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1.
Molecular characterization of Brazilian isolates of orf virus   总被引：4，自引：0，他引：4  
Outbreaks of an epidermic disease suggesting parapox virus infections have been observed in all major herds of sheep and goats from different geographical areas of Brazil. Clinical samples (dried scabs) were collected and orf virus was isolated and characterized by electron microscopy in previous work. In order to characterize these viruses at the molecular level, a modified methodology for genomic DNA extraction directly from scabs was used and such DNA was used to derive the restriction enzyme digestion patterns for clinical samples from three distinct geographic origins. Pulsed field gel electrophoresis was used to separate restriction enzyme DNA fragments and heterogeneity among isolates from different geographic areas could be observed on stained gels. The HindIII-G DNA fragment from orf-A virus genome was cloned and hybridized to DNA of other orf virus isolates. Further heterogeneity was confirmed by these hybridizations.  相似文献   

2.
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3.
Five cases of orf virus infection in Korean black goats were diagnosed in our laboratory between 2010 and 2011. One orf virus (ORF/2011) was isolated from an ovine testis cell line (OA3.Ts) for use as a vaccine candidate. Sequences of the major envelope protein and orf virus interferon resistance genes were determined and compared with published reference sequences. Phylogenetic analyses revealed that orf viruses from Korean black goats were most closely related to an isolate (ORF/09/Korea) from dairy goats in Korea. This result indicates that the orf viruses might have been introduced from dairy goats into the Korean black goat population.  相似文献   

4.
Orf virus encodes a range of immuno-modulatory genes that interfere with host anti-virus immune and inflammatory effector mechanisms. The function of these reflects the pathogenesis of orf. The orf virus interferon resistance protein (OVIFNR) and virus IL-10 (vIL-10) inhibit interferon production and activity. In addition the vIL-10 suppresses inflammatory cytokine production by activated macrophages and keratinocytes. The virus GM-CSF inhibitory factor (GIF) is a novel virus protein that binds to and inhibits the biological activity of GM-CSF and IL-2. Together, these immuno-modulators target key effector mechanisms of host anti-virus immunity to allow time for virus replication in epidermal cells.  相似文献   

5.
Thirteen orf virus (ORFV) isolates from natural outbreaks in sheep and goats belonging to different geographical regions of India were analysed on the basis of ORF108 (a homologue of poxviral A32 gene), which is known to encode for ATPase and involved in virion DNA packaging. Comparative sequence analysis of ATPase proteins revealed highly conserved N-terminal region with five different motifs [Walker A, Walker B, A32L specific motifs (III and IV) and a novel AYDG (motif-V)] among all poxviruses and divergent carboxyl terminus with either single or double RGD sequences among all Indian ORFV isolates. A homology model and secondary structure predictions of N-terminal region of ORFV A32 revealed that most of the poxviruses including ORFV ATPase protein belong to a distinct clade of the HerA/FtsK super family of DNA packaging proteins. Despite differences in host cell specificity and poxvirus infections among animals, DNA packaging motor domain of poxviruses presumed to share remarkable similarities as indicated by the presence of conserved ATPase motifs in the present investigation. The study also indicated the circulation of heterogeneous strains of ORFV in India and possibilities of differentiation of ORFV strains based on C-terminal heterogeneity.  相似文献   

6.
Immunomodulation by virulence proteins of the parapoxvirus orf virus   总被引：4，自引：0，他引：4  
Three orf virus putative virulence proteins are described that exhibit immunomodulatory functions. The OVIFNR gene at the left terminus of the viral genome encodes an interferon resistance protein with homology to the E3L gene of vaccinia virus. OVIFNR functions by preventing a dsRNA-dependent kinase from inhibiting virus and cell protein synthesis as part of the interferon-induced anti-viral state within infected cells. The orf virus orthologue of the ovine interleukin-10 (vIL-10) gene is located at the right terminus of the viral genome. Both vIL-10 and host (ovine) IL-10 function in vitro as inhibitors of pro-inflammatory cytokine production by keratinocytes and macrophages, and both inhibit IFN-gamma production from activated peripheral blood lymphocytes. Both the orf virus vIL-10 and ovine IL-10 stimulate mast cell and thymocyte proliferation. In this respect the orf virus IL-10 differs from Epstein Barr virus IL-10 which does not exhibit cell proliferative activity. Finally, the orf virus GM-CSF inhibitory factor gene (GIF) at the right terminus of the viral genome encodes an inhibitor of GM-CSF that also binds IL-2. Together, these viral proteins are capable of inhibiting key components of the ovine anti-virus immune and inflammatory response.  相似文献   

7.
Monoclonal antibodies were produced against orf virus-specified cell surface proteins in an attempt to develop reagents capable of differentiating between members of the Parapoxviridae. Two immunization protocols were used to induce an anti-orf response in BALB/c mice, one of which resulted in virus replication in the recipient. The monoclonal antibodies produced were tested for crossreactivity with bovine papular stomatitis virus (BPS) and milker's node virus (MNV) by indirect immunofluorescence assay (IFA) and immunoblotting. The results indicate that significant antigenic overlap exists between isolates of orf, MNV and BPS, even at the level of specificity provided by monoclonal antibodies. One monoclonal antibody reacted strongly in IFA with orf virus isolates, very weakly with MNV, and not at all with BPS. On immunoblots this same antibody recognized a 40-43 kDa protein in orf virus-infected cells, and also a 45-48 kDa protein in cells infected with MNV or BPS virus. The data suggest that it may be possible to define parapoxvirus strains on the basis of small variations in specific virus-directed cell surface proteins.  相似文献   

8.
A genomic library of infectious laryngotracheitis virus (ILTV) DNA BamH1 fragments was prepared and two cloned fragments were evaluated for their potential as probes for the detection of ILTV infected cells. The virus was purified by a modified sucrose density gradient procedure for the isolation of pure ILTV DNA. A genomic library was constructed using BamH1-digested ILTV DNA and pGEM7 as a vector. A 1.1 kb cloned BamH1 fragment of ILTV DNA was tested in a slot or dot blot assay for the detection of ILTV infected cells. The limit of detection for this probe was at least 0.12 ng of pure ILTV DNA. The probe was able to identify both chicken embryo liver (CELi) cells and choriallantoic membranes infected with ILTV. Chicken embryo liver cells infected with several field isolates and a vaccine strain of ILTV were positive by dot blot analysis using this probe. Some qualitative differences in the degree of hybridization to cells infected by different ILTV isolates were observed. Uninfected cells and cells infected with fowlpox virus, turkey herpesvirus, Marek's disease virus or Newcastle disease virus were negative by the same assay. Compared with the 1.1 kb fragment, a larger 6 kb cloned BamH1 fragment of ILTV DNA showed a stronger hybridization signal to DNA from ILTV infected cells.  相似文献   

9.
Since 1981 a domesticated muskoxen herd had been successfully vaccinated against papillomatosis with homogenated, glutaraldehyde inactivated papilloma tissue. In the fall of 1985 a new clinical outbreak of disease occurred, affecting previously infected as well as vaccinated animals. The purification of parapox virions directly from papilloma tissue and orf scabs collected in a local sheep farm was followed by restriction endonuclease analysis of viral DNA. The morphological identity of purified virus was controlled by electron microscopy. Comparison of restriction endonuclease digests (10 different enzymes) by gel electrophoresis demonstrated that the muskoxen parapoxvirus from the new outbreak 1985 differed considerably from the 2 other isolates (muskoxen 1981 and local orf). The latter viruses demonstrated a high degree of homology, but differences were evident after digestion with the enzyme EcoRI. During metrizamide gradient purification minor bands containing morphologically intact virions were isolated in addition to the major fractions. The restriction enzyme digests indicated that the virions of the minor bands differed from those in the major bands.  相似文献   

10.
Severe persistent orf in young goats.   总被引：1，自引：0，他引：1  
Orf (contagious ecthyma) is a viral disease of small and wild ruminants, humans, and less frequently other species. In sheep and goats, the disease is characterized by the formation of vesiculo-proliferative lesions in the skin of lips and nostril. Here, a form of generalized orf in 16 goat kids from 2 different locations in west Texas is described. The disease was characterized by multifocal, severe, proliferative dermatitis that persisted from about 2 months of age until the goat kids were euthanized 3 months later. All affected goats were Boer or Boer crosses under 1 year of age. The mean immunoglobulin concentration in sera of affected goats was elevated compared with healthy control goats. Severe to moderate lymphadenomegaly of the nodes draining the areas of the skin affected with orf lesions was present in all 16 goat kids. Suppurative arthritis, chronic fibrinous pneumonia, and premature thymic involution were found in 3, 5, and 7 of the goat kids, respectively. The skin lesions of 3 goat kids were infested with larvae of the opportunistic black garbage fly (Ophira sp.). The orf virus was identified in skin lesions by isolation in Marbin-Darby ovine kidney cells, electron microscopy, and amplification of viral DNA by polymerase chain reaction. The orf virus was not detected in peripheral blood or lymph node mononuclear cells of any of the goats. Cross-neutralization experiments showed that an ovine orf virus antiserum raised in sheep was more effective in neutralizing a sheep orf virus isolate than a caprine orf virus isolate. The clinical and epidemiological characteristics of these orf cases may be the result of susceptibility factors within some individuals of the Boer breed of goats.  相似文献   

11.
为了建立鉴别绵羊痘病毒(SPPV)、山羊痘病毒(GTPV)和羊口疮病毒(ORFV)的多重PCR检测方法,针对GenBank中3种病毒的基因组序列,合成了3对引物,通过优化多重PCR反应条件,建立了鉴别检测3种病毒的多重PCR方法。特异性试验表明,应用该方法可分别扩增出3种病毒对应的目的片段,对大肠埃希菌、沙门菌、猪圆环病毒2型(PCV2)、猪细小病毒(PPV)、Vero细胞、正常羊组织的DNA和灭菌双蒸水均无扩增;敏感性试验表明,该方法最低检测量分别为30.46pg/μL的绵羊痘病毒、28.9pg/μL的山羊痘病毒和26.94pg/μL的羊口疮病毒基因组DNA;应用本方法对85份临床病料进行检测,结果与其他已建立的单项PCR检测方法结果一致,说明该方法可以用于临床上SPPV、GTPV和ORFV的鉴别诊断。  相似文献   

12.
Orf virus (ORFV), the type species of Parapoxvirus, is responsible for contagious ecthyma in sheep and goats. In the present report, sequence analysis of major envelope gene (B2L) of four Indian orf virus isolates originating two each from sheep and goats was carried out. These recent isolates belonged to different outbreaks that occurred in Kumaon hills and adjoining plains during 2004-2005. Preliminary screening of the scab samples was carried out by diagnostic PCR. Full-length B2L gene encoding for immunogenic major envelope protein from all the four ORFV isolates was amplified by PCR and the amplicons (1206 bp) were cloned and sequenced. Comparative sequence analysis revealed an open reading frame of 1137 nucleotides (nt) encoding a polypeptide of 378 amino acids (aa). Indian isolates were highly related amongst themselves with sequence identity of over 97% at the nt and aa level. Further, they showed 97-98% sequence identity with sequences of other ORFV isolates from around the world; while 94-95 and 82.7-83.8% sequence identity was observed, respectively, with pseudocowpox and bovine papular stomatitis viruses--the other members of the genus. Phylogenetic analysis also showed that these Parapoxviruses from sheep and goats are closely related to other orf viruses reported worldwide.  相似文献   

13.
Eight field isolates of bovid herpesvirus type 4 (BHV-4) were examined by restriction analysis and Southern blot hybridization with respect to their relatedness to one another and to the BHV-4 prototype strain DN-599. Isolates were obtained from cattle exhibiting a range of disease states including abortion, pneumonia, enteritis, metritis, and vaginal blisters. Initial growth studies of all 9 viruses were performed and revealed that the overall rate of virus growth was slow when compared with that of other herpesviruses. Infection with each virus also resulted in the formation of large fused cells, which in addition to the slow growth rate, indicated that the isolates were of the cytomegalovirus type. Further studies to characterize and compare the various BHV-4 isolates were undertaken by obtaining cell-free virus from infected cell populations. Viral isolates were purified and used as a source of BHV-4 DNA. Purified DNA, representing each of the 8 field isolates and the prototype strain DN-599, were each cleaved with 3 restriction enzymes and were separated by agarose-gel electrophoresis, and the resultant fragment patterns were compared. In general, genomic fragments of the field isolates corresponded to those generated by cleavage of DN-599 DNA, with the exception of the abortion-associated isolate 83-3572. Additional minor differences were also seen between DN-599 DNA and DNA from the other field isolates, but the overall restriction patterns were similar. To confirm that all isolates were members of the BHV-4 type, hybridization studies were performed using DN-599.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

14.
The primary cause of contagious ecthyma is the orf virus, the parapoxvirus prototype. It is a viral problem observed in goat and sheep flocks in Iran, causing economic loss. Orf is a zoonosis with little epidemiological investigation present in Iran. The current research aims at determining the status of this virus, and a PCR was used as a confirmatory instrument. We sampled 668 goats and sheep and various breeding systems. Besides, the orf prevalence was studied, and vaccination efficacy was determined. Moreover, the potential risk factors surveyed for infection with ecthyma were identified. Samples were taken from goat and sheep flocks in the present cross-sectional research, and PCR was used for testing orf DNA. A checklist including animals’ general information was completed. Data were analyzed using univariate tests (chi-square and t-tests) and multivariable binary logistic regression analysis. Three hundred one (45%) goats and sheep detected orf DNA. The age of 70% of positive cases was below one month. Ecthyma infection was significantly higher in imported breeds (87.3%) than indigenous (39.3%). Ninety-six percent of infected goats and sheep in the present work were not vaccinated against ecthyma. The high prevalence of the orf virus was confirmed among goat and sheep flocks in Iran. It is necessary to train ranchers regarding sanitary actions, quarantine, and application of orf vaccination plans.  相似文献   

15.
Heartwater is a serious tick-borne disease of ruminants caused by the rickettsial organism Ehrlichia (Cowdria) ruminantium. A diagnostic test, targeting the pCS20 genomic region and using PCR amplification and probe hybridization, detects E. ruminantium infection in ticks and animals. However, only the pCS20 sequence of the Crystal Springs E. ruminantium isolate is available and the existence of sequence variation amongst different E. ruminantium isolates has not been determined. Primers were designed from the published pCS20 sequence to obtain sequences of the pCS20 region of various E. ruminantium isolates. These primers were unable to amplify the pCS20 region from genomic Welgevonden DNA and genome walking was used to characterize the pCS20 region. This technique showed that the published pCS20 sequence is from a chimeric clone. Sequences of the pCS20 region of 14 different E. ruminantium isolates were determined after amplification with newly designed primers. Sequencing data indicated that West African E. ruminantium isolates are highly conserved, whereas more variation occurs amongst the southern African isolates. These results facilitated the design of a short pCS20 probe and a large PCR target that improved the sensitivity of the E. ruminantium detection assay.  相似文献   

16.
Equine arteritis virus (EAV), a non-arthropod borne togavirus, has been shown to have a global distribution. To date, no major antigenic variation has been demonstrated between EAV isolates from different geographic origins. In this study, the genomic RNA of EAV isolates obtained from horses of different breeds in various countries around the world was oligonucleotide fingerprinted. Comparisons of these fingerprints were used to determine the extent of genomic variation among such isolates. Comparisons among isolates from North American horses revealed, for the most part, oligonucleotide homologies of less than 60%. Only 29 of the 98 comparisons revealed greater than 60% oligonucleotide homology. Nonetheless, several comparisons indicated a close epidemiologic relationship between isolates from horses of different breeds located in different states. Though all European isolates were of Standardbred origin and were from horses located in northern European countries, the majority had oligonucleotide homologies of less than 60%. Where oligonucleotide homology was apparent, it was, with one exception, greater than 70%. The two isolates from New Zealand had 93.2% oligonucleotide homology. This is indicative of an extremely close epidemiologic relationship. Comparisons between EAV isolates from around the world revealed oligonucleotide homologies between viruses from North America, Europe and New Zealand. In several instances, this homology was greater than 70% and in one case greater than 80%. No oligonucleotide homology was evident in comparisons involving the virus from South Africa. The high level of genomic conservation between certain EAV isolates of disparate geographic origins may reflect dissemination of the virus associated with the international movement of horses. The extent of genomic variation demonstrated between most of the EAV isolates used in this study confirms the need for further investigation of genomic heterogeneity among strains of this virus before techniques that rely upon nucleic acid hybridization can be effectively applied as diagnostic procedures.  相似文献   

17.
RD114 virus is an endogenous xenotropic Type C retrovirus of domestic cat. Previously, it had been shown that genomic DNA of cat contained approximately 20 copies of RD114-related sequences. Only one encoded for the replication-competent RD114 virus. The endogenous sequences exhibited substantial sequence conservation within the gag and pol genes and LTRs, but were characterized by deletions and substitutions within the env region. The endogenous cat retroviral element ECE2 was isolated by screening a genomic DNA library of cat liver DNA with an env-specific cDNA of RD114 virus. It contained an env region which differed from all RD114-related sequences so far isolated and was homologous to the corresponding region of replication-competent RD114 with regard to their restriction map and partial sequence analysis (p20). Otherwise, ECE2 had a deletion of approximately 1 kbp in the putative pol gene and, therefore, did not represent the locus of inducible RD114 virus.  相似文献   

18.
Forty-six isolates of Taylorella equigenitalis were analysed by pulsed-field gel electrophoresis (PFGE) after separate digestion of the genomic DNA with ApaI and with NotI. The isolates had been obtained from horses in six European countries and were classified into 18 genotypes. In Belgium, 2 genotypes were detected in 2 isolates, in England 9 among 15, in Finland 2 in 2, in France 2 among 10, in Sweden 3 among 5, and in Switzerland 3 among 12. Two English isolates and 4 French isolates gave identical PFGE profiles to those of Kentucky 188 from the United States. A common genotype was found in 5 isolates from Belgium and England and also in 10 isolates from France and Switzerland. The analysis of genomic DNA from 12 isolates of T. equigenitalis obtained from male horses in France, Sweden and Switzerland gave no evidence of a sex-related difference in the genomic DNA. Genomic DNA from 11 streptomycin (STM)-susceptible isolates obtained in Sweden and Switzerland were classified into four genotypes by PFGE. Each of the six genotypes determined among the 17 isolates from these two countries had single phenotypes for resistance or susceptibility to STM.  相似文献   

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20.
During the last decade several outbreaks of classical swine fever (CSF) occurred in Germany in domestic pigs and in wild boar, respectively. Two major epidemics which also affected other EU Member States were recorded. To support epidemiological investigations genetic typing was applied and virus isolates originating from different outbreaks in Germany were assigned to groups and virus types. Two genomic regions were selected for the phylogenetic analysis, namely 150 nucleotides from the 5' non-translated region (5'-NTR) and 190 nucleotides from the E2 glycoprotein gene. All German CSF virus isolates of the nineties (Group 2) were distinct from former reference strains (Group 1). Within Group 2 both genomic regions allowed to distinguish three subgroups, namely 2.1, 2.2 and 2.3. Within subgroup 2.3 five virus types could be discriminated using the 5'-NTR sequences. These are 2.3*Uelzen and 2.3*Spreda, mainly with isolates from Lower Saxony, as well as 2.3*Rostock, 2.3*Güstrow and 2.3*Spante, mainly with isolates from Eastern Germany. Analysis of the E2 gene fragment allowed a better discrimination between single isolates, but only two virus types could be defined: 2.3*MV/BB, comprising the isolates from Eastern Germany, and 2.3*NI, with the isolates from Lower Saxony. Genetic typing allowed to discriminate between isolates involved in different CSF epidemics, and was useful for tracing the origin and spread of CSF viruses. Due to the close relationship between German CSF virus isolates, epidemiological data are a prerequisite for the interpretation of the results obtained by genetic typing. In addition, at least both genomic regions suggested here should be analysed to determine the identity of a new isolate.  相似文献   

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