Frog virus 3 (FV3) is the type species member of the genus Ranavirus (family Iridoviridae). To better understand the molecular mechanisms involved in the replication of FV3, including transcription of its highly methylated DNA genome, we have determined the complete nucleotide sequence of the FV3 genome. The FV3 genome is 105903 bp long excluding the terminal redundancy. The G + C content of FV3 genome is 55% and it encodes 98 nonoverlapping potential open reading frames (ORFs) containing 50-1293 amino acids. Eighty-four ORFs have significant homology to known proteins of other iridoviruses, whereas twelve of these unique FV3 proteins do not share homology to any known protein. A microsatellite containing a stretch of 34 tandemly repeated CA dinucleotide in a noncoding region was detected. To date, no such sequence has been reported in any animal virus.

The genome sequence of Yaba-like disease virus (YLDV), an unclassified member of the yatapoxvirus genus, has been determined. Excluding the terminal hairpin loops, the YLDV genome is 144,575 bp in length and contains inverted terminal repeats (ITRs) of 1883 bp. Within 20 nucleotides of the termini, there is a sequence that is conserved in other poxviruses and is required for the resolution of concatemeric replicative DNA intermediates. The nucleotide composition of the genome is 73% A+T, but the ITRs are only 63% A+T. The genome contains 151 tightly packed open reading frames (ORFs) that either are > or =180 nucleotides in length or are conserved in other poxviruses. ORFs within 23 kb of each end are transcribed toward the termini, whereas ORFs within the central region of the genome are encoded on either DNA strand. In the central region ORFs have a conserved position, orientation, and sequence compared with vaccinia virus ORFs and encode many enzymes, transcription factors, or structural proteins. In contrast, ORFs near the termini are more divergent and in seven cases are without counterparts in other poxviruses. The YLDV genome encodes several predicted immunomodulators; examples include two proteins with similarity to CC chemokine receptors and predicted secreted proteins with similarity to MHC class I antigen, OX-2, interleukin-10/mda-7, poxvirus growth factor, serpins, and a type I interferon-binding protein. Phylogenic analyses indicated that YLDV is very closely related to yaba monkey tumor virus, but outside the yatapoxvirus genus YLDV is more closely related to swinepox virus and leporipoxviruses than to other chordopoxvirus genera.

Autoantibodies directed against a wide range of normal tissue antigens have been found in the sera of patients with autoimmune diseases. It is generally thought that different and specific autoantibodies react with different tissues but the possibility exists that some autoantibodies may react with common antigens found in different tissues and organs. Recently, we showed that mice infected with reovirus developed a polyendocrine disease with autoantibodies to the pancreas, anterior pituitary, thymus and gastric mucosa. Using hybridoma technology, we obtained a number of monoclonal autoantibodies which reacted with antigens in single organs. We now report the production and pattern of reactivity of seven multiple organ-reactive monoclonal autoantibodies. By using antibody-affinity columns, autoantigens also have been isolated and their molecular weights determined. The results suggest that monoclonal multiple organ-reactive autoantibodies react either with the same molecule present in several organs or with common antigenic determinants on different molecules in multiple organs. In either case, the existence of multiple organ-reactive antibodies may be a partial explanation for multiple organ autoimmunity.

A class of secreted poxvirus tumor necrosis factor (TNF)-binding proteins has been isolated from Tanapox-infected cell supernatants. The inhibitor bound to a TNF-affinity column and was identified as the product of the 2L gene. Sequence analysis of 2L family members from other yatapoxviruses and swinepox virus yielded no sequence homology to any known cellular gene. The expressed Tanapox virus 2L protein bound to human TNF with high affinity (Kd ‫؍‬ 43 pM) and exhibits an unusually slow off-rate. However, 2L is unable to bind to a wide range of human TNF family members. The 2L protein can inhibit human TNF from binding to TNF receptors I and II as well as block TNF-induced cytolysis. Thus, Tanapox virus 2L represents an inhibitor of human TNF and offers a unique strategy with which to modulate TNF activity.T he tumor necrosis factor (TNF) superfamily of ligands plays critical roles in both adaptive and innate immunity. These ligands form a trimeric structure and bind to members of the TNF receptor (TNFR) superfamily that mediate the signaling events that regulate diverse responses, particularly inflammation and apoptosis (1). During a viral infection, TNF, the prototypic ligand for the family, is induced as an early response of the immune system to an invading pathogen (2). TNF is expressed by activated macrophages as a membrane-associated ligand, which subsequently is cleaved and secreted as a trimer. The proinflammatory effects of TNF are mediated through the cell surface TNFRI and, to a lesser extent, TNFRII (3). Binding of the TNF trimer causes conformational reorganization of the receptors and subsequent recruitment of signaling proteins such as TNFR-associated factor 2 (TRAF2) and Fas-associated death domain (FADD) (3). The TNF͞TNFR signal transduction pathway is such an important aspect of the antiviral response that a number of viruses have evolved targeted strategies to specifically disrupt it.Poxviruses employ a wide range of strategies to modulate, avoid, or diminish the various antiviral immune responses, including the secretion of cytokine-and chemokine-binding proteins and the production of cytokine and growth factor homologs (4-7). The best characterized of these strategies are the secreted homologs of the TNFRs that most likely were derived by the acquisition of an ancestral host TNFR cDNA and subsequent deletion of the transmembrane domain and cytoplasmic tail (8). The first characterized viral homolog of TNFR was the secreted T2 protein from Shope fibroma virus, which binds both TNF and lymphotoxin-␣ (LT-␣) just as its cellular counterpart (9, 10). Deletion of the related T2 gene from myxoma virus greatly attenuated the virus in vivo (11).Other poxvirus-secreted TNFRs were described for monkeypox virus, variola virus, vaccinia virus, ectromelia virus, and cowpox virus (4,8,12). Cowpox virus is atypical among poxviruses because it encodes multiple, unique, soluble TNFRs designated as cytokine response modifier (Crm)B (13), CrmC (14), CrmD (15), and CrmE (16). Each of these cowpox virus TNFRs has...

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Colorectal cancers are significant causes of morbidity and mortality and existing therapies often perform poorly for individuals afflicted with advanced disease. Oncolytic virotherapy is an emerging therapeutic modality with great promise for addressing this medical need. Herein we describe the in vivo testing of recombinant variants of the tanapoxvirus (TPV). Recombinant viruses were made ablated for either the 66R gene (encoding a thymidine kinase), the 2L gene (encoding a TNF-binding protein), or both. Some of the recombinants were armed to express mouse chemotactic protein 1 (mCCL2/mMCP-1), mouse granulocyte-monocyte colony stimulating factor (mGM-CSF), or bacterial flagellin (FliC). Tumors were induced in athymic nude mice by implantation of HCT 116 cells and subsequently treated by a single intratumoral injection of one of the recombinant TPVs. Histological examination showed a common neoplastic cell type and a range of immune cell infiltration, necrosis, and tumor cell organization. Significant regression was seen in tumors treated with virus TPV/Δ2L/Δ66R/fliC, and to a lesser extent the recombinants TPV/Δ2L and TPV/Δ66R. Our results suggest that oncolytic recombinants of the TPV armed with activators of the innate immune response may be effective virotherapeutic agents for colorectal cancers in humans and should be explored further to fully realize their potential.

Previous reports [Flintoff, W. F., Davidson, S. V., & Siminovitch, L. (1976) Somatic Cell Genet. 2, 245--261; Gupta, R. S., Flintoff, W. F., & Siminovitch, L. (1977) Can. J. Biochem. 55, 445--452] described a series of Chinese hamster ovary cells that were resistant to the cytotoxic action of methotrexate and contained a dihydrofolate reductase that was less sensitive to inhibition by the drug than wild-type enzyme. In this study, binding of labeled methotrexate to the reductase--NADPH complex and separation of free and bound drug by filtration through Sephadex G--25 have been used to demonstrate that clonal isolates of these resistant cells contain a dihydrofolate reductase varying between 2.5- and 6-fold lower in affinity for the drug than the wild-type enzyme. The apparent dissociation constant for the wild-type enzyme is 0.5 x 10(-9) M. Using two-dimensional polyacrylamide gel electrophoresis, 11 independently selected resistant isolates have been shown to contain a reductase with a similar overall net charge as the wild-type enzyme. Reductase purified from either wild-type or resistant cells contains two components after isoelectric focusing in polyacrylamide gels. The major component represents about 90% of the total protein and has a pI of about 8.0. The minor component representing about 10% of the reductase protein has a pI between 7.2 and 7.6.

We have compared a number of properties of the well-characterized iridovirus, frog virus 3, with two other iridoviruses from amphibia, bullfrog edema virus and Lucké triturus virus, and with a piscine iridovirus, goldfish virus (GFV), to provide information for developing taxonomic classification of these viruses and establishing their ecological niche. Purified virions had similar size and shape (icosahedral) for each virus, and the genomic DNAs of each virus were methylated by a virus-induced DNA methyltransferase. The three amphibian viruses replicated equally well in fish (FHM), hamster (BHK), and human (WI-38) cell monolayer with identical cytopathology, while GFV failed to replicate in these cell lines. However, GFV replicated albeit at a slow rate, in a goldfish cell line; there was no detectable replication by the amphibian viruses in these cells. The amphibian iridoviruses had virtually similar DNA sequences, while those of GFV were markedly different. Analyses of virus-induced polypeptides in infected cells corroborated the DNA analyses; the polypeptides of the amphibian viruses were similar and distinct from those of the fish virus. Nongenetic reactivation could only be accomplished between the three amphibian viruses but not with the piscine virus. Based on these data, we suggest taxonomic and nomenclature designations of amphibian and piscine iridoviruses.