Reindeer papillomavirus transforming properties correlate with a highly conserved E5 region.

A papillomavirus was isolated from the epithelial layer of a cutaneous fibropapilloma on a Swedish reindeer (Rangifer tarandus). Reindeer papillomavirus (RPV) is morphologically indistinguishable from other papillomaviruses, but the restriction enzyme cleavage pattern of its genome is different. No sequence homology was detected between RPV DNA and the DNAs of bovine papillomavirus type 1 (BPV-1) and avian papillomavirus when hybridization was performed under stringent conditions. However, the RPV genome hybridized to the genome of the European elk papillomavirus and the deer papillomavirus under stringent conditions. A physical map of the RPV genome was constructed, and selected regions of the genome, covering the open translational reading frame (ORF) E5 and part of the E1 and L1 ORFs, were studied by nucleotide sequence analysis. The results made it possible to align the RPV genome with the genome of BPV-1. The E5 ORF of RPV has the potential to encode a 44-amino-acid, exceptionally hydrophobic polypeptide which is very similar to the E5 polypeptides of BPV-1 and deer and European elk papillomaviruses. RPV is oncogenic for hamsters and transforms C127 mouse cells in vitro. Several virus-specific mRNAs were detected in RPV-transformed C127 cells.     

Analysis of a restriction endonuclease map for a rabbit papillomavirus DNA

A restriction endonuclease map was constructed for rabbit papillomavirus DNA. Analysis of the viral genome by cleavage with specific endonucleases, when compared with bovine papillomavirus types 1 and 2 genome maps, revealed similarities among the three genomes. A number of cleavage sites and their relative order on each genome were found to be conserved.     

表达猴免疫缺陷病毒Env蛋白的重组鸡痘病毒的构建和筛选

目的:构建并筛选表达猴免疫缺陷病毒(SIV)Env蛋白的重组鸡痘病毒(FPV),并对其进行鉴定。方法:设计引物,通过PCR技术扩增SIV env基因,将其连接到pMD18-T载体上,测序正确后将其克隆入本实验室自行构建的FPV穿梭载体pTKET中,获得重组质粒pTKET-SIV env,然后将其与FPV282E4株共转染原代鸡胚成纤维细胞进行同源重组,以增强型绿色荧光蛋白为筛选标记,通过噬斑筛选获得重组病毒,应用PCR、RT-PCR、Western印迹对重组病毒进行鉴定和遗传稳定性分析。结果:通过10次噬斑筛选,PCR检测表明目的基因已整合到重组FPV基因组中,RT-PCR、Western印迹结果表明SIV Env蛋白在感染细胞内表达且具有抗原性;连续传代20次,PCR、RT-PCR、Western印迹均能检测到外源基因的整合、转录和表达,且未能扩增出FPV-TK基因,表明重组病毒遗传稳定性良好,且病毒已经纯化。结论:获得表达SIV Env蛋白的重组FPV,为进一步免疫试验研究奠定了基础。     

Nucleotide sequence and genome organization of canine parvovirus.

The genome of a canine parvovirus isolate strain (CPV-N) was cloned, and the DNA sequence was determined. The entire genome, including ends, was 5,323 nucleotides in length. The terminal repeat at the 3' end of the genome shared similar structural characteristics but limited homology with the rodent parvoviruses. The 5' terminal repeat was not detected in any of the clones. Instead, a region of DNA starting near the capsid gene stop codon and extending 248 base pairs into the coding region had been duplicated and inserted 75 base pairs downstream from the poly(A) addition site. Consensus sequences for the 5' donor and 3' acceptor sites as well as promotors and poly(A) addition sites were identified and compared with the available information on related parvoviruses. The genomic organization of CPV-N is similar to that of feline parvovirus (FPV) in that there are two major open reading frames (668 and 722 amino acids) in the plus strand (mRNA polarity). Both coding domains are in the same frame, and no significant open reading frames were apparent in any of the other frames of both minus and plus DNA strands. The nucleotide and amino acid homologies of the capsid genes between CPV-N and FPV were 98 and 99%, respectively. In contrast, the nucleotide and amino acid homologies of the capsid genes for CPV-N and CPV-b (S. Rhode III, J. Virol. 54:630-633, 1985) were 95 and 98%, respectively. These results indicate that very few nucleotide or amino acid changes differentiate the antigenic and host range specificity of FPV and CPV.     

鸡痘病毒复制非必需区的选择对重组鸡痘病毒免疫效力的影响

母源抗体的干扰是重组鸡痘病毒(FPV)活载体疫苗至今未能得到大规模推广应用的主要原因,而选择适当的FPV复制非必需区可能是解决这一问题的方法之一。根据已发表的美国致病株FPV的基因组设计两对引物,用PCR方法扩增FPV假定复制非必需区的两个侧翼区FPV1和FPV2 ,利用此假定复制非必需区构建FPV表达载体pP12LS及表达ZJ1株新城疫病毒(NDV)F基因的转移载体pP12LSF。pP12LSF与2 82E4株FPV共转染鸡胚成纤维细胞(CEF) ,经数轮蓝斑筛选得到纯化的重组病毒rFPV_FSC。重组FPV在CEF上连续传2 0代仍具有良好的遗传稳定性。对重组FPV进行免疫效力试验,在SPF鸡上,重组病毒rFPV_FSC和与之仅有复制非必需区差异的rFPV_FSB均能抵抗NDV强毒的攻击,提供10 0 %的保护。但在有母源抗体的商品鸡上,rFPV_FSC与rFPV_FSB的免疫效力却有显著差异,保护率分别为10 0 %和6 1 5 4 % ,rFPV_FSC的免疫效力与NDV常规油苗相当。试验结果表明,母源抗体对重组FPV的免疫效力有一定的影响,而选择合适的FPV复制非必需区是克服母源抗体并提高重组FPV免疫效力的有效策略之一     

含有禽网状内皮组织增生病病毒基因组片段的天然重组禽痘病毒的研究

将国内5个不同生产厂家来源的禽痘弱毒疫苗株和1株禽痘野毒株在鸡胚成纤维细胞(CEF)上连续传5代,用禽网状内皮组织增生病病毒(Reticuloendotheliosis virus,REV)特异性的单克隆抗体进行间接免疫荧光试验(Immunonuoreseellee assay,IFA),均检测不到传染性REV。但以6株禽痘病毒(FPV)感染的第2代和第5代细胞基因组提取物为模板,通过PCR均能扩增出REV的长末端重复序列(LTR)和囊膜蛋白(env)基因片段。用特异性核酸探针作分子斑点杂交(Dot blot),结果显示所扩增的PCR条带为特异的REV—LTR和REV—env)基因片段。实验结果表明,国内的一些痘病毒疫苗和野毒株基因组中,已稳定地整合进了REV的基因组成分。     

Reticuloendotheliosis virus sequences within the genomes of field strains of fowlpox virus display variability

Nine field strains of fowlpox virus (FPV) isolated during a 24-year span from geographically diverse outbreaks of fowlpox in the United States were screened for the presence of reticuloendotheliosis virus (REV) sequences in their genomes by PCR. Each isolate appeared to be heterogeneous in that either a nearly intact provirus or just a 248- or 508-nucleotide fusion of portions of the integrated REV 5' and 3' long terminal repeats (LTRs) was exclusively present at the same genomic site. In contrast, four fowlpox vaccines of FPV origin and three originating from pigeonpox virus were genetically homogeneous in having retained only the 248-bp LTR fusion, whereas two other FPV-based vaccines had only the larger one. These remnants of integrated REV presumably arose during homologous recombination at one of the two regions common to both LTRs or during retroviral excision from the FPV genome. Loss of the provirus appeared to be a natural event because the tripartite population could be detected in a field sample (tracheal lesion). Moreover, the provirus was also readily deleted during propagation of FPV in cultured cells, as evidenced by the detection of truncated LTRs after one passage of a plaque-purified FPV recombinant having a "genetically marked" provirus. However, the deletion mutants did not appear to have a substantial replicative advantage in vitro because even after 55 serial passages the original recombinant FPV was still prevalent. As to the in vivo environment, retention of the REV provirus may confer some benefit to FPV for infection of poultry previously vaccinated against fowlpox.     

貂肠炎病毒的核苷酸序列和基因组结构

本试验测定了已克隆的貂肠炎病毒(MEV)复制型(RF)DNA的核苷酸序列,确定MEV基因组全长约为5064个核苷酸(nucleotides,nt),推测了3'端和5'端结构,在5'端非编码区有3个51 nt的重复。MEV基因组序列与犬细小病毒(CPV)、猫细小病毒(FPV)有很高的同源性,结构基因区的同源性分别达99.1%和99.9%,但在5'端非编码区有较大差异。MEV基因组结构与CPV和FPV基本一致,有两个大的开放阅读框架,分别编码688和722个氨基酸。在map unit(m.u.)3.7和m.u.39处有两个启动子,在m.u.97处有poly A位点。NS2、VP1和VP2的mRNA都发生剪接。     

Cloning and characterization of a papillomavirus associated with papillomas and carcinomas in the European harvest mouse (Micromys minutus).

Individuals in a colony of European harvest mice (Micromys minutus) were diagnosed with a variety of skin tumors including papillomas, trichoepitheliomas, and sebaceous carcinomas. Papillomavirus group-specific antigens and viruslike particles were detected in the papillomas. A 7.6-kilobase supercoiled circular DNA, which was cleaved once by EcoRI, was visualized in papilloma extracts by low-stringency Southern blot hybridization with a bovine papillomavirus type 2 probe. The molecule was cloned in pUC18, and a restriction map was generated. The molecule was shown to be colinear with the genome of human papillomavirus type 1a by partial sequence analysis. The DNA hybridized to human papillomavirus type 1a, rabbit oral papillomavirus, and the genome of Mastomys natalensis papillomavirus at Tm - 33 degrees C but not to the DNAs of 13 other papillomaviruses. Transformation of NIH 3T3 or C127I cells by tail papilloma extracts or transfected viral DNA was not observed. All 17 tumors examined contained large amounts of viral DNA in a supercoiled, unintegrated form as revealed by Southern blot hybridization. Furthermore, many extracts (25 of 35) from normal organs and skin of individuals with lesions elsewhere on their bodies contained viral DNA. This represents the first reported molecular cloning of a papillomavirus genome from a mouse species.     

Genome organization and nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer.

The 7,909-nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer, has been determined and used to deduce the corresponding genome arrangement. Extensive sequence homologies and other genetic features are shared with the related oncogenic virus, human papillomavirus type 16, especially in the major reading frames. A surprising difference was found in the noncoding region of human papillomavirus type 33 as, unlike all other sequenced papillomaviruses, it contains a perfect 78-base pair tandem repeat.     

Langerhans' cells and subtypes of human papillomavirus in cervical intraepithelial neoplasia.

There is strong circumstantial evidence that human papillomavirus is a cofactor in the development of cervical neoplasia. Systemic immunosuppression has also been implicated. A study was therefore carried out examining the relation between subtypes of human papillomavirus and local immunocompetent cells in the cervix. Colposcopically directed punch biopsy specimens were taken from normal cervix and from histologically proved cervical intraepithelial neoplasia for immunohistochemical studies. Human papillomavirus genome probing was performed on the abnormal specimens. A relation was apparent between decreased Langerhans'' cells and moderate to high copy numbers of human papillomavirus type 16. The reduction in Langerhans'' cells was significant for human papillomavirus type 18 even at low copy numbers. Conversely, the absence of human papillomavirus was associated with increased numbers of Langerhans'' cells in cervical intraepithelial neoplasia. These findings suggest that the proposed oncogenic potential of human papillomavirus type 16 and human papillomavirus type 18 in particular may be mediated by a specific effect on the afferent limb of the immune response.     

传染性喉气管炎病毒gB基因和新城疫病毒F基因在重组禽痘病毒中共表达

在重组禽痘病毒中表达多个禽类病原的主要免疫原基因是构建多价基因工程疫苗的前提,但相关研究很少。在表达传染性喉气管炎病毒(ILTV)gB基因重组禽痘病毒的转移载体的基础上,构建了含有ILTV gB基因和新城疫病毒(NDV)F基因的重组禽痘病毒转移载体pSY-gB-F,采用脂质体转染禽痘病毒感染的鸡胚成纤维(CEF)细胞后,通过蓝斑试验筛选出重组禽痘病毒(rFPv-gB-F),并进行了6轮蚀斑纯化。Western-blot试验和间接免疫荧光试验证明ILTV gB基因和NBVF基因在rFPV-gB-F感染的CEF细胞中获得表达。为传染性喉气管炎、新城疫与鸡痘活载体多价疫苗的研制奠定基础。     

Characterization of a novel close-to-root papillomavirus from a Florida manatee by using multiply primed rolling-circle amplification: Trichechus manatus latirostris papillomavirus type 1

By using an isothermal multiply primed rolling-circle amplification protocol, the complete genomic DNA of a novel papillomavirus was amplified from a skin lesion biopsy of a Florida manatee (Trichechus manatus latirostris), one of the most endangered marine mammals in United States coastal waters. The nucleotide sequence, genome organization, and phylogenetic position of the Trichechus manatus latirostris papillomavirus type 1 (TmPV-1) were determined. TmPV-1 is the first virus isolated from the order of Sirenia. A phylogenetic analysis shows that TmPV-1 is only distantly related to other papillomavirus sequences, and it appears in our phylogenetic tree as a novel close-to-root papillomavirus genus.     

Avian papillomaviruses: the parrot <Emphasis Type="Italic">Psittacus erithacus</Emphasis> papillomavirus (PePV) genome has a unique organization of the early protein region and is phylogenetically related to the chaffinch papillomavirus

Background  

An avian papillomavirus genome has been cloned from a cutaneous exophytic papilloma from an African grey parrot (Psittacus erithacus). The nucleotide sequence, genome organization, and phylogenetic position of the Psittacus erithacus papillomavirus (PePV) were determined. This PePV sequence represents the first complete avian papillomavirus genome defined.