Koi herpesvirus represents a third cyprinid herpesvirus (CyHV-3) in the family Herpesviridae.

The sequences of four complete genes were analysed in order to determine the relatedness of koi herpesvirus (KHV) to three fish viruses in the family Herpesviridae: carp pox herpesvirus (Cyprinid herpesvirus 1, CyHV-1), haematopoietic necrosis herpesvirus of goldfish (Cyprinid herpesvirus 2, CyHV-2) and channel catfish virus (Ictalurid herpesvirus 1, IcHV-1). The genes were predicted to encode a helicase, an intercapsomeric triplex protein, the DNA polymerase and the major capsid protein. The results showed that KHV is related closely to CyHV-1 and CyHV-2, and that the three cyprinid viruses are related, albeit more distantly, to IcHV-1. Twelve KHV isolates from four diverse geographical areas yielded identical sequences for a region of the DNA polymerase gene. These findings, with previously published morphological and biological data, indicate that KHV should join the group of related lower-vertebrate viruses in the family Herpesviridae under the formal designation Cyprinid herpesvirus 3 (CyHV-3).

Genomic studies of the Lucké tumor herpesvirus (RaHV-1).

Ranid herpesvirus 1 (RaHV-1) is the etiological agent of the Lucké renal adenocarcinoma of the North American leopard frog Rana pipiens. Construction of cosmid libraries containing RaHV-1 DNA inserts allowed the derivation of a BamHI map for the viral genome. Summation of fragment sizes indicates that the genome is 217 kbp in size, a value in accordance with the most recent published estimate (220 kbp) obtained by field-inversion gel electrophoresis. The DNA sequence of the 39,757-bp insert in 1 cosmid (cos54) was determined and was predicted to contain 21 complete and 3 partial genes. In all, 12 genes have distant counterparts in a fish herpesvirus (ictalurid herpesvirus 1) and are present in 2 blocks, 1 of which is relatively inverted. This indicates that RaHV-1 belongs to the fish virus lineage of the herpesvirus family rather than to the lineage populated by mammalian and avian viruses. The remaining 12 genes in cos54 lack counterparts in any other herpesvirus. One of these encodes a putative DNA (cytosine-5) methyltransferase. This raises the possibility that biological processes induced in the host by RaHV-1 might involve methylation of cellular DNA by the viral enzyme.

Lucké renal adenocarcinoma, an anuran neoplasm: studies at the interface of pathology, virology, and differentiation competence.

The northern leopard frog, Rana pipiens, is vulnerable to a herpesvirus-induced renal tumor. The Lucké renal adenocarcinoma is metastatic as a function of temperature. The cloning procedure of nuclear transplantation has been used to study the differentiation potential of the tumor genome. This paper summarizes current studies of the pathology, virology, and differentiation competence of the Lucké tumor.

Pronephric carcinoma: chromosomes of cells rescued from apoptosis by an oncogenic herpesvirus detected with a polymerase chain reaction.

The amphibian pronephros is fated to die during early development. Pronephric cells undergo apoptosis and their function is replaced by the mesonephros, which becomes the functional kidney of the adult frog. Tadpoles of the northern leopard frog, Rana pipiens, were inoculated with a Lucké tumour herpesvirus (LTV) preparation. Most of the animals developed typical Lucké renal carcinomas at metamorphosis. Fewer developed carcinomas of the pronephric cell type. A pronephric carcinoma, rescued from apoptosis by the herpesvirus, was harvested from a post-metamorphic frog. The tumour was judged to be pronephric by its anatomical location (in the anterior part of the body) and because both mesonephric kidneys were intact and tumour-free upon removal of the tumour mass. A tumour fragment was fixed for histological examination, which confirmed that the tissue was a renal carcinoma. A further fragment was subjected to short-term culture in order to produce metaphase cells for cytogenetical analysis. Based upon silverstained nucleolar organizing region numbers, 14 of 15 metaphase cells were estimated to have the diploid number (2N = 26) of chromosomes and a karyotype was constructed which did not appear to differ from that of normal cells. A single cell was estimated to be tetraploid (4N = 52). This is the first report of chromosomes of a pronephric Lucké carcinoma. LTV replicates only in tumour tissue maintained in the cold. Because the frog in this study had been maintained in the laboratory at 22 degrees C for about 10 months, no viruses would have been detectable with electron microscopy. However, the presence of Lucké herpesvirus DNA was detected in tumour homogenates by polymerase chain reaction amplification of a 1.2 kbp Hind III restriction fragment of the LTV DNA. The presence of LTV DNA provided assurance that the rescued pronephric tumour was indeed a Lucké carcinoma.

Fate of herpesvirus DNA in embryos and tadpoles cloned from Lucké renal carcinoma nuclei.

The Lucké renal carcinoma of the northern leopard frog, Rana pipiens, has a herpesvirus aetiology. Lucké tumour nuclei inserted into enucleated frogs' eggs produce development to the swimming tadpole stage. Tissue from these tumour nuclear transplant animals can be induced to survive and differentiate further by allografting to normal tadpoles. We wished to ascertain whether the aetiological agent, the Lucké tumour herpesvirus (LTV), persists in the animals produced by tumour nuclear transplantation. The polymerase chain reaction was used to amplify a 1.2 kbp Hind III restriction fragment of LTV DNA in whole animal homogenates prepared from tumour nuclear transplant tadpoles and normal tadpoles fertilized in vitro. The LTV fragment was not present in the majority (31 of 34) of the cloned animals derived from tumour nuclei, nor was it present in any of five normal tadpoles. Either the 1.2 kbp fragment of LTV DNA was eliminated from most of the cloned animals during the massive reprogramming of the neoplastic genome initiated by insertion of the tumour nuclei into egg cytoplasm, or the nuclei selected for transplantation were primarily those lacking this fragment of LTV DNA. The limited development of the tumour nuclear tadpoles was probably not due to the presence of these viral sequences, but rather reflected the limited plasticity of the tumour cell genome as assayed by nuclear transplantation. Failure to detect the 1.2 kbp fragment of LTV DNA in the majority of mitotic progeny of the Lucké tumour genome does not imply that other parts of the viral genome do not persist.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of DNA sequences of the Lucké herpesvirus in normal and neoplastic kidney tissue of Rana pipiens.

The Lucké tumour herpesvirus (LTV) is the aetiological agent of the Lucké renal adenocarcinoma of the northern leopard frog, Rana pipiens. LTV virions can be detected by electron microscopy in renal adenocarcinomata after prolonged exposure to low temperature. Tumours maintained at warm temperatures do not contain viral particles. To gain insight into the processes of viral infection, replication and oncogenesis, evidence was sought for the presence of LTV DNA in warm renal tumours and normal renal tissue. The polymerase chain reaction was used to amplify a Hind III restriction fragment of LTV DNA in tissue homogenates. LTV DNA was detected in a significant percentage of normal kidneys as well as in "virus-free" warm tumours and virus-containing cold tumours.

Cytogenetic analysis of triploid renal carcinoma in Rana pipiens.

To provide a cytogenetic marker for nuclear transplantation experiments, triploid Rana pipiens embryos were produced. These embryos were injected with Lucké tumor herpesvirus. The chromosome profile of a renal carcinoma that developed in one of these triploid embryos was compared to the chromosomal profiles of a naturally occurring diploid renal carcinoma and a diploid renal tumor maintained as serial anterior eye chamber allografts for over 7 yr. Examination of Ag-NOR-stained chromosome spreads from the putative triploid and naturally occurring putative diploid tumor revealed the expected results. The vast majority of the chromosome spreads, 54/57 (95%) and 6/7 (86%), respectively, displayed euploid chromosome and Ag-NOR profiles: 3N = 39 with three Ag-NORs at the secondary constrictions in the long arm of chromosome 10 (10q) and 2N = 26 with two Ag-NORs in 10q. Chromosome profiles from the long-term allografted tumor were highly aneuploid (82%) and, based on their Ag-NOR content, displayed variations in their 2N, 3N, and 4N numbers. These data indicate that the majority of recently transformed triploid Lucké tumor cells can provide donor nuclei suitable for the characterization of developmental potential.

Changes in nucleolar and ribosomal RNA of the frog kidney after malignant transformation by the Lucke tumor herpesvirus.

Malignant transformation of the frog kidney by the Lucke herpesvirus changes the nucleotide base composition of normal kidney nucleolar and ribosomal RNA. In the Lucke tumor there is a moderate decline in guanylic acid and a sharp decline in adenylic acid levels. Conversely, there is a sharp increase in cytidylic acid and uridylic acid levels in the tumor cells. However, there was an increase in the G + C content of nucleolar and ribosomal RNA over that obtained from the normal kidney cells. Nearly identical quantitative changes in the base composition of each RNA species were measured for the adult (spontaneous) mesonephric carcinoma and a Lucke-herpesvirus-induced pronephric tumor cell line; similar correspondence was obtained for the normal adult mesonephros and a normal pronephric cell line.

Temperature-dependent metastasis of the Lucke renal carcinoma and its significance for studies on mechanisms of metastasis.

Metastasis is temperature dependent in the renal adenocarcinoma of the North American leopard frog, Rana pipiens. Widespread, multiple, metastatic colonies occur in tumor-bearing frogs kept at 28 degrees C for 50 days while tumor-bearing frogs kept at 7 degrees C for 98 days or more have either no secondary deposits or they have only an occasional small metastatic nodule. An attractive aspect of the frog tumor is that invasion and metastasis can be permitted or inhibited by the manipulation of temperature alone-no exogenous chemicals or drugs are required for the effect. Because of this, biological variables which reproducibly and specifically associate with metastasis permissive conditions when ambient temperature is cycled between permissive and inhibitory values are strong candidates for being causal elements in the multistep process leading to metastasis. Intravascularly injected labelled renal tumor cells reached all organs studied in as little as 15 minutes at both metastasis restrictive and permissive temperature. The results with tumor cell inoculation dispose of the possibility that failure of metastasis in chilled animals is due to cold-induced changes in blood flow. Histologically typical metastatic colonies developed in frogs, kept at the permissive temperature, after injection with disaggregated tumor cells which were previously cryopreserved. Frog tumors elaborate type I collagenase in a temperature dependent manner. Type IV collagenase has been demonstrated as well. Tumor cell detachment in vitro, assembly and disassembly of tumor cell cytoplasmic microtubules, and invasion in vitro, are all temperature dependent.

Temperature-dependent dissociation of Lucké renal adenocarcinoma cells.

Fragments of Lucké renal adenocarcinoma were subjected to dissociation by rapid shaking after exposure to a divalent cation-free electrolyte solution, with or without 5 X 10(-4) M ethylenediaminetetraacetate (EDTA), at 7 degrees C and 28 degrees C. More cells detached at 28 degrees C than at 7 degrees C. Dissociation of cells from normal mesonephros fragments was minimal at both temperatures. It has been shown else-where that this frog tumor elaborates collagenase in a temperature-dependent manner. More collagenase is detected at 30 degrees C than at 7 degrees C. Normal kidney elaborates low levels of collagenase at both temperatures. Because our results suggested the possibility that some dissociation of the tumor cells may have been attributable to tumor-elaborated collagenase, we studied the effect of two collagenase inhibitors on dissociation. Both EDTA at high concentration and cysteine inhibit collagenase and both diminished tumor-cell dissociation.

Cytoplasmic microtubules of normal and tumor cells of the leopard frog. Temperature effects.

The cytoplasmic microtubule complex (CMTC) was examined in monolayer cultures of normal tadpole mesonephros, primary renal adenocarcinoma, and an established cell line derived from a pronephric renal adenocarcinoma (PNKT-4B) of the leopard frog, Rana pipiens. Immunocytochemistry revealed typical arrays of microtubules extending from the cytocentrum to the cell periphery in all three cell types when cultured at 28 degrees C; similar results were obtained at 20 degrees C. However, the CMTC was disorganized in both tumor types, in contrast to the retention of a typical CMTC in normal tissue cultured at 7 degrees C. The response of PNKT-4B cells differed from that of normal tadpole mesonephros when treated with the microtubule inhibitor drug nocodazole. At 28 degrees C, PNKT-4B and tadpole mesonephros cells lost their CMTC with nocodazole treatment, and both were able to reconstitute CMTC when nocodazole was removed. Similarly, both lost CMTC organization with nocodazole and culture at 70 degrees C. However, while normal cells could effect a recovery at 7 degrees C after the removal of nocodazole, PONKT-4B cells were unable to restructure CMTC under the same conditions. Metastasis in the frog renal adenocarcinoma is temperature-dependent, with an elevated prevalence of metastasis in tumor-bearing frogs maintained at 28 degrees C. Few metastatic colonies are detected in tumor-bearing frogs maintained at a low temperature (7 degrees C). Other studies have indicated that microtubules, which are essential for cell motility, play an important role in the invasion by tumor cells of normal tissue fragments in vitro. The effects of temperature on metastasis of the Lucke renal adenocarcinoma are consistent with temperature-mediated changes in tumor-cell CMTC.

The etiology of renal-cell carcinoma.

Experimental renal-cell carcinoma can be induced by many different chemical carcinogens; dimethyl nitrosoamine has been most studied. The disease so induced in experimental animals closely resembles the spontaneous disease in man in histopathology, course, and other characteristics. Two agents that are probably etiological of renal-cell cancer in man are tobacco and the analgesic, phenacetin; however, these materials can account for only a minority of the cases. The predominance of males in adult renal carcinoma might be explained by the more efficient metabolic activation of carcinogens by renal enzymes that are induced by male hormones. Mouse experiments support this hypothesis. Studies utilizing human kidney tissues that would test the hypothesis in man can and should be done. No obvious clues have emerged to explain the wide geographic differences in incidence of renal carcinoma. No group of industrial workers, or of others with a unique environment, has yet been described that has an especially high incidence of renal-cell carcinoma. A minority of renal carcinomas are familial. They represent a number of different diseases, one of which is associated with the von Hippel-Lindau disease. The hereditary renal-cell carcinoma of the Ecker rat, which is transmitted as an autosomal dominant, provides a useful laboratory model for hereditary carcinoma of man. Recently, two human families with renal-cell carcinoma were described in which there were unique chromosomal abnormalities associated with the disease. Such changes have been linked with oncogene activation in the instance of other tumors. Further studies of chromosomal abnormalities in renal-cell carcinoma will probably define a common pattern of chromosomal rearrangements. While estrogen readily induces renal-cell carcinoma in hamsters other species, including man, appear resistant. An excess of renal-cell carcinoma has not been reported in men on chronic estrogen therapy for prostatic carcinoma, nor has it been associated with the DES syndrome. A virus etiology for renal-cell carcinoma in man comparable to that of the Lucke tumor in frogs is unlikely on epidemiologic, ultrastructural morphologic, and other grounds. There is nothing suggesting horizontal transmission in the human disease, and a unique excess of renal-cell carcinomas in immunosuppressed patients or patients with the acquired immunodeficiency syndrome (AIDS) is not apparent. There is overwhelming evidence that renal adenomas represent early adenocarcinomas, or at least precursor lesions; certainly they are closely related to renal-cell carcinomas.(ABSTRACT TRUNCATED AT 400 WORDS)

Herpesviruses in metastatic Lucké renal adenocarcinoma.

The etiologic agent of the renal adenocarcinoma of leopard frogs, Rana pipiens, is the Lucké tumor herpesvirus (LTHV). The virus is easily detected with thin section electron microscopy in primary tumors of frogs which have been exposed to a cold environment. Several spontaneous metastatic nodules and a large primary tumor were detected at autopsy of a frog which had been maintained at 4 degrees C for 73 days. LTHV was found not only in the primary tumor, as previously reported, but also was present in metastatic tumor cells in the liver, fat body, and bladder. The presence of LTHV in metastatic cells demonstrates that the differentiated state of primary Lucké tumor cells is retained in its metastatic colonies even at the fine structure level revealed by electron microscopy.

Effect of thymectomy on development of Lucké renal adenocarcinomas in virus-infected leopard frog tadpoles.

Thymectomy of very young leopard frog (Rana pipiens) larvae dramatically reduced in vitro lymphocyte responses to the nitrogen phytohemagglutinin-P and prolonged survival of Lucké tumor alloimplants. However, tumor incidence in premetamorphic or metamorphosing frogs that had received injections as embryos of Lucké tumor herpesvirus (LTHV) was the same in thymectomized and normal immunocompetent animals. This result suggests that T-cell-mediated cytotoxicity does not prevent development of Lucké tumors in immunocompetent larvae and that if tumor-specific transplantation antigens appear, they may induce tolerance rather than destructive immunity in normal LTHV-injected larvae.

Reduced prevalence of the Lucké renal adenocarcinoma in populations of Rana pipiens in Minnesota.

Northern leopard frogs (Rana pipiens) afflicted with the Lucké renal adenocarcinoma virtually disappeared from Minnesota in the autumn of 1977. Frogs from four sites in Minnesota counties (Polk, Otter Tail, Kandiyohi, and Scott) with a previously high prevalence of Lucké renal tumor were studied. In the past decade, prevalence averaged 4.2% in 29 collections (total, 1,870 frogs). No tumors were detected in 685 frogs autopsied in the autumn of 1977 by the method of previous studies. Frog collections, each comprised of 20 or more individuals, were compared for the presence or absence of tumor-bearing frogs. Significantly fewer collections contained tumor-bearing frogs in the autumn of 1977 than did collections of previous years.

Detection of Lucké herpes virus antigens in infected frog pronephric cells.

A frog pronephric cell line, infected with herpes virus derived from Lucké renal carcinomas of Rana pipiens was examined for the presence of Lucké herpes virus antigens. Non-infected pronephric cells were controls. Antiserum to purified Lucké tumor herpes virus was applied in blind tests to the normal and virus infected cells. Both cytoplasmic and nuclear fluorescence was found in the herpes virus infected cells after indirect immunofluorescence with the antiserum. Infected cells cultivated at the optimum growth temperature of 25 degrees C or maintained at 9 degrees C, a temperature inducive to herpes virus replication, showed equivalent fluorescence reactions. No fluorescence was found in the normal pronephric cell line. Examination of parallel herpes infected cells showed cytopathic effect in monolayers by light microscopy, and nuclear or cytoplasmic immunofluorescence. Electron microscopic examination revealed proviral elements in nuclei and sparsely scattered herpes virus coincident with cytoplasmic fluorescence.