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Host reactivity to bovine papilloma virus-induced fibroblastomasBarthold, Stephen W. January 1974 (has links)
Thesis--University of Wisconsin--Madison. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.
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MOLECULAR ASPECTS OF CELLULAR TRANSFORMATION BY BOVINE PAPILLOMAVIRUS TYPE 1.ABRAHAM, JOHN MARTIN. January 1983 (has links)
The bovine papillomaviruses (BPV) are capable of transforming cells from a wide range of species both in vivo and in vitro. The 69% portion of the BPV-1 viral genome that contains the transforming region, as well as the 31% portion, were cloned in the pBR322 plasmid vector. An extensive restriction endonuclease map of the transforming region was prepared. Using the cloned transforming region as a ³²P probe, BPV-1 coded mRNA transcripts from transformed cells were detected and sized using the northern blot technique. The largest open reading frame of the transforming region of the BPV-1 genome was sequenced using the Maxam and Gilbert chemical method and the amino acid sequence of a protein that this region could code for was presented.
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LOCALIZATION AND COMPLETE NUCLEOTIDE SEQUENCE OF THE BOVINE PAPILLOMAVIRUS TYPE-2 LATE REGION. POLYPEPTIDE COMPOSITION OF BPV-2.POTTER, HAROLD LEE, JR. January 1984 (has links)
This investigation was designed to partially characterize the genome of bovine papillomavirus type-2 (BPV-2). A closely related virus, bovine papillomavirus type-1 (BPV-1), has been extensively studied. Additionally, many members of the human papillomaviruses (HPV) are being investigated in great detail. However, only a few molecular biology studies of BPV-2 have been reported. Therefore, it seemed appropriate to initiate an investigation whose results would provide a good basis for continued studies of BPV-2. A detailed physical map of BPV-2 DNA was prepared by restriction endonuclease digestion of BPV-2 DNA. A comparison between the genomes of BPV-2 and BPV-1 in regard to location and number of cleavage sites of seven enzymes was performed. This comparison revealed similarities between the two genomes and indicated the probable location of the BPV-2 late region by direct extrapolation from the BPV-1 genomic map. This area was completely sequenced by the chemical method and the results confirmed it to indeed be the BPV-2 late region. Comparative analysis between the BPV-2 late region and the BPV-1 late region demonstrated identical genetic organization to exist along with a high degree of nucleotide sequence conservation. Also, the polypeptide composition of BPV-2 was determined and similarities with these and BPV-1 polypeptides were noted. Lastly, a discussion of the BPV-2 polypeptides as potential products of the BPV-2 late region is presented.
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CHARACTERIZATION OF THE BOVINE PAPILLOMA VIRUS TYPE-1 GENOME AND TRANSFORMATION OF MAMMALIAN CELLS.MORGAN, DON MITCHELL. January 1982 (has links)
The papillomaviruses do not appear to be capable of passage in cultured cell lines despite numerous attempts to identify conditions permissive for their propagation. These viruses induce benign tumors in animals (warts, papillomas) by unknown mechanisms. A broad range of animal species is susceptible to papillomavirus-induced tumorogenesis. The basic molecular mechanisms of papillomavirus replication, transcription, and translation to produce virus-specific products in cells are unknown. Since the viruses do not reproduce in vitro, no conditional-lethal and deletion mutants like those characterized in other systems exist. Thus, progress in understanding the papillomavirus-host cell interactions leading to neoplasia has been severely hindered. This dissertation is concerned with biochemical analysis of bovine papillomavirus type 1 (BPV-1) DNA and RNA in BPV-1 transformed and tumor cells. The specific conditions of infection resulting in stable lines of BPV-1 transformed cells are described. Colonies of BPV-1 transformed cells exhibiting anchorage-independent growth in agarose-containing medium were isolated and cloned cell lines were established. Formation of tumors in a rabbit following inoculation with BPV-1 is reported and represents the first evidence of BPV-induced tumorogenesis in this animal species. The BPV-1 transformed and tumor cells are characterized with respect to the quantity and physical state of the BPV-1 genome in the cells. The BPV-1 DNA is present in high copy numbers in free, non-integrated supercoiled and nicked open-circular forms. No evidence of integrated BPV-1 sequences is noted. This is unusual since all other characterized DNA tumor viruses require covalent integration of at least a portion of the viral DNA in the cellular genome during transformation. A detailed restriction endonuclease cleavage map of the BPV-1 genome is presented, representing a more complete physical characterization of the viral genome. The first evidence of BPV-1 specific transcripts in transformed cells is reported. These results should aid in functional characterizations of the BPV-1 genome, particularly the determination of the specific regions of the BPV-1 DNA transcribed during transformation of cells, analogous to early regions defined in other DNA tumor virus systems, and analysis of post-transcriptional processing mechanisms involved in the synthesis of BPV-1 specific messenger RNA.
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THE EFFECTS OF RETINOIC ACID ON MOUSE CELLS TRANSFORMED BY BOVINE PAPILLOMAVIRUS TYPE-1.HENLEY, MARILYN JEAN. January 1984 (has links)
The purpose of this research was to determine the effects of retinoic acid on bovine papillomavirus (BPV) transformed cells. Since BPV transformed cells are able to form colonies in agar and their untransformed counterparts are not, of particular interest was the effect of retinoic acid on this marker. Retinoic acid inhibited the growth of colony forming cells, but the extent of inhibition varied among several cloned cell lines. Retinoic acid inhibited the proliferation of BPV transformed cells and the extent of this inhibition also varied. The copy number of the virus was determined for each of three cell lines by liquid reassociation experiments. The copy number did not change when the cells were treated with RA for a prolonged period of time. Gel electrophoresis and Southern blotting of extrachromosomal DNA from the cell lines revealed the presence of unit length BPV DNA in all three transformed cell lines. The amount of BPV DNA per cell was decreased when the cells were treated with 10⁻⁵ M RA for three days.
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The molecular cloning and expression of the BPV-2 L-2 open reading frame in Escherichia coli.Rippe, Richard Allen. January 1988 (has links)
The bovine papilloma virus type 2 (BPV-2) L2 open reading frame (ORF) was cloned into a λ pL promoter expression vector. This clone was shown to express a fusion protein which comprised 75% of the BPV-2 ORF linked to the first 13 N-terminal amino acids of the λ cIl gene product. Antisera was generated against this fusion protein and subsequently used to identify the L2 gene product as a 64,000 dalton protein in BPV-2 virions. It was also demonstrated that the L2 viral protein was present in full caps ids, but only in very limited amounts in empty caps ids. Densitometer analysis indicated that the L2 protein comprised only 8% of the total L1 + L2 "Coomassie blue stainable" protein in full capsids. The antisera was also used to demonstrate that the BPV-2 L2 gene product is antigenically related to the BPV-1 L2 gene product. Finally, an attempt was made to determine the location of the L2 gene product within the capsid structure. Hemagglutination inhibition and enzyme-llnked-immunosorbent- assay data both indicate that the L2 protein is exposed on the surface of the capsid. Immune electron microscopy data was inconclusive in determining the location of the L2 gene product.
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Genetic analysis of human papillomavirus in a cohort of women in routine care in Northern South AfricaRikhotso, Rixongile Rhenny 18 May 2019 (has links)
MSc (Microbiology) / Department of Microbiology / BACKGROUND: Human papillomavirus (HPV) is a common sexually transmitted virus known to be a causative agent of cervical cancer (CC), one of the most frequent cancers in women worldwide. HPV is a double stranded DNA virus of approximately 7,900 bp; belonging to Papillomaviridae family. To date, about 202 low risk (LR) and high risk (HR) HPV genotypes have been identified. However, available vaccines against HPV infection are designed based on the most common known genotypes. Therefore, it is critical to understand the scope and diversity of HPV genotypes in all geographical locations which can help to inform the design and development of future vaccines.
OBJECTIVE: The objective of this study was to describe the burden and diversity of HPV genotypes in a cohort of women in routine care in northern South Africa.
METHODS: Eighty seven women consented to participate in the study and each provided a specimen for analysis. With the help of qualified health care practitioners, Aptima Cervical Specimen Collection and Transport Kit (Hologic, San Diego, CA) was used to collect cervical specimens from each study participant following the manufacturer’s procedure. Total DNA was purified from the cervical pellet using QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The purified DNA was then subjected to a single round conventional PCR in a reaction volume of 100 μl to amplify HPV L1 gene comprising of approximately 450 bp. A portion of each PCR amplicon from each participant was denatured, hybridized and genotyped using the Linear Array HPV genotyping Test Kit (Roche Molecular Systems, Inc. Branchburg, NJ USA). The kit is designed to detect 37 HPV genotypes (genotypes 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83,
84, IS39 and CP6108). To detect the HPV genotypes, the Linear Array (LA) reference guide was used for results interpretation following the manufacturer’s instructions. The other portion of each of the amplicons was subjected to next generation sequencing (NGS) using the Illumina MiniSeq platform. Using the Nextera XT DNA Library preparation kit, an initial input of 1ng genomic DNA was tagmented, cleaned up, normalized and pooled. The pooled library was then denatured with 0.1
N NaOH and diluted into a final volume of 500 μl at 1.8 pM then sequenced using the Local Run Manager option following the manufacturer’s instructions. The generated sequence data was downloaded into fastaQ format and analysed using Genious 11.0.5 software.
RESULTS: Of the 87 participants, the overall proportion of women harbouring HPV DNA by linear array (LA) PCR was 23% (n=20). Of the 20, 16 (80%) were living with HIV. However, this difference was not significant (p=0.077). Genotyping data generated by Roche LA method was successful for all the 20 positive amplicons. In this study, 27 (73%) of the 37 HPV genotypes incorporated in the Roche Linear Array method were detected. The detected genotypes include: types 84, 83, 81, 73, 72, 71, 70, 69, 68, 66, 62, 61, 59, 54, 53, 52, 51, 45, 42, 39, 35, 26, 18, 16, 6, IS39 and CP6108. Most women (15/20;75%) harboured multiple infections compared to single infection. In terms of genotypes distribution, the most frequent genotypes detected LR HPV types in increasing order of frequency included HPV type 61 and 83 (12%), 62 (36%) and 81 (43%). On the other hand, HPV type 66, 53, 52, 51, 18 and 16 were the most common genotypes detected HR HPV types.
In contrast, although genotyping data was successfully generated from 15 of 20 women (75%), NGS technology was seen to be more sensitive compared to Roche LA method. Nearly all the detected genotypes identified by the commercial kit were detected by NGS. In addition, NGS detected 10 namely: HPV types 11, 31, 33, 40,
55, 56, 58, 64, 67, and 82 that were not detected by the LA yet incorporated in the kit. Moreover, it was observed that NGS identified additional 6 HPV types including HPV types 2, 27, 30, 35, 85 and 102 not incorporated in the Roche LA kit. A similar distribution of HPV multiple infections was observed in the study population, however, high frequency of 93% (14 of 15) was detected by NGS. The proportion of women harbouring one or more of the 22 LR HPV types was 100% (n=15).The most frequent LR genotypes in increasing order of frequency was HPV type 62 and 70 (27%), 6 (40%) and 11 (47%). HPV types 40, 42, 54, 72, 64, and 81 were the least detected genotypes with n=1 (7%) each. Furthermore, the common combination observed among the participants was type 6 and 11. In contrast, the most frequent detected genotypes in the study population by NGS under the HR HPV types in increasing order of frequency include type 35 (21%), 39, 56 and 82 (29%), 68 (36%) and 51 (50%). In addition, HPV types 26, 31, 45, 53, 56, 58 and 66 were the least
detected genotypes n=1 (7%) in the study population. HPV 39 and 68 were observed as the common combination detected under HR HPV types. Following genotyping by LA and NGS, the demographic and clinical data of all the 20 positive subjects by PCR were subjected to statistical analysis to determine the association between HPV positive DNA status and associated risk factors. Smoking status (p=0.000), age at first sexual intercourse (p=0.011), vaccination status (p=0.000), gender of sexual partner (p=0.000), highest level of education (p=0.004), marital status (p=0.008) and number of sexual partners (p=0.000) were found to be having a positive statistical association.
CONCLUSION: Amplification of targeted HPV DNA from cervical specimens demonstrated the presence of HPV infection in the study cohort, with a proportion of 23%. The findings illustrate that there is a diversity of HPV genotypes prevalent in the study population as shown by Roche LA and NGS methods. However, the NGS method was observed to be more sensitive than Roche LA in detecting HPV genotypes. Furthermore, NGS identified 6 additional HPV types not incorporated in the Roche LA. Thus, there are genotypes that may be present in the study population that the Roche commercial kit may fail to detect. Therefore, is it imperative to use both genotyping methods to confirm HPV genotypes. / NRF
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