Molecular cloning of viral DNA from human genital warts.

The DNA of human papilloma virus type 6 (HPV 6) has been cloned in Escherichia coli K-12 by using pBR322 as vector. The DNA was cloned at the BamHI and EcoRI cleavage sites. This DNA was mapped by employing further restriction endonucleases and by terminal labeling. No major differences were noted as compared to HPV 6 DNA originating directly from a genital wart. The existence of at least two DNA subtypes (HPV 6a and 6b) became apparent.     

Molecular cloning, refined physical map and heterogeneity of methylation sites of papilloma virus type 1a DNA

The entire genome of human papilloma virus type 1a was cloned in Escherichia coli using the plasmid pBR322 as vector. The integrity and the homogeneity of the viral DNA thus obtained was confirmed by restriction endonucleases analysis. Viral DNA isolated from a single wart was partially methylated at only one out of the four HpaII sites, d(C-C-G-G). Recognition sites for Bg/I, Bg/II, PstI and PvuII restriction endonucleases were located on the cloned genome.     

Evidence for multiple vegetative DNA replication origins and alternative replication mechanisms of bovine papillomavirus type 1

By following up the chance detection in the electron microscope of a DNA replication intermediate within a preparation of bovine papillomavirus (BPV-1) DNA isolated from purified virus particles, information was obtained about the mechanism of BPV-1 genome replication during the final stages of virus multiplication in naturally infected bovine wart tissue. The structure of viral replication intermediates was investigated by electron microscopic analysis of viral DNA linearized by digestion with restriction endonucleases which cleave the circular BPV-1 chromosome at defined sites. Both Cairns and rolling circle-type molecules were identified. Furthermore, replication eyes were widely distributed within the viral genome, indicating that vegetative BPV-1 DNA replication origins are largely uncoupled from previously described plasmid maintenance sequence elements.     

Bovine papilloma virus transcription: polyadenylated RNA species and assessment of the direction of transcription

The bovine papilloma virus type 1 (BPV-1)-specific RNA species were identified in virus-induced bovine warts, hamster tumors, and transformed hamster and mouse cells. In each case two major species were present (1.1 and 1.3 kilobases [kb]). Also two species of 1.6 and 1.8 kb appearing in variable amounts were found. Only in the keratinized periphery of the warts, where virus replication takes place, was it possible to reveal an additional 2-kb RNA species. In this tissue, however, the 1.6-kb species was not detected. The basal part of a bovine wart contained an additional minor, 2.9-kb, BPV-1-specific RNA sequence. By hybridization with purified defined BPV-1 DNA fragments it was shown that most of the coding sequences of the 2-kb species were transcribed from a region between 0.02 and 0.19 map units. The majority of the coding sequences of the smaller species in transformed cells were located in the region between 0.31 and 0.61 map units. The putative 5' ends mapped between 0.72 and 0.96 map units. Oligodeoxythymidylic acid-primed [(32)P]cDNA was synthesized from various RNA preparations to generate probes for the detection of 3' termini of the polyadenylated BPV-1 RNAs. By hybridization across the BPV-1 genome only one signal between the map positions 0.30 and 0.40 was obtained when RNA from transformed cells and from a tumor was used as a template. In contrast, RNA from the periphery of a wart led to the detection of an additional signal which was confined to the region between 0.96 and 1.00 map units. From the arrangement of both the 3' termini and the coding areas along the viral genome it appears that several RNA species are transcribed from one DNA strand.     

Presence of human papillomavirus DNA sequences in cervical intraepithelial neoplasia.

Twenty two patients referred to a district colposcopy clinic because of an abnormal cervical cytology report or a suspicious cervix and found to have a cervical epithelial abnormality were studied. The techniques of cytology, histology, immunohistochemistry, and DNA-DNA hybridisation were used to detect infection by human papillomavirus. Using an indirect immunoalkaline phosphatase technique human papillomavirus antigen was found in biopsy specimens from six of the 22 patients and DNA of papillomavirus type 6 in biopsy specimens from 13 of these women, including four out of six whose histological diagnosis was cervical intraepithelial neoplasia grade 3. In eight cases where cytological, colposcopical, and histological investigations all indicated the presence of wart virus infection, papillomavirus type 6 DNA was found in seven. Papillomavirus type 6 DNA was found in more than half of the proved cases of cervical intraepithelial neoplasia. The presence of this viral DNA in women with no cervical abnormality is to be studied.     

HPV-2E2蛋白(A338V)突变体对E2蛋白DNA结合能力增强的研究

HPV-2是引起皮肤寻常疣的常见HPV型别,病毒E2蛋白可抑制病毒早期启动子的活性。我们曾经报道来自一例巨大寻常疣患者的HPV-2突变E2蛋白对病毒早期启动子活性的抑制作用明显减弱,该E2蛋白在其C末端的DNA结合区域带有A338V的点突变。本研究利用原核表达系统表达纯化了突变E2(A338V)和HPV-2原毒株的羧基端和全长蛋白。电泳迁移率实验结果显示,E2蛋白可与带有E2蛋白特异性结合位点的寡核苷酸探针形成复合物,突变E2蛋白比原毒株E2蛋白的DNA结合能力强。这提示DNA结合能力的增强可能为E2蛋白对病毒启动子活性影响的分子基础,与患者出现罕见巨大寻常疣这一临床表型关联。     

Enhancement of bleomycin-mediated DNA damage by epidermal microsomal enzymes

The role of epidermal microsomal enzymes in catalyzing bleomycin-mediated chain breakage in calf-thymus DNA and in DNA isolated from neonatal rat epidermis was studied. Aerobic incubation of bleomycin with epidermal microsomes, epidermal or calf-thymus DNA and NADPH caused substantial chain breakage of the DNA which was dependent upon concentrations of drug, microsomal protein and NADPH. The reactive oxygen scavenger superoxide dismutase, the metal chelator EDTA, and cytochrome c each inhibited the enzyme-mediated chain breakage reaction. Scavengers of hydrogen peroxide and hydroxyl radicals, including catalase and benzoate and inhibitors of microsomal cytochrome P-450-dependent monooxygenases such as 1-benzylimidazole, metyrapone and alpha-naphthoflavone, had no inhibitory effects on bleomycin-mediated DNA chain breakage. In contrast, ascorbic acid significantly enhanced DNA damage by bleomycin. These studies indicate that mammalian epidermis possesses membrane-bound enzyme activity capable of enhancing bleomycin-mediated chain breakage of DNA and that oxidation/reduction of adventitious iron and generation of reactive oxygen participate in the reaction. These responses in the epidermis could directly relate to the mechanism of action of intralesional injections of bleomycin which are used quite effectively in the management of recalcitrant human warts. Either epidermal or wart virus DNA or both could be targets for this pharmacologic effect of the drug which is augmented by epidermal microsomal enzymes.     

Human papillomavirus infection in the cervix of pregnant females in Hong Kong

Routine cytological examination and HPV-DNA probing were performed on cervical cells obtained from 215 pregnant Chinese females. The procedure utilized the specific binding of a mixture of complementary [32P]-labelled RNA probes to membrane-bound target HPV DNA types 6, 11, 16, 18, 31, 33 and 35. Positive results for HPV-DNA were obtained in 11/215 (5%) of the cases. The cervical smears were routinely processed and in 3/215 (1.5%) cases a cytological diagnosis of wart virus infection was made. All three were included in the 11 probe-positive cases. Using similar molecular biological techniques, pregnant Western females have an incidence of HPV infection of the cervix which is up to five times that of the present series. This is an unexpected finding in view of the fact that the incidence of cervical cancer is higher in Chinese women than American women.     

Multiple alleles for resistance and susceptibility modulate the defense response in the interaction of tetraploid potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>) with <Emphasis Type="Italic">Synchytrium endobioticum</Emphasis> pathotypes 1, 2, 6 and 18

The obligate biotrophic, soil-borne fungus Synchytrium endobioticum causes wart disease of potato (Solanum tuberosum), which is a serious problem for crop production in countries with moderate climates. S. endobioticum induces hypertrophic cell divisions in plant host tissues leading to the formation of tumor-like structures. Potato wart is a quarantine disease and chemical control is not possible. From 38 S. endobioticum pathotypes occurring in Europe, pathotypes 1, 2, 6 and 18 are the most relevant. Genetic resistance to wart is available but only few current potato varieties are resistant to all four pathotypes. The phenotypic evaluation of wart resistance is laborious, time-consuming and sometimes ambiguous, which makes breeding for resistance difficult. Molecular markers diagnostic for genes for resistance to S. endobioticum pathotypes 1, 2, 6 and 18 would greatly facilitate the selection of new, resistant cultivars. Two tetraploid half-sib families (266 individuals) segregating for resistance to S. endobioticum pathotypes 1, 2, 6 and 18 were produced by crossing a resistant genotype with two different susceptible ones. The families were scored for five different wart resistance phenotypes. The distribution of mean resistance scores was quantitative in both families. Resistance to pathotypes 2, 6 and 18 was correlated and independent from resistance to pathotype 1. DNA pools were constructed from the most resistant and most susceptible individuals and screened with genome wide simple sequence repeat (SSR), inverted simple sequence region (ISSR) and randomly amplified polymorphic DNA (RAPD) markers. Bulked segregant analysis identified three SSR markers that were linked to wart resistance loci (Sen). Sen1-XI on chromosome XI conferred partial resistance to pathotype 1, Sen18-IX on chromosome IX to pathotype 18 and Sen2/6/18-I on chromosome I to pathotypes 2,6 and 18. Additional genotyping with 191 single nucleotide polymorphism (SNP) markers confirmed the localization of the Sen loci. Thirty-three SNP markers linked to the Sen loci permitted the dissection of Sen alleles that increased or decreased resistance to wart. The alleles were inherited from both the resistant and susceptible parents.     

Anogenital warts contain several distinct species of human papillomavirus.

Anogenital warts from 26 patients were examined for the presence of human papillomavirus (HPV). Although no whole, intact virus could be identified, varying amounts of nonintegrated HPV DNA were detected in 18 tissue specimens (70%) by employing both an agarose gel-ethidium bromide staining method and the Southern blot hybridization procedure. When hybridization analysis was performed under stringent conditions, six anogenital warts were observed to contain HPV genomic sequences related to either of the cutaneous viruses HPV type 1 (HPV-1) or HPV-2. In 12 tissue samples lacking sequence homology to either HPV-1 or HPV-2 under stringent conditions, HPV-related sequences were detected when the hybridization was performed under less stringent conditions, indicating that an HPV distinct from both HPV-1 and HPV-2 is also associated with these lesions. This anogenital HPV also appeared to be distinct from the other characterized types of HPV. These data indicate that at least three HPVs are associated with anogenital wart disease.     

Electron microscopy of cells showing viral cytopathic effects in Papanicolaou smears

A technique is described whereby cells showing cytologic changes suggestive of virus infection by light microscopy can be processed further for examination in the electron microscope so that virus particles present in the cell can be visualized directly. We present the results of electron microscopy of over 100 Papanicolaou-stained smears processed this way. The morphologic changes in the cells and the ultrastructural appearances of the virus particles are demonstrated. This technique is particularly valuable for retrospective studies of mounted cytologic or histologic material. It has also proven to be a valuable research tool in the study of human polyomavirus and human wart virus infection.     

A sequence-independent strategy for detection and cloning of circular DNA virus genomes by using multiply primed rolling-circle amplification

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with phi29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 x 10(4)-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information.     

Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle

Human papillomaviruses (HPVs) are small circular DNA viruses that cause warts. Infection with high-risk anogenital HPVs, such as HPV type 16 (HPV16), is associated with human cancers, specifically cervical cancer. The life cycle of HPVs is intimately tied to the differentiation status of the host epithelium and has two distinct stages: the nonproductive stage and the productive stage. In the nonproductive stage, which arises in the poorly differentiated basal epithelial compartment of a wart, the virus maintains itself as a low-copy-number nuclear plasmid. In the productive stage, which arises as the host cell undergoes terminal differentiation, viral DNA is amplified; the capsid genes, L1 and L2, are expressed; and progeny virions are produced. This stage of the viral life cycle relies on the ability of the virus to reprogram the differentiated cells to support DNA synthesis. Papillomaviruses encode multiple oncoproteins, E5, E6, and E7. In the present study, we analyze the role of one of these viral oncogenes, E5, in the viral life cycle. To assess the role of E5 in the HPV16 life cycle, we introduced wild-type (WT) or E5 mutant HPV16 genomes into NIKS, a keratinocyte cell line that supports the papillomavirus life cycle. By culturing these cells under conditions that allow them to remain undifferentiated, a state similar to that of basal epithelial cells, we determined that E5 does not play an essential role in the nonproductive stage of the HPV16 life cycle. To determine if E5 plays a role in the productive stage of the viral life cycle, we cultured keratinocyte populations in organotypic raft cultures, which promote the differentiation and stratification of epithelial cells. We found that cells harboring E5 mutant genomes displayed a quantitative reduction in the percentage of suprabasal cells undergoing DNA synthesis, compared to cells containing WT HPV16 DNA. This reduction in DNA synthesis, however, did not prevent amplification of viral DNA in the differentiated cellular compartment. Likewise, late viral gene expression and the perturbation of normal keratinocyte differentiation were retained in cells harboring E5 mutant genomes. These data demonstrate that E5 plays a subtle role during the productive stage of the HPV16 life cycle.     

AN ELECTRON MICROSCOPE STUDY OF THE DEVELOPMENT OF SV40 VIRUS

Kidney cells, predominantly from Cercopithecus monkeys but also from baboons, were infected in vitro with the SV40 virus. The infectious cycle was studied with the electron microscope by means of thin sections of cells fixed from 3 hours up to 11 days after infection. The frequency of virus formation and various nuclear and cytoplasmic lesions in relation to the infection are described. The virus particles appear in the nucleus in close contact with the chromatin. In a small number of cells they have been observed as early as 10 to 12 hours after infection, but most often they appear 24 to 48 hours afterward. Their mean diameter is 33 mµ. They have no membrane and are frequently arranged as crystal-like structures. In addition to the appearance of virus, one observes various lesions in the nucleoplasm and particularly in the nucleolus, which shows an early hypertrophy and produces unusual, dense condensations in contact with the nucleolonema. The importance of these nucleolar lesions and the relationship between the SV40 virus and the polyoma, common wart, and Shope papilloma viruses are discussed.     

Human papillomaviruses in anogenital warts in children: typing by in situ hybridisation.

OBJECTIVE--To identify the types of human papillomaviruses found in anogenital warts in children and to relate these to clinical and social information. DESIGN--In situ hybridisation using biotin labelled DNA probes to 11 types of human papillomavirus was performed on biopsy specimens from 17 children with anogenital warts. SETTING--Nuffield department of pathology and the department of dermatology, Oxford. PATIENTS--Children in one group were referred by general practitioners or paediatricians to the dermatology department, where biopsies were performed. The other children were seen in four different hospitals, and biopsy specimens were submitted to the laboratory at the physician''s or pathologist''s request. RESULTS--Of the 17 biopsy specimens, 10 contained cells positive with a probe to a genital human papillomavirus type (types 6 or 11), while six were positive with a skin virus type (types 2 or 3). One was negative. The virus type present bore no relation to the site or appearance of the warts. The virus type did, however, appear to correlate with groups of children. Skin types were commoner in older children (over 4 years), in those with a relative who had skin warts, and in children with warts elsewhere; there was no relation with the child''s sex and no suspicion of sexual abuse in these children. These circumstances suggested non-sexual transmission, such as autoinoculation. In contrast, genital types were commoner in girls, in children under 3 years, in children with relatives with genital warts, and in those with no warts elsewhere. Nevertheless, there was suspicion or evidence of sexual abuse in only half these children, suggesting that other routes of transmission--for example, perinatal--might have been implicated. CONCLUSION--Anogenital warts in children may contain either skin or genital wart virus type. Although the type of human papillomavirus present may give some indication of the likely mode of transmission, this can be interpreted only in conjunction with all available clinical and social information. The type of virus does not provide proof of the presence or absence of sexual transmission.     

Ultrastructural characteristics of hybrids derived from normal and hand wart keratinocytes after serial passages

Somatic hybrids realized between mouse fibroblasts 3T3.4E and normal human keratinocytes or hand wart keratinocytes were examined from the 6th to the 30th passages by scanning and transmission electron microscopy. Whatever the passage, hybrid cells showed a fibroblastic morphology but, as keratinocytes, they had the capability to stratify. Branched mitochrondria were observed in hybrids whereas normal mitochondria were present in mouse fibroblasts. In human keratinocytes, most of the mitochondria were normal but sometimes few of them were branched. In wart hybrids heterogeneous nucleoli were detected instead of normal nucleoli in normal keratinocyte hybrids, 3T3.4E cells and human keratinocytes.     

HECATE2 acts with GLABROUS3 and Tu to boost cytokinin biosynthesis and regulate cucumber fruit wart formation

Warty fruit in cucumber (Cucumis sativus L.) is an important quality trait that greatly affects fruit appearance and market value. The cucumber wart consists of fruit trichomes (spines) and underlying tubercules, in which the existence of spines is prerequisite for tubercule formation. Although several regulators have been reported to mediate spine or tubercule formation, the direct link between spine and tubercule development remains unknown. Here, we found that the basic Helix-Loop-Helix (bHLH) gene HECATE2 (CsHEC2) was highly expressed in cucumber fruit peels including spines and tubercules. Knockout of CsHEC2 by the CRISPR/Cas9 system resulted in reduced wart density and decreased cytokinin (CTK) accumulation in the fruit peel, whereas overexpression of CsHEC2 led to elevated wart density and CTK level. CsHEC2 is directly bound to the promoter of the CTK hydroxylase-like1 gene (CsCHL1) that catalyzes CTK biosynthesis, and activated CsCHL1 expression. Moreover, CsHEC2 physically interacted with GLABROUS3 (CsGL3, a key spine regulator) and Tuberculate fruit (CsTu, a core tubercule formation factor), and such interactions further enhanced CsHEC2-mediated CsCHL1 expression. These data suggested that CsHEC2 promotes wart formation by acting as an important cofactor for CsGL3 and CsTu to directly stimulate CTK biosynthesis in cucumber. Thus, CsHEC2 can serve as a valuable target for molecular breeding of cucumber varieties with different wart density requirements.

Fruit wart is a key external quality trait, and CsHECATE2 promotes wart formation by interacting with a spine regulator and a tubercule factor to directly stimulate cytokinin biosynthesis in cucumber     

2种块菌的物种分子鉴定与系统地位分析

以采自内蒙古贺兰山地区的2种块菌为实验材料,分别运用形态学和分子生物学方法进行物种鉴定,并通过与GenBank数据库信息比对,利用Mega5.1软件构建系统进化树,分析其进化关系并确定2种块菌的进化地位。结果表明:两菌株子囊果都为地下生;其中,菌株order-a为灰黄色,近球形到不规则形,较硬,表面被覆微绒毛;菌株tuber-b呈黄褐色,质地均匀,表面有疣状物,顶端呈凹陷状,全株带有干果般特殊的香气。经分子生物学方法分析认为菌株order-a为块菌属的一种,tuber-b为冬块菌(Tuber brumale),并初步确定为内蒙古新记录种。