Long-term culture of human immunodeficiency virus type 1 resulting in loss of glycosylation sites.

Cultures of human immunodeficiency virus type 1 (HIV-1) provided a model for the study of mutations in the absence of host antibodies. Replicate cultures of biological and molecular clones of HIV-1 were passaged weekly for 30 or 34 weeks. Eight regions of HIV-1 genomic RNA were analyzed by means of single-strand conformation polymorphism analysis and nucleotide sequencing. Six mutations were detected in the biological clones. Two were G-->A substitutions. The frequency of mutations was higher in V1 compared to that in other regions (P = 0.01). Three mutations involved loss of potential glycosylation sites in V1. These results show that mutations in the viral genome may result from selection by factors other than host immune pressures.

Co-receptor usage was more predictive than NSI/SI phenotype for HIV replication in macrophages: is NSI/SI phenotyping sufficient?

A monocyte-derived macrophage (MDM) culture assay was used to define the replication kinetics of HIV isolates. Ten-day-old MDMs were infected with HIV. Supernatants were collected and assayed for HIV p24 on days 3, 7, 10, and 14 post-infection (PI). In this assay, SF162 (macrophage tropic, NSI) produced increasing amounts of HIV p24 antigen with increasing time in culture. BRU (nonmacrophage tropic, SI) infection resulted in low levels of HIV p24 antigen with no increase in production during the culture period. A panel of 12 clinical isolates was evaluated. All isolates produced detectable levels of HIV p24 antigen in MDMs. However, the NSI viruses had significantly higher log10 HIV p24 antigen values at all times PI (P < 0.01). Co-receptor usage was determined for all 12 isolates (8 NSI and 4 SI). All SI isolates used CXCR4 for entry; two used CXCR4 only, one used CXCR4, CCR5, and CCR3, and one was a mixture of two isolates using CXCR4 and CCR5. None of the NSI viruses used CXCR4 for entry. All used CCR5 as their predominant co-receptor. Of the eight NSI isolates, three used CCR5 only, two used CCR5 and CCR2b, one used CCR5 and CCR3, and one used CCR5, CCR3, and CCR2b. Log10 HIV p24 antigen production on day 14 PI for viruses that used CCR5+CCR3 (3.79 + 1.40) was greater than for viruses that used CCR5+CCR2b (3.22 + 1.55) or CCR5 (3.32 + 1.49), and all were greater than those that used CXCR4 only (1.69 + 0.28), regardless of SI phenotype (P < 0.05). Thus, in these primary isolates, macrophage tropism and replication kinetics were closely linked to CCR5 utilization, whereas SI capacity was closely linked to CXCR4 utilization. Furthermore, viruses, which could use CCR5 and CCR3 for entry, had a replication advantage in macrophages, regardless of SI phenotype.

[Cerebrospinal fluid serotonin concentration in pyogenic and tick-borne encephalomeningitis]. / Stezenie serotoniny w plynie mózgowo-rdzeniowym w przebiegu ropnych i kleszczowych zapalen opon mózgowo-rdzeniowych i mózgu.

Cerebrospinal fluid (CSF) serotonin was determined in 38 patients with pyogenic meningitis and tick-borne encephalitis (TBE) before and after treatment. Increase of CSF serotonin concentration was observed in acute phase of pyogenic meningits and normalized after treatment.

Selection of appropriate HIV-1 genomic regions for single-strand conformation polymorphism analysis of the diversity, modification, and transmission of HIV-1 quasispecies.

Single-strand conformation polymorphism (SSCP) analysis is a useful tool for studying viral quasispecies. Four regions within the HIV-1 genome were studied by means of SSCP analysis with the aim of determining which regions were the most informative for the study of HIV-1 transmission or for detection of changes in HIV-1 quasispecies populations. Nested polymerase chain reaction (PCR) was used to amplify V1, V2, V3 of the env gene, and the p2 region in the gag gene. In total, 114 plasma specimens from 79 individuals were tested, including serial specimens from 10 mother-infant pairs that were provided by the Women and Infants Transmission Study (WITS). HIV-1 in specimens that were PCR-positive with primer pair SK38/SK39 showed different percentages of positive signals with primer pairs for the four regions: V1, 63%; V2, 83%; V3, 88%, and p2, 100%. HIV-1 sequences in the p2 target region displayed the greatest degree of polymorphism. Analysis of serial specimens showed that the V1 target region was the most variable of the four regions studied and was the most appropriate region for monitoring changes in quasispecies populations. Of the four regions studied, p2 was the most informative for the study of HIV transmission, as shown by analysis of samples from documented cases of mother to infant HIV-1 transmission.

Transendothelial migration of lymphocytes from HIV-1-infected donors: a mechanism for extravascular dissemination of HIV-1.

To identify factors that cause HIV-1 to establish perivascular foci of infected cells, we studied the transendothelial migration of blood mononuclear leukocytes (MNL) from 76 HIV+ patients and 41 controls. The fraction of patients' lymphocytes that migrated across endothelial cell monolayers in vitro was significantly increased (p < or = 0.03) compared with that of control donors. Migration of patients' CD4+ T cells was particularly enhanced, whereas the migration of monocytes did not differ between patients and controls. Lymphocyte migration correlated with expression of CD11a/CD18 and CD49d/CD29 and with the quantity of TNF-alpha produced as MNLs migrated through the endothelium. Measurement of HIV-1 proviral DNA copies in the patients' MNLs (n = 26) suggested that in half the cases virus-infected cells accumulated preferentially amidst the migratory leukocytes. We observed the same behavior with normal donor MNLs infected, in vitro, with each of 4 strains of HIV-1. The number of HIV-1 proviral DNA copies per million MNLs was 40 to 178 times higher in the migratory population than in the original population added to the endothelium. To test whether only certain strains of HIV-1 stimulate transendothelial migration of infected cells, we used single strand conformation polymorphism analysis to identify quasispecies of HIV-1 in the MNLs. If all strains of HIV-1 were equal in their ability to stimulate transendothelial migration, we expected to find no differences in the quasispecies present in the original and migratory cell populations. In fact the quasispecies differed in 14 of 19 paired samples, suggesting that only certain HIV-1 quasispecies promote transendothelial migration of infected cells.

Kernel density analysis of variable and conserved regions of the envelope proteins of human immunodeficiency virus type 1 and associated epitopes.

A new statistical approach to the study of conservation of amino acid and nucleotide sequences based on kernel density analysis is described that enables analysis of both conserved and highly variable HIV-1 protein sequences. The amino acid sequences of HIV-1 env proteins in 63 isolates were analysed to determine, first, whether the designations of regions identified in 1987 as conserved (C1-C6) or variable (V1-V5) were still valid. Even though the data base used was nine times larger, the designations that were based on seven isolates from five patients remain correct. Second, the new approach enabled the quantifications of the degree of conservation in reported B or T cell epitopes. Using this approach, highly conserved epitopes located in both gp41 and gp120 were identified.

Analysis of sewage effluent for human immunodeficiency virus (HIV) using infectivity assay and reverse transcriptase polymerase chain reaction.

Environmental survival of human immunodeficiency virus type 1 (HIV-1) is an important public health concern. Survival of HIV in waste water is of particular interest to those who work at treatment facilities and to the general public who have contact with rivers or ocean water receiving treated sewage effluent. Other researchers have reported that HIV can be detected in waste water. Their studies, however, detected homologous nucleic acid sequences but did not attempt to determine infectivity. The current study tested primary and secondary effluent from a major metropolitan sewage agency for the presence of HIV-1 using reverse transcriptase polymerase chain reaction (RT-PCR), HIV-1 p24 antigen enzyme-linked immunosorbent assay, and infectivity testing. For RT-PCR, primers SK38/SK39 and M667/AA55 were used to identify HIV-1 RNA sequences from concentrated and extracted sewage samples. Infectivity assays employed donor peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin. Coxsackievirus B4, echovirus 7, and poliovirus 1, enteroviruses normally present in sewage, were tested for replication in PBMCs. Poliovirus 1 was found to infect the PBMCs. To eliminate other enteroviruses that may also infect the PBMCs and interfere with HIV-1 testing, concentrated sewage was treated with human immunoglobulin (free of HIV antibodies) and poliovirus antisera before infectivity assays were performed. All treated sewage samples tested negative for HIV-1 by all methods used. HIV-1 seeded into sewage, however, remained infectious in the assay, indicating that the sewage water sample did not interfere with HIV infectivity nor was it toxic to the PBMCs.

Single-strand conformation polymorphism study of human immunodeficiency virus type 1 RNA and DNA in plasma, peripheral blood mononuclear cells, and their virologic cultures.

Single-strand conformation polymorphism (SSCP) analysis was applied to human immunodeficiency virus type 1 (HIV-1) nucleic acids in plasma and peripheral blood mononuclear cells (PBMC) from 16 patients and to 15 PBMC cocultures and 6 plasma cultures prepared from the specimens. Two hypervariable regions were analyzed: in the gag gene and part of the V3 loop. Random paired matching of SSCP patterns between HIV-1 RNA and provirus DNA was tested, from plasma and PBMC from the same blood specimen, supernatant and PBMC from the same PBMC coculture, supernatant and PBMC from the same plasma culture, provirus DNA in cocultured PBMC and the PBMC inoculum, and HIV-1 RNA in a plasma culture supernatant and in the plasma inoculum. Paired matching was nonrandom for both regions in the first three situations and for gag in the fourth, with P < or = .01; matching was random for gag in the last situation. The HIV-1 env target region produced in culture diverged from that in the inoculum in 18 of 21 instances.

Anti-idiotype-based vaccines against biological toxins.

Biological toxins produced by living organisms represent one of the major sources of contamination of stored grain and agricultural products, and other food sources. The majority of these biological toxins are highly lethal, nonproteinaceous low-molecular-weight chemical compounds which exert their potent toxicity through a variety of mechanisms. Because of their small size, they generally do not induce a significantly high affinity protective antibody response upon toxin exposure, even when conjugated to large protein carriers which enhance their immunogenicity. Moreover, the very toxic nature of biological toxins precludes their use as immunogens in the induction of protective immunity. To circumvent this difficulty, an attempt was made to develop antibody (anti-idiotype)-based vaccines against a protein synthesis inhibitor, the trichothecene mycotoxin T-2, and the sodium channel blockers tetrodotoxin and saxitoxin. Protective monoclonal antitoxin antibodies were first generated and then used to induce specific monoclonal anti-idiotype antibodies. Specific anti-idiotype antibodies were assessed for their ability to induce in vivo protective immunity against toxicity.

Effect of sunlight on survival of indicator bacteria in seawater.

The stability of the natural populations of fecal coliforms and fecal streptococci in raw sewage diluted 1:1,000 in seawater or phosphate-buffered water at 24 +/- 2 degrees C was markedly affected by the absence or presence of sunlight. In the absence of sunlight, these bacteria survived for days, whereas in the presence of sunlight 90% of the fecal coliforms and fecal streptococci were inactivated within 30 to 90 min and 60 to 180 min, respectively. The bactericidal effect of sunlight was shown to penetrate glass, translucent polyethylene, and at least 3.3 m of clear seawater, suggesting that the visible rather than the ultraviolet light spectrum of sunlight was primarily responsible for the observed bactericidal effect. However, these same sewage-borne bacteria were relatively resistant to the bactericidal effect of sunlight when diluted in fresh mountain stream waters. These results indicate that the presence of sunlight is a major factor controlling the survival of fecal coliforms and fecal streptococci in seawater.