M. D. Daniel

Importance of the nef gene for maintenance of high virus loads and for development of AIDS

When rhesus monkeys were infected with a form of cloned SIVmac239 having a premature stop signal at the 93rd codon of nef, revertants with a coding codon at this position quickly and universally came to predominate in the infected animals. This suggests that there are strong selective forces for open functional forms of nef in vivo. Although deletion of nef sequences had no detectable effect on virus replication in cultured cells, deletion of nef sequences dramatically altered the properties of virus in infected rhesus monkeys. Our results indicate that nef is required for maintaining high virus loads during the course of persistent infection in vivo and for full pathologic potential. Thus, nef should become a target for antiviral drug development. Furthermore, the properties of virus with a deletion in nef suggest a means for making live-attenuated strains of virus for experimental vaccine testing.

Identification of a nef allele that causes lymphocyte activation and acute disease in Macaque monkeys

Residues 17 and 18 in nef of SIVmac239 were changed from RQ to YE to create a translated sequence of SRPSGDLYERLLRARGETYGRLLGEVEDGYSQSP from residues 10-43. The YXXL motifs in this context match very well with consensus sequences for SH2 binding domains and are similar to ones present in nef of the acutely lethal pathogen SIVpbj14. The YE variant of SIVmac239, unlike SIVmac239 but like SIVpbj14, replicated well in resting peripheral blood mononuclear cell cultures, caused extensive T lymphocyte activation, and produced an acute disease in rhesus and pigtailed monkeys characterized by severe diarrhea, rash, and extensive lymphoid proliferation in the gastrointestinal tract. The YEnef gene transformed NIH 3T3 cells in culture. Both 239nef and YEnef were found to associate with src in cotransfected COS cells, and both 60 kDa src and 34 kDa nef were phosphorylated at tyrosine in these cells. The extent of tyrosine phosphorylation of 239nef was considerably less than that of YEnef in these assays. These findings identify an important determinant of the SIVpbj14 phenotype, and they provide evidence of a role for nef in signal transduction and cellular activation.

Long-term persistent infection of macaque monkeys with the simian immunodeficiency virus.

Juvenile rhesus macaques 6 to 18 months of age were experimentally infected by intravenous inoculation with the simian immunodeficiency virus (SIV), the T cell-tropic retrovirus of monkeys related to the human acquired immunodeficiency syndrome (AIDS) virus HIV. The SIV used for inoculation was grown either in normal human peripheral blood lymphocytes in the presence of interleukin 2 or in the human tumour cell line HUT-78. Eight of the macaques died 129 to 352 days post-inoculation with a variety of clinical and pathological findings paralleling those of AIDS in humans. However eight other animals became persistently infected for prolonged periods; these eight macaques remained alive at 537 and 820 days post-inoculation despite persistent lymphadenopathy and our continued ability to isolate SIV. The ability of these monkeys to survive infection correlated directly with the strength of their antibody response to SIV. Infection was also established in macaques using approximately 100 tissue culture infectious doses of HUT-78-grown SIV. There was no correlation between the dose of virus inoculum and either the strength of the antibody response or clinical outcome. These results demonstrate that SIV infection of macaques can be used not only to study acute AIDS but also to mimic the long-term persistent infection seen in carriers of HIV.

Reactivity of primate sera to foamy virus Gag and Bet proteins.

In order to establish criteria for the serodiagnosis of foamy virus infections we investigated the extent to which sera from infected individuals of human and primate origin react with structural and non-structural virus proteins in immunoblot assays. Using lysates from infected cells as the source of virus antigen, antibodies were preferentially detected against the Gag proteins and the non-structural Bet protein. Both the Gag precursor molecules of 70 and 74K apparent M(r) and the cytoplasmic 60K M(r) Bet protein were found to be phosphorylated, the latter being synthesized in large amounts in infected cells. Rabbit antiserum raised against recombinant human foamy virus (HFV) Gag major capsid protein cross-reacted with foamy viruses of chimpanzee, gorilla, orang-utan, rhesus monkey and African green monkey origin. This was reflected by a broad cross-reactivity of the respective monkey sera to the Gag proteins of the various foamy virus isolates. Cross-reactivity of antisera against the Bet protein was restricted to viruses from man and the great apes. Recombinant Gag and Bet proteins expressed in prokaryotes or in insect cells were readily recognized by foamy virus-positive primate sera. Screening serum samples from chimpanzees with HFV Gag and Bet proteins expressed by recombinant baculoviruses revealed that 18 out of 35 (52%) were positive for Gag antibodies. Of these, 13 (72%) showed antibodies against the Bet protein, indicating that Bet antigen is of value in serological screening for foamy virus infections.

EXPERIMENTAL TRANSMISSION OF MACAQUE AIDS BY MEANS OF INOCULATION OF MACAQUE LYMPHOMA TISSUE

Acquired immuno-deficiency syndrome (AIDS) of macaques, an animal model for human AIDS, was transmitted to previously healthy macaque monkeys by means of inoculation of either tissue or a cell-free filtrate of a macaque lymphoma. The recipients showed evidence of profound lymphocyte dysfunction or died with infections from such opportunistic agents as Candida albicans, Cryptosporidium, and cytomegalovirus.

Acute Lymphocytic Leukemia in Owl Monkeys Inoculated with Herpesvirus saimiri

Our study demonstrates for the first time that Herpesvirus saimiri can induce acute lymphocytic leukemia in owl monkeys (Aotus trivirgatus) and that malignant lymphoma can be induced in this species of nonhuman primates by the inoculation of the virus by various routes (intravenous, sub-cutaneous, and intradermal).

Isolation of Herpes-T virus from a spontaneous disease in squirrel monkeys (Saimiri sciureus)

Herpes-T virus was isolated from 2 of 4 clinically ill squirrel monkeys. The clinical manifestations of the disease in the monkeys was characterized by oral and labial lesions. From one of two animals sacrificed for histopathological examination, Herpes-T virus was isolated from the tongue and salivary gland. Herpes-T was isolated from the oral swab of one of the two live monkeys on the day of arrival in our laboratory and again on the 4th day. The anal swabs collected from both these monkeys failed to yield virus. The sera of these monkeys collected on the day of arrival showed a neutralization index (NI) of 1.0 and 1.5 against Herpes-T virus. The convalescent sera, collected two weeks later, showed a NI of 3.5 and 4.0 respectively for the same virus. Clear plaques ranging in size from 2 to 3 mm were produced in chick fibroblast and rabbit kidney primary monolayers. This is the first report of the isolation of Herpes-T virus from naturally infected squirrel monkeys. This data further supports that the squirrel monkey is a natural host of Herpes-T virus.

Tumour induction with DNA of oncogenic primate herpesviruses

THE viruses Herpesvirus saimiri and H. ateles are highly oncogenic agents of New World primates. The viruses are not pathogenic in the natural host species (squirrel and spider monkeys, respectively), but induce malignant neoplastic diseases of the lymphatic system in various other primates1,2 and in rabbits3. Marmoset monkeys (Saguinus sp.) are the most susceptible animals to tumour induction, as infection with these viruses invariably results in rapidly progressing malignant lymphoma or acute lymphocytic leukaemia within a few weeks4,5. H. saimiri and H. ateles are related viruses; they share common antigens6 and some homologous DNA sequences7. DNA from both viruses is infectious in permissive owl monkey kidney (OMK) cell cultures8. We report here that the isolated DNA from these two herpesviruses can also cause malignant tumours in animals.

Herpesvirus saimiri. III. Plaque Formation Under Multi Agar, Methyl Cellulose and Starch Overlays

Summary Herpesvirus saimiri produced plaques in cultures of fetal squirrel monkey heart, lung, intestine, and kidney and in owl monkey kidney and marmoset kidney cell cultures. The plaque populations in all cell types were heterogeneous in respect to size. Harvests of single plaques produced heterogeneous plaque populations on serial plating. Plaques were most numerous under methyl cellulose. Under agar, plaques were less numerous but were larger. Protamine sulfate and, to a limited degree, DEAE-dextran stimulated plaque production under agar while arginine had no effect. Plaques also formed under starch but were difficult to visualize. The number of plaques was directly proportional to the virus dilution, was also proportional to the volume of the inoculum, and was reduced by specific antiserum.

Targeted inhibition of immunodeficiency virus replication in lymphocytes through retroviral mediated gene transfer

SummaryTo test the feasibility of gene therapy for AIDS patients, an animal model is needed to evaluate the efficacy and safety of this approach. Antiviral genes (encoding antisense RNA or viral protein) derived from Simian immunodeficiency virus (SIV) were efficiently targeted into CD4+ lymphocytes through retroviral-mediated gene transfer. After challenging with infectious viruses, the transduced lymphocytes that received antiviral genes were not only protected from SIV infection, but also from infection with HIV, for at least 25 days. Furthermore, little or no cytolytic effect (syncytium formation) was observed in the protected cells. These data demonstrated that SIV or HIV replication could be effectively blocked by antisense sequence(s) or negative dominant factors which were introduced into targeted cells through retroviral-mediated gene transfer.