151The major extracellular proteases of Aeromonas hydrophila NRC 505 and ATCC 7966 are a thermostable metallo-protease (TSMP) and a thermolabile serine-protease (TLSP), respectively. The purified enzymes differed in sensitivity to heating at 56 "C for 30 min, in response to the inhibitors EDTA and phenylmethylsulphonyl fluoride (PMSF), and were antigenically distinct. When crude extracellular products (ECP) of 47 strains of A . hydrophila were screened, 27 strains produced both TSMP and TLSP. TSMP was the only extracellular protease produced by 19 strains, whereas only A . hydrophila ATCC 7966 produced TLSP as its sole protease. This distribution of protease enzymes among strains explains conflicting reports from studies in which single strains of A . hydrophila were examined. The TLSP of A. hydrophila was similar to the protease of A.-salmonicida; Either or both TSMP and TLSP were produced by some strains of A. sobria and A'. caviae.

Previously, we described the derivation of a pathogenic strain of simian-human immunodeficiency virus (SHIV(KU-2)) consisting of the tat, rev, vpu, and env genes of HIV-1 (strain HXB2) in a genetic background of SIV(mac)239 that causes AIDS and productive infection of the CNS in rhesus macaques (Macca mulatta) (Raghavan et al., 1997, Brain Pathol. 7, 851-861). We report here on the characterization of a molecular clone of SHIV(KU-2), designated SHIV(KU-2MC4), that caused CD4(+) T cell loss as well as neurological and renal disease in macaques. DNA sequence analysis of selected SIV regions of SHIV(KU-2MC4) revealed 10 nucleotide changes in the LTR, whereas Gag, Vif, Vpr, Vpx, and Nef had 1, 1, 1, 2, and 13 predicted amino acid substitutions, respectively, compared to SIV(mac)239. DNA sequence analysis of HIV-1 derived regions of SHIV(KU-2MC4) revealed 2, 1, 2, and 18 predicted amino acid substitutions in the Tat, Rev, Vpu, and Env proteins, respectively, when compared to SHIV-4. Unlike the parental SHIV-4, which is not tropic for macrophages, SHIV(KU-2MC4) replicated efficiently in macrophage cultures as determined by p27 assays. However, despite the numerous changes in the Env protein and newly acquired tropism for macrophages, SHIV(KU-2MC4), like the parental SHIV-4, used CXCR4 exclusively as its coreceptor for entry into susceptible cells. Inoculation of SHIV(KU-2MC4) into two rhesus macaques resulted in severe infection in which the numbers of circulating CD4(+) T cells in the blood declined rapidly by 2 weeks postinoculation and virus producing cells in the peripheral blood mononuclear cells were identified throughout the course of infection. At the time of euthanasia (20 and 22 weeks), both macaques had lost a significant amount of weight and had no circulating CD4(+) T cells. In addition, one macaque developed intension tremors and uncoordinated movements. Virological examination of tissues at necropsy revealed active virus replication in both lymphoid and nonlymphoid tissues such as the lung and brain. Histological examination revealed that the induced immunodeficiency was associated with lymphoid depletion of the lymph nodes and spleen, opportunistic infections, lentiviral encephalitis, and severe glomerulosclerosis of the kidney. This molecular clone will serve as the basis for analyzing the molecular determinants through which SHIV(KU-2) causes severe CD4(+) T cell loss, neurological disease, and SHIV nephropathy in rhesus macaques.

We recently reported that a chimeric simian/human immunodeficiency virus (SHIVKU-1) developed in our laboratory caused progressive depletion of CD4+ T lymphocytes and AIDS within 6 months of inoculation into pig-tailed macaques (M. nemestrina). None of the pig-tailed macaques showed productive SHIV infection in the central nervous system (CNS). In this report, we show that by further passage of the pathogenic virus in rhesus macaques [M. mulatta], we have derived a new strain of SHIV (SHIVKU-2) that has caused AIDS and productive CNS infection in 3 of 5 rhesus macaques infected with the virus. Productive replication of SHIV in the CNS was clearly shown by high infectivity titers and p27 protein levels in brain homogenates, and in 2 of the 3 rhesus macaques this was associated with disseminated, nodular, demyelinating lesions, including focal multinucleated giant cell reaction, largely confined to the white matter. These findings were reminiscent of HIV-1 associated neurological disease, and our immunohistochemical and in situ hybridization data indicated that the neuropathological lesions were associated with the presence of SHIV-specific viral antigens and nucleic acid respectively. However, the concomitant reactivation of opportunistic infections in these macaques suggested that such pathogens may have influenced the replication of SHIV in the CNS, or modified the neuropathological sequelae of SHIV infection in the rhesus species, but not in pig-tailed macaques. Our findings in the two species of macaques highlight the complexities of lentiviral neuropathogenesis, the precise mechanisms of which are still elusive.

We have examined both the sequence changes in the LTR, gag, vif, vpr, vpx, tat, rev, vpu, env, and nef genes and the cell tropism of a cell-free stock of chimeric simian-human immunodeficiency virus (SHIV) isolated from the cerebrospinal fluid of a pig-tailed macaque (PNb) that developed AIDS. This virus (SHIVKU-1) is highly pathogenic when inoculated into other macaques. DNA sequence analysis of PCR-amplified products revealed a total of 5 nucleotide changes in the LTR while vif had 2 consensus amino acid changes. The gag, vif, and vpx had no consensus amino acid substitutions, whereas vpr had 1 consensus substitution. The tat and rev genes of the HXB2 region of SHIVKU-1 had 2 and 1 consensus amino acid changes, respectively. The vpu gene of the HXB2 region of SHIV, which originally had an ACG at the beginning of the gene, reverted to an initiation ATG codon and in addition contained a consensus amino acid substitution at position 69 of this protein. As expected, the majority of the nucleotide substitutions were found in the env and nef genes. Thirteen and 5 amino acid changes were predicted for the corresponding Env and Nef proteins, respectively. In addition, one-third of the env gene clones isolated from the SHIVKU-1 stock had a 5-amino-acid deletion in the V4 region. Using three independent assays, we determined that the changes in the SHIVKU-1 were associated with an increase in the efficiency of replication in macrophages. The strikingly few consensus changes in the virus suggest that conversion of this virus to one capable of causing AIDS in pig-tailed macaques was associated with relatively few changes in the viral envelope and/or accessory genes. These results will provide the basis for the development of a pathogenic, molecular clone of SHIV capable of causing AIDS in pig-tailed macaques.

No abstract