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It has long been established that group A human adenoviruses (HAdV-A12, -A18, and -A31) can cause tumors in newborn rodents, with tumorigenicity related to the presence of a unique spacer region located between conserved regions 2 and 3 within the HAdV-A12 early region 1A (E1A) protein. Group B adenoviruses are weakly oncogenic, whereas most of the remaining human adenoviruses are nononcogenic. In an attempt to understand better the relationship between the structure of the AdE1A spacer region and oncogenicity of HAdVs, the structures of synthetic peptides identical or very similar to the adenovirus 12 E1A spacer region were determined and found to be α-helical using nuclear magnetic resonance (NMR) spectroscopy. This contrasts significantly with some previous suggestions that this region is unstructured. Using available predictive algorithms, the structures of spacer regions from other E1As were also examined, and the extent of the predicted α-helix was found to correlate reasonably well with the tumorigenicity of the respective virus. We suggest that this may represent an as-yet-unknown binding site for a partner protein required for tumorigenesis. IMPORTANCE This research analyzed small peptides equivalent to a region within the human adenovirus early region 1A protein that confers, in part, tumor-inducing properties to various degrees on several viral strains in rats and mice. The oncogenic spacer region is α-helical, which contrasts with previous suggestions that this region is unstructured. The helix is characterized by a stretch of amino acids rich in alanine residues that are organized into a hydrophobic, or “water-hating,” surface that is considered to form a major site of interaction with cellular protein targets that mediate tumor formation. The extent of α-helix in E1A from other adenovirus species can be correlated to a limited degree to the tumorigenicity of that virus. Some serotypes show significant differences in predicted structural propensity, suggesting that the amino acid type and physicochemical properties are also of importance.

Marek’s disease virus (MDV) is an alphaherpesvirus associated with Marek’s disease (MD), a highly contagious neoplastic disease of chickens. MD serves as an excellent model for studying virus-induced T-cell lymphomas in the natural chicken hosts. Among the limited set of genes associated with MD oncogenicity, MDV-miR-M4, a highly expressed viral ortholog of the oncogenic miR-155, has received extensive attention due to its direct role in the induction of lymphomas. Using a targeted CRISPR-Cas9-based gene editing approach in MDV-transformed lymphoblastoid cell lines, we show that MDV-miR-M4, despite its critical role in the induction of tumors, is not essential for maintaining the transformed phenotype and continuous proliferation. As far as we know, this was the first study in which precise editing of an oncogenic miRNA was carried out in situ in MD lymphoma-derived cell lines to demonstrate that it is not essential in maintaining the transformed phenotype., MicroRNAs (miRNAs) are small noncoding RNAs with profound regulatory roles in many areas of biology, including cancer. MicroRNA 155 (miR-155), one of the extensively studied multifunctional miRNAs, is important in several human malignancies such as diffuse large B cell lymphoma and chronic lymphocytic leukemia. Moreover, miR-155 orthologs KSHV-miR-K12-11 and MDV-miR-M4, encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV) and Marek’s disease virus (MDV), respectively, are also involved in oncogenesis. In MDV-induced T-cell lymphomas and in lymphoblastoid cell lines derived from them, MDV-miR-M4 is highly expressed. Using excellent disease models of infection in natural avian hosts, we showed previously that MDV-miR-M4 is critical for the induction of T-cell lymphomas as mutant viruses with precise deletions were significantly compromised in their oncogenicity. However, those studies did not elucidate whether continued expression of MDV-miR-M4 is essential for maintaining the transformed phenotype of tumor cells. Here using an in situ CRISPR/Cas9 editing approach, we deleted MDV-miR-M4 from the MDV-induced lymphoma-derived lymphoblastoid cell line MDCC-HP8. Precise deletion of MDV-miR-M4 was confirmed by PCR, sequencing, quantitative reverse transcription-PCR (qRT-PCR), and functional analysis. Continued proliferation of the MDV-miR-M4-deleted cell lines demonstrated that MDV-miR-M4 expression is not essential for maintaining the transformed phenotype, despite its initial critical role in the induction of lymphomas. Ability to examine the direct role of oncogenic miRNAs in situ in tumor cell lines is valuable in delineating distinct determinants and pathways associated with the induction or maintenance of transformation in cancer cells and will also contribute significantly to gaining further insights into the biology of oncogenic herpesviruses. IMPORTANCE Marek’s disease virus (MDV) is an alphaherpesvirus associated with Marek’s disease (MD), a highly contagious neoplastic disease of chickens. MD serves as an excellent model for studying virus-induced T-cell lymphomas in the natural chicken hosts. Among the limited set of genes associated with MD oncogenicity, MDV-miR-M4, a highly expressed viral ortholog of the oncogenic miR-155, has received extensive attention due to its direct role in the induction of lymphomas. Using a targeted CRISPR-Cas9-based gene editing approach in MDV-transformed lymphoblastoid cell lines, we show that MDV-miR-M4, despite its critical role in the induction of tumors, is not essential for maintaining the transformed phenotype and continuous proliferation. As far as we know, this was the first study in which precise editing of an oncogenic miRNA was carried out in situ in MD lymphoma-derived cell lines to demonstrate that it is not essential in maintaining the transformed phenotype.

The Herpesviridae conserved infected-cell protein 27 (ICP27) is essential for cell culture-based replication of most herpesviruses studied. For members of the Alphaherpesvirinae, ICP27 regulates the expression of many viral genes, including expression of pUL44 (gC), pUL47 (VP13/14), and pUL48 (VP16). These three viral proteins are dysregulated during Marek's disease alphaherpesvirus (MDV) replication in cell culture. MDV replicates in a highly cell-associated manner in cell culture, producing little to no infectious virus. In contrast, infectious cell-free MDV is produced in specialized feather follicle epithelial (FFE) cells of infected chickens, in which these three genes are abundantly expressed. This led us to hypothesize that MDV ICP27, encoded by gene UL54, is a defining factor for the dysregulation of gC, pUL47, and pUL48 and, ultimately, ineffective virus production in cell culture. To address ICP27's role in MDV replication, we generated recombinant MDV with ICP27 deleted (vΔ54). Interestingly, vΔ54 replicated, but plaque sizes were significantly reduced compared to those of parental viruses. The reduced cell-to-cell spread was due to ICP27 since plaque sizes were restored in rescued viruses, as well as when vΔ54 was propagated in cells expressing ICP27 in trans In chickens, vΔ54 replicated, induced disease, and was oncogenic but was unable to transmit from chicken to chicken. To our knowledge, this is the first report showing that the Herpesviridae conserved ICP27 protein is dispensable for replication and disease induction in its natural host.IMPORTANCE Marek's disease (MD) is a devastating oncogenic disease that affects the poultry industry and is caused by MD alphaherpesvirus (MDV). Current vaccines block induction of disease but do not block chicken-to-chicken transmission. There is a knowledge gap in our understanding of how MDV spreads from chicken to chicken. We studied the Herpesviridae conserved ICP27 regulatory protein in cell culture and during MDV infection in chickens. We determined that MDV ICP27 is important but not required for replication in both cell culture and chickens. In addition, MDV ICP27 was not required for disease induction or oncogenicity but was required for chicken-to-chicken transmission. This study is important because it addresses the role of ICP27 during infection in the natural host and provides important information for the development of therapies to protect chickens against MD.

Human papillomavirus 58 (HPV58) is found in 10 to 18% of cervical cancers in East Asia but is rather uncommon elsewhere. The distribution and oncogenic potential of HPV58 variants appear to be heterogeneous, since the E7 T20I/G63S variant is more prevalent in East Asia and confers a 7- to 9-fold-higher risk of cervical precancer and cancer. However, the underlying genomic mechanisms that explain the geographic and carcinogenic diversity of HPV58 variants are still poorly understood. In this study, we used a combination of phylogenetic analyses and bioinformatics to investigate the deep evolutionary history of HPV58 complete genome variants. The initial splitting of HPV58 variants was estimated to occur 478,600 years ago (95% highest posterior density [HPD], 391,000 to 569,600 years ago). This divergence time is well within the era of speciation between Homo sapiens and Neanderthals/Denisovans and around three times longer than the modern Homo sapiens divergence times. The expansion of present-day variants in Eurasia could be the consequence of viral transmission from Neanderthals/Denisovans to non-African modern human populations through gene flow. A whole-genome sequence signature analysis identified 3 amino acid changes, 16 synonymous nucleotide changes, and a 12-bp insertion strongly associated with the E7 T20I/G63S variant that represents the A3 sublineage and carries higher carcinogenetic potential. Compared with the capsid proteins, the oncogenes E7 and E6 had increased substitution rates indicative of higher selection pressure. These data provide a comprehensive evolutionary history and genomic basis of HPV58 variants to assist further investigation of carcinogenic association and the development of diagnostic and therapeutic strategies. IMPORTANCE Papillomaviruses (PVs) are an ancient and heterogeneous group of double-stranded DNA viruses that preferentially infect the cutaneous and mucocutaneous epithelia of vertebrates. Persistent infection by specific oncogenic human papillomaviruses (HPVs), including HPV58, has been established as the primary cause of cervical cancer. In this work, we reveal the complex evolutionary history of HPV58 variants that explains the heterogeneity of oncogenic potential and geographic distribution. Our data suggest that HPV58 variants may have coevolved with archaic hominins and dispersed across the planet through host interbreeding and gene flow. Certain genes and codons of HPV58 variants representing higher carcinogenic potential and/or that are under positive selection may have important implications for viral host specificity, pathogenesis, and disease prevention.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). Similar to other herpesviruses, KSHV has two life cycles, latency and lytic replication. In latency, the KSHV genome persists as a circular episome in the nucleus of the host cell and only a few viral genes are expressed. In this review, we focus on oncogenic, antiapoptotic, and immunomodulating properties of KSHV-encoded homologues of cellular interferon regulatory factors (IRFs)—viral IRF1 (vIRF1) to vIRF4—and their possible role in the KSHV-mediated antiviral response, apoptosis, and oncogenicity.

Adenoviruses (AdVs) contain genes coding for proteins with transforming potential, and certain AdV serotypes have been shown to induce tumors in rodents. However, data on the possible oncogenicity of AdVs in humans are scarce. We have therefore employed a real-time quantitative PCR (RQ-PCR) assay permitting highly sensitive detection of all 51 currently known human AdV serotypes to screen more than 500 tumor specimens derived from 17 different childhood cancer entities including leukemias, lymphomas, and solid tumors. Most tumor entities analyzed showed no evidence for the presence of AdV sequences, but AdV DNA was detected by RQ-PCR in different brain tumors including 25/30 glioblastomas, 22/30 oligodendrogliomas, and 20/30 ependymomas. Nonmalignant counterparts of AdV-positive brain tumors, including specimens of ependymal cells, plexus choroideus, and periventricular white matter, were screened for control purposes and revealed the presence of AdV DNA in most specimens tested. Identification of the AdV types present in positive malignant and nonmalignant brain tissue specimens revealed predominantly representatives of species B and D and, less commonly, C. To exclude contamination as a possible cause of false-positive results, specimens with AdV sequences detectable by PCR were subsequently analyzed by in situ hybridization, which confirmed the PCR findings in all instances. The central nervous system appears to represent a common site of AdV infection with virus persistence, thus providing the first evidence for the possible contribution of AdVs to the multistep process of tumor pathogenesis in brain tissue.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that induces T-cell lymphomas in poultry. We report the construction of bacterial artificial chromosome (BAC) clones of the highly oncogenic RB-1B strain by inserting mini-F vector sequences into the U S 2 locus. MDV reconstituted from two BAC clones induced rapid-onset lymphomas similar to those induced by the wild-type virus. Virus reconstituted from another BAC clone that showed a 7.7-kbp deletion in the internal and terminal unique long repeat regions was nononcogenic, suggesting that the deleted region may be associated with oncogenicity. The generation of the oncogenic BAC clones of MDV is a significant step in unraveling the oncogenic determinants of this virus.

In adenovirus-infected cells, binding of E1B-55kDa and E4orf6 to the tumor suppressor protein p53 inhibits its transcriptional activity and causes rapid turnover of the protein. To investigate the requirements of the E1B-E4orf6 complex to modulate p53 function, we generated an E4orf6 mutant that failed to associate functionally and physically with E1B-55kDa but still interacted with p53. We confirm that E4orf6 and E1B-55kDa reduce p53 transactivation individually and show that their combined inhibition is additive rather than synergistic. Furthermore, we found that downregulation of p53's expression level, but not transcriptional inhibition of p53, depends on a functional E1B-E4 complex. A functional interaction of E1B-55kDa with p53, on the other hand, is a prerequisite for both transcriptional repression and downregulation of p53. The separation of these two functions will enable further dissection of the requirements for oncogenicity by the E4orf6 protein.

RB1BUS6 lacgpt , a Marek’s disease virus (MDV) mutant having a disrupted glycoprotein D (gD) homolog gene, established infection and induced tumors in chickens exposed to it by inoculation or by contact. Lymphoblastoid cell lines derived from RB1BUS6 lacgpt -induced tumors harbored only the mutant virus. These results provide strong evidence that an intact gD homolog gene is not essential for oncogenicity or horizontal transmission of MDV.

Mutant forms of herpesvirus saimiri (HVS) subgroup C strain 488 with deletions in either STP-C488 or Tip were constructed. The transforming potentials of the HVS mutants were tested in cell culture and in common marmosets. Parental HVS subgroup C strain 488 immortalized common marmoset T lymphocytes in vitro to interleukin-2-independent growth, but neither of the deletion mutants produced such growth transformation. Wild-type HVS produced fatal lymphoma within 19 to 20 days of experimental infection of common marmosets, while HVS ΔSTP-C488 and HVS ΔTip were nononcogenic. Virus was repeatedly isolated from the peripheral blood of marmosets infected with mutant virus for more than 5 months. These results demonstrate that STP-C488 and Tip are not required for replication or persistence, but each is essential for transformation in cell culture and for lymphoma induction in common marmosets.

Association of c-Rel with the inhibitor of kappaB-alpha (IkappaB-alpha) protein regulates both cellular localization and DNA binding. The ability of v-Rel, the oncogenic viral counterpart of avian c-Rel, to evade regulation by p40, the avian IkappaB-alpha protein, contributes to v-Rel-mediated oncogenesis. The yeast two-hybrid system was utilized to dissect Rel:IkappaB-alpha interactions in vivo. We find that distinct domains in c-Rel and v-Rel are required for association with p40. Furthermore, while the ankyrin repeat domain of p40 is sufficient for association with c-Rel, both the ankyrin repeat domain and the PEST domain are required for association with v-Rel. Two amino acid differences between c-Rel and v-Rel that are principally responsible for PEST-dependent association of v-Rel with p40 were identified. These same amino acids were principally responsible for PEST-dependent cytoplasmic retention of v-Rel by p40. The presence of mutations in c-Rel that were sufficient to confer PEST-dependent association of the mutant c-Rel protein with p40 did not increase the weak oncogenicity of c-Rel. However, the introduction of these two c-Rel-derived amino acids into v-Rel markedly reduced the oncogenicity of v-Rel. Deletion of the NLS of either c-Rel or v-Rel did not abolish association with p40, but did confer PEST-dependent association of c-Rel with p40. Surprisingly, deletion of the nuclear localization signal in v-Rel did not affect oncogenicity by v-Rel. Analysis of several mutant c-Rel and v-Rel proteins demonstrated that association of Rel proteins with p40 is necessary but not sufficient for cytoplasmic retention. These results are not consistent with the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by the same mechanism. Rather, these results support the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by distinct mechanisms.

Oncogenicity tests have revealed that a nonmutated erbB oncogene induces renal adenocarcinoma in addition to erythroblastosis. The erbB oncogene is a truncated form of the chicken epidermal growth factor receptor that lacks the extracellular ligand-binding domain. Previously, the nonmutated erbB oncogene has been reported to cause only erythroblastosis. The expansion of the disease potential of erbB to additional neoplasms has been associated with mutations (truncations, deletions, and point mutations) within the erbB gene. Our results indicate that a nonmutated virally expressed erbB oncogene (REB-c) causes a 100% incidence of renal neoplasia.

The incompatibility group W plasmid pSa suppresses Agrobacterium tumefaciens oncogenicity (J. Loper and C. Kado, J. Bacteriol. 139:591-596, 1979). The oncogenic suppressive activity was localized to a 3.1-kb region of pSa by Tn5 mutagenesis and deletion analysis. Within this fragment, a 1.1-kb subclone bearing oncogenic suppressive activity was subjected to further characterization. Nucleotide sequencing of the 1.1-kb fragment revealed a 570-bp open reading frame (ORF1) that has a coding capacity for a protein of 21.1 kDa. Sequencing of flanking regions revealed a second ORF (ORF2) located 3 bp upstream of ORF1, with a coding capacity for a protein of 22.8 kDa. Gene fusions of these ORFs to a T7 phi 10 expression system in Escherichia coli resulted in the synthesis of polypeptides of the predicted sizes. An E. coli promoter consensus sequence was not found in the expected positions in the region preceding ORF1. However, several sequences with similarity to the consensus -10 sequence of the A. tumefaciens vir gene promoters were found upstream of ORF1. Potential translational start signals are upstream of ORF1 and ORF2. These sequences showed no significant similarity at the nucleotide or amino acid levels with those in available data bases. However, the C-terminal portion of the ORF1 protein is rich in hydrophobic residues. Perhaps oncogenicity suppression is effected by an association of this protein with the Agrobacterium membrane such that T-DNA transfer is blocked.

bic is a novel gene identified at a common retroviral integration site in avian leukosis virus-induced lymphomas and has been implicated as a collaborator with c- myc in B lymphomagenesis. It lacks an extensive open reading frame and is believed to function as an untranslated RNA (W. Tam, Gene 274:157-167, 2001; W. Tam, D. Ben-Yehuda, and W. S. Hayward, Mol. Cell. Biol. 17:1490-1502, 1997). The oncogenic potential of bic , particularly its ability to cooperate with c- myc in oncogenesis, was tested directly by expressing c- myc and bic , either singly or in pairwise combination, in cultured chicken embryo fibroblasts (CEFs) and in chickens using replication-competent retrovirus vectors. Coexpression of c- myc and bic in CEFs caused growth enhancement of cells. Most importantly, chick oncogenicity assays demonstrated that bic can cooperate with c- myc in lymphomagenesis and erythroleukemogenesis. The present study provides direct evidence for the involvement of untranslated RNAs in oncogenesis and provides further support for the role of noncoding RNAs as riboregulators.

Chickens susceptible to infection with subgroup E viruses were inoculated with four independent isolates of Rous-associated virus type 60 (RAV-60) that are subgroup e recombinants of endogenous and exogenous virus. Neoplasms developed in each inoculated group. Therefore, nontransforming viruses of subgroup E can induce lymphoid leukosis at a moderate rate compared with RAV-0, a subgroup E endogenous virus, suggesting that oncogenicity is not a viral envelope (env)-related characteristic. Since the common (c) regions of the RAV-60s examined were of exogenous origin, we suggest that the c region rather than env is important for a high rate of induction of lymphoid leukosis and related neoplasms.

RNA C-type viruses are known to exist in an unexpressed form in all mouse cells. These endogenous viruses can be activated from cells in tissue culture under certain experimental conditions. The biologic activity in vivo of one class of chemically-activated C-type virus has been studied. This virus is shown to induce a specific tumor, lymphatic leukemia, in mice of a low leukemic incidence strain.

We isolated a monoclonal antibody that immunoprecipitated two proteins of 22 and 27 kilodaltons (kDa) from nononcogenic adenovirus type 5 early region 1 (E1)-transformed rat cells but not from oncogenic adenovirus type 12 E1-transformed rat cells. In a variety of adenovirus-transformed cells including cells transformed by E1A and the c-H-ras oncogene, we found a perfect, inverse correlation between the presence of these two proteins and the oncogenicity of these cells in syngeneic immunocompetent rats. Characterization of the two proteins revealed that they occur in a large (700-kDa) complex and that the 27-kDa protein is identical to the already known 27-kDa (28-kDa) heat shock protein hsp27. The suppression of the hsp27 protein in oncogenic cells is further demonstrated by the fact that its mRNA is absent even after heat-shock induction.

We incubated 31 newly established Marek's disease tumor cell lines at 41 degrees C for 48 h after subculturing and then examined them to determine the spontaneous rates of expression of viral internal antigen(s), viral membrane antigen(s), and virus isolation. All but two of the lines were isolated from tumors induced by clone-purified Marek's disease virus strain JM-10, GA-5, RB-1B, and BC-1A in nine different genetic strains of chickens with defined histocompatibility antigens. The line-to-line variations in the rates of spontaneous expression for the antigens or virus rescue were great, but the levels of expression were very low in most cases. The median rates of expression for viral internal antigen, viral membrane antigen, and virus isolation were 32, 8, and 2 positive cells per 10(5) cells, respectively (ranges, 0 to 20,280, 0 to 22,990, and 0 to 220 positive cells per 10(5) cells, respectively). The ratio of viral internal antigen expression to virus isolation was extremely variable and often high, whereas the ratio of viral internal antigen to viral membrane antigen expression was more consistent and generally low. The virus strain which induced the cell line influenced the level of virus genome expression, but the cell genotype did not. Cell lines transformed by JM-10 virus, which exhibited low oncogenicity, had significantly (p less than 0.01) higher rates of expression than cell lines transformed by CA-5 and RB-1B viruses, which exhibited high oncogenicity. Treatment with iododeoxyuridine or incubation at 37 degrees C induced increased rates of expression in most lines but not in all lines. The degree of enhanced expression was inversely proportional to the rate of spontaneous expression.

A test is reported for the in vivo detection of the replicative ability of murine leukemia viruses (MLV) in the BALB/c mouse strain. Growth in the spleen was assayed by the complement-fixation assay for MLV group-specific antigen after injection of newborn mice. Low doses of laboratory-derived and wild-type MLV from cell-culture and animal-grown sources were detected as early as 7 to 14 days postinoculation. The sensitivity of this in vivo test compared favorably with in vitro assays for MLV. In vivo detection of MLV replication was correlated with long-term oncogenicity.

A family of recombinant mink cell focus-forming viruses (MCF) was derived by inoculation of NFS mice with a Friend murine leukemia virus, and their genomes were analyzed by RNase T1-resistant oligonucleotide fingerprinting. The viruses were obtained from the thymuses and spleens of preleukemic and leukemic animals and were evaluated for dualtropism and oncogenicity. All these isolates induced cytopathic foci on mink cells but could be classified into two groups based on their relative infectivities for SC-1 (mouse) or mink (ATCC CCL64) cells. One group of Friend MCFs (F-MCFs) (group I) exhibited approximately equal infectivities for SC-1 and mink cells, whereas a second group (group II) infected mink cells 1,000- to 10,000-fold more efficiently than SC-1 cells. Structural analyses of the F-MCFs revealed that group I and group II viruses correlated with recombination of Friend murine leukemia virus with two distinct, but closely related, endogenous NFS proviral sequences. No correlation was found between the type of F-MCF and the tissue of origin or the disease state of the animal. Furthermore, none of the F-MCF isolates were found to be oncogenic in NFS/N or AKR/J mice. F-MCFs of both groups underwent extensive substitution of ecotropic sequences, involving much of the gag and env genes of group I F-MCFs and most of the gag, pol, and env genes of group II F-MCFs. All F-MCF isolates retained the 3' terminal U3 region of Friend murine leukemia virus. Comparison of the RNAs of the F-MCFs with RNAs of MCFs derived from NFS.Akv-1 or NFS.Akv-2 mice indicated that the F-MCFs were derived from NFS proviral sequences which are distinct from the sequences contained in NFS.Akv MCF isolates. This result suggested that recombination with particular endogenous proviral sequences to generate MCFs may be highly specific for a given murine leukemia virus.

Takemoto , K. K. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), R. L. Kirschstein, and K. Habel . Mutants of simian virus 40 differing in plaque size, oncogenicity, and heat sensitivity. J. Bacteriol. 92: 990–994. 1966.—Three mutants of simian virus 40 were isolated on the basis of the type of plaques produced in primary cultures of African green monkey kidney cells and designated as L (large), S (small), and M (minute) strains. Significant differences in oncogenicity for hamsters were observed, with the 50% oncogenic dose being 10 4.5 for the L, 10 5.2 for the S, and 10 5.8 for the M strains. All three strains were capable of transforming human diploid cells (W138 strain). At temperatures up to 41 C, the S and M mutants were capable of multiplying to titers almost equivalent to those obtained at 37 C. In contrast, infectious virus was not produced when cells were infected with the L mutant and were incubated at temperatures above 39 C, although complement-fixing viral and tumor antigens were formed. The temperature-sensitive phase of replication of the L strain was shown to be a late stage in viral maturation or assembly.

A cell line derived from a murine sarcoma virus (Moloney pseudotype)-induced tumor has been established. It retains oncogenicity, releases both sarcoma and leukemia viruses, and has virus-induced cellular antigens.

A variety of biological products which included live and inactivated viral vaccines, inactivated rickettsial and bacterial vaccines, toxoids, and a multiple bacterial antigen product did not appear to be oncogenic for newborn hamsters following a single subcutaneous inoculation. The incidence of spontaneous tumors was approximately 4.7%, and this figure was not significantly altered by the inoculation of any of the test materials.