Viral reactivation and pseudotype production in an in vitro superinfection system with two different strains of HIV-1.

Viral production and variability of HIV-1 is normally high in vivo causing the necessary conditions for cellular superinfection. In order to evaluate the superinfection dynamics in vitro, H9HTLVIIIB cell line was superinfected with HIVMN. Superinfected cells showed nearly 50% cell mortality at day 1 post-superinfection (ps), which increased significantly up to day 4 ps. Superinfecting genome was detectable until day 10 ps. The superinfecting strain was found in the supernatant only on day 1 ps, but was recovered up to day 4 ps by coculture with non-infected cells. The existing strain (HIVHXB2) was recovered throughout the studied period. Pseudotype formation by the HIVHXB2 genome and envelope proteins of the superinfecting strain (HIVMN) was observed from day 1 to 6 ps. Viral production was increased by 1.7 LOG in superinfected cells from day 1 ps. Both viral production increase and pseudotype formation could be relevant for HIV pathogenesis in vivo.

Oxytocin and prostaglandins F2alpha and E2 do not enhance HIV antigen production in vitro.

Oxytocin and prostaglandins (PGs) are hormones involved in labor and are used clinically for its induction. In this study the effect of oxytocin, PGF(2alpha), and PGE(2) on Humour immunodeficiency virus-1 production in acutely and persistently infected cells was measured. No significant effect on p24 antigen production was found with oxytocin or PGs, except for a transient decrease in persistently infected cells treated with 1 micro M PGF(2alpha). These results showed that oxytocin and PGs could be used clinically for labor induction without any direct enhancement in viral production. Besides, the results with PGF(2alpha) at the highest concentration studied may indicate a pharmacological effect.

Bioactive oleanolic acid saponins and other constituents from the roots of Viguiera decurrens.

The bisdesmoside oleanolic acid saponin, 3-0-(methyl-beta-D-glucuronopyranosiduronoate)-28-0-beta-D-glucopyranosyl-oleanolate along with nine known compounds (two diterpenic acids, one chromene, three triterpenes, one steroidal glycoside, and two monodesmoside oleanolic acid saponins), were obtained from Viguiera decurrens roots. The chemical structure of the bisdesmoside oleanolic saponin was determined by chemical and NMR spectral evidence. A mixture of monodesmoside saponins displayed cytotoxic activity against P388 and COLON cell lines (ED50= 2.3 and 3.6 microg/ml, respectively). Two of the known compounds showed insecticidal activity against the Mexican bean beetle larvae (Epilachna varivestis).

HIV-2 genetic variation and DNA load in asymptomatic carriers and AIDS cases in Guinea-Bissau.

The purpose of this study was to document which genetic subtypes of HIV-2 are present in Guinea-Bissau and to investigate whether asymptomatic HIV-2 carriers and AIDS patients carry distinct genetic variants. A secondary aim was to correlate proviral DNA load to clinical and immunologic status of the patients. Thirty-eight asymptomatic HIV-2 carriers and 11 AIDS patients from Bissau, Guinea-Bissau were included in a cross-sectional study in which HIV-2 env V3 sequences, HIV-2 DNA load, and CD4-positive (CD4+) lymphocyte counts were determined. Phylogenetic analyses showed that all investigated subjects carried subtype A HIV-2 variants and that the sequences from AIDS patients and asymptomatic carriers did not form distinct subclusters in the tree. As expected, patients with AIDS had significantly higher median HIV-2 DNA load than did asymptomatic carriers (4.6 vs. 2.0 log10 HIV-2 DNA copies/10(6) CD4+ lymphocytes). Our study indicates that the HIV-2 epidemic in Guinea-Bissau is almost exclusively caused by subtype A HIV-2 variants and that the HIV-2 infections among the asymptomatic carriers and AIDS cases included in the study do not have distinct epidemiologic histories.

PIP: HIV-2 is associated with AIDS but is less pathogenic than HIV-1. HIV-2 is endemic in West Africa, with the highest prevalence in Guinea-Bissau; epidemiologic studies in the country have found HIV-2 seroprevalence in the general population to be 8-10%. HIV-2 seroprevalence increases with age and peaks near age 50-59 years, although the mean age of HIV-2-associated AIDS cases is near 40 years. Only scattered cases of HIV-2 have been reported outside of West Africa, with some concentration in Portugal, France, and India. 38 asymptomatic HIV-2 carriers and 11 AIDS patients from Bissau were included in a cross-sectional study in which HIV-2 env V3 sequences, HIV-2 DNA load, and CD4-positive lymphocyte counts were determined. Phylogenetic analyses showed that all investigated subjects carried subtype A HIV-2 variants and that the sequences from AIDS patients and asymptomatic carriers did not form distinct subclusters in the tree. Subjects with AIDS had significantly higher median HIV-2 DNA load than did asymptomatic carriers.

Different integrated and unintegrated HIV-1 DNA after superinfection and cell to cell transmission.

Efficient superinfection of H9HTLVIIIB cell line (persistently infected with HIVHXB2 strain) with HIVMN strain is reported. The superinfecting viral DNA was found in the chromosomic and extrachromosomic fractions at early stages, but at 48 hours post superinfection, it remained mainly unintegrated. Interestingly, superinfected cells only produced HIVHXB2 in the supernatant and no increase of viral yield of this persistent virus was observed. Remarkably, virions of both strains. HIVHXB2 and HIVMN, were recovered after cocultivating superinfected cells with MT2 cell line. In the extrachromosomic fractions of seven different superinfected subclons of H9HTLVIIIB, viral DNA of the superinfecting HIVMN strain predominated while in the chromosomic fraction, the proportion of superinfecting viral DNA differed. The study of the presence of different integrated and unintegrated genomes in a single cell could be crucial in the understanding of HIV biology.

Different integrated and unintegrated HIV-1 DNA after superinfection and cell to cell transmission

Efficient superinfection of H9HTLVIIIB cell line (persistently infected with HIVHXB2 strain) with HIVMN strain is reported. The superinfecting viral DNA was found in the chromosomic and extrachromosomic fractions at early stages, but at 48 hours post superinfection, it remained mainly unintegrated. Interestingly, superinfected cells only produced HIVHXB2 in the supernatant and no increase of viral yield of this persistent virus was observed. Remarkably, virions of both strains, HIVHXB2 and HIVMN, were recovered after cocultivating superinfected cells with MT2 cell line. In the extrachromosomic fractions of seven different superinfected subclons of H9HTLVIIIB, viral DNA of the superinfecting HIVMN strain predominated while in the chromosomic fraction, the proportion of superinfecting viral DNA differed. The study of the presence of different integrated and unintegrated genomes in a single cell could be crucial in the understanding of HIV biology. (AU)

Different integrated and unintegrated HIV-1 DNA after superinfection and cell to cell transmission

Efficient superinfection of H9HTLVIIIB cell line (persistently infected with HIVHXB2 strain) with HIVMN strain is reported. The superinfecting viral DNA was found in the chromosomic and extrachromosomic fractions at early stages, but at 48 hours post superinfection, it remained mainly unintegrated. Interestingly, superinfected cells only produced HIVHXB2 in the supernatant and no increase of viral yield of this persistent virus was observed. Remarkably, virions of both strains, HIVHXB2 and HIVMN, were recovered after cocultivating superinfected cells with MT2 cell line. In the extrachromosomic fractions of seven different superinfected subclons of H9HTLVIIIB, viral DNA of the superinfecting HIVMN strain predominated while in the chromosomic fraction, the proportion of superinfecting viral DNA differed. The study of the presence of different integrated and unintegrated genomes in a single cell could be crucial in the understanding of HIV biology.

Molecular epidemiology and MT-2 cell tropism of Russian HIV type 1 variant.

Twenty-two HIV-1-infected Russian individuals were studied to gain better insight on the genetic and biological characteristics of HIV-1 variants present in Russia. The V3 domain of the HIV-1 envelope was directly sequenced from cultured peripheral blood mononuclear cells (PBMCs). Phylogenetic analyses were used to determine the HIV-1 genetic subtype and to study transmission patterns. Virus isolates were obtained from PBMCs and the biological phenotype was determined by coculture with MT-2, CEM, and Jurkat-tat cells. Twelve homo- and bisexual men carried subtype B variants, whereas 6 heterosexually infected individuals carried subtype F virus. A heterosexual man infected in the Ivory Coast and a nosocomially infected young female carried subtype A and G virus, respectively. Interestingly, phylogenetic analyses suggested that subtype B may have entered Russia on at least four occasions and, even more surprisingly, that the relatively rare subtype F has entered Russia on at least three occasions. Two suspected transmission clusters of subtype F were supported by the phylogenetic analyses, whereas one suspected subtype B transmission cluster was not. Positively charged amino acids in positions 311 and 325 of V3 loop have been shown to be important determinants for the MT-2-positive biological phenotype of virus isolates. Interestingly, we found that the direct PBMC sequences from individuals with MT-2-positive isolates displayed specific neutral, instead of positively charged, amino acids, in these positions. This indicates that it may be more difficult to predict the biological phenotype of HIV-1 using sequences from uncultured PBMCs than from virus isolates.

Bioactive polyacetylenes from Bidens pilosa.

From the methanolic extract of the whole plants of Bidens pilosa the new beta-D-glucopyranosyloxy-3-hydroxy-6(E)-tetradecen-8, 10,12-triyne besides a known polyine has been isolated and identified mainly by IR and NMR methods. The new compound showed overgrowing action against normal and transformed human cell lines in culture.

Biological phenotype of HIV type 2 isolates correlates with V3 genotype.

The biological phenotype of HIV-2 isolates can be divided into two groups, rapid/high and slow/low, based on the ability to infect CD4+ tumor cell lines. Similar differences in the biological phenotype of HIV-1 isolates are largely determined by the charge of two specific amino acids in the V3 loop of the envelope protein gp120. In this study we have sequenced the V3 loop and flanking regions of 14 HIV-2 isolates from Guinea-Bissau and the Ivory Coast and correlated the results to the biological phenotype of the isolates. The sequences were obtained by PCR amplification of DNA from peripheral blood mononuclear cells infected with the different isolates, followed by direct sequencing of the amplified products. Eleven other HIV-2 isolates with known V3 sequence and biological phenotype were also included. Thirteen of the 14 new isolates were classified as subtype A of HIV-2 and one as subtype B. The V3 loop of rapid/high HIV-2 isolates differed significantly from slow/low isolates in that it was more heterogeneous in sequence and had higher net charge. Mutations at two specific amino acid positions (313 and 314), often to positively charged amino acids, were also significantly associated with the rapid/high phenotype. There were no sequence differences between rapid/high and slow/low isolates in the regions that flank the V3 loop. Our findings indicate that there may be a high degree of similarity in the molecular features that underlie the biological phenotypes of HIV-1 and HIV-2 isolates.

Biological and molecular characterization of subtype D, G, and A/D recombinant HIV-1 transmissions in Sweden.

HIV-1 can be subdivided into at least nine genetic subtypes (A through H and O), but in Europe and the United States there is an almost complete dominance of subtype B. In this study three Swedish HIV-1 transmission chains of subtypes other than subtype B have been biologically and molecularly characterized. The three index cases were African men. The p17 gag and env V3 regions of the HIV-1 genome were directly sequenced from uncultured lymphocytes. Phylogenetic analyses showed that the HIV-1 variants with each transmission group were genetically closely related, supporting the epidemiological information. The individuals in transmission groups I (n = 3) and II (n = 2) carried subtype G and D virus, respectively. Interestingly, all four individuals in transmission group III displayed a recombinant genotype with subtype D p17 gag sequence and subtype A V3 sequence. The biological phenotype of virus isolates (rapid/high, syncytium-inducing; or slow/low, non-syncytium-inducing) correlated with the clinical stage of the infected individual. The study also suggested that the correlation between biological phenotype and V3 genotype that has been established for subtype B HIV-1 variants may be valid also for other subtypes. This study demonstrates that HIV-1 variants of subtypes other than B, including a subtype A/D recombinant, are being transmitted in Europe.