White monkey syndrome in infant baboons (Papio species).

Over 23 months, zinc toxicosis was diagnosed in 35 baboons aged 5-12 months in one galvanized metal and concrete cage complex with conditions that led to excessive exposure to environmental zinc. Clinical signs included reduced pigmentation of hair, skin, and mucous membranes (whiteness), alopecia, dehydration, emaciation, cachexia, dermatitis, diarrhea and, in six cases, severe gangrenous dermatitis of extremities. The syndrome was characterized by pancytopenia, elevated zinc and low copper serum concentrations, low vitamin D and bone-specific alkaline phosphatase levels, and atypical myelomonocytic proliferation of bone marrow. This syndrome emphasizes the importance of proper husbandry and cage design and indicates the potential of infant baboons as a model to study the effects of excessive zinc on development. This is the first report describing the epidemiologic and clinical presentation of zinc toxicosis in infant baboons in captivity.

Expression of the interleukin-18 gene from rhesus macaque by the simian immunodeficiency virus does not result in increased viral replication.

Interleukin-18 (IL-18), previously known as interferon-gamma (IFN-gamma)-inducing factor (IGIF), is a proinflammatory cytokine expressed by activated macrophages that acts in synergy with IL-12 as an important amplifying factor for IFN-gamma production and Th1 development. To study the effect of IL-18 on a lentiviral infection, we cloned the IL-18 gene from a rhesus macaque and constructed replication-competent simian immunodeficiency virus (SIV) that expressed either the precursor pro-IL-18 (SIV(IL-18)) or the mature form (SIV(mIL-18)) of IL-18. The predicted amino acid sequence for rhesus IL-18 had 96% homology with the human one, differing in only 8 of 193 residues. SIV(IL-18) and SIV(mIL-18) replicated more slowly than control viruses in the CEM x 174 cell line and resulted in the development of chronically infected cell lines that expressed high levels of infectious SIV. The cell line generated by SIV(IL-18) released large quantities of IL-18 into the supernatant, whereas the one obtained from SIV(mIL-18) showed the accumulation of IL-18 in the cytoplasm. Similarly, SIV(IL-18) and SIV(mIL-18) replicated more slowly than the unmodified viral vector in rhesus peripheral blood mononuclear cells (PMBC), but only SIV(IL-18) expressed biologically active IL-18. These experiments show that the precursor form of IL-18 is necessary for the efficient release of the cytokine and that IL-18 does not promote increased replication of SIV in rhesus PBMC.

Cytokine expression, natural killer cell activation, and phenotypic changes in lymphoid cells from rhesus macaques during acute infection with pathogenic simian immunodeficiency virus.

We studied the innate and adaptive immune system of rhesus macaques infected with the virulent simian immunodeficiency virus isolate SIVmac251 by evaluating natural killer (NK) cell activity, cytokine levels in plasma, humoral and virological parameters, and changes in the activation markers CD25 (interleukin 2R ¿IL-2R alpha chain), CD69 (early activation marker), and CD154 (CD40 ligand) in lymphoid cells. We found that infection with SIVmac251 induced the sequential production of interferon-alpha/beta (IFN-alpha/beta), IL-18, and IL-12. IFN-gamma, IL-4, and granulocyte-macrophage colony-stimulating factor were undetected in plasma by the assays used. NK cell activity peaked at 1 to 2 weeks postinfection and paralleled changes in viral loads. Maximum expression of CD69 on CD3(-)CD16(+) lymphocytes correlated with NK cytotoxicity during this period. CD25 expression, which is associated with proliferation, was static or slightly down-regulated in CD4(+) T cells from both peripheral blood (PB) and lymph nodes (LN). CD69, which is normally present in LN CD4(+) T cells and absent in peripheral blood leukocyte (PBL) CD4(+) T cells, was down-regulated in LN CD4(+) T cells and up-regulated in PBL CD4(+) T cells immediately after infection. CD8(+) T cells increased CD69 but not CD25 expression, indicating the activation of this cellular subset in PB and LN. Finally, CD154 was transiently up-regulated in PBL CD4(+) T cells but not in LN CD4(+) T cells. Levels of antibodies to SIV Gag and Env did not correlate with the level of activation of CD154, a critical costimulatory molecule for T-cell-dependent immunity. In summary, we present the first documented evidence that the innate immune system of rhesus macaques recognizes SIV infection by sequential production of proinflammatory cytokines and transient activation of NK cytotoxic activity. Additionally, pathogenic SIV induces drastic changes in the level of activation markers on T cells from different anatomic compartments. These changes involve activation in the absence of proliferation, indicating that activation-induced cell death may cause some of the reported increase in lymphocyte turnover during SIV infection.

Phylogenetic comparison of the S3 gene of United States prototype strains of bluetongue virus with that of field isolates from California.

To better define the molecular epidemiology of bluetongue virus (BTV) infection, the genetic characteristics and phylogenetic relationships of the S3 genes of the five U.S. prototype strains of BTV, the commercially available serotype 10 modified live virus vaccine, and 18 field isolates of BTV serotypes 10, 11, 13, and 17 obtained in California during 1980, 1981, 1989, and 1990 were determined. With the exception of the S3 gene of the U.S. prototype strain of BTV serotype 2 (BTV 2), these viruses had an overall sequence homology of between 95 and 100%. Phylogenetic analyses segregated the prototype U.S. BTV 2 strain to a unique branch (100% bootstrap value), whereas the rest of the viruses clustered in two main monophyletic groups that were not correlated with their serotype, year of isolation, or geographical origin. The lack of consistent association between S3 gene sequence and virus serotype likely is a consequence of reassortment of BTV gene segments during natural mixed infections of vertebrate and invertebrate hosts. The prototype strain of BTV 13, which is considered an introduction to the U.S. like BTV 2, presents an S3 gene which is highly homologous to those of some isolates of BTV 10 and especially to that of the vaccine strain. This finding strongly suggests that the U.S. prototype strain of BTV 13 is a natural reassortant. The different topologies of the phylogenetic trees of the L2 and S3 genes of the various viruses indicate that these two genome segments evolve independently. We conclude that the S3 gene segment of populations of BTV in California is formed by different consensus sequences which cocirculate and which cannot be grouped by serotype.

Construction and characterization of replication-competent simian immunodeficiency virus vectors that express gamma interferon.

We report the construction and characterization of several replication-competent simian immunodeficiency virus (SIV) vectors with a deletion in the viral nef gene (SIV(delta nef)) that express gamma interferon (IFN-gamma). The expression of the cytokine gene was controlled either by the simian virus 40 early promoter or by the SIV 5' long terminal repeat regulatory sequences, utilizing the nef gene splice signals. To enhance the expression of IFN-gamma, the two in-frame nef start codons were mutated without altering the Env amino acid sequence (SIV(HyIFN)). Plasmids containing full-length proviral genomes were used to obtain high-titer stocks of each recombinant virus in cell cultures. Expression of IFN-gamma by SIV(HyIFN) reached levels as high as 10(6) U/ml after 11 days in culture. The IFN-gamma gene was unstable and sustained deletions after serial passage of SIV(delta nef) vectors in CEM-X-174 cells. The degree of instability appears to depend on size and orientation of the insert and the expression of IFN-gamma. Only one virus, SIV(HyIFN), expressed detectable levels of IFN-gamma up to the sixth passage. Prospects for the use of IFN-gamma and other lymphokines to enhance the safety and efficacy of live attenuated vaccines are discussed.

Immune response of rhesus macaques to recombinant simian immunodeficiency virus gp130 does not protect from challenge infection.

Simian immunodeficiency virus (SIV) infection of rhesus macaques is a model for human immunodeficiency virus (HIV) infection in humans. Inactivated and modified live whole-virus vaccines have provided limited protective immunity against SIV in rhesus macaques. Because of safety concerns in the use of inactivated and live whole-virus vaccines, we evaluated the protective immunity of vaccinia virus recombinants expressing the surface glycoprotein (gp130) of SIVmac and subunit preparations of gp130 expressed in mammalian cells (CHO). Three groups of animals were immunized with recombinant SIV gp130. The first group received SIV gp130 purified from genetically engineered CHO cells (cSIVgp130), the second group was vaccinated with recombinant vaccinia virus expressing SIVmac gp130 (vSIVgp130), and the third group was first primed with vSIVgp130 and then given a booster immunization with cSIVgp130. Although anti-gp130 binding antibodies were elicited in all three groups, neutralizing antibodies were transient or undetectable. None of the immunized animals resisted intravenous challenge with a low dose of cell-free virus. However, the group primed with vSIVgp130 and then boosted with cSIVgp130 had the lowest antigen load (p27) compared with the other groups. The results of these studies suggest that immunization of humans with HIV type 1 surface glycoprotein may not provide protective immunity against virus infection.

Vaccinia virus recombinants expressing chimeric proteins of human immunodeficiency virus and gamma interferon are attenuated for nude mice.

We have developed a method for attenuating vaccinia virus recombinants by expressing a fusion protein of a lymphokine and an immunogen. Chimeric genes were constructed that coded for gamma interferon (IFN-gamma) and structural proteins of the human immunodeficiency virus type 1 (HIV-1). In this study, we describe the biological and immunological properties of vaccinia virus recombinants expressing chimeric genes of murine or human IFN-gamma with glycoprotein gp120, gag, and a fragment of gp41. All fusion proteins retained the antigenic characteristics of both IFN-gamma and HIV as shown by immunoblot analysis. However, the antiviral activity of IFN-gamma could be demonstrated only for the IFN-gamma-gag fusion protein. In contrast, the attenuating activity of IFN-gamma for nude mice was retained by all of the recombinants, albeit at various rates. Unlike the antiviral activity, the attenuating activity of IFN-gamma was not species specific. Implications for the development of attenuated live recombinant vaccines for AIDS are discussed.

Protection conferred by TrpE fusion proteins containing portions of the C-terminal region of capsid protein VP1 of foot-and-mouth disease virus.

Major immunogenic sites of foot-and-mouth disease virus (FMDV) have been mapped to the C-terminal third of capsid protein VP1; we studied the immunogenicity of a series of TrpE-FMDV fusion proteins containing this region of FMDV strain O1 Campos. Fusion protein TrpE-dCN, which contains a dimer of VP1 amino acid sequences consisting of amino acids 200 to 213 linked by a diproline spacer to amino acids 141 to 158 (200-213 approximately P-P-G approximately 141-158), induced the best response. A single inoculation of guinea-pigs with 100 micrograms TrpE-dCN elicited high levels of neutralizing antibodies and protected all the animals against challenge infection with homologous virus. Although the closely related FMDV strains O1 Campos and O1 Caseros induced high levels of cross-protection, TrpE-dCN-vaccinated guinea-pigs were poorly protected against challenge infection with heterologous FMDV strain O1 Caseros. Nucleotide sequence analysis revealed that amino acid differences at residues 149 and 152 were critical for the induction of cross-protection and that neutralizing epitopes not present in TrpE-dCN are likely to be responsible for conferring a high level of cross-protection between FMDV strains O1 Campos and O1 Caseros.