (LSD) is caused by LSD virus which is a member of the Capripoxvirus (CaPV) genus. Although PCR provides for a rapid and sensitive diagnosis, it has limited use due to its complexity in terms of cost, time and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, specific and cost-effective method with a diagnostic accuracy similar to PCR. To compare the detection rate (DR) of two LAMP assays versus PCR for the detection of CaPV. This study used 105 apparently health animals (AHA) and 59 clinically sick animals (CSA). PCR and LAMP assays (LAMP1 and LAMP 2) were compared for detection of CaPV from AHA and CSA using blood and tissue samples. The detection was confirmed by sequencing of PCR positive samples. Analytical sensitivity and specificity of LAMP assays also were assessed. The DR in CSA was 13.6% for PCR whereas for LAMP it was 39.0% and 25.4% for LAMP 1 and 2 methods, respectively. In AHA, the LAMP assay DR was 14.3% and 1.9% for LAMP 1 and 2, respectively. Phylogenetic tree analysis confirmed the identity of CaPV. Analytic sensitivity showed a detection limit of 8 copies/μL. The analytic specificity test showed no cross detection with other infectious agents. Good sensitivity and specificity results for LAMP assay support its application in the routine diagnosis of LSD, whereas its ability to detect LSDV in apparently healthy animals shows its usefulness in identifying populations at risk of LSD.

Goatpox outbreak at a high altitude goat farm of Mizoram: possibility of wild life spill over to domestic goat population.

In this study, pox-like outbreaks in goat population was investigated that occurred in a high altitude goat farm located in Mizoram, a hilly state of North eastern India. The outbreak initially involved the serows, an wild animal belonging to the family Bovidae, subfamily Caprinae and genus Capricornis, the state animal of Mizoram. Later, the disease affected the domestic goat population. The disease was diagnosed on the basis of gross lesions and PCR amplification of partial P32 gene of capripox virus. The virus was isolated in vero cells. The full length P32 gene was sequenced and phylogenetic tree was constructed. It was revealed that the capripox virus isolated from the outbreak was closely related to the Chinese strain of goatpox virus at both amino acid and nucleotide level. To the authors' knowledge, this is the first report on isolation and characterization of capripoxvirus from north eastern region of India.

Phylogenetic analysis of sheep pox virus (SPPV) virion core protein P4a gene revealed extensive sequence conservation among capripox viruses

In the present study, virion core protein P4a gene was PCR amplified from sheep pox virus (SPPV) Jaipur isolate and Roumanian Fanar (RF) vaccine strain adapted and propagated in lamb testis/vero cells. Gene specific primers were designed for amplification of P4a gene. Amplified P4a gene fragment was sequence characterized and 808 bp sequence was compared across SPPV, GTPV and LSDV isolates available in GenBank database which revealed extensive sequence conservation of 97% to 100% within pox virus groups. Sheep pox virus Jaipur isolate was found closely placed with Roumaninan Fanar (RF) and TU isolates. Further, phylogenetic analysis of P4a gene sequence indicated three distinct clusters of Capripox viruses with GTPV interestingly placed closely to LSDV group.

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

BackgroundSheeppox (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.ResultsA 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.ConclusionThe 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.

Expression of cytokines following vaccination of goats with a recombinant capripoxvirus vaccine expressing Rift Valley fever virus proteins

The mosquito-borne Rift Valley fever virus (RVFV) causes severe diseases in domesticated animals including cattle, sheep, camels and goats. Capripoxviruses (CPV) are suitable vectors for multivalent vaccine development. A recombinant rKS1-based CPV expressing the gene encoding the viral glycoprotein Gn of RVFV has been shown to induce protection in mice and sheep. The aim of this study was to evaluate the immunogenicity induced by this candidate vaccine in goats, and the level of cytokines produced by RVFV-specific Th1 and Th2 lymphocytes. The results of this study suggest that Th2 mediates immunity mainly through the significant production of IL4, which, coupled with a decrease in IFN-γ, may be involved in the replication of the capripoxvirus expressing the GN of RVFV. CD4+ cells may play the role of helper cells in B cell responses and neutralizing antibody production in the anti-CPV humoral response, leading to strong immunity against RVFV.

Genetic analysis of L1R myristoylated protein of Capripoxviruses reveals structural homogeneity among poxviruses

Sheeppox virus (SPPV) and goatpox virus (GTPV) are members of the genus Capripoxvirus (CaPV) of the family Poxviridae. CaPVs are responsible for important contagious diseases of small ruminants that are enzootic to the Indian sub-continent, Central and Northern Africa and the Middle East. In the present study, the sequence and phylogenetic analysis of the L1R gene of sixteen CaPV isolates (seven SPPV and nine GTPV) from India were performed along with 3D homology modeling of the L1R protein. L1R is a myristoylated protein responsible for virion assembly and being present on intracellular mature virion (IMV) surface, it is also a potent target for eliciting neutralizing antibodies. Sequence analysis of CaPV L1R gene revealed an ORF of 738bp with >99% and >96% identity within species and between species, respectively, at both nucleotide as well as amino acid levels. Phylogenetic analysis displayed distinct clusters of members of genus Capripoxvirus, as GTPV, SPPV and LSDV. L1R at the protein level showed various species-specific signature residues that may be useful for future grouping or genotyping of CaPV members. CaPV L1R was predicted to possess myristoylation motif GAAASIQTTVNTLNEKI and a potential N-glycosylation site at amino acid residue 50 (Asn). Despite of different host specificity in poxviruses, comparative sequence analysis of L1R proteins revealed highly conserved nature with presence of myristoylation motif (GXXXS) and six cysteine residues forming three disulfide bonds among all poxviruses. The conserved and immunogenic nature of the CaPV L1R gene may prove to be a potential candidate/target for developing molecular diagnostics including recombinant protein based assays and prophylactics for the control of CaPV diseases in tropical countries like India.

Capripoxvirus antibodies detection: Relationship between the two methods alpha and beta of virus neutralisation test.

Virus neutralisation test (VNT) of capripoxvirus (CaPVs) was studied to assess the post-vaccination (vaccine effectiveness) or post-infection antibodies level using two methods: alpha-VNT and beta-VNT which are generally carried out to measure the Neutralising Index (N.I.) and the serum Antibody titre (TAb) respectively. The authors have demonstrated that a positive correlation exists between N.I. and TAb values, this study aimed to add more evidence to this correlation by establishing a graph and its mathematical equations. We found that: N.I. = (1.489 Log TAb) + 1.331; this serves as a base to calculate N.I. using TAb values measured by beta-VNT without going through alpha-VNT and vice versa. At the end of this study, we evaluated the equation accuracy by two parameters; the deviation (d) and the error percentage, which were d = 0.2 and error (%) = 8%, respectively.

New generation vaccines against bluetongue virus

Over the last three decades, a variety of approaches have been investigated to develop new types of bluetongue virus (BTB) vaccines, ranging from baculovirus-expressed subunit vaccines to live vector vaccines. DNA vaccines against BTV consist of DNA plasmid expressing different BTV proteins after inoculation of the animals. The recombinant viral vector vaccines against BTV are based on recombinant viruses that express desired BTV antigens in the host upon inoculation. Viruses such as vaccinia, modified vaccinia Ancara (MVA), capripox, canarypox, herpes, myxoma and fowlpox viruses have been used as vectors of BTV genes. The reverse genetics (RG) systems for BTV are useful tools for BTV vaccine development. Disabled infectious single-cycle (DISC) vaccines make it possible to restore virus replication and can be used for differentiating infected from vaccinated animals (DIVA). These vaccines are based on the production of a modified virus with a deletion in one or more genes that are essential for virus replication. Another approach for BTV vaccine development using RG is the disabled infectious single-animal (DISA) vaccine, generated by deletion of NS3/NS3a expression. DISC and DISA vaccines can mimic the natural tropism of the virus and can express BTV proteins at the site of infection. Important advantages of these new generation vaccines over the conventional BTV vaccines are their high efficacy as well as the possibility of applying them for DIVA. At present, there are a number of novel laboratory-scale BTV vaccines that could meet vaccine profiles required for different field situations. However, further development and licensing of these vaccine candidates for many BTV serotypes is needed in order to prepare for future BT outbreaks. To date, all novel BTV vaccines described in this paper are still under laboratory testing. They are not available commercially, and the time of their application in the field is still indefinite. .

Modeling the spread of capripoxvirus among livestock and optimal vaccination strategies

Capripox is an important transboundary animal disease that is endemic across Africa, the Middle East, and some parts of Asia. The disease is highly contagious and considered to be a major obstacle causing significant economic loses in many agricultural areas. In this study, a mathematical model is developed to describe the transmission dynamics of capripoxvirus (CaPV) among livestock. This proposed model incorporates direct and indirect transmission of CaPV, together with two vaccination strategies, to investigate their effects on the severity of outbreaks and the prevalence of CaPV among the livestock population. The results suggest that ratio of potential vectors to livestock, successful probability of infection, vaccination rates, waning rate of vaccine-conferred protection, and virus introduction time play crucial roles in determining the outbreak severity and the prevalence level. The results also show that it is optimal to vaccinate newborns at the maximum effort throughout the control program and moderately increase vaccination rate for a susceptible pool to reach its maximum level after the outbreak.

Development of real-time and lateral flow dipstick recombinase polymerase amplification assays for rapid detection of goatpox virus and sheeppox virus

BackgroundGoatpox virus (GTPV) and sheeppox virus (SPPV), which belong to the Capripoxvirus (CaPV), are economically important pathogens of small ruminants. Therefore, a sensitive, specific and rapid diagnostic assay for detection of GTPV and SPPV is necessary to accurately and promptly control these diseases.MethodsRecombinase polymerase amplification (RPA) assays combined with a real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA LFD assay) were developed targeting the CaPV G-protein-coupled chemokine receptor (GPCR) gene, respectively.ResultsThe sensitivity of both CaPV real-time RPA assay and CaPV RPA LFD assay were 3 × 102 copies per reaction within 20 min at 38 °C. Both assays were highly specific for CaPV, with no cross-reactions with peste des petits ruminants virus, foot-and-mouth disease virus and Orf virus. The evaluation of the performance of these two assays with clinical sample (n = 107) showed that the CaPV real-time RPA assay and CaPV RPA LFD assay were able to specially detect SPPV or GTPV present in samples of ovine in liver, lung, kidney, spleen, skin and blood.ConclusionsThis study provided a highly time-efficient and simple alternative for rapid detection of GTPV and SPPV.

A G-protein-coupled chemokine receptor: A putative insertion site for a multi-pathogen recombinant capripoxvirus vaccine strategy

Capripoxviruses (CaPVs) have been shown to be ideal viral vectors for the development of recombinant multivalent vaccines to enable delivery of immunogenic genes from ruminant pathogens. So far, the viral thymidine kinase (TK) gene is the only gene used to generate recombinants. A putative non-essential gene encoding a G-protein-coupled chemokine receptor subfamily homologue (GPCR) was targeted as an additional insertion site. Peste des petits ruminants (PPR) was chosen as a disease model. A new recombinant CaPV expressing the viral attachment hemagglutinin (H) of the PPR virus (PPRV) in the GPCR insertion site (rKS1-HPPR-GPCR) was generated in the backbone North African isolate KS1 strain of lumpy skin disease virus (LSDV). Comparison with the recombinant CaPV expressing the H of PPRV in the TK gene (rKS1-HPPR-TK) shown to induce protection against both PPR and LSD in both sheep and goats was assessed. The suitability of the GPCR gene to be a putative additional insertion site in the CaPV genome is evaluated and discussed.

Prevalence, distribution, and risk factor for sheep pox and goat pox (SPGP) in Algeria.

A cross-sectional study using a tested questionnaire was carried out across Algeria between January and June 2014. Our investigation demonstrated that of the 150 flocks visited, 21 were positive for sheep pox and goat pox (SPGP) with an overall flock prevalence of 14% (95% CI 11.08-16.92%) suggesting that SPGP is endemic in Algeria. Our results showed also that the disease appears only in sheep and no case affecting goats has been reported. For the risk factor analysis, univariate analysis of variables followed by a multiple logistic regression identified steppe region (OR = 1.81, 95% CI 0.87-2.57; P = 0.037), large flocks (OR = 2. 19, 95% CI 1.02-3.36; P = 0.027), and transhumance (OR = 3.98, 95% CI 2.59-5.34; P = 0.021) as risk factors in the spread of the disease. Furthermore, our study revealed that the use of vaccination as preventive measures in the selected flocks decreased the odds for SPGP positivity by 5.78 (95% CI 2.22-9.34; P < 0.001) times compared to non vaccinated flocks. In conclusion, our findings documented an evidence of a widespread distribution and endemic establishment of the SPGP in Algerian sheep population despite the annual vaccination program. Consequently, the vaccination must cover all the Algerian sheep population to improve animal welfare and reduce economic losses associated with outbreak episodes.

Investigation of malignant form of sheep pox outbreak in fattening lambs in Mandya, Karnataka

Sheep pox is an endemic and economically important viral disease of small ruminants in India. The present study describes detailed investigation of sheep pox disease outbreak in fattening lambs. The disease outbreak occurred in month of January in a village of Mandya District of Karnataka state in the organized farm. Despite the presence of goats in the same flock, the disease affected sheep only. The clinical signs and post-mortem findings were suggestive of malignant form of sheep pox, which is more frequently recorded in lambs. The duration of disease was 30 days with morbidity and mortality rates of 50.91% and 19.09%, respectively. The capripox virus was identified in the clinical and morbid samples by partial P32 gene based PCR. The sheep pox virus was confirmed by virus isolation and sequencing of full length P32 gene. The sheep poxvirus isolate sequence from the present outbreak, genetically resembled with sheep poxvirus isolates from India and other countries. In conclusion, based on clinical, isolation, PCR and sequence analysis the outbreak was attributed to sheep poxvirus, which highlights the need of at least two separate vaccines for control of sheep and goat pox disease.

Immune response characteristics of Capri pox virus vaccines following emergency vaccination of cattle against lumpy skin disease virus

In this research immune response characteristics of two available heterologous vaccines including Gorgan goat pox virus (GPV) and Romanian sheep pox virus (SPV) vaccines against lumpy skin disease have been examined, by using the monitoring of humoral and cell-mediated immune responses in vaccinated calves in the field. The evaluation of humoral immune response showed that the neutralizing antibody titers in both vaccinated groups started at day 7 post-vaccination, then reached to the protective level at day 21 post-vaccination and persisted till 35 day post-vaccination. The neutralizing antibody titers in GPV-vaccinated calves (GVC) the ratio was higher than SPV-vaccinated calves (RVC), and on days 21 and 35 post-vaccination were significantly different (p

Detection, identification, and differentiation of sheep pox virus and goat pox virus from clinical cases in Giza Governorate, Egypt.

Aim:To isolate, identify, and differentiate Capripoxviruses (CaPV) (sheep pox virus and goat pox virus) infections by egg inoculation, transmission electron microscopy (TEM), and 30 kDa RNA polymerase subunit gene-based polymerase chain reaction (PCR) (RPO30) in clinically affected animals in Hawamdia township of Giza Governorate, Egypt.Materials and Methods:A total of 37 scab samples were collected from clinically suspected field cases of sheep pox and goat pox. These samples were collected during (2014-2015) during different outbreaks of sheep pox and goat pox from Hawamdia township of Giza Governorate, Egypt. The samples were subjected to egg inoculation, TEM, and (RPO30) gene-based PCR. By using the egg inoculation: Previously prepared 37 scab samples (n=23 sheep and n=14 goats) were inoculated on the chorioallantoic membrane of specific pathogen free (SPF) embryonated chicken eggs (12 days old age). In the presence of the suitable percentage of humidity and candling, the inoculated eggs were incubated at 37°C. By using the TEM: Samples showed positive pock lesions on the chorioallantoic membranes, were fixed in glutaraldehyde, then processed and sectioned for TEM. Using the (RPO30) gene-based PCR assay, 30 of positive samples after egg inoculation (n=19 sheep and n=11 goats) were screened.Results:Using the egg inoculation, a characteristic pock lesions for poxviruses were seen in 30/37 (n=19 sheep and n=11 goats) (81.08%). Using the TEM, examination of the positive samples after egg inoculation revealed positive result in 23/30 (n=15 sheep and n=8 goats) (76.66%). The positive results represented by the presence of negatively stained oval-shape virus particles. Using the (RPO30) gene-based PCR assay, out of 30 total of positive samples after egg inoculation (n=19 sheep and n=11 goats) were screened, 27 (90%) samples (n=17 sheep and n=10 goats) were positive. The given band sizes of sheep and goats were 172 and 152 bp, respectively.Conclusion:PCR assay depended on RPO30 gene can be used lonely for the detection, identification, and differentiation of CaPVs. RPO30 gene-based PCR assay in combination with gene sequencing helps in molecular epidemiological studies of CaPV infection.

Epidemiological Study of Lumpy Skin Disease Outbreaks in North-western Iran

Lumpy skin disease (LSD) is a highly contagious transboundary disease of cattle with major economic losses. This study was undertaken to address the emergence and epidemiological features of LSD in four north-western provinces of Iran. These provinces have extensive borders with others country including Iraq, Turkey, Azerbaijan and Armenia. A population of 683 cattle from 91 farms were examined during LSD outbreak in Iran during 2014-2016. The information of the farms including the population size, gender, age, vaccination status, clinical signs and the number of death because of LSD were recorded in the designed questionnaires. A number of 234 blood samples were collected randomly from animals with and without clinical signs of LSD. DNA was extracted from blood samples, and they were used for amplifying a fragment of 434bp in size coupled with restriction fragment length polymorphism (RFLP) for molecular detection of lumpy skin disease virus (LSDV). The estimated prevalence, cumulative mortality and case fatality were 17.9%, 3.5% and 19.7%, respectively. There was no significant difference in occurrence of the disease between male and female cattle. LSD occurrence in age groups above 5years old and below 6months old showed highest and lowest relative frequencies, respectively. Vaccination was significantly decreased the occurrence of clinical disease. The developed PCR-RFLP technique was able to differentiate between LSDV, sheep pox virus (ShPV) and goat pox virus (GPV). It was concluded that LSD was entered into Iran probably from Iraq via uncontrolled animal movements along common land borders between two countries. Developed PCR-RFLP could be used as a rapid and inexpensive method for differentiating Capripoxviruses (CaPVs).