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Plaque purified Coxsackie B4 virus (CB4) caused a persistent infection of rat insulinoma (RIN) cells which lasted for the 70 day observation period. Infectious virus was produced and no cytopathic effect was observed. Indirect immunofluorescence and infectious center assays demonstrated that a majority of RIN cells were infected. Defective interfering particles were not demonstrated. Despite persistent infection, insulin synthesis by RIN cells was not altered.

We describe the molecular and biological properties of BK virus (BKV)-IR, a new BKV variant isolated from a human tumor of pancreatic islets. BKV-IR bears a 253-base-pair (bp) deletion and an 80-bp insertion in the early region of the genome. The deletion abolishes the expression of small-t antigen. The inserted sequences, grouped in four clusters, produce rearrangements in the first and second enhancer elements. They are bound by 12-bp direct repeats and could form a 217-base stem-loop structure suggestive of an insertion sequence. As compared with wild-type BKV, BKV-IR transformed hamster cells with a reduced efficiency and induced ependymomas in hamsters at a lower frequency and with a longer latency period. Tumors induced by BKV-IR, however, showed features of higher malignancy. The possible role of the insertion sequence-like element in transformation by BKV-IR is discussed.

BK virus (BKV) DNA was detected by Southern blot hybridization in 19 out of 74 (25.6%) human brain tumors and in 4 out of 9 (44.4%) human tumors of pancreatic islets. BKV DNA was free, in an episomal state and generally in a low copy number (0.2 to 2 genome equivalents per cell). Only occasional tumors contained 10 to 20 genome copies per cell. Viral DNA sequences integrated into cellular DNA were not detected. A number of tumors expressed BKV-specific RNA and T antigen. By transfection of total tumor DNA into human embryonic fibroblasts, viruses with the biological and antigenic properties of BKV were rescued from 6 brain tumors and from 2 tumors of pancreatic islets. Restriction endonuclease mapping of the genomes of the rescued viruses showed that they differ from wild-type BKV. They are all similar to each other and to BKV-IR, a virus previously rescued from a human tumor of pancreatic islets, suggesting the possible association of a BKV variant with specific types of human neoplasms. The significance of the relationship of these BKV variants to human tumors and their possible etiologic role in human oncogenesis are discussed.

BK virus (BKV)-induced tumors in hamsters were investigated for the presence of viral DNA by the blot-transfer hybridization technique. Several viral genomes per cell were found in tumor tissues and in their derived cell lines and clones. Most of the detected viral genomes were integrated into the cellular DNA, but some tumors also contained free viral DNA sequences. Integration patterns were different from each other, and many different integration sites were available on the cellular or on the viral DNA or on both. Typical features of integration patterns were found in ependymomas, which were the most frequent (72%) among BKV-induced tumors. Readily detectable viral DNA sequences were only found in neoplastic tissues, but traces of BKV DNA were also present in the apparently normal portion of the brain of an animal that had developed an ependymoma and in the brain (but not in the liver) of another animal 15 days after virus inoculation. A cell line and a single-cell clone derived from a tumor had hybridization patterns considerably simpler that the pattern of the original tumor, lacking several integrated viral genomes and all free viral sequences.

Newborn hamsters were inoculated intracerebrally with BK virus. Between 3 and 6 months after inoculation, they developed papillary ependymoma (8 hamsters, 42%) or functional malignant islet cell tumors of the pancreas (insulinoma, 8 hamsters), or both (1 hamster). Both tumors contained an antigen reactive to SV40 T-antibody, suggesting that at least a part of BK virus genomen has been integrated into the tumor cells. No infectious virus was detected in the extract of these tumors.

The ultrastructural morphology of the clonal insulin-producing cell line, RINm5F, was investigated. Virus-like particles, probably C type viruses, were identified both intra- and extracellularly. Because these particles could not be found in the original transplantable tumor, it is probable that viruses were induced at some later stage in the development of the RINm5F cell line. All investigators using the RINm5F cells should be aware of the fact that these cells may contain one or several types of viruses, and of the possibility that these particles may interfere with a variety of cellular functions.

Newborn hamsters were inoculated intracerebrally with a series of purified and concentrated BK virus samples originating from a single stock of Gardner's original strain. Most (60-100%) of the hamsters developed various tumors 3-9 months later. The frequent types of tumors were ventricular tumors (choroid plexus papillomas and ependymomas: 7-53%), malignant insulinomas (0-92%), and osteosarcomas (0-50%). The T-antigen was positive in 59 of 60 tumors tested, but the virus was rescued by the cell fusion method from only 1 of 11 cell lines derived from these tumors. The incidence of insulinomas varied greatly with the virus sample; the two samples that showed the highest incidence (47 and 92%) originated from one parental virus stock, and all the other samples with the lower incidences (0-9%) originated from another ancestral stock. These results suggest the presence of a BK virus mutant(s) differing in capacity to induce insulinoma. A functional insulinoma cell line was thus established.

The ability to introduce foreign DNA into the genome of mice offers unique opportunities to produce new models of disease process. Recent experiments have shown that integration and expression of simian virus 40 (SV40) T antigen genes and the murine mammary tumour virus (MMTV)-myc genes in transgenic mice can lead to the development of neoplasia in a remarkably tissue-specific manner. In the case of SV40-bearing mice, tumours consistently develop in the choroid plexus. In the accompanying paper, we show that the 72-base pair (bp) enhancer in the SV40 genome is instrumental in directing tumorigenesis to the choroid plexus. However, when the enhancer is deleted from a construction also containing the metallothionein-human growth hormone fusion gene (SV delta e-MGH), an entirely new pattern of pathology results. The present report focuses on transgenic mice carrying this construct; they develop demyelinating peripheral neuropathies, hepatocellular carcinomas and islet cell adenomas.

BK virus (BKV) DNA was detected by blot hybridization in a human adenoma of pancreatic islets from patient I.R. BKV DNA was free, and no evidence was found of viral sequences integrated into cellular DNA. Virus was rescued by transfection of human embryonic fibroblasts with tumor DNA. The DNA from rescued virus (BKV-IR) was different from wild-type BKV DNA by restriction endonuclease mapping. The genome of BKV-IR is 235 base pairs (bp) shorter than the genome of wild-type BKV. This alteration originates from a deletion of approximately 300 bp involving HindIII fragments B and D, and an insertion of 70 bp in the region of HindIII fragment C. Transformation of hamster kidney cells was induced by total tumor DNA as well as by BKV-IR and BKV-IR DNA. No antibodies to BKV tumor (T) antigen were detected in the patient's serum by immunofluorescence. The significance of episomal BKV DNA in a human tumor is discussed.