Intracellular targeting of walleye dermal sarcoma virus Orf A (rv-cyclin).

Walleye dermal sarcoma virus (WDSV) induces tumors and allows or possibly directs tumor regression. WDSV encodes a putative cyclin homologue, Orf A, and six variant Orf A transcripts have been identified. Northern analysis indicated that a 3.3-kb transcript, encoding full-length Orf A, is the predominant transcript in developing, but not regressing, tumors. Three Orf A proteins, one full-length and two amino-truncated forms, were expressed in mammalian and piscine cells, and their intracellular locations were determined. The full-length form was nuclear and concentrated in interchromatin granule clusters, defined by colocalization with SC-35. The amino-truncated forms were cytoplasmic. Fusion of amino-terminal portions of Orf A to a heterologous protein demonstrated that residues 1-112 were necessary for nuclear localization. Mutation of aa K80 and/or E110 disrupted nuclear localization, suggesting a mechanism similar to that of cellular A- and D-type cyclins for its nuclear import.

Assessment of bovine leukemia virus transcripts in vivo.

Reverse transcriptase PCR (RT-PCR) consistently detected bovine leukemia virus transcripts in fresh cells, and competitive RT-PCR enumerated these transcripts. The detection of transcripts in limited numbers of tumor cells indicated that expression occurs in a minority of cells. The data suggest that individual cells contain hundreds of copies of the tax/rex transcript in vivo.

Three closely related herpesviruses are associated with fibropapillomatosis in marine turtles.

Green turtle fibropapillomatosis is a neoplastic disease of increasingly significant threat to the survivability of this species. Degenerate PCR primers that target highly conserved regions of genes encoding herpesvirus DNA polymerases were used to amplify a DNA sequence from fibropapillomas and fibromas from Hawaiian and Florida green turtles. All of the tumors tested (n = 23) were found to harbor viral DNA, whereas no viral DNA was detected in skin biopsies from tumor-negative turtles. The tissue distribution of the green turtle herpesvirus appears to be generally limited to tumors where viral DNA was found to accumulate at approximately two to five copies per cell and is occasionally detected, only by PCR, in some tissues normally associated with tumor development. In addition, herpesviral DNA was detected in fibropapillomas from two loggerhead and four olive ridley turtles. Nucleotide sequencing of a 483-bp fragment of the turtle herpesvirus DNA polymerase gene determined that the Florida green turtle and loggerhead turtle sequences are identical and differ from the Hawaiian green turtle sequence by five nucleotide changes, which results in two amino acid substitutions. The olive ridley sequence differs from the Florida and Hawaiian green turtle sequences by 15 and 16 nucleotide changes, respectively, resulting in four amino acid substitutions, three of which are unique to the olive ridley sequence. Our data suggest that these closely related turtle herpesviruses are intimately involved in the genesis of fibropapillomatosis.

Detection of a novel bovine lymphotropic herpesvirus.

Degenerate PCR primers which amplify a conserved region of the DNA polymerase genes of the herpesvirus family were used to provide sequence evidence for a new bovine herpesvirus in bovine B-lymphoma cells and peripheral blood mononuclear cells (PBMC). The sequence of the resultant amplicon was found to be distinct from those of known herpesvirus isolates. Alignment of amino acid sequences demonstrated 70% identity with ovine herpesvirus 2, 69% with alcelaphine herpesvirus 1, 65% with bovine herpesvirus 4, and 42% with bovine herpesvirus 1. Phylogenetic analysis placed this putative virus within the tumorigenic Gammaherpesvirinae subfamily, and it is tentatively identified as bovine lymphotropic herpesvirus. This novel agent was expressed in vitro from infected PBMC, and cell-free supernatants were used to transfer infection to a bovine B-cell line, BL3. Analysis, with specific PCR primers, of DNA from bovine PBMC and lymphoma cells identified infection in blood of 91% of adult animals (n = 101), 63% of lymphomas (n = 32), and 38% of juveniles (n = 13). Of the adults, herpesvirus infection was present in 94% of animals that were seropositive for bovine leukemia virus (BLV) (n = 63) and in 87% of BLV-seronegative animals (n = 38). Of the seropositive group, 17 animals exhibited persistent lymphocytosis, and 100% of these were herpesvirus positive by PCR. A role for bovine lymphotropic herpesvirus as a cofactor in BLV pathogenesis is considered.

A deletion in the proximal untranslated pX region of human T-cell leukemia virus type II decreases viral replication but not infectivity in vivo.

The function of untranslated (UT) nucleotide sequences in the proximal portion of the pX region of the human T-cell leukemia virus (HTLV) family of retroviruses remains enigmatic. Previous studies have shown that these sequences are not necessary for the expression of viral proteins or for the induction, transmission, or maintenance of the transformed cell type in vitro. To determine the effect of the UT region in vivo, separate groups of rabbits were inoculated with lethally irradiated, stable clones of the human B-lymphoblastoid cell line, 729, transfected with either a full-length wild-type HTLV-II clone (pH6neo) or a mutant clone containing a 324-bp deletion in the proximal UT portion of pX (pH6neo delta UT[6661-6984]), or nontransfected 729 cells. All rabbits inoculated with either wild-type or pX-deleted HTLV-II developed a similar profile and titer of serum antibodies against HTLV-II antigens, as determined by Western immunoblots, by 4 weeks postinoculation (PI). Antibody titers, as determined by enzyme immunoassay, were similar between the two groups of rabbits and increased over the 18-week period of study. All rabbits were killed at 18 weeks PI, and spleen, peripheral blood lymphocytes (PBMC), bone marrow, and mesenteric lymph node were assayed for HTLV-II tax/rex sequences by quantitative polymerase chain reaction. Virus was detected in all tissues tested from all rabbits inoculated with 729pH6neo cells containing wild-type HTLV-II, which contained between 1.4 and 0.3 mean copies of provirus per cell. In contrast, the distribution and number of provirus copies were more limited in rabbits inoculated with 729pH6neo delta UT(6661-6984) cells containing UT-deleted HTLV-II; in most tissues, there was a fivefold to sevenfold reduction in mean provirus copies per cell as compared with rabbits inoculated with wild-type HTLV-II. All rabbits inoculated with control 729 cells remained negative for HTLV-II infection, as determined by the same techniques. It was concluded that UT sequences in the proximal portion of HTLV-II are not necessary for infection but confer increased replicative capacity in vivo.

Prospective characterization of the clinicopathologic and immunologic features of an immunodeficiency syndrome affecting juvenile llamas.

The clinicopathologic and immunologic features of 15 llamas affected with juvenile llama immunodeficiency syndrome (JLIDS) are described. Healthy adult (n = 10) and juvenile (n = 10) llamas served as controls. JLIDS llamas were characterized by wasting, and clinically apparent, repeated infections were frequently observed. The median age at which a health problem was first perceived was 11.6 months. All 15 affected llamas died or were killed, and JLIDS was confirmed at necropsy. The median duration of illness was 3.5 months. Lymphocyte blastogenesis assays showed suppressed responses (particularly to Staphylococcus sp. Protein A) in JLIDS llamas. No evidence of retroviral infection was detected. Mild, normocytic, normochromic, non-regenerative anemia, low serum albumin concentration and low to low-normal globulin concentrations were typically found on initial clinical evaluation. Lymph node biopsies showed areas of paracortical depletion. All llamas affected with JLIDS had low serum IgG concentrations, pre-vaccination titers against Clostridium perfringens C and D toxoids of < or = 1:100, and no titer increase following vaccination.

Chronic generalized obliterative arteriopathy in cattle: a sequel to sheep-associated malignant catarrhal fever.

Malignant catarrhal fever (MCF) in cattle is generally associated with a short clinical course and a high case fatality rate (90-95%). The lesions in cattle that survive acute MCF for a prolonged period or appear to recover have not been documented. In a naturally occurring outbreak of MCF in a herd of beef cattle in Wyoming, 7 of 84 yearling heifers (8.3% of replacement herd) and 2 of 230 cows (0.9% of cow herd) developed clinical signs of pyrexia, mucopurulent discharge, bilateral keratitis, and weight loss following contact with ewes that had lambed 34-62 days earlier. Six of 9 affected cattle were examined postmortem following clinical signs (CS) that developed 2-150 days earlier. Three cattle with CS for < or = 39 days had lesions of regional lymphadenopathy and widespread severe segmental lymphoid arteritis-phlebitis that were typical of acute MCF, and proliferative intimal lesions were present in a small proportion of arteries at days 20 and 39 of CS. By contrast, 3 cattle that survived to 90, 105, and 150 days after clinical onset had distinctive arterial lesions in multiple organs, characterized by proliferative concentric fibrointimal plaques, disrupted inner elastic lamina, focally atrophic tunica media, and vasculitis of variable severity. Immunohistochemical and ultrastructural examination of intimal plaques identified the predominant cellular component to be smooth muscle cells with a contractile phenotype. No viral structures were seen. Serologic studies, using a competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) that detects antibody to an epitope broadly conserved among isolates of the MCF virus, found that 2 chronically affected cattle were serologically positive between days 42 and 100 of CS, with seroconversion in 1 animal between days 52 and 73 of CS. Seroprevalence was 7.9% in the 76 remaining healthy animals of the replacement heifer herd and 40% (75% in adult sheep and 4% in lambs) in the in-contact sheep flock 77 days after onset of CS in the index case. This episode suggests that, in addition to the common and well recognized acute form of MCF in cattle, this viral infection encompasses a disease spectrum that includes chronic disease and partial to "complete" clinical recovery, and in recovered animals chronic obliterative arteriopathy is the preeminent lesion.

The herpes simplex virus 1 UL15 gene encodes two proteins and is required for cleavage of genomic viral DNA.

Previous studies have shown that a ts mutant [herpes simplex virus 1 (mP)ts66.4] in the UL15 gene fails to package viral DNA into capsids (A. P. W. Poon and B. Roizman, J. Virol. 67:4497-4503, 1993) and that although the intron separating the first and second exons of the UL15 gene contains UL16 and UL17 open reading frames, replacement of the first exon with a cDNA copy of the entire gene does not affect viral replication (J.D. Baines, and B. Roizman, J. Virol. 66:5621-5626, 1992). We report that (i) a polyclonal rabbit antiserum generated against a chimeric protein consisting of the bacterial maltose-binding protein fused in frame to the majority of sequences contained in the second exon of the UL15 gene reacted with two proteins with M(r) of 35,000 and 75,000, respectively, in cells infected with a virus containing the authentic gene yielding a spliced mRNA or with a virus in which the authentic UL15 gene was replaced with a cDNA copy. (ii) Insertion of 20 additional codons into the C terminus of UL15 exon II caused a reduction in the electrophoretic mobility of both the apparently 35,000- and 75,000-M(r) proteins, unambiguously demonstrating that both share the carboxyl terminus of the UL15 exon II. (iii) Accumulation of the 35,000-M(r) protein was reduced in cells infected and maintained in the presence of phosphonoacetate, an inhibitor of viral DNA synthesis. (iv) The UL15 proteins were localized in the perinuclear space at 6 h after infection and largely in the nucleus at 12 h after infection. (v) Viral DNA accumulating in cells infected with herpes simplex virus 1(mP)ts66.4 and maintained at the nonpermissive temperature was in an endless (concatemeric) form, and therefore UL15 is required for the cleavage of mature, unit-length molecules for packaging into capsids.

Pathogenicity of molecularly cloned bovine leukemia virus.

To delineate the mechanisms of bovine leukemia virus (BLV) pathogenesis, four full-length BLV clones, 1, 8, 9, and 13, derived from the transformed cell line FLK-BLV and a clone construct, pBLV913, were introduced into bovine spleen cells by microinjection. Microinjected cells exhibited cytopathic effects and produced BLV p24 and gp51 antigens and infectious virus. The construct, pBLV913, was selected for infection of two sheep by inoculation of microinjected cells. After 15 months, peripheral blood mononuclear cells from these sheep served as inocula for the transfer of infection to four additional sheep. All six infected sheep seroconverted to BLV and had detectable BLV DNA in peripheral blood mononuclear cells after amplification by polymerase chain reaction. Four of the six sheep developed altered B/T-lymphocyte ratios between 33 and 53 months postinfection. One sheep died of unrelated causes, and one remained hematologically normal. Two of the affected sheep developed B lymphocytosis comparable to that observed in animals inoculated with peripheral blood mononuclear cells from BLV-infected cattle. This expanded B-lymphocyte population was characterized by elevated expression of B-cell surface markers, spontaneous blastogenesis, virus expression in vitro, and increased, polyclonally integrated provirus. One of these two sheep developed lymphocytic leukemia-lymphoma at 57 months postinfection. Leukemic cells had the same phenotype and harbored a single, monoclonally integrated provirus but produced no virus after in vitro cultivation. The range in clinical response to in vivo infection with cloned BLV suggests an important role for host immune response in the progression of virus replication and pathogenesis.

Seroprevalence of bovine immunodeficiency-like virus and bovine leukemia virus in a dairy cattle herd.

To determine the prevalence of single vs. dual infection with bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV), sera (n = 95) from a dairy cattle herd were analyzed for anti-BIV and anti-BLV antibodies by an enzyme linked immunosorbent assay. Twenty-one percent (20/95) of samples were BIV-seropositive, while 52% (49/95) of the same samples were BLV-seropositive. A significantly greater percentage of BIV-seronegative samples were BLV-seropositive, 57% (43/75), than were BIV-seropositive samples, 30% (6/20). There was no significant correlation between data ranked from least to greatest amount of anti-viral antibody. Five cattle had persistent lymphocytosis (PL); all five were BLV-seropositive and two were BIV-positive. The mean anti-BLV titer was significantly greater in PL cattle, as compared at non-PL cattle, whereas there was no significant difference between the mean anti-BIV titer in PL cattle, as compared with non-PL cattle. These results provide additional information on the seroprevalence of naturally occurring BIV infection, and indicate that BIV can exist independent of other common infectious agents, such as BLV. Further, the results suggest that infection with BIV is not associated with an increased rate of infection with other infectious agents such as BLV.

Comparative biological responses of rabbits infected with human T-lymphotropic virus type I isolates from patients with lymphoproliferative and neurodegenerative disease.

An experimental rabbit model was used to determine host responses to infection by various human T-lymphotropic virus type-I (HTLV-I) strains. Seven groups of 4 to 5 rabbits each were inoculated with lethally-irradiated HTLV-I-infected cell lines derived from patients with adult T-cell leukemia/lymphoma or from patients with HTLV-I-associated myelopathy. Four separate control groups of 2 rabbits each were inoculated with similarly prepared HTLV-I-negative cells derived from rabbits or humans. Anti-viral antibody responses were assessed by immunoblot assay and hematologic parameters were measured using automated cell counters and cytologic staining. The virologic status of challenged rabbits was determined by co-culture and HTLV-I antigen capture assay, as well as by polymerase chain reaction (PCR) amplification of HTLV-I DNA from peripheral blood mononuclear cells (PBMC) or tissues. The HTLV-I inocula could be separated into groups based upon their infectivity to rabbits: highly infectious strains elicited intense serologic responses and were detected frequently in tissues by antigen and PCR assays, while other strains were moderately to poorly infectious, induced weak antibody responses and were infrequently detected by antigen and PCR assays. Overall, PBMC appeared to have the greatest quantity of HTLV-I containing cells, while bone marrow was a poor source of virus. No clinical or hematologic abnormalities were evident during the 24-week course of infection. Taken together, our results suggest there is heterogeneity in the biological response to HTLV-I infection which is, in part, dependent on the infecting strain of virus.

Infectious transmission of human T-cell lymphotropic virus type II in rabbits.

To determine the susceptibility of rabbits to experimental infection with human T-cell lymphotropic virus type-II (HTLV-II), four separate groups of four weanling rabbits each were inoculated intravenously with lethally irradiated HTLV-II-infected human cell lines Mo-T (HTLV-IIMo-infected T cells), WIL-NRA (an Epstein-Barr virus [EBV]-transformed B-lymphoblastoid cell line infected with HTLV-IINRA), 729pH6neo (an EBV-transformed lymphoblastoid cell line transfected with a molecular clone of HTLV-IIMo), or G12.1 (HTLV-II-infected T cells from a Panamanian Guaymi Indian). Two additional groups of four rabbits each were similarly inoculated with control uninfected 729 or HuT 78 cells. Early and persistent seroconversion to HTLV-II core antigen p24, as determined by Western immunoblot, occurred in all HTLV-II-inoculated rabbits and was most intense in rabbits inoculated with G12.1 cells; seroreactivity to other HTLV-II gag or env antigens occurred later, with less intensity, or not in all inoculated rabbits. Peripheral blood mononuclear cells (PBMC) and other lymphoid cells from HTLV-II-inoculated rabbits produced minimal p24 in vitro, as determined by enzyme immunosorbent capture assay. Virus was more readily detected by polymerase chain reaction amplification of HTLV-II pol sequences; this occurred most frequently in rabbits inoculated with Mo-T cells, and most frequently in PBMC as compared with other tissues tested (bone marrow, brain, and liver). No evidence of disease occurred in HTLV-II-inoculated rabbits observed for as long as 24 weeks. All control rabbits remained negative for evidence of HTLV-II infection, as determined by the same procedures. These results provide the first evidence of HTLV-II infection in a species other than humans, and demonstrate the usefulness of the rabbit as an animal model to study the biologic response to different isolates of this human retrovirus.

Isolation of bovine leukemia virus infected endothelial cells from cattle with persistent lymphocytosis.

Incubation of adherent cells derived from peripheral blood mononuclear cells of cattle naturally infected with bovine leukemia virus (BLV) led to the establishment of three, persistently infected, primary cell cultures. These cultures were obtained exclusively from animals exhibiting persistent lymphocytosis, and not from uninfected or infected, hematologically normal cattle. The cells contained monoclonally integrated, full length BLV provirus, indicating that each culture resulted from clonal expansion of a single cell. They expressed high levels of all BLV specific mRNAs and showed intracellular reactivity to antibodies directed to viral gag and env proteins. Viral particle morphogenesis was highly restricted as determined by low levels of reverse transcriptase activity in cell supernatants and the paucity of viral particles on the cell surface. Analysis of cellular antigenic determinants, using monoclonal antibodies to bovine leukocyte differentiation and major histocompatibility complex antigens, was inconclusive. Cytochemical, morphologic, and ultrastructural analyses were consistent with endothelial cells and they exhibited the distinctive functional capacity of endothelial cells derived from specialized postcapillary venules, which constitute sites of lymphocyte extravasation. These data suggest that infection of these endothelial cells may be involved in the development of persistent lymphocytosis in BLV-infected animals.

In vivo transcription of the bovine leukemia virus tax/rex region in normal and neoplastic lymphocytes of cattle and sheep.

Expression of bovine leukemia virus (BLV) has been considered to be blocked at the transcriptional level in vivo, since viral RNA species are not readily detected in freshly isolated leukocytes from BLV-infected animals. However, the presence of a persistent antiviral antibody response in infected animals suggests that some degree of virus expression must occur in vivo. The purpose of this study was to determine whether BLV RNA species could be detected by using the polymerase chain reaction in normal or neoplastic lymphoid cells freshly isolated from naturally or experimentally BLV-infected cattle and sheep, respectively. Primers designed to detect a 2.1-kb doubly spliced BLV tax/rex-specific mRNA were used to amplify cDNA copies of RNA derived from infected animals. The amplified viral product was then detected with a radiolabeled BLV tax/rex-specific probe. BLV-specific RNA was detected readily in freshly isolated peripheral blood leukocytes derived from BLV-seropositive cattle or sheep with persistent lymphocytosis and less readily in peripheral blood leukocytes from BLV-seropositive but hematologically normal animals. BLV-specific RNA was also detected in fresh samples of BLV-induced lymphosarcomas. Normal and neoplastic lymphoid cells from BLV-seronegative animals were uniformly negative under similar conditions. These primers also amplified the same viral product from genomic DNA derived from BLV-seropositive animals, providing further evidence for in vivo transcription and suggesting that BLV RNA-dependent DNA polymerase is capable of reverse transcribing the 2.1-kb mRNA in vivo. The demonstration of transcriptional products of BLV in vivo proves that viral latency in BLV infection is incomplete.

Persistent infection of rabbits with HTLV-I: patterns of anti-viral antibody reactivity and detection of virus by gene amplification.

Two groups of rabbits were inoculated on the day of birth or at 4 weeks of age with a human T-cell leukemia virus type-I (HTLV-I)-infected and transformed rabbit cell line (Ra-I). Rabbits seroconverted to HTLV-I, as determined by indirect immunofluorescence, by 3 weeks after inoculation and remained persistently seropositive during a 22-month period of observation. Seroconversion did not occur in saline-inoculated controls. Using Western immunoblotting and radio-immunoprecipitation, persistent seroconversion occurred against viral antigens p24, p55 and gp68, while reactivity to p19 was variable between rabbits. Using the polymerase chain reaction (PCR) with HTLV-I gag and pol primer pairs, HTLV-I sequences were demonstrable in peripheral blood mononuclear cells and other tissues collected at 70 and 90 weeks after inoculation. DNA extracts from normal rabbit tissue remained negative under the same conditions. No qualitative or quantitative changes in leukocytes or erythrocytes were detected in the infected rabbits and no clinical signs could be directly attributed to infection with HTLV-I.

The correlation between the direct and indirect detection of bovine leukemia virus infection in cattle.

Ninety-three cattle from a herd naturally infected with bovine leukemia virus (BLV) were tested for the presence of BLV infection by two indirect indicators, anti-BLV antibodies and lymphocytosis, and two direct indicators, BLV provirus and BLV gp51 antigen expression in peripheral blood mononuclear cells (PBMC). Forty-eight percent (45/93) of the cattle were seropositive, and of these, 53% (24/45) were provirus-positive. Freshly isolated PBMC were negative for gp51 antigen expression, but 11 cattle were positive following short-term culture of their PBMC; 10 of these were seropositive/provirus-positive cattle, and one was a seropositive/provirus-negative cow. Lymphocytosis was present in eight cattle, all of which were seropositive/provirus-positive/gp51-positive. Four cattle were provirus-positive, but negative for all other indicators of BLV infection; a second blood sample was collected from three of these cattle at a later date, at which time two of the three had seroconverted. These results suggest that depending on the stage of the infection, the pathogenesis of BLV in cattle may involve fundamental differences in the host-viral relationship, including the number of cells infected or the number of copies of integrated provirus per cell, regulation of expression of viral antigens, induction of the anti-viral immune response, and the polyclonal or monoclonal proliferation of lymphocytes.

Purification of 2',5'-oligoadenylate synthetase from rabbit reticulocytes.

The 2',5'-oligoadenylate synthetase (2-5A synthetase) from rabbit reticulocytes has been purified to apparent homogeneity. The purification procedure consists of (NH4)2SO4 fractionation (30-50% cut), specific binding of the 2-5A synthetase to and elution from the affinity matrix of polyinosinic-polycytidylic-cellulose, another (NH4)2SO4 precipitation step, and finally chromatography on DEAE-cellulose. Upon electrophoresis in sodium dodecyl sulfate polyacrylamide gel (10%), the purified enzyme migrates as a single polypeptide with an apparent molecular weight of 110,000 daltons. A sedimentation coefficient of 5.8S is obtained by glycerol density gradient centrifugation. The synthesis of 2',5'-oligoadenylate by the purified enzyme is dependent on the presence of double-stranded (ds) RNA, in the absence of which the enzyme is highly unstable. Biochemical characteristics of the purified enzyme have been defined.