Genetic diversity and relationships among Venezuelan equine encephalitis virus field isolates from Colombia and Venezuela.

During field studies of enzootic Venezuelan equine encephalitis (VEE) viruses associated with epizootic emergence, a large number of virus isolates were made in sylvatic foci of Venezuela and Colombia. To rapidly characterize these isolates, antigenic subtypes were determined by means of immunofluorescence and by single-strand conformational polymorphism (SSCP) analysis by use of an 856-bp fragment from the P62 gene, which we used to distinguish genetic variants. Representative isolates were sequenced to assess the sensitivity of SSCP to detect genetic differences. The SSCP analysis distinguished isolates differing by as little as 1 nucleotide; overall, differences of > or = 1 nucleotide were recognized 89% of the time, and the sensitivity to distinguish strains that differed by only 1 or 4 nucleotides was 17 and 57%, respectively. Phylogenetic analyses of representative sequences showed that all recent isolates from the Catatumbo region of western Venezuela and the middle Magdalena Valley of Colombia were closely related to epizootic subtype IAB and IC strains; strains from Yaracuy and Miranda States were more distantly related. Cocirculation of the same virus genotype in both Colombian and Venezuelan foci indicated that these viruses are readily transported between enzootic regions separated by > 300 km. The SSCP analysis appears to be a simple, fast, and relatively efficient method of screening VEE virus isolates to identify meaningful genetic variants.

Detection of genetic diversity among bovine immunodeficiency virus population by single-strand conformation polymorphism analysis.

Serial virus specimens rescued from rabbits, experimentally infected with bovine immunodeficiency (BIV) strain R29, were monitored for changes in quasispecies population, using the single-strand conformation polymorphism (SSCP) analysis. The generation of characteristic SSCP patterns enables the rapid differentiation of BIV variants derived from the conserved part on the env region of the BIV genome, reducing the need for expensive and time-consuming direct sequencing analyses. Our results showed genetic polymorphism among a number of sampled BIV population in experimentally infected rabbits. At least three SSCP patterns (BIV quasispecies) were detected. The SSCP analysis allows for an easy, sensitive, and rapid screening of genetic variants of the virus and the assessment of variation at a number of tissue target sites. These variations may relate to cell-type targets and/or disease progression, and could be significant to our understanding of lentiviral pathogenesis.

Selective in vitro protection of SIVagm-induced cytolysis by ajoene, [(E)-(Z)-4,5,9-trithiadodeca-1,6,11-triene-9 oxide].

We studied the effect of synthetic ajoene on simian immunodeficiency virus (SIVagm)-mediated cell fusion and subsequent virus-induced cytolysis. Our data indicate that this compound is a strong antifusion agent with a 50% syncytium inhibitory concentration (SIC50%) value of about 2.9 microM. We suggest that ajoene interacts with the cell-specific integrin molecules and sterically hinders the association between fusion (or other co-receptors) and the CD4-gp120 complex at the cell surface of SIV-infected cells. Although ajoene was maximally effective in suppressing syncytium formation during the early period (ie, up to 6 h) of the fusion process, when the compound was recurrently added to the co-cultures, the inhibitory effect was regained and further cell death was markedly delayed. This indicates that ajoene was also effective after the initial cell-to-cell contact stage. These data suggest that ajoene may be a promising approach for the treatment of SIV/human immunodeficiency virus (HIV) infections.

Infection of rabbits with R29 strain of bovine immunodeficiency virus: virulence, immunosuppression, and progressive mesenteric lymphadenopathy.

To assess the value of bovine immunodeficiency virus (BIV) infection as a model for human immunodeficiency virus (HIV) infection in man, we studied the impairment of certain immunologic functions in New Zealand white rabbits experimentally infected with an uncloned virulent isolate of the virus, BIV R29. Serum samples were tested by Western blot for the presence and persistence of antibody production. The T- and B-lymphocyte function was studied by evaluation of the blastogenic responsiveness to concanavalin A (Con A) and to dextran sulfate (DxS). All infected rabbits seroconverted to BIV antigens within 2 to 4 weeks postinfection (p.i.) The BIV was isolated from the peripheral blood lymphocytes (PBLs) of 13 of 17 rabbits (77%) early in the infection and also from 5 of 17 hyperplastic mesenteric lymph nodes (29%) and 10 of 17 spleens (59%) during the chronic stage of infection. Seven of 17 BIV-infected rabbits (41%) developed marked immunodepression 2 to 5 months p.i., and later, 5 exhibited a rapidly progressive disease with anorexia, weight loss, neurologic impairment, splenomegaly, and mesenteric lymphadenopathy. These data underline the value of the BIV model for studying HIV pathogenesis in vivo and the development of interventional strategies for AIDS.

Anti-HIV activity of extracts from Calendula officinalis flowers.

Extracts of dried flowers from Calendula officinalis were examined for their ability to inhibit the human immunodeficiency virus type 1 (HIV-1) replication. Both organic and aqueous extracts were relatively nontoxic to human lymphocytic Molt-4 cells, but only the organic one exhibited potent anti-HIV activity in an in vitro MTT/tetrazolium-based assay. In addition, in the presence of the organic extract (500 micrograms/mL), the uninfected Molt-4 cells were completely protected for up to 24 h from fusion and subsequent death, caused by cocultivation with persistently infected U-937/HIV-1 cells. It was also found that the organic extract from Calendula officinalis flowers caused a significant dose- and time-dependent reduction of HIV-1 reverse transcription (RT) activity. An 85% RT inhibition was achieved after a 30 min treatment of partially purified enzyme in a cell-free system. These results suggested that organic extract of flowers from Calendula officinalis possesses anti-HIV properties of therapeutic interest.

In vitro suppression of HIV-1 replication by ajoene [(e)-(z)-4,5,9-trithiadodeca-1,6,11-triene-9 oxide].

Studies were performed to establish whether synthetic ajoene exhibited differential inhibitory activity against human immunodeficiency virus (HIV)-1 (IIIB) and to clarify the mechanism of its antiviral effects. Our results demonstrate that ajoene protected acutely infected Molt-4 cells against HIV-1 and blocked further destruction of CD4 T-cells in vitro. Ajoene showed dose-dependent inhibition, with 50% cytotoxic concentration (CTC50%) and 50% effective inhibitory concentration (EIC50%) values of 1.88 microM and about 0.35 microM, respectively, when the test compound was added before or after HIV-1 infection and incubation carried out at 37 degrees C for 4 days. Ajoene proved relatively more active than dextran sulfate in blocking HIV-1 virus-cell attachment. The mode of anti-HIV action of ajoene can be ascribed to the inhibition of early events of viral replication, particularly virus adsorption.

Different effects of phorbol ester derivates on human immunodeficiency virus 1 replication in lymphocytic and monocytic human cells.

The mode of action of the phorbol 12,13-dibutyrate (PDBu) and phorbol 12-myristate 13-acetate (PMA) on the human immunodeficiency virus 1 (HIV-1) replication in human lymphocytes and monocytes was studied. PDBu and PMA appear to have similar effects on the regulation of HIV-1 replication in acutely infected cells. Here we show a significantly increased replication of HIV-1 induced by PDBu and PMA in Molt-4 and Jurkat cells, but a reduced replication in THP-1 and U-937 cells. Moreover, quantitatively different activity of the two derivatives in relation to HIV-1 replication was observed. PDBu proved to be a stronger stimulator or suppressor of HIV-1 replication as compared to PMA. Although the precise mechanism of the activation of HIV-1 replication by phorbol ester derivatives is not clear, it can be assumed that the hydrophilycity of PDBu may cause its stronger effect.

Possible role of bovine immunodeficiency virus in bovine paraplegic syndrome: evidence from immunochemical, virological and seroprevalence studies.

Bovine paraplegic syndrome (BPS) is a debilitating cattle disease of unknown origin that is characterized by leukocytosis, lymphocytopenia and monocytopenia. The major clinical signs are difficulties in locomotion affecting hind limbs, hypoalgesia in the hind quarters, posterior paralysis and death within 72 to 96 hours after recumbency. To investigate the aetiological basis of BPS, we examined a possible association of the syndrome with infection by bovine immunodeficiency virus (BIV), a lentivirus implicated in immune system dysfunction and central nervous system lesions in cattle. Serum samples (n = 1,278) were collected from both healthy and BPS-prevalent cattle herds in Venezuela, and organ extracts were prepared from euthanized animals (n = 11) suspected of having BPS. Sera were analysed for reactivity to recombinant BIV and bovine leukaemia virus gag precursor proteins by immunoblot procedures. Serum reactivity to BIV ranged from 12 to 66% between groups of BPS prevalent herds. The percentage of samples reactive to BLV antigen was much lower (2 to 17%). Rabbits inoculated with extracts from BPS-afflicted animals exhibited an anamnestic immune response to BIV antigens as well as the presence of BIV gag antigens in their tissues. We present evidence for a possible association between BPS disease and a viral agent related to BIV. The role of BIV, in combination with malnutrition, in BPS is discussed.

Acquired immune dysfunction in rabbits experimentally infected with an infectious molecular clone of the bovine immunodeficiency virus (BIV127).

To investigate the effect of bovine immunodeficiency virus (BIV) infection on the rabbit immune system, we studied the proliferative responses of peripheral blood lymphocytes (PBLs) of rabbits experimentally inoculated with BIV. All BIV127-inoculated rabbits seroconverted after 6 weeks and remained seropositive over a prolonged period of time. Assays for specific lymphocyte reactivity to concanavalin A (Con A) were performed monthly for over 1 year. One-hundred percent of infected rabbits developed abnormally low T cell responses, as measured by Con A stimulation. By 3 months postinoculation, the PBL response to Con A was diminished and remained depressed for 6 months. All animals were clinically asymptomatic within 14 months of BIV inoculation. By 15 and 16 months postinoculation, two of three infected rabbits exhibited recurrent lowering of the T cell responsiveness including a decrease in absolute PBL counts. One of these animals died unexpectedly. Our results further confirmed that a functional impairment of lymphocytes was induced early in the course of BIV infection, prior to clinical disease. These findings suggested that BIV infection may mimic asymptomatic infection of human immunodeficiency virus (HIV) and provided further evidence of the importance of BIV-induced disease in rabbits as a relevant model for the study of AIDS.

[Natural products from the tropical rain forest of Venezuela as inhibitors of HIV-1 replication]. / Productos naturales del bosque húmedo tropical de Venezuela, inhibidores de la replicación de VIH-1.

More than 100 plant extracts from the Amazonian rain forest of Venezuela were evaluated for their cytotoxicity and inhibitory activity against the human immunodeficiency virus type-1 (HIV-1). Aqueous extracts from Fomitella supina (S # 0389-4), Phellinus rhabarbarinus (S # 0389-7), Trichaptum perrottetti (S # 0389-8) and Trametes cubensis (S # 0389-13), Polyporaceae exhibited strong anti-HIV-1 activity, without toxicity for Molt-4 lymphocytic cells. Our results demonstrated, that the compound(s) acted by mechanism of direct virion inactivation and by inhibition of syncytium formation in an in vitro culture system. These results support the suggestion that the test extracts specifically act at the level of CD4-gp120 binding. The active components of these extracts is at present unknown, but anti-AIDS agents, such as those found in this study, individually or in combination, may be of therapeutic relevance.

Use of immunogold assay for a rapid evaluation of antigens of spotted fever group rickettsiae.

Rabbit-antihuman IgG, yolk immunoglobulins (IgY) and monoclonal antibodies against Rickettsia conorii were adsorbed to colloidal gold. Thus obtained conjugates were found to be active and responded specifically to the agent of the Mediterranean Spotted fever. The suitability of the immunogold assay as a alternative to other known immunoenzymatic or immunofluorescent methods for assessing the presence and/or activity of rickettsial antigens is discussed.