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1.
Rziha H Henkel M Cottone R Bauer B Auge U Götz F Pfaff E Röttgen M Dehio C Büttner M 《Journal of biotechnology》2000,83(1-2):137-145
Orf virus (OV) is an epitheliotropic poxvirus and belongs to the genus Parapoxvirus (PPV). PPV, especially OV, is regarded as a promising candidate for an expression vector. Among available live vaccines only strain D1701 represents a highly attenuated OV strain with clearly reduced pathogenicity. Therefore, we started to identify potentially non-essential genes or regions of D1701, which might be suitable for insertion and expression of foreign genes. The present contribution reviews some of the progress using the vegf-e (homologue of the mammalian vascular endothelial growth factor) gene locus for the generation of recombinant D1701. The vegf-e gene of D1701 is dispensable for virus growth in vitro and in vivo, and represents a major virulence determinant of OV. It is shown that foreign genes can be inserted and functionally expressed in the vegf-e locus, also leading to the induction of a specific immune response in the non-permissive host. Furthermore, it is reported that adaptation to VERO cells led to the deletion of three further regions of the OV D1701 genome, which seems to be combined with additional virus attenuation in sheep. Molecular analysis of this OV D1701 variant allows the identification of new, potentially non-essential sites in the viral genome. 相似文献
2.
Homologs of vascular endothelial growth factor are encoded by the poxvirus orf virus. 总被引:5,自引:1,他引:4
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A gene encoding a polypeptide with homology to mammalian vascular endothelial growth factors (VEGFs) has been discovered in the genome of orf virus (OV), a parapoxvirus that affects sheep and goats and, occasionally, humans. The gene is transcribed abundantly early in infection and is found immediately outside the inverted terminal repeat at the right end of the genome. In the NZ2 strain of OV (OV NZ2), the gene encodes a polypeptide with a molecular size of 14.7 kDa, while in another strain, OV NZ7, there is a variant gene that encodes a polypeptide of 16 kDa. The OV NZ2 and OV NZ7 polypeptides show 22 to 27% and 16 to 23% identity, respectively, to the mammalian VEGFs. The viral polypeptides are only 41.1% identical to each other, and there is little homology between the two genes at the nucleotide level. Another unusual feature of these genes is their G+C content, particularly that of OV NZ7. In a genome that is otherwise 63% G+C, the OV NZ2 gene is 57.2% G+C and the OV NZ7 gene is 39.7% G+C. The OV NZ2 gene, but not the OV NZ7 gene, is homologous to the mammalian VEGF genes at the DNA level, suggesting that the gene has been acquired from a mammalian host and is undergoing genetic drift. The lesions induced in sheep and humans after infection with OV show extensive dermal vascular endothelial proliferation and dilatation, and it is likely that this is a direct effect of the expression of the VEGF-like gene. 相似文献
3.
Iketani Y Inoshima Y Asano A Murakami K Shimizu S Sentsui H 《Microbiology and immunology》2002,46(4):285-291
The possibility of persistent parapoxvirus (PPV) infection was investigated by serologically and genetically using cattle infected with the virus experimentally and naturally. Three cattle were inoculated with the virus subcutaneously at several spots in the lips and abdominal regions. Small papules developed in the inoculated regions, and antibodies to the virus developed and continued persistently. One animal, from which one PPV had been previously isolated, was also subjected to serological and viral detection tests as a naturally infected case. Two of these four cattle were injected with dexamethasone (DM), and one was injected with interferon-gamma (IFN-gamma). The viral genome was rarely detected from the peripheral blood leukocytes in the ordinary condition, but frequently when the animals were injected with IFN-gamma. The viral genome was also detected from the lymph nodes as these PPV infected animals were euthanized. These results indicated that cattle were infected with PPV subclinically and persistently, and the virus was activated in stressed or immunosuppressed animals. The virus would be harbored in the lymphotic tissues of the animals when they show no clinical symptoms. 相似文献
4.
The structure of a putative scaffolding protein of immature poxvirus particles as determined by electron microscopy suggests similarity with capsid proteins of large icosahedral DNA viruses
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Hyun JK Coulibaly F Turner AP Baker EN Mercer AA Mitra AK 《Journal of virology》2007,81(20):11075-11083
Orf virus, the prototype parapoxvirus, is responsible for contagious ecthyma in sheep and goats. The central region of the viral genome codes for proteins highly conserved among vertebrate poxviruses and which are frequently essential for viral proliferation. Analysis of the recently published genome sequence of orf virus revealed that among such essential proteins, the protein orfv075 is an orthologue of D13, the rifampin resistance gene product critical for vaccinia virus morphogenesis. Previous studies showed that D13, arranged as "spicules," is necessary for the formation of vaccinia virus immature virions, a mandatory intermediate in viral maturation. We have determined the three-dimensional structure of recombinant orfv075 at approximately 25-A resolution by electron microscopy of two-dimensional crystals. orfv075 organizes as trimers with a tripod-like main body and a propeller-like smaller domain. The molecular envelope of orfv075 shows unexpectedly good agreement to that of a distant homologue, VP54, the major capsid protein of Paramecium bursaria Chlorella virus type 1. Our structural analysis suggests that orfv075 belongs in the double-barreled capsid protein family found in many double-stranded DNA icosahedral viruses and supports the hypothesis that the nonicosahedral poxviruses and the large icosahedral DNA viruses are evolutionarily related. 相似文献
5.
Angélica Dal Zotto Mónica Balzarini Juan M. Raigón Mirta N. Rossini Daniel A. Ducasse 《Journal of Phytopathology》2014,162(1):55-60
A Plum pox virus (PPV) isolate detected in a Japanese plum orchard in Pocito (San Juan, Argentina) was transmitted mechanically to Prunus tomentosa and Nicotiana benthamiana. DAS‐ELISA and DASI‐ELISA indicated the virus presence and serological relationship with D‐strain isolates; IC‐RT‐PCR amplified a 1.2‐kb fragment of the virus genome encoding the CP‐3′ nc region. The analysis of the sequence showed the presence of the DAG motif at the 5′ end of the capsid protein and the Rsa I and Alu I sites at the 3′ end. The phylogenetic relationships and multiple alignment with PPV isolates from NCBI database indicated greatest (+98%) homology with the D strain and close identity with MNAT1 ( AF360579 ) USA peach isolate. The sequence analysed showed two amino acid mutations towards the 5′ N‐terminus of CP (the most variable region) with respect to a consensus of PPV D‐strain isolates. This is the first molecular characterization of 3′terminal genome region of PPV isolate to confirm D strain in a Japanese plum from Argentina. 相似文献
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Kensaku Maejima Masayoshi Hashimoto Yuka Hagiwara-Komoda Akio Miyazaki Masanobu Nishikawa Ryosuke Tokuda Kohei Kumita Noriko Maruyama Shigetou Namba Yasuyuki Yamaji 《Molecular Plant Pathology》2020,21(4):475-488
Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection. 相似文献
8.
Murine gammaherpesvirus 68 (MHV-68) has been developed as a model for the human gammaherpesviruses Epstein-Barr virus and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV), which are associated with several types of human diseases. Open reading frame 45 (ORF45) is conserved among the members of the Gammaherpesvirinae subfamily and has been suggested to be a virion tegument protein. The repression of ORF45 expression by small interfering RNAs inhibits MHV-68 viral replication. However, the gene product of MHV-68 ORF45 and its function have not yet been well characterized. In this report, we show that MHV-68 ORF45 is a phosphorylated nuclear protein. We constructed an ORF45-null MHV-68 mutant virus (45STOP) by the insertion of translation termination codons into the portion of the gene encoding the N terminus of ORF45. We demonstrated that the ORF45 protein is essential for viral gene expression immediately after the viral genome enters the nucleus. These defects in viral replication were rescued by providing ORF45 in trans or in an ORF45-null revertant (45STOP.R) virus. Using a transcomplementation assay, we showed that the function of ORF45 in viral replication is conserved with that of its KSHV homologue. Finally, we found that the C-terminal 23 amino acids that are highly conserved among the Gammaherpesvirinae subfamily are critical for the function of ORF45 in viral replication. 相似文献
9.
Michel Ravelonandro Ralph Scorza Hily Jean Michel Pascal Briard 《Plant Cell, Tissue and Organ Culture》2014,118(2):347-356
Plum transformed with an intron hairpin RNA CP (ihpRNA-CP) was resistant to plum pox virus (PPV) infection through the specific process of RNA silencing involving both small interfering-RNA (siRNA) and a methylated virus transgene. Silencing specifically targeted the PPV genome and led to the degradation of viral RNA in the model plant species Nicotiana benthamiana and the natural Prunus domestica host. Plums inoculated with the five major PPV strains, three widespread PPV strains (D, M, and Rec), and the atypical EA strain did not allow systemic spread of PPV in greenhouse-grown transgenic ihRNA-CP plum over multiple cycles of vegetative growth and cold-induced dormancy. PPV ihRNA-CP N. benthamiana displayed an immunity reaction and also allowed for the testing of PPV-C, a strain that was unable to infect P. domestica. This stable resistance demonstrated in plum based on the accumulation of siRNA can prevent PPV infection and can also act as a “curative” when PPV is inoculated through graft inoculation, through a recovery reaction. Regardless PPV strain variability based on geography, host species, epidemiology and serotypes of the CP protein and substitutions of nucleotides at the NH2-terminus of CP of the major five PPV strains tested, we show that the use of a PPV-CP intron hairpin (ihp) RNA is an effective strategy to specifically target the PPV genome. We provide methods and tools that demonstrate a reliable path towards developing PPV resistance suitable for protecting stone fruit orchards. 相似文献
10.
Infection by the parapoxvirus orf virus causes proliferative skin lesions in which extensive capillary proliferation and dilation are prominent histological features. This infective phenotype may be linked to a unique virus-encoded factor, a distinctive new member of the vascular endothelial growth factor (VEGF) family of molecules. We constructed a recombinant orf virus in which the VEGF-like gene was disrupted and show that inactivation of this gene resulted in the loss of three VEGF activities expressed by the parent virus: mitogenesis of vascular endothelial cells, induction of vascular permeability, and activation of VEGF receptor 2. We used the recombinant orf virus to assess the contribution of the viral VEGF to the vascular response seen during orf virus infection of skin. Our results demonstrate that the viral VEGF, while recognizing a unique profile of the known VEGF receptors (receptor 2 and neuropilin 1), is able to stimulate a striking proliferation of blood vessels in the dermis underlying the site of infection. Furthermore, the data demonstrate that the viral VEGF participates in promoting a distinctive pattern of epidermal proliferation. Loss of a functional viral VEGF resulted in lesions with markedly reduced clinical indications of infection. However, viral replication in the early stages of infection was not impaired, and only at later times did it appear that replication of the recombinant virus might be reduced. 相似文献
11.
MicroRNA-guided processing impairs Plum pox virus replication, but the virus readily evolves to escape this silencing mechanism 总被引:1,自引:0,他引:1
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Since the discovery of microRNA (miRNA)-guided processing, a new type of RNA silencing, the possibility that such a mechanism could play a role in virus defense has been proposed. In this work, we have analyzed whether Plum pox virus (PPV) chimeras bearing miRNA target sequences (miR171, miR167, and miR159), which have been reported to be functional in Arabidopsis, were affected by miRNA function in three different host plants. Some of these PPV chimeras had clearly impaired infectivity compared with those carrying nonfunctional miRNA target sequences. The behaviors of PPV chimeras were similar but not identical in all the plants tested, and the deleterious effect on virus infectivity depended on the miRNA sequence cloned and on the site of insertion in the viral genome. The effect of the miRNA target sequence was drastically alleviated in transgenic plants expressing the silencing suppressor P1/HCPro. Furthermore, we show that virus chimeras readily escape RNA silencing interference through mutations within the miRNA target sequence, which mainly affected nucleotides matching the 5'-terminal region of the miRNA. 相似文献
12.
A gene encoding a polypeptide with homology to interleukin-10 (IL-10) has been discovered in the genome of orf virus (OV) strain NZ2, a parapoxvirus that infects sheep, goats, and humans. The predicted polypeptide sequence shows high levels of amino acid identity to IL-10 of sheep (80%), cattle (75%), humans (67%), and mice (64%), as well as IL-10-like proteins of Epstein-Barr virus (63%) and equine herpesvirus (67%). The C-terminal region, comprising two-thirds of the OV protein, is identical to ovine IL-10, which suggests that this gene has been captured from its host sheep during the evolution of OV. The IL-10-like gene is transcribed early. Conditioned medium from COS cells transfected with a eukaryotic expression vector containing the OV IL-10-like gene showed the same biological activity as ovine IL-10 in a murine thymocyte proliferation assay. OV IL-10 is likely to be important in immune evasion by OV, since IL-10 is a multifunctional cytokine that has inhibitory effects on nonspecific immunity and Th1 effector function. 相似文献
13.
Transgenic or plant expression vector-mediated recombination of Plum Pox Virus 总被引:1,自引:0,他引:1
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Different mutants of an infectious full-length clone (p35PPV-NAT) of Plum pox virus (PPV) were constructed: three mutants with mutations of the assembly motifs RQ and DF in the coat protein gene (CP) and two CP chimeras with exchanges in the CP core region of Zucchini yellow mosaic virus and Potato virus Y. The assembly mutants were restricted to single infected cells, whereas the PPV chimeras were able to produce systemic infections in Nicotiana benthamiana plants. After passages in different transgenic N. benthamiana plants expressing the PPV CP gene with a complete (plant line 4.30.45.) or partially deleted 3'-nontranslated region (3'-NTR) (plant line 17.27. 4.), characterization of the viral progeny of all mutants revealed restoration of wild-type virus by recombination with the transgenic CP RNA only in the presence of the complete 3'-NTR (4.30.45.). Reconstitution of wild-type virus was also observed following cobombardment of different assembly-defective p35PPV-NAT together with a movement-defective plant expression vector of Potato virus X expressing the intact PPV-NAT CP gene transiently in nontransgenic N. benthamiana plants. Finally, a chimeric recombinant virus was detected after cobombardment of defective p35PPV-NAT with a plant expression vector-derived CP gene from the sour cherry isolate of PPV (PPV-SoC). This chimeric virus has been established by a double recombination event between the CP-defective PPV mutant and the intact PPV-SoC CP gene. These results demonstrate that viral sequences can be tested for recombination events without the necessity for producing transgenic plants. 相似文献
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15.
The X protein of borna disease virus serves essential functions in the viral multiplication cycle
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The X gene of Borna disease virus (BDV) encodes a nonstructural 10-kDa protein that can interact with viral polymerase cofactor P, thus regulating polymerase activity. It remained unknown whether X is essential for virus multiplication. All our attempts to generate mutant BDV with a nonfunctional X gene proved unsuccessful. However, a mutant virus with an inactive X gene was able to replicate in Vero cells if an artificial gene cassette encoding X was inserted at a site near the 5' end of the viral genome. These results indicate that X performs essential viral functions. 相似文献
16.
Prevention of virus persistence and protection against immunopathology after Borna disease virus infection of the brain by a novel Orf virus recombinant
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The Parapoxvirus Orf virus represents a promising candidate for novel vector vaccines due to its immune modulating properties even in nonpermissive hosts such as mouse or rat. The highly attenuated Orf virus strain D1701 was used to generate a recombinant virus (D1701-VrVp40) expressing nucleoprotein p40 of Borna disease virus, which represents a major antigen for the induction of a Borna disease virus-specific humoral and cellular immune response. Infection with Borna disease virus leads to distinct neurological symptoms mediated by the invasion of activated specific CD8+ T cells into the infected brain. Usually, Borna disease virus is not cleared from the brain but rather persists in neural cells. In the present study we show for the first time that intramuscular application of the D1701-VrVp40 recombinant protected rats against Borna disease, and importantly, virus clearance from the infected brain was demonstrated in immunized animals. Even 4 and 8 months after the last immunization, all immunized animals were still protected against the disease. Initial characterization of the immune cells attracted to the infected brain areas suggested that D1701-VrVp40 mediated induction of B cells and antibody-producing plasma cells as well as T cells. These findings suggest the induction of various defense mechanisms against Borna disease virus. First studies on the role of antiviral cytokines indicated that D1701-VrVp40 immunization did not lead to an enhanced early response of gamma or alpha interferon or tumor necrosis factor alpha. Collectively, this study describes the potential of the Orf virus vector system in mediating long-lasting, protective antiviral immunity and eliminating this persistent virus infection without provoking massive neuronal damage. 相似文献
17.
Both excision and replication of cloned autonomous parvovirus DNA require the NS1 (rep) protein. 总被引:13,自引:11,他引:2
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When a bacterial plasmid containing the entire genome of LuIII virus except for the terminal 18 nucleotides from the right end is transfected into HeLa cells, the viral DNA is rescued and replicated, with production of infectious virus. This experimental system was used to examine the viral proteins and cis elements required for the excision and replication of viral DNA. The deletion of the entire NS1 gene provided a viral genome that was excised from the plasmid and replicated only when an NS1 gene was provided in trans. A frameshift mutation in the NS2 intron that truncates NS1 prevented excision and replication. Deletion of the left-end terminal inverted repeat or the right-end inverted repeat prevented excision of viral DNA from that end but not from the wild-type terminus. The viral terminus excised from the plasmid was protected from a processive degradation process, which began on the vector portion of the plasmid. The inhibitor of DNA polymerases alpha and delta, aphidicolin, blocked the excision reaction. 相似文献
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The entire cloned human adenovirus type 5 (Ad5) genome is known to be able to generate infectious virus after transfection into 293 cells when the both ends of the genome are exposed by digestion with appropriate restriction enzymes. However, when one or both ends of the genome are tagged with nucleotides and are not intact, whether the tagged end of the viral genome was remained tagged or corrected to be intact during the generation of viral clones has been unclear and, if such oligonucleotide removal occurs, how does the virus remove these tagged sequences and thereby restore its proper structure? Here, we show in our semi‐quantitative study that the generation efficiency of virus clones decreases depending on the length of nucleotide tags at the both ends and that both the oligonucleotide tags were precisely removed during virus generation with restoration of the proper terminal sequences. Interestingly the viral genome of which one end was tagged, while the other was attached about 12‐kb sequences, did generate intact viral clones at a reduced but significant efficiency. From these results, we here propose a possible mechanism whereby the terminal‐protein‐deoxycytidine complex enters from the enzyme‐cleaved end and reaches deoxyguanine at the initiating position of DNA synthesis in vivo. A replication origin at one end, embedded deeply in double‐stranded DNA, can be activated by two cycles of one‐directional full‐length DNA synthesis initiated by the other exposed replication origin about 30 kilobases away. We also describe new cassette cosmids which can use not only PacI but also BstBI for construction of an adenovirus vector, without reducing construction efficiency. 相似文献