The viral envelope is a major determinant for the induction of lymphoid and myeloid tumours by avian leukosis virus subgroups A and J, respectively.

Among the six envelope subgroups of avian leukosis virus (ALV) that infect chickens, subgroups A (ALV-A) and J (ALV-J) are the most pathogenic and widespread among commercial chicken populations. While ALV-A is predominantly associated with lymphoid leukosis (LL) and less frequently with erythroblastosis (EB), ALV-J mainly induces tumours of the myeloid lineage. In order to examine the basis for the lineage specificity of tumour induction by these two ALV subgroups, we constructed two chimeric viruses by substituting the env genes into the reciprocal proviral clones. The chimeric HPRS-103(A) virus carrying the subgroup A env gene is identical to ALV-J prototype virus HPRS-103 except for the env gene, and the chimeric RCAS(J) virus carrying the subgroup J env gene is identical to the parent replication-competent ALV-A vector RCAS except for the env gene. In experimentally inoculated chickens, HPRS-103(A) virus induced LL and EB similar to ALV-A isolates such as RAV-1, while RCAS(J) virus induced myeloid leukosis (ML) and EB, similar to ALV-J, suggesting that the env gene is the major determinant for the lineage-specific oncogenicity. There were genetic differences in susceptibility to tumour induction between line 0 and line 15(I) chickens, indicating that in addition to the env gene, other viral or host factors could also serve as determinants for oncogenicity. Induction of both LL and ML by the two chimeric viruses occurred through the activation of c-myc, while the EB tumours were induced by activation of the c-erbB oncogene.

Acutely transforming avian leukosis virus subgroup J strain 966: defective genome encodes a 72-kilodalton Gag-Myc fusion protein.

Avian leukosis virus subgroup J (ALV-J), the most recent member of the avian retroviruses, is predominantly associated with myeloid leukosis in meat-type chickens. We have previously demonstrated that the acutely transforming virus strain 966, isolated from an ALV-J-induced tumor, transformed peripheral blood monocyte and bone marrow cells in vitro and induced rapid-onset tumors, suggesting transduction of oncogenes (L. N. Payne, A. M. Gillespie, and K. Howes, Avian Dis. 37:438-450, 1993). In order to understand the molecular basis for the rapid transformation and tumor induction, we have determined the complete genomic structure of the provirus of the 966 strain. The sequence of the 966 provirus clone revealed that its genome is closely related to that of HPRS-103 but is defective, with the entire pol and parts of the gag and env genes replaced by a 1,491-bp sequence representing exons 2 and 3 of the c-myc gene. LSTC-IAH30, a stable cell line derived from turkey monocyte cultures transformed by the 966 strain of ALV-J, expressed a 72-kDa Gag-Myc fusion protein. The identification of the myc gene in 966 virus as well as in several other ALV-J-induced tumors suggested that the induction of myeloid tumors by this new subgroup of ALV occurs through mechanisms involving the activation of the c-myc oncogene.

Equid herpesvirus 1: platelets and alveolar macrophages are potential sources of activated TGF-B1 in the horse.

Cell mediated responses to Equid herpesvirus 1 (EHV-1) are of short duration in vivo and require considerable expansion to be detected in vitro. Raised serum levels of active transforming growth factor B (TGF-B1) have been shown to depress proliferative T cell responses in experimental infections with EHV-1 in ponies. The present work indicates that latent transforming growth factor B (TGF-B1) is present in circulating platelets, lymph node, bronchial epithelium and alveolar macrophages. Activation of platelets in vitro by thrombin resulted in the release of latent TGF-B1 from platelets, with a pg level of conversion to active TGF-B1, but virus alone did not activate TGF-B1. Exposure of circulating leucocytes to EHV-1 in vivo or in vitro does not result in detection of active TGF-B1 above residual levels that could be attributed to activation of platelets by manipulation. However, alveolar macrophages obtained by lavage at autopsy yield both latent and active TGF-B1 in ng quantities. Bronchial epithelium, and mesenteric lymph node leucocytes had equivalent levels of latent TGF-B1, but horses varied as to whether these tissues were a source of activated TGF-B1 and as to whether EHV-1 activated TGF-B1.

Detection of latency-associated transcripts of equid herpesvirus 1 in equine leukocytes but not in trigeminal ganglia.

Results from Southern hybridization and PCR amplification experiments using a randomly synthesized reverse transcription-PCR product showed that peripheral blood leukocytes from horses showing no clinical signs of disease expressed a putative latency-associated transcript antisense to and overlapping the 3' end of the equid herpesvirus 1 (EHV-1) immediate-early gene (gene 64). A PCR product derived from this transcript has > or =96% identity with the published EHV-1 sequence. In situ hybridization studies of equine bronchial lymph nodes corroborated these findings and are consistent with reactivation data (D. A. Smith, A. Hamblin, and N. Edington, unpublished data), indicating that EHV-1 latency is established predominantly in CD5+/CD8+ leukocytes.

Nucleotide sequence of equine MxA cDNA.

A 2.6 kb cDNA species has been isolated from a cDNA library prepared from interferon-alpha stimulated equine peripheral blood leucocytes and the nucleotide sequence determined. The cDNA has a single open reading frame potentially encoding a 660 amino acid polypeptide showing a high degree of homology with known mammalian Mx proteins, including the possession of three consensus GTP-binding motifs. The protein has a calculated pI = 6.1 and in accordance with proposed nomenclature we have designated it equine MxA.

Human papillomavirus type 16 DNA expresses a replication modulation factor in cos-1 cells.

Plasmid DNA including the simian virus 40 (SV40) origin of replication undergoes uncontrolled, runaway replication in cos-1 cells owing to the intracellular production of SV40 large T antigen. Covalent linkage of such plasmids with human papillomavirus type 16 (HPV-16) DNA was found to prevent runaway replication. Replication control was found to be dependent on the presence of HPV-16 DNA sequences including the E1 open reading frame and part of the non-coding region.

Analysis of human papillomavirus type 16 open reading frame E7 immortalizing function in rat embryo fibroblast cells.

The E7 open reading frame of human papillomavirus type 16 (HPV-16) encodes a protein that can immortalize primary rat cells, cooperate with the ras oncoprotein to transform low passage rat cells and transform established rodent cells to anchorage independence. The immortalizing and cooperation functions have been investigated using a series of point mutations that introduce single amino acid changes into the E7 protein in two distinct regions. Certain mutations altering amino acids conserved between the E7 protein of genital HPV types, the adenovirus E1a protein and simian virus 40 large T antigen abolished the ability of the E7 protein to immortalize or cooperate with ras in a focus forming assay. Mutations in a consensus sequence for a casein kinase II recognition site, which is also shared by E1a and large T, reduced immortalizing activity, but did not affect the ability to cooperate with ras. Single mutations disrupting cysteine motifs, which form putative zinc-binding sites in the second region, reduced the activity of the E7 protein, whereas double mutants, in which neither of the cysteine motifs remained intact, showed no or very low activity. The activity of the mutants in immortalization and cooperation assays was essentially the same as their transforming activities in NIH 3T3 cells. This indicates that these three functions of E7 map to overlapping domains which cannot be separated by these mutations in the region of E1a/large T homology or the cysteine motifs.

Human papillomavirus types 6 and 16 in cooperation with Ha-ras transform secondary rat embryo fibroblasts.

Using a focus assay we have shown that the entire human papillomavirus type 16 (HPV-16) genome is capable of cooperating with an activated ras oncogene to transform secondary rat embryo fibroblast (REF) cells as indicated by focus formation of unselected cells. However, this assay failed to detect any similar activity with either the whole HPV-6 genome or with subgenomic fragments. In contrast, transformed colonies appeared when G418 (geneticin)-resistant colonies were selected after cotransfection with activated ras DNA and either the entire HPV-6 genome or subgenomic fragments containing the E6/E7 open reading frames (ORFs) of HPV-6 or HPV-16. The transformation assessment was based on the development of a ras-transformed appearance in G418-resistant colonies. The appearance of this morphology did not imply the ability of transformed cells to produce colonies in semi-solid agarose (anchorage-independent growth), and extended culture for about 10 to 20 population doublings was necessary before transfected cells exhibited anchorage-independent growth. Transformation of REF cells was not observed with the E5 ORF of HPV-16 under the control of an exogenous promoter (the long terminal repeat of Rous sarcoma virus) in cooperation with activated ras DNA. No transformation was observed using an activated myc oncogene with either HPV-6 or HPV-16 DNA.

Prevalence of human papillomavirus type 16 DNA sequences in cervical intraepithelial neoplasia and invasive carcinoma of the cervix.

The frequency of human papillomavirus (HPV) type 16 in premalignant and malignant lesions of the cervix was investigated and compared with the detection of HPV type 6. In cervical intraepithelial neoplasia (CIN) grades I-III HPV 6 was detected in 28% and HPV 16 in 62% of patients whereas 90% of malignant lesions contained HPV 16 only. In the CIN lesions there was an increase in HPV 16 detection as the severity of disease increased while the level of detection of HPV 6 decreased. Only three (18%) of the cervices that were colposcopically and histologically normal contained HPV genomes; although two of these three women had either a history of genital warts or a sexual partner with penile warts.

Human papillomavirus type 16 recombinant DNA is maintained as an autonomously replicating episome in monkey kidney cells.

Human papillomavirus type 16 (HPV16) DNA cloned in the expression vector pSV2-neo has been shown to be maintained in transfected monkey kidney cells as an autonomously replicating episome at a level of 2 to 10 copies per cell. Integration of pSV2-neo/HPV16 DNA with the host genome occurred in transfected mouse fibroblasts. Neither type of cell appeared to be phenotypically transformed.

Arginine metabolism in infected cell cultures as a marker character for the differentiation of orthopoxviruses.

Arginine has been shown to be essential for the replication of several orthopoxviruses in mouse sarcoma 180 cells and in chick embryo fibroblast cultures. Both host systems are characterized by their inabilities to utilize citrulline for the biosynthesis of arginine due to deficiencies in the requisite cellular enzymes and cell multiplication is absolutely dependent on the availability of exogenous arginine. Virus replication in such cells maintained with citrulline results from the induction of virus-specific enzymes. Significant virus yields in the absence of exogenous arginine or citrulline can arise from the replenishment of intracellular amino acid pools by increased utilization of arginyl residues in cellular proteins. The extent of the phenotypic expression of these characters in infected cells permitted significant discrimination between the viruses examined. Distinctions could be drawn between rabbitpox, ectromelia, cowpox, buffalopox and vaccinia strains. However, cowpox could not be distinguished from other viruses isolated from diseased animals in European zoos.

Persistence of DNA sequences of BK virus and JC virus in normal human tissues and in diseased tissues.

Available evidence suggests that BK virus (BKV) and JC virus (JCV) persist in the kidneys of healthy individuals after primary infection and may reactivate when the host's immune response is impaired. Data supporting this hypothesis are presented. A previous study had shown BKV to be present in the kidneys of eight (57%) of 14 subjects. In the present study, which extended the investigation to a total of 30 subjects, BKV DNA was found in the renal tissues of 10 (33%) subjects, and JCV DNA was found in the renal tissues of three (10%) subjects. The viral DNA detected appeared not to be integrated with host DNA and to be isolated in foci. Investigation of normal and diseased brain tissue, including tissue from six subjects with multiple sclerosis, failed to reveal the presence of either JCV DNA or BKV DNA.

The persistence of papovavirus BK DNA sequences in normal human renal tissue.

Evidence has accumulated indicating that BK virus, following an inapparent primary infection, persists in the renal organs of normal healthy individuals and reactivates upon immunosuppression. Data to support this hypothesis are presented and suggest that BK virus DNA sequences are present at very low levels in the kidneys of more than 50% of the population and that this persistence is localized in several foci within these organs.