To investigate the prevalence of bovine papillomavirus (BPV) in bovine papilloma and healthy skin, DNA extracted from teat papillomas and healthy teat skin swabs was analysed by PCR using the primer pairs FAP59/FAP64 and MY09/MY11. Papillomavirus (PV) DNA was detected in all 15 papilloma specimens using FAP59/FAP64 and in 8 of the 15 papilloma specimens using MY09/MY11. In swab samples, 21 and 8 of the 122 samples were PV DNA positive using FAP59/FAP64 and MY09/MY11, respectively. Four BPV types (BPV-1, -3, -5 and -6), two previously identified putative BPV types (BAA1 and -5) and 11 putative new PV types (designated BAPV1 to -10 and BAPV11MY) were found in the 39 PV DNA-positive samples. Amino acid sequence alignments of the putative new PV types with reported BPVs and phylogenetic analyses of the putative new PV types with human and animal PV types showed that BAPV1 to -10 and BAPV11MY are putative new BPV types. These results also showed the genomic diversity and extent of subclinical infection of BPV.

SUMMARYFive cell lines, SKG-I, SKG-II, SKG-IIIb, QG-U and QG-H derived from cervical carcinomas of Japanese patients, were examined for the presence of human papillomavirus (HPV) DNA and the expression of viral mRNA. The DNA of HPV type 16 was shown to be linked covalently with SKG-IIIb, QG-U and QG-H cell DNA, and HPV 18 DNA with SKG-I and SKG-II cell DNA. Although different regions of the HPV genome were integrated in these cell lines, the non-coding region and an early region including the E6 and E7 open reading frames (ORFs) were conserved in all cell lines. The complete genome of HPV 16 was found in QG-H cells by digestion of the DNA with a single-cut restriction enzyme. The other early region ORFs E 1, E2, E4 and E5 were interrupted by flanking host cell DNA, suggesting that the integration into host cell DNA occurs preferentially in this region. HPV-specific mRNA species were detected in all five cell lines. In the three cell lines containing the HPV 16 genome, mRNAs hybridized with the early region of the genome, covering the entire E6 and E7 ORFs and a minor part of the E10RF, although the amount and size of the major mRNAs varied in these cell lines. These mRNAs did not hybridize with the late region of the HPV genome containing the LI and L20RFs. In SKG-II, SKG-IIIb and QG-H cells we also detected c-myc and c-Ha-ras mRNA expression at about nine times the level of that in normal cells.The human papillomaviruses (HPVs) induce epithelial proliferative lesions of skin or mucosa and have been classified into more than 40 types on the basis of their DNA sequence homology.

Six bovine papillomavirus (BPV) types and 16 putative BPV types have been reported previously. Here, the complete genome sequence of BAPV6, a novel putative BPV type isolated from cattle in Japan, was determined by using multiple-primed rolling-circle amplification. The genome consisted of 7412 bp (G+C content of 46 mol%) that encoded five early (E1, E2, E4, E6 and E7) and two late (L1 and L2) genes, but did not encode the E5 gene. The E6 protein contained a non-consensus CxxC(x)33CxxC and a consensus CxxC(x)29CxxC zinc-binding domain, and the E7 protein lacked the LxCxE motif. The nucleotide sequence of the L1 open reading frame (ORF) was related most closely (57–58 %) to the L1 ORF of member(s) of the genera Betapapillomavirus, Gammapapillomavirus and Pipapillomavirus. Phylogenetic analysis based on the complete L1 ORF suggests that BAPV6 should be classified in a novel genus in the family Papillomaviridae as BPV-7.

SUMMARYThe presence of human papillomavirus (HPV) type 16 DNA in biopsies from precancerous lesions and from early lesions of human cervical cancer, and the integration of virus DNA into host cell DNA were analysed by dot blot and Southern blot hybridizations. HPV 16 DNA was detected in 23~ of mild dysplasias, 329/o of moderate dysplasias, 55 ~ of severe dysplasias and 62 ~ of carcinomas in situ by dot blot hybridization. Digestion of the DNA with restriction enzymes PstI and BamHI followed by Southern blot analysis revealed the presence of some typical restriction fragments of HPV 16 DNA in most virus-positive samples. In addition, we detected submolar fragments which might represent virus-cell junction sequences in 869/oo of dysplasias, suggesting that the integration of HPV 16 DNA could occur in the precancerous stage.Human papillomavirus (HPV) type 16 and type 18 DNAs are frequently found in biopsies from precancerous and malignant cervical lesions (Boshart et al., 1984;Crum et al., 1985;Lehn et al., 1985;Tomita et al., 1986), with HPV 16 DNA being more common than HPV 18 DNA in these lesions (Diirst et al., 1983 ;Boshart et al., 1984;Yoshikawa et al., 1985). HPV 16 DNA has been cloned from an invasive cervical carcinoma (Dtirst et al., 1983) and the complete nucleotide sequence was determined (Seedorf et al., 1985). Recently, Diirst et al. (1985) reported the physical state of HPV 16 DNA in some malignant turnouts and showed that the integration of HPV 16 genome into the host cell DNA occurs with a head-to-tail viral genome arrangement. Their report includes a discussion of HPV integration in connection with the causative event in malignant transformation.Dysplasias are considered to be precancerous lesions and are classified as mild, moderate and severe (Koss, 1978). In order to see whether the integration of HPV 16 DNA into host cell DNA occurs in precancerous lesions, biopsy samples from mild, moderate and severe dysplasias and from cervical cancers were screened for the presence of HPV 16 DNA by dot blot hybridization. Virus-specific restriction fragments were analysed by Southern blot hybridization. We report here the detection of submolar fragments, which might be virus-cell junction sequences, in most dysplasias and carcinomas in situ that harbour HPV 16 DNA.Biopsy samples were collected under colposcopy and kept at -70 °C. High molecular weight DNA was extracted and purified from samples that had been histopathologically confirmed (Tomita et al., 1986). For dot blot hybridization, about 7.5 p.g of the purified DNA was denatured, neutralized, then spotted onto a nitrocellulose filter and hybridized with 32p-labelled cloned HPV 16 DNA in 6 x SSC at 68 °C for 24 h. The specific activity of the HPV 16 DNA was 108 to 2 x 108 c.p.m./~tg. The filter was washed extensively with 0

Human papillomavirus type 16 (HPV16) genome DNA and its transcripts in biopsied cervical neoplasias were analyzed by simultaneous extraction of DNA and RNA from one biopsied sample. Southern blot analysis revealed that 5 of 20 cervical intraepithelial neoplasias (CINs) contained HPV16 DNAs existing primarily as episomes and two of seven invasive carcinomas harbored HPV16 genome sequences integrated into the host DNA. Northern (RNA) blot analysis showed that the HPV16 genome sequences were transcriptionally active in the five CINs, as well as in the two invasive carcinomas. The pattern of HPV16-specific transcripts in the CINs was uniform, and the major transcripts were 4.2, 2.2, 1.6, and 1.4 kilobases in size. However, the pattern of HPV16-specific transcripts in the invasive carcinomas was variable and different from that in CINs, suggesting that the alteration of transcriptional pattern might play a key role in the development of malignancy.