Cowpox Research Articles

The XIX century smallpox prevention in Naples and the risk of transmission of human blood-related pathogens.

Vaccines are the most successful strategy developed in Medicine to prevent and even eradicate the most dreadful epidemic infectious diseases. The history of smallpox vaccination in Naples is quite unique. Although Galbiati established the retro-vaccination (1803) and developed the “calf” lymph vaccine, recognized and implemented since 1864 as the optimal smallpox vaccine in the following hundred years, Naples general population was mainly vaccinated with “human” lymph from abandoned children until 1893. Mini-epidemics of syphilis and serum hepatitis were periodically reported as results of arm-to-arm procedure. The risk of transmission of blood-related pathogens was higher in Naples where >80% of abandoned children, used as repository of cowpox virus, were dying in their first year of life. Recent vaccinology standards finally eliminated the risk of adventitious contaminating pathogens. Implementation of hepatitis B vaccination since 1991 eventually contributed to current HBV prevalence in Campania region <1%, within the range of the European Countries.

The clinical effects of cowpox vaccination in inflammatory skin extract in the treatment ofdiabetic peripheral neuropathy

Objective To study the effectiveness of vaccinia vaccination rabbit inflammatory skin extracts on blood homocysteine（Hcy） and insulin-like growth factor-1（IGF-1） in patients with diabetic peripheral neuropathy. Methods 100 patients with diabetic peripheral neuropathy were randomly divided into the two groups,the observa-tion group（n=50 cases） and the control group（n=50 cases）.The observation group was treated through vaccinia vaccination rabbit inflammatory skin extracts, while the control group was treated through mecobalamin.They were treated for 15 days.Median nerve,peroneal motor nerve conduction velocity （MCV） and sensory nerve conduction velocity （ SCV） were determined before and after treatment.Blood IGF-1 and Hcy were detected.Results TSS were significantly lowered after treatment （t=9.772,13.624,all P〈0.01）.Compared with the control group,the observa-tion group was significantly decreased （t=3.925,P〈0.05）.After treatment,the median nerve,peroneal nerve MCV and SCV were significantly increased （t=5.103,3.019,4.998,2.928,5.128,3.112,5.286,3.118,all P〈0.05）. Compared with the control group,theobservation group was observed to increase more significantly（t=2.826,2.743, 3.268,3.096,all P〈0.05）.After treatment,serum IGF-1 were significantly elevated,and compared with the control group after treatment,post-treatment observation group increased more significantly（t=4.179,P〈0.05）.After treat-ment plasma Hcy were significantly lower than that of the control group after treatment,the observation group after treatment significantly decreased（t=3.165,P〈0.05）.Conclusion Vaccinia vaccination rabbit inflammatory skin extracts can improve blood Hcy and IGF-1 in patients with diabetic peripheral neuropathy. Key words: Diabetic neuropathies; Viral vaccines; Tissue extracts; Cysteine

A negative feedback modulator of antigen processing evolved from a frameshift in the cowpox virus genome.

Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing.

Study of the Inhibitory Action of Marboran on the Replication Cycle of Cowpox Virus

Research Project The project undertaken involved a study of the effects of the inhibitor N-methyl isatin thiosemicarbazone (Marboran) on the replication cycle of cowpox virus in monolayers of baby hamster kidney cells (BHK). The cytotoxic effects of Marboran on the BHK cells were studied and were found to be directly related to the time of exposure to the inhibitor with a maximum tolerated dose of Marboran of 30/UM/ml. after 7 days. This drug concentration was subsequently used to avoid the occurrence of a cytotoxic effect of Marboran masking any cytopathic effect (CPE) of the virus when both inhibitor and virus were used in conjunction.

Exploring interaction of TNF and orthopoxviral CrmB protein by surface plasmon resonance and free energy calculation.

Inhibition of the activity of the tumor necrosis factor (TNF) has become the main strategy for treating inflammatory diseases. The orthopoxvirus TNF-binding proteins can bind and efficiently neutralize TNF. To analyze the mechanisms of the interaction between human (hTNF) or mouse (mTNF) TNF and the cowpox virus N-terminal binding domain (TNFBD-CPXV), also the variola virus N-terminal binding domain (TNFBD-VARV) and to define the amino acids most importantly involved in the formation of complexes, computer models, derived from the X-ray structure of a homologous hTNF/TNFRII complex, were used together with experiments. The hTNF/TNFBD-CPXV, hTNF/TNFBD-VARV, mTNF/TNFBD-CPXV, and mTNF/TNFBD-VARV complexes were used in the molecular dynamics (MD) simulations and MM/GBSA free energy calculations. The complexes were ordered as hTNF/TNFBD-CPXV, hTNF/TNFBD-VARV, mTNF/TNFBD-CPXV and mTNF/TNFBD-VARV according to increase in the binding affinity. The calculations were in agreement with surface plasmon resonance (SPR) measurements of the binding constants. Key residues involved in complex formation were identified.

Real-time PCR assay for specific detection of cowpox virus

The species cowpox virus (CPXV), genus Orthopoxvirus (OPV), consists of isolates highly variable in their biological properties and their genotypes. A TaqMan PCR assay for the specific detection of CPXV DNA based on sequences of the ORF D11L has been developed recently. (Gavrilova et al., 2010; Shchelkunov et al., 2011); however, a rather limited panel of CPXV stains has been used. When a much larger panel of 47 CPXV DNAs has been tested, three strains could not be amplified at all because of large deletions in their respective ORF D11L. In addition, a deletion of 23bp led to low-efficiency detection of five other CPXV strains. To solve this problem a new primer/probe combinations was selected based on sequences of ORF D8L, and a new real-time PCR method for (i) a genus-specific detection of OPVs and (ii) a simultaneous CPXV-specific differentiation is described in this study. The specificity and sensitivity were assessed by analyzing DNA of 67 strains belonging to human-pathogenic OPV species, including variola virus, as well as specimens of CPXV-infected mice.

Incongruencies in Vaccinia Virus Phylogenetic Trees

Over the years, as more complete poxvirus genomes have been sequenced, phylogenetic studies of these viruses have become more prevalent. In general, the results show similar relationships between the poxvirus species; however, some inconsistencies are notable. Previous analyses of the viral genomes contained within the vaccinia virus (VACV)-Dryvax vaccine revealed that their phylogenetic relationships were sometimes clouded by low bootstrapping confidence. To analyze the VACV-Dryvax genomes in detail, a new tool-set was developed and integrated into the Base-By-Base bioinformatics software package. Analyses showed that fewer unique positions were present in each VACV-Dryvax genome than expected. A series of patterns, each containing several single nucleotide polymorphisms (SNPs) were identified that were counter to the results of the phylogenetic analysis. The VACV genomes were found to contain short DNA sequence blocks that matched more distantly related clades. Additionally, similar non-conforming SNP patterns were observed in (1) the variola virus clade; (2) some cowpox clades; and (3) VACV-CVA, the direct ancestor of VACV-MVA. Thus, traces of past recombination events are common in the various orthopoxvirus clades, including those associated with smallpox and cowpox viruses.

Identification and characterization of a phage display-derived peptide for orthopoxvirus detection

Fast and reliable diagnostic assays are required for a resilient detection of clinical infections or biothreat-relevant pathogens. While PCR has proven to be the gold standard for nucleic acid detection, the identification of pathogen particles is still challenging and depends on the availability of well-characterized, chemically stable, and selective recognition molecules. Here, we report the screening of a phage display random peptide library for vaccinia virus-binding peptides. The identified peptide was extensively characterized using peptide-probe ELISA, surface plasmon resonance, nLC-MS/MS, Western Blot, peptide-based immunofluorescence assay, and electron microscopy. Following identification, the phage-free, synthetic peptide, designated αVACVpep05, was shown to bind to vaccinia virus and other orthopoxviruses. We can demonstrate that the highly conserved orthopoxvirus surface protein D8 is the interaction partner of αVACVpep05, thus enabling the peptide to bind to other orthopoxviruses, including cowpox virus and monkeypox virus, viruses that cause clinically relevant zoonotic infections in humans. The process of phage display-mediated peptide identification has been optimized intensively, and we provide recommendations for the identification of peptides suitable for the detection of further pathogens. The peptide described here was critically characterized and seems to be a promising reagent for the development of diagnostic platforms for orthopoxviruses. We believe that our results will help to promote the development of alternative, nonantibody-based synthetic detection molecules for further pathogens.

Transcutaneous applications for vaccination and immunotherapy

Although Edward Jenner applied the first vaccines by scratching cow pox material into the skin, the profound immunological properties of the skin have become evident through research and discoveries only in the last 20 years. The immunological cells in the epidermis and the dermis are suitable targets for transcutaneous vaccination and immunotherapy. However, as the skin represents a natural barrier for topically administered large molecules, novel methods to overcome this barrier function have been described. There are chemical, biochemical and physical methods, many of which are pain-free and therefore especially suitable for children. Also for adults non-invasive methods of vaccination and immunotherapy are attractive as self-administration is feasible. Future products are currently undergoing clinical tests which provide promising results.

Cowpox virus protein CPXV012 eludes CTLs by blocking ATP binding to TAP.

CD8(+) CTLs detect virus-infected cells through recognition of virus-derived peptides presented at the cell surface by MHC class I molecules. The cowpox virus protein CPXV012 deprives the endoplasmic reticulum (ER) lumen of peptides for loading onto newly synthesized MHC class I molecules by inhibiting the transporter associated with Ag processing (TAP). This evasion strategy allows the virus to avoid detection by the immune system. In this article, we show that CPXV012, a 9-kDa type II transmembrane protein, prevents peptide transport by inhibiting ATP binding to TAP. We identified a segment within the ER-luminal domain of CPXV012 that imposes the block in peptide transport by TAP. Biophysical studies show that this domain has a strong affinity for phospholipids that are also abundant in the ER membrane. We discuss these findings in an evolutionary context and show that a frameshift deletion in the CPXV012 gene in an ancestral cowpox virus created the current form of CPXV012 that is capable of inhibiting TAP. In conclusion, our findings indicate that the ER-luminal domain of CPXV012 inserts into the ER membrane, where it interacts with TAP. CPXV012 presumably induces a conformational arrest that precludes ATP binding to TAP and, thus, activity of TAP, thereby preventing the presentation of viral peptides to CTLs.

Molecular characterization and phylogenetics of Fennoscandian cowpox virus isolates based on the p4c and atip genes.

BackgroundCowpox virus (CPXV), a rodent-borne Orthopoxvirus (OPV) that is indigenous to Eurasia can infect humans, cattle, felidae and other animals. Molecular characterization of CPXVs isolated from different geographic locations is important for the understanding of their biology, geographic distribution, classification and evolution. Our aim was to characterize CPXVs isolated from Fennoscandia on the basis of A-type inclusion (ATI) phenotype, restriction fragment length polymorphism (RFLP) profiles of atip gene fragment amplicon, and phylogenetic tree topology in conjunction with the patristic and genetic distances based on full length DNA sequence of the atip and p4c genes.MethodsATI phenotypes were determined by transmission electron microcopy and RFLP profiles were obtained by restriction enzyme digestion of the atip gene fragment PCR product. A 6.2 kbp region spanning the entire atip and p4c genes of Fennoscandian CPXV isolates was amplified and sequenced. The phylogenetic affinity of Fennoscandian CPXV isolates to OPVs isolated from other geographic regions was determined on the basis of the atip and p4c genes.ResultsFennoscandian CPXV isolates encoded full length atip and p4c genes. They produce wild type V+ ATI except for CPXV-No-H2. CPXVs were resolved into six and seven species clusters based on the phylogeny of the atip and p4c genes respectively. The CPXVs isolated from Fennoscandia were grouped into three distinct clusters that corresponded to isolates from Norway, Sweden and Finland.ConclusionCPXV is a polyphyletic assemblage of six or seven distinct clusters and the current classification in which CPXVs are united as one single species should be re-considered. Our results are of significance to the classification and evolution of OPVs.

N-phenethyl-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamides as possible anti-viral agents

Based on the preliminary calculated screening performed by the PASS programme the synthesis of the group of the corresponding N-phenethyl-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamides as potential antiviral agents has been carried out by the interaction of ethyl esters of 2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acids with 2-arylethylamines in boiling ethanol. Their structure has been confirmed by the data of elemental analysis and NMR 1H spectroscopy. Of the Herpesviruses family such viruses as Herpes Simplex Viruses Type 1 and 2, Varicella-Zoster Virus, Epstein-Barr Virus, as well as Cytomegalovirus have been involved in the screening research. Influenza Virus was presented by two types: A (subtypes H1N1, H3N2, H5N1) and B. The group of viruses affecting different sections of the respiratory tract included Parainfluenza Virus, SARS Virus, Rhinovirus, Adenovirus and Respiratory Syncytial Virus. Besides, Measles Virus, Vaccinia Virus and related Cowpox Virus, Hepatitis B and C Viruses, as well as Venezuelan Equine Encephalitis Virus were involved. The group of viruses causing febrile states of varying severity in human was presented by Rift Valley Fever Virus, West Nile Virus, Yellow Fever Virus, Dengue Virus and Tacaribe Virus. According to the results of the tests performed N-(4-chlorophenethyl)- 2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide has been recommended for further research. Having a low cytotoxicity this compound revealed a high antiviral activity in relation to the West Nile fever virus agent.

Identification of 10 Cowpox Virus Proteins That Are Necessary for Induction of Hemorrhagic Lesions (Red Pocks) on Chorioallantoic Membranes

Cowpox viruses (CPXV) cause hemorrhagic lesions ("red pocks") on infected chorioallantoic membranes (CAM) of embryonated chicken eggs, while most other members of the genus Orthopoxvirus produce nonhemorrhagic lesions ("white pocks"). Cytokine response modifier A (CrmA) of CPXV strain Brighton Red (BR) is necessary but not sufficient for the induction of red pocks. To identify additional viral proteins involved in the induction of hemorrhagic lesions, a library of single-gene CPXV knockout mutants was screened. We identified 10 proteins that are required for the formation of hemorrhagic lesions, which are encoded by CPXV060, CPXV064, CPXV068, CPXV069, CPXV074, CPXV136, CPXV168, CPXV169, CPXV172, and CPXV199. The genes are the homologues of F12L, F15L, E2L, E3L, E8R, A4L, A33R, A34R, A36R, and B5R of vaccinia virus (VACV). Mutants with deletions in CPXV060, CPXV168, CPXV169, CPXV172, or CPXV199 induced white pocks with a comet-like shape on the CAM. The homologues of these five genes in VACV encode proteins that are involved in the production of extracellular enveloped viruses (EEV) and the repulsion of superinfecting virions by actin tails. The homologue of CPXV068 in VACV is also involved in EEV production but is not related to actin tail induction. The other genes encode immunomodulatory proteins (CPXV069 and crmA) and viral core proteins (CPXV074 and CPXV136), and the function of the product of CPXV064 is unknown. It has been known for a long time that cowpox virus induces hemorrhagic lesions on chicken CAM, while most of the other orthopoxviruses produce nonhemorrhagic lesions. Although cowpox virus CrmA has been proved to be responsible for the hemorrhagic phenotype, other proteins causing this phenotype remain unknown. Recently, we generated a complete single-gene knockout bacterial artificial chromosome (BAC) library of cowpox virus Brighton strain. Out of 183 knockout BAC clones, 109 knockout viruses were reconstituted. The knockout library makes possible high-throughput screening for studying poxvirus replication and pathogenesis. In this study, we screened all 109 single-gene knockout viruses and identified 10 proteins necessary for inducing hemorrhagic lesions. The identification of these genes gives a new perspective for studying the hemorrhagic phenotype and may give a better understanding of poxvirus virulence.

Poxviral infection elicits human neutralizing antibodies recognizing diverse epitopes of the major vaccinia virus surface protein D8 (VIR4P.1016)

Abstract Immunization with vaccinia virus (VACV) confers cross-protective immunity to variola, the causative agent of smallpox, and other clinically important poxviral species, such as cowpox and monkeypox viruses. Vaccine-mediated protection has been correlated with elicitation of broad neutralizing antibody (Ab) responses, however the specificity of human neutralizing antibodies to poxviruses is poorly understood. We used a highly-optimized human hybridoma technology to generate large panels of anti-VACV monoclonal Abs (mAbs) from vaccinia-immunized subjects or from subjects who had naturally acquired monkeypox infection. Immunologic characterization and DNA sequencing of the panel revealed 16 individual mAbs that recognized the immunodominant surface protein antigen D8. Several of anti-D8 mAbs possessed neutralizing activity against VACV, cowpox and monkeypox viruses. These Abs exhibited mainly a complement-dependent pattern of neutralization and they formed at least four cross-blocking groups, suggesting the existence of several non-overlapping neutralizing epitopes for D8. The results suggest that D8 is an important target for human neutralizing Abs generated following poxviral infection or vaccination, and suggest the existence of novel epitopes targeted by anti-D8 Abs that contribute to cross-protective immunity following smallpox vaccination.

Fatal Cowpox Virus Infection in Cotton-Top Tamarins (Saguinus oedipus) in Germany

Cowpox virus (CPXV) was isolated from a fatal outbreak among cotton-top tamarins. Samples from healthy common marmosets in contact were also CPXV genome positive. The CPXV isolated from the cotton-top tamarins exhibited a unique hemagglutinin sequence. Pathogenicity investigations using a Wistar rat model characterized the isolate as low pathogenic.

Direct detection of green fluorescent protein in histological preparations of recombinant vaccinia virus in mice (1050.9)

Green fluorescent protein (GFP)‐expressing recombinant viruses have proven to be invaluable tools for the in‐vivo monitoring of viral kinetics and disease progression in infectious disease models. Histopathological evaluation of the ex‐vivo tissues by way of immunohistochemical (IHC) staining for viral antigens further assists in the characterization of disease pathology, but can be insensitive, nonspecific, and time consuming. In this study, we determine if the direct detection of GFP signal provides a suitable alternative to IHC staining in GFP‐expressing tissues. Fixation and histological processing protocols were developed in a GFP‐expressing 293T cells and then optimized and validated in a GFP‐recombinant rat model. The optimized protocol was then applied to GFP‐recombinant cowpox virus (VACV‐TK‐GFP) infected mouse lungs in addition to lungs infected with a wild‐type virus strain (VACV‐WR). While animals infected with both virus strains manifested lesions reactive to anti‐Vaccinia IHC, GFP fluorescence was only detected in lesions from animals infected with the recombinant virus strain. The method was further validated through IHC staining of both virus strains with anti‐GFP antibodies, which were only reactive in tissues infected with VACV‐TK‐GFP. Digital alignment of sections showed a strong correlation between GFP fluorescence and IHC staining for GFP and viral proteins suggesting direct‐detection of fluorescent proteins is a promising alternative to traditional methods.

A Role for H/ACA and C/D Small Nucleolar RNAs in Viral Replication

We have employed gene-trap insertional mutagenesis to identify candidate genes whose disruption confer phenotypic resistance to lytic infection, in independent studies using 12 distinct viruses and several different cell lines. Analysis of >2,000 virus-resistant clones revealed >1,000 candidate host genes, approximately 20 % of which were disrupted in clones surviving separate infections with 2–6 viruses. Interestingly, there were 83 instances in which the insertional mutagenesis vector disrupted transcripts encoding H/ACA-class and C/D-class small nucleolar RNAs (SNORAs and SNORDs, respectively). Of these, 79 SNORAs and SNORDs reside within introns of 29 genes (predominantly protein-coding), while 4 appear to be independent transcription units. siRNA studies targeting candidate SNORA/Ds provided independent confirmation of their roles in infection when tested against cowpox virus, Dengue Fever virus, influenza A virus, human rhinovirus 16, herpes simplex virus 2, or respiratory syncytial virus. Significantly, eight of the nine SNORA/Ds targeted with siRNAs enhanced cellular resistance to multiple viruses suggesting widespread involvement of SNORA/Ds in virus–host interactions and/or virus-induced cell death.

Elimination of A-type inclusion formation enhances cowpox virus replication in mice: Implications for orthopoxvirus evolution

Some orthopoxviruses including cowpox virus embed virus particles in dense bodies, comprised of the A-type inclusion (ATI) protein, which may provide long-term environmental protection. This strategy could be beneficial if the host population is sparse or spread is inefficient or indirect. However, the formation of ATI may be neutral or disadvantageous for orthopoxviruses that rely on direct respiratory spread. Disrupted ATI open reading frames in orthopoxviruses such as variola virus, the agent of smallpox, and monkeypox virus suggests that loss of this feature provided positive selection. To test this hypothesis, we constructed cowpox virus mutants with deletion of the ATI gene or another gene required for embedding virions. The ATI deletion mutant caused greater weight loss and higher replication in the respiratory tract than control viruses, supporting our hypothesis. Deletion of the gene for embedding virions had a lesser effect, possibly due to known additional functions of the encoded protein.

Merits and Demerits of Vaccination for Patients with Neurological Diseases

When it comes to vaccination, you might have heard about Edward Jenner when you were students. Based on experimental findings revealing that individuals with a history of cowpox virus infection were less likely to be infected with smallpox, and smallpox infection only led to mild infectious symptoms without resulting in death, researchers investigated the application of vaccines to treatment. It appears that experiments performed by other physicians caused numerous deaths. Jenner vaccinated humans against smallpox using cowpox virus. This attempt, currently referred to as vaccination, has proved to be successful and saved many people’s lives. Consequently, in May 1980, the WHO declared that smallpox had been eliminated.Thus, appropriately used vaccination is highly effective and markedly benefits society. However, I think that we need to obtain accurate information on vaccination, such as the individuals for whom each type of vaccination should be indicated, the timing of its use, its limitations, and side effects. As is the case with any drug, no vaccine achieves the same effects in every person, and the beneficial and side effects of vaccines may differ according to each individual’s immune state. The purposes of vaccine use also differ according to the target person and area.In vaccination, foreign substances (nonself antigens) that do not exist in the human body are administered through the skin, nose, or mouth, or using intramuscular or intravenous injections, in order to induce the involuntary activation of an individual’s immune function. In other words, vaccine is the key player to perturb the immunological milieu and therefore initiates break tolerance.Therefore, vaccination may cause severe side effects, which are sometimes fatal.In this public lecture on vaccination, other speakers have reported the latest relevant information, but I will describe the neurological side effects of influenza vaccine. I am also going to talk about the neurological side effects of cervical cancer vaccine, which has been a hot topic in Japan recently, the new reporting system in post-vaccination inflammatory CNS demyelinating event such as ADEM, and the history of therapeutic vaccination against multiple sclerosis, which is my main research theme. Also I discuss the failed vaccination trial against Alzheimer’s disease, which is becoming increasingly prevalent worldwide, including Japan. I enclosed lead out in these studies, and how to tackle issues associated with vaccination, whose frequency is expected to increase globally. We have learned that a risk (疫eki; plague ) and a benefit (益eki; value) are two sides of the same coin. To protect ourselves from disease, we need to properly understand immunity and excessive immunological responses.

Genome Annotation and Comparative Genomics of ORF Virus

ORF virus (ORFV), the etiological agent of contagious pustular dermatitis in small ruminants, belongs to members of the genus Parapoxvirus of the Poxviridae. The genome of the ORFV is dsDNA of 139,962 bp which has about 89% coding region, 63% GC content and codes 130 proteins. There are four unique genes within the genome revealed by homology search of them two posses’ strong regulatory region and transmembrane helices. One of the ORF-039 contains signal peptide indicating the possibilities to be secretory protein coding gene. Comparative genomic analysis reveals significant differences in Bovine Papular Stomatitis Virus (BPSV) strain BV-AR02 and ORFV strain OV-SA00, and these may account for differences in host range. Interspecies sequence variability is observed in all functional classes of genes but is the highest in putative virulence/host range genes. Notably, ORFV contains genes which are homologous of Vaccinia virus. Phylogenetic analysis reveals that although divergent, ORFV virus is distinct from other known mammalian cowpox virus. An improved understanding of Parapoxvirus (PPV) biology will permit the engineering of novel vaccine viruses and expression vectors with enhanced efficacy and greater versatility. The novel vaccine will have a significant role in the economy of a country through the control of disease in an economically important and small ruminant caused by ORFV.