Different models, different trees: the geographic origin of PTLV-I.

Using nucleotide sequences from three genomic regions of the human and simian T-cell lymphotropic virus type I (HTLV-I/STLV-I)-consisting of 69 sequences from a 140-bp segment of the pol region, 98 sequences from a 503-bp segment of the LTR, and 154 sequences from a 386-bp segment of the env region-we tested two hypotheses concerning the geographic origin and evolution of STLV-I and HTLV-I. First, we tested the assumption of equal rates of evolution along STLV-I and HTLV-I lineages using a likelihood ratio test to ascertain whether current levels of genomic diversity can be used to determine ancestry. We demonstrated that unequal rates of evolution along HTLV-I and STLV-I lineages have occurred throughout evolutionary time, thus calling into question the use of pairwise distances to assign ancestry. Second, we constructed phylogenetic trees using multiple phylogenetic techniques to test for the geographic origin of STLV-I and HTLV-I. Using the principle of likelihood, we chose a statistically justified model of evolution for each data set. We demonstrated the utility of the likelihood ratio test to determine which model of evolution should be chosen for phylogenetic analyses, revealing that using different models of evolution produces conflicting results, and neither the hypothesis of an African origin nor the hypothesis of an Asian origin can be rejected statistically. Our best estimates of phylogenetic relationships, however, support an African origin of PTLV for each gene region.

Phylogenetic analysis of simian T-lymphotropic virus Type I (STLV-I) in common chimpanzees (Pan troglodytes): evidence for interspecies transmission of the virus between chimpanzees and humans in Central Africa.

Serum and peripheral blood leukocytes from the chimpanzees (Pan troglodytes) of the colony of the Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, NIH, were tested for the presence of STLV-I-specific antibodies and proviral DNA. Antibodies were determined by gelatin particle agglutination and Western blot (WB) assays utilizing HTLV-I antigens. Proviral DNA was detected by four PCR assays targeting three different regions of STLV-I genome: the fragments of the env and pol genes and LTR. Twenty of twenty-two DNA samples from WB-positive animals were PCR positive. None of the DNA samples from WB-negative (n = 5) and WB-indeterminate (n = 4) animals was PCR positive. The results of the nested and double nested env PCR tests were fully concordant; the seminested LTR PCR test was much less sensitive. The DNA sequences from the env (483 bp) and the pol (200 bp) genes and LTR (705 bp) were determined for six, two, and two chimpanzee STLV-I isolates, respectively. Phylogenetic analysis revealed that chimpanzee STLV-I isolates can be attributed to three clades. The first of these clades (SS-PTR1/CSA) included STLV-I isolates from the chimpanzees and West African subspecies of African green monkeys (Cercopithecus a. sabaeus). The other clades (S-PTR2 and S-PTR3) included STLV-I isolates only from chimpanzees. However, both S-PTR2 and S-PTR3 clustered together with Central African HTLV-I comprising the human/simian clade (HS-HSA/PTR). This pattern of phylogenetic clustering suggests that interspecies transmission of STLV-I occurred between chimpanzees and African green monkey subspecies as well between chimpanzees and human populations in Central Africa.

B-cell lymphomagenesis in SIV-immunosuppressed cynomolgus monkeys.

B-cell lymphomas developed frequently (approx. 40%) in SIVsm (SMM3) immunosuppressed monkeys and were mostly extranodal, aggressive and all associated with an EBV-related simian herpes virus operationally designated herpes virus Macaca fascicularis (HVMF-I). Lymphoma tissues from 21 monkeys were studied by PCR and DNA PAGE for mono/oligoclonality of the VDJ-rearranged IgH genes. Most lymphomas (n = 15) showed a monoclonal and approximately 1/3 (n = 6) an oligoclonal VDJ rearrangement pattern. The time after infection to tumor presentation was significantly shorter for oligoclonal than for monoclonal lymphomas, suggesting that oligoclonal selection frequently precedes the outgrowth of a single malignant clone. Comparison of the VDJ rearrangements in an established lymphoma cell line and the original, oligoclonal lymphoma tissue indicated in vitro selection of one HVMF-infected clone. Longitudinal studies of sequential lymph-node biopsies showed that the malignant lymphoma clone in 3 out of 8 lymphomas could be identified as a predominant clone in lymph nodes 2-12 months after SIV infection and 6-10 months before clinical presentation of the lymphomas. VDJ-rearranged DNA corresponding to that of the lymphomas was also detected in most sera at the time of lymphoma manifestation but not in corresponding PBL preparations. Clearly, the SIVsm AIDS model in cynomolgus monkeys represents a powerful tool for biological and clinical studies of herpes-virus-associated lymphomagenesis in immunosuppressed states.

[Malignant lymphoma in rabbits induced by the administration of herpes virus-containing material from brown macaques]. / Zlokachestvennye limfomy u krolikov, vyzvannye vvedeniem materiala, soderzhashchego virus gerpesa burykh makakov.

Oncogenicity for rabbits of lymphotropic herpesvirus of M. arctoides (HVMA) isolated by us earlier from lymphoid cells of lines MAL-1-3 has been shown in the course of studies. Inoculation of animals both with HVMA-containing cells and cell-free virus caused the development of generalized malignant lymphomas of prolymphocytic-lymphoblastic character leading the animals to death. The molecular hybridization method permitted revealing DNA sequences related to the DNAs of B-lymphotropic herpesviruses of primates in the cells of HVMA-containing cultures, primarily-induced tumours, and in a cell line established from rabbit tumour. This fact is indicative of etiological role of HVMA in the development of malignant lymphomas in rabbits. The question about the origin of the C type particles found in MAL-1-3 and OK-1 cells remains open.

Genetics of primate lymphomas in a baboon (Papio hamadryas) colony of Sukhumi, USSR.

A lymphoma outbreak occurred at the Sukhumi Center in the Late 1960's, when 12 baboons were inoculated with blood from human leukemia patients. Out of a total of 3219 animals, 218 contracted lymphoma and died. This outbreak appears to be primarily horizontal in transmission. Genetic investigations reveal that: (1) inbreeding does not increase risk of lymphoma; (2) there is a weak association between the PGM2 locus and lymphoma; (3) based upon path analysis, there is evidence of a significant transmissible component (genetic predisposition) passed from the parental to the next generation.

[Modelling of malignant lymphoma in rabbits using primate oncogenic viruses. Preliminary report]. / Modelirovanie zlokachestvennoi limfomy na krolikakh s pomoshch'iu onkogennykh virusov primatov. Predvaritel'noe soobshchenie.

Inoculation of rabbits with permanent B-cell line cultures obtained from Stumptailed Macaques M. arctoides (MAL) which contain lymphotropic herpesvirus and C-type particles has led to the development of generalized lymphomas. The lymphoma cells had rabbit karyotype and did not contain surface an cytoplasmic immunoglobulins. Permanent suspension of lymphoid cell line independent of growth factors was obtained from rabbit lymphoma. The serum of a rabbit with lymphoma transmitted from another rabbit with MAL-induced lymphoma did not react with virus-negative human Raji cells when tested in immunofluorescence. But this serum reacted positively with cytoplasmic antigens of simian 594S-F9 cells (producing lymphotropic herpesvirus and two types of retroviruses, namely, endogenous C-type and HTLV-I-like) and human C91-PL cells (producing HTLV-I). The results obtained demonstrated high oncogenicity of the viruses produced by simian permanent cellular MAL lines for rabbits.

Antibodies reacting with human T-lymphotropic retrovirus (HTLV-I) or related antigens in lymphomatous and healthy hamadryas baboons.

The sera of lymphomatous and healthy hamadryas baboons of the main, lymphoma-prone Sukhumi stock were tested for antibodies reacting with HTLV-I antigens in the indirect immunofluorescence test. Antibodies of this specificity were found in all but one of 58 lymphomatous baboons and in 45% of 177 healthy ones. The prevalence of HTLV-I reactive antibodies in lymphoma-free baboon populations (including 118 Sukhumi "forest" stock animals and 195 baboons imported in 3 groups from Ethiopia) was consistently lower (5-8%). The specificity of baboon antibodies reacting with HTLV-I or a related agent is supported by the following evidence: Concordant reactivity pattern of baboon sera with several HTLV-I-positive and-negative human cell lines; elimination of baboon sera anti-HTLV reactivity by absorption with purified HTLV-I, but not by other retroviruses; significant correlation between immunofluorescence titers of baboon sera and their reactivity in enzyme-linked immunosorbent assay with purified HTLV-I; competition of baboon anti-HTLV with monoclonal antibodies GIN-14 for binding of the epitope on p19HTLV-I. The prevalence of anti-HTLV positives in the main Sukhumi stock increased by age, reaching its maximum (approx. 80%) at 5-15 years, and showed no significant sex-related variation. The level of anti-HTLV antibodies in lymphomatous baboons and in age-, sex- and population-matched healthy ones did not differ. However, in pre-lymphoma sera these antibodies reached significantly higher levels than in sera of lymphomatous baboons (obtained in the terminal stage) or of matched, healthy controls.

Immunoprecipitation of Epstein-Barr virus (EBV)-specific proteins by prelymphomatous and normal baboon sera containing antibodies reactive with EBV early antigen.

Carbon (14C)-amino acid mixture-labelled antigen extracted from 12-0-tetradecanoyl-phorbol-13-acetate (TPA) and sodium n-butyrate-induced P3HR-1 cells was immunoprecipitated with sera from normal, prelymphomatous and lymphomatous baboons, as well as with sera from human controls. Most of the Epstein-Barr virus (EBV)-specific proteins that were precipitated by human sera were also precipitated by baboon sera. A major difference between prelymphomatous and normal control baboon sera was that 5 out of 6 the former and none of the latter immuno-precipitated an EBV-early antigen-specific protein with Mr 31,000.

Seroepizootiology of the herpesvirus Papio (HVP) infection in healthy baboons (Papio hamadryas) of high- and low-lymphoma risk populations.

Seroepizootiology of Herpesvirus Papio (HVP) infection was studied in three groups of healthy hamadryas baboons (Papio hamadryas): the main Sukhumi (high-lymphoma) stock, forest Sukhumi (lymphoma-free) stock and newly imported wild animals. The prevalence to HVP infection, as judged by anti-VCA-HVP positivity, was approximately the same in both Sukhumi stocks (86% and 90% respectively) and it was significantly lower in the pooled group of newly imported baboons. It is interesting that prevalence of HVP infection in the different independent groups varied markedly (35-79%). Geometric mean titers of positives in all groups were approximately the same. The prevalence of HVP infection was age-dependent. It increased during the first years of life reaching the maximum (about 100%) at the age of 5 years being stable up to the age of 18 years and "decreased" at very old ages (over 18 years). The prevalence of HVP infection in newly imported baboons increased with age up to 71% in a group of the "oldest" monkeys and did not plateau. No significant sex differences in anti-HVP titers were found. Anti-EA-HVP-positive (with one exception) and anti-HUPNA-positive animals were found only in the main Sukhumi stock. Thus, "serologic activity" against HVP infection was the highest in the ligh-lymphoma stock.

Inbreeding, heterozygosity, and lymphoma risk among the baboons (Papio hamadryas) of Sukhumi, USSR.

This paper describes the spread of lymphoma through a baboon (Papio hamadryas) colony in the Institute of Experimental Pathology and Therapy at Sukhumi, USSR. In the late 1960s, Soviet scientists inoculated 12 baboons with cells from hospitalized human leukemia patients, causing the death of a total of 135 animals between 1967 and 1978. The death rate from lymphoma averages almost 12 baboons per year in the Sukhumi colony. Genetic investigations of these baboons revealed the following: 1) Six blood protein markers out of 16 systems (38%) tested were polymorphic; 2) the average inbreeding coefficient for the entire colony (N = 1,226) was 0.027 (exclusion of baboons with F values equal to 0.0 raised the mean inbreeding coefficient to 0.096); 3) no relationship between inbreeding and risk of lymphoma was noted; and 4) there was an apparent association between both PGM loci and the incidence of lymphoma at the 0.005 levels of significance. This association was further supported by the significantly lower incidence of PGM2 (2-1) genotype in baboons with high anti-VCA-HVP titers.

[Etiological aspects of leukemias in primates including man]. / Nekotorye aspekty étiologii leikozov primatov, vkliuchaia cheloveka.

Attempts have been made to induce viral leukemia in monkeys (Papio hamadryas and Macaca arctoides) by inoculating them with blood from humans with different types of leukemias. In hamadryas baboons, the disease spread horizontally. By today 218 P. hamadryas and 5 M. arctoides monkeys had died of malignant lymphoma. The following viruses have been isolated from sick monkeys: lymphotropic baboon herpes virus (HVP), endogenous baboon C type viruses--xenotropic (BILN), and ecotropic (EVPG). C type oncovirus called "plasmic", which differs immunologically from the endogenous one, was also detected in the blood of sick animals. Altogether 165 sera from baboons, different species of macacas and chimpanzee were examined by the immunofluorescent technique for antibodies to HTLV virus isolated recently from sick humans with T cell leukemia/lymphoma. Antibodies to HTLV virus were detected only in monkeys (P. hamadryas and M. arctoides) with malignant lymphomas or in those which had been in close contact with them. Possible origin of simian HTLV-like virus is discussed. It originates either from leukemic patients or there is a family of primate HTLV like viruses related to the occurrence of leukemia.

Increased antibody responses to Herpes virus papio (HVP) antigens in pre-lymphomatous baboons (Papio hamadryas) of the Sukhumi high lymphoma stock.

Antibody responses to Herpes virus papio (HVP) antigens were studied in 21 pre-lymphoma baboons (which subsequently died of malignant lymphoma), 21 paired controls, i.e. age-, sex- and population-matched healthy baboons, and 185 randomly selected healthy baboons of the same population. The sera were all collected at the same time and were tested blind in the fixed-cell indirect immunofluorescence test against HVP viral capsid antigen (VCA)-positive, early antigen (EA)-positive cell targets before and after absorption with HVP. Eleven of the pre-lymphoma sera were anti-EA-positive whereas none of the paired controls contained anti-EA. Anti-VCA titers of pre-lymphoma sera were higher than those of paired controls in thirteen cases. Only in four cases were anti-VCA titers of pre-lymphoma sera lower than those of paired controls. Qualitatively, the same results were obtained when anti-VCA and anti-EA titers of pre-lymphoma sera were compared with respective mean population values. The differences between pre-lymphoma group and control groups, especially in the case of anti-EA, were statistically highly significant. Thus, elevated anti-HVP titers in healthy baboons of the Sukhumi lymphoma-prone stock can be considered as a marker of high risk for development of malignant lymphoma.

Immunological investigation of the prelymphoma period in baboons of the Sukhumi monkey stock.

In the "pre-lymphoma" period, some high-risk baboons develop changes in their immunity which is expressed, in particular, in the imbalnace of T-lymphocyte subpopulations, that is, increase in the number of cells of T gamma subpopulation which are carriers of suppressive functions in man. As a reflection of immunological disturbances, increase in the level of antibodies against HVP, apparently as a result of HVP activation, is observed in baboons in the "pre-lymphoma" period.

Enzyme-linked immunosorbent assay for detection of antibodies to Epstein-Barr virus antigens.

Enzyme-Linked Immunosorbent Assay (ELISA) was standardized for measurement of antibody activity of reference human and baboon (Papio hamadryas) sera to soluble Epstein-Barr virus (EBV) antigens. A comparison with the immunofluorescent (IF) method showed that ELISA detects antibody specifically and sensitivity. In ELISA, Herpesvirus Papio (HVP) nuclear antigen (HUPNA) positive baboon serum reacted with EBV nuclear antigen (EBNA), as a further indication of the antigenic similarity between HVP and EBV. Forty-two baboon sera were tested with EBV antigens in both ELISA and IF test. The results showed an agreement between the two methods and also that by the use of EBV antigens, ELISA measures anti-HVP activity of baboon sera. ELISA did not reveal significant difference in antibody activity of 23 baboons with lymphoma and that of 24 healthy baboons. Results provide further data that ELISA can be used effectively in the field of EBV serology.

[Study of virus-associated lymphomas in primates using a model of malignant lymphoma of the baboon]. / Izuchenie virusassotsiirovannykh gemoblastozov primatov na modeli zlokachestvennoi limfomy pavianov.

An outbreak of malignant lymphoma was registered in a population of hamadryas baboons. Animals inoculated with blood from patients with various forms of leukemia were planted into the stock 10 years ago. 218 out of 3219 animals have already died from lymphoma. The morphological pattern of horizontally-spreading lymphoma was similar to basic cell patterns of Hodgkin's and non-Hodgkin lymphoma in man. As far as the immunologic classification of tumor lymphoid cells is concerned, baboon lymphomas were of B-cell, T-cell and neither T- nor B-cell types. Xenotropic and ecotropic endogenous oncoviruses and lymphotropic herpesvirus of the baboon were isolated from cell cultures of healthy and sick animals. The said herpesvirus was closely related to a human one, both belonging to a family of primate lymphotropic herpesviruses. Plasma residue of the sick animals contained C-type oncovirus which immunologically differed from that of baboon endogenous oncovirus. Blood serum of nearly all sick baboons and those in the stock at high risk for lymphoma contained antibodies which entered into immunofluorescence reaction with antigens of HTLV virus-producing cells. Similar antibodies at a low titer concentration were identified only in individual lymphoma-free baboons in control. The relationship between HTLV virus and C-type oncovirus as well as their relevance to human hemoblastosis are being under study.

Functional complementation between Epstein-Barr virus and herpesvirus papio.

The possibility of complementation between herpesvirus papio (HVP) and Epstein-Barr virus (EBV) was investigated. Strain 594S-F9 of HVP, unlike strain P3HR-1 of EBV, is not capable of inducing virus antigen synthesis in the EBV genome-carrying, nonproducer lymphoma cell line Raji. The effects of dual infection with these viruses were studied. With untreated viruses, the percentage of cells positive for viral antigens was equal to or slightly less than that in cultures infected with P3HR-1 virus only. However, if UV-irradiated P3HR-1 virus was employed in the dual infection, the relative number of virus antigen-positive cells was enhanced over cultures infected with P3HR-1 virus alone. These results suggest functional complementation between EBV and HVP.

Common antigenic determinants on structural proteins p10--12 of PMFV and MPMV type D retroviruses.

PMFV, a type D retrovirus isolated from a malignant human embryo cell line, was compared with Mason-Pfizer monkey virus (MPMV) in a sensitive tannic acid enhanced indirect immunodiffusion test. In addition to the previously shown common antigens, both viruses contain identical group-specific antigenic determinants on their p 10--12 as demonstrated with a specific p 10--12 MPMV test system. Interspecies mammalian type C virus antigens were not detected in highly concentrated PMFV preparations.

Prevalence of antibodies to soluble antigen of Epstein-Barr virus-producing cells (P3HR-1) in the sera of hamadryas baboons of the high lymphoma incidence stock.

Soluble antigen of P3HR-1 cells (SA-P3HR-1) was identified in indirect double immunodiffusion enhanced with tannic acid using serum of a nasopharyngeal carcinoma patient containing high-titer antibodies to Epstein-Barr virus (EBV) antigens. SA-P3HR-1 was nonidentical to soluble antigen of Raji cells. Human and baboon sera containing antibodies to all the known antigen of EBV and HVP respectively were anti-SA-3HR-1-positive. Human and baboon sera containing antibodies to all the known antigens of EBV and herpesvirus Papio (HVP) were also anti-SA-P3HR-1-negative. Prevalence of anti-SA-P3HR-1 was very high in two groups of the high-lymphoma incidence stock of hamadryas baboons of the Sukhumi monkey colony. 54% (15 of 28) and 38% (13 of 34) of clinically lymphomatous and clinical normal monkeys, respectively, were anti-SA-P3HR-1-positive.. Only 1 of 30 normal baboons studied, living in the forest and having no contacts with the baboons in the main stock of the Sukhumi monkey colony, was anti-SA-P3HR-1-positive (3%).