Prevalence and epidemiological traits of HIV infections in populations with high-risk behaviours as revealed by genetic analysis of HBV.

The prevalence and epidemiological traits of human immunodeficiency virus (HIV)/hepatitis B virus (HBV) infections in high-risk populations (HRPs) remained unclarified in Japan. We determined the prevalence of HIV, HBV and Treponema pallidum (TP) and the viral genotypes in HRPs who attended primary sexually transmitted infection (STI) clinics in Osaka province during 2006-2011. Of 7898 specimens, 133 (1·7%) were HIV positive, which was significantly higher than the figures reported by Japanese Red Cross (0·0019%) and public health centres (0·27%) in Japan. The frequency of HIV-1 subtype B was 88·7%, followed by CRF01_AE (2·3%) and C (0·8%), which were almost identical to the national trend. HBV seroprevalence was surprisingly high in the HIV-positive group (63·2%), which was significantly higher than that in the HIV-negative group (25·6%). By contrast, there was no statistical correlation between HIV and TP infection. Interestingly, the distinct HBV genotypes Ae and G were prevalent in the HIV-positive population (60·0% and 20·0%, respectively), although both were rarely detected during nationwide surveillance. The transmission of HIV and HBV appeared to occur largely within a closed community early in life. Of note, about one-quarter of HIV-positive cases would have remained untested if health professionals had not motivated individuals to undergo HIV testing. This is the first evidence-based assessment of HIV positivity and HIV/HBV co-infection in HRPs at primary STIs in Japan and the effect of the involvement of health professionals in the diagnosis of HIV infections in asymptomatic carriers. The genotyping of HBV provided valuable information for understanding HIV epidemical traits.

A novel substance purified from Perilla frutescens Britton inhibits an early stage of HIV-1 replication without blocking viral adsorption.

Pf-gp6, a 6 kDa anti-degranulation glycoprotein purified from the extract of Perilla frutescens, was examined for its antiviral activity against HIV-1 and HIV-2 in vitro. HIV-1-induced cytopathic effect and proviral DNA synthesis were inhibited in the presence of Pf-gp6. The 50% inhibitory concentrations of Pf-gp6 for various HIV-1 strains, including clinical isolates and CCR5-using (R5) HIV-1, ranged between 1.3 and 71.0 microg/ml, depending on the combination of viral strain and host cell. Furthermore, Pf-gp6 did not directly inactivate infectious viral particles. A time-of-addition experiment revealed that Pf-gp6 lost its activity before zidovudine but after the CXCR-4 antagonist AMD3100 during the early stage of viral infection. Although the pinpoint target of Pf-gp6 remains to be elucidated, it may interfere with a step between viral entry and reverse transcription.

Anti-HIV-1 phorbol esters from the seeds of Croton tiglium.

Five phorbol diesters, together with three known ones, were isolated from a MeOH extract of the seeds of Croton tiglium, and their structures were determined by spectroscopic methods and selective hydrolysis of acyl groups. These compounds were assessed for their abilities to inhibit an HIV-induced cytopathic effect (CPE) on MT-4 cells and to activate protein kinase C (PKC) associated with tumor-promoting action. 12-O-Acetylphorbol-13-decanoate and 12-O-decanoylphorbol-13-(2-methylbutyrate) effectively inhibited the cytopathic effect of HIV-1 [complete inhibitory concentration (IC100) values of 7.6 ng/ml and 7.81 microg/ml, and minimum cytotoxic concentration (CC0) value of 62.5 and 31.3 microg/ml, respectively]; however, 12-O-acetylphorbol-13-decanoate showed no activation of PKC at concentrations of 10 and 100 ng/ml. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was found to be not only the most potent inhibitor of HIV-1-induced CPE (IC100 value of 0.48 ng/ml), but also the most potent activator of PKC (100% activation at 10 ng/ml).

Anti-HIV-1 and anti-HIV-1-protease substances from Ganoderma lucidum.

A new highly oxygenated triterpene named ganoderic acid alpha has been isolated from a methanol extract of the fruiting bodies of Ganoderma lucidum together with twelve known compounds. The structures of the isolated compounds were determined by spectroscopic means including 2D-NMR. Ganoderiol F and ganodermanontriol were found to be active as anti-HIV-1 agents with an inhibitory concentration of 7.8 micrograms ml-1 for both, and ganoderic acid B, ganoderiol B, ganoderic acid C1, 3 beta-5 alpha-dihydroxy-6 beta-methoxyergosta-7,22-diene, ganoderic acid alpha, ganoderic acid H and ganoderiol A were moderately active inhibitors against HIV-1 PR with a 50% inhibitory concentration of 0.17-0.23 mM.

[Detection of genotypic and phenotypic drug-resistant HIV-1 in patients receiving antiretroviral therapy].

We have analyzed the sequences of HIV-1 reverse transcriptase and protease genes in peripheral blood mononuclear cells obtained from patients receiving antiretroviral therapy to evaluate the drug resistance-associated mutations. Of 84 HIV-1-infected individuals treated with reverse transcriptase inhibitors, 43 (51.2%) have been found to carry amino acid substitutions predicted to acquire drug-resistances. One to 3 mutations at amino acid residues reported to be associated with protease inhibitor-resistance were detected in more than 80% of protease inhibitor-naive patients. However, these pre-existing mutations did not seem to raise a real resistance after the initiation of therapy with protease inhibitors. Phenotypic resistance assay was performed with 6 clinical isolates to compare with genotypic resistance. In most of the cases, phenotype was correlated with genotypic changes, however, two strains which were isolated from patients having no experience of chemotherapy showed a decrease in susceptibility to several drugs without any resistance-related mutations detected in their genes. Taken together, determination of phenotypic resistance is necessary, especially when a newly-infected patients starts antiviral therapy.

[Evaluation of antiretroviral chemotherapy based on the profile of virus isolation from HIV-1 infected individuals].

It is well-known that the biological characteristics of HIV-1 persistently infected in the host have often changed into a rapid growth in vitro, T-cell line tropism and marked syncytium inducing (SI) ability accompanied by the progress of clinical stages from AC to ARC or AIDS. We have developed a follow-up diagnostic test using the clinical markers based on the virus phenotypes mentioned above, and reported that the test was significantly useful for determining the clinical status of HIV infected individuals. Recently, highly active antiretroviral therapy (HAART) was introduced into HIV-1 chemotherapy, and has been reported to be a markedly effective treatment for HIV infected individuals. In this study, we carried out an investigation to see whether the follow-up diagnosis was useful even after introducing the HAART in Japan in 1997. The results by the laboratory diagnosis on 139 HIV infected individuals who were clinically observed over a long period showed that the positive rate of virus isolation and MT-4 cell tropism in the isolates during the two years between 1997 and 1998 were significantly lower than that of the nine years from 1988 to 1996 before the implementation of HAART. In addition, we obtained data that the effect of HAART reflects the biological profiles of virus isolation more than the CD4+ T cell counts. These results suggest that data of clinical examination using virus isolation as a parameter are useful not only for predicting the development of AIDS but also for evaluating the effects of HAART, particularly in patients showing no association between the CD4+ T cell counts and the plasma viral load.

[Follow-up of HIV carriers by a laboratory method. I: Virus isolation is useful for prognostication of the onset of symptoms].

We followed 54 HIV-1 carriers (44 asymptomatic carriers and 10 AIDS patients) by virus isolation and immunological examination and evaluated their usefulness for prognostication of the onset of symptoms. From 37 carriers (27 asymptomatic carriers and 10 symptomatic), 132 HIV-1 strains were isolated; the virus isolation rate was 60% in the asymptomatic carriers (AC) but 100% in the symptomatic. In the AC, the isolation rate was 54.5% in the group showing stable in the CD4+ level but 95.5% in the group showing a decrease in the CD4+ level. With progression of the disease, the culture time required for virus isolation was shortened, and the percentage of isolates showing infectivity to the T-cell line (MT-4 cells) increased. These findings suggest that the virus in the body is changed with progression of the disease to that showing rapid replication, T-cell tropic, and high pathogenicity. Indeed, progression of the disease was observed in all carriers in whom a highly pathogenic virus was detected; some developed the disease within 1 year, some showed temporary recovery in the CD4+ level after AZT administration followed by progression to ARC, and others showed a rapid decrease in the CD4+ level. In contrast, in carriers with only slightly pathogenic virus, the CD4+ level was maintained for a long period. These results suggest that the detection of a highly pathogenic virus is one of the most reliable marker for the prognostication of the onset of the disease. The detection of HIV-1 antigen in the plasma and a decrease in the gag antibody were also associated with the progression of the disease. However, the reliability of these markers seems to be lower than that of virus isolation.

[Prognosis and evaluation of drug therapy by V3 and RT gene analysis of HIV-1].

We have reported on the investigation of 54 HIV-1 patients concerning the isolation and clinical marker in the preceding paper. We have attempted the analysis of the V3 and RT genes. HIV-1 from a patient who had rapidly taken a turn for the worse had basic amino acid at position 11 (Arg) and lost an acidic amino acid at position 25 (Gln) of V3. This sequence pattern was a distinguished feature of a virus with a rapid-high, syncytium inducing (SI) and T-cell-line tropic phenotype. In contrast, patients with no or mild clinical symptoms had sequences characterized as slow-low, non-synsytium inducing (NSI) and macrophage tropic. We then investigated the appearance of resistant to AZT and ddI. It was shown that the virulent virus obtained drug resistant variants faster than the wild type by analysis of the RT gene. We consider that these data concerning virus isolation and gene analysis are useful for prognosis and strategy for clinical therapy.

Novel method of inactivation of human immunodeficiency virus type 1 by the freeze pressure generation method.

It has been reported that high-pressure (over 600 MPa) treatment at room temperature inactivates human immunodeficiency virus type 1 (HIV-1), and it has recently been shown that the high pressure generated by the expansion of water due to freezing (freeze pressure generation method, or FPGM) has an inactivating effect on bacteria and fungi. In this study, we examined the effects of treatment by FPGM on HIV-1. A sturdy vessel filled with water and securely closed with a lid was kept at 0 degrees C to -30 degrees C. High pressures of 200 MPa and 250 MPa were generated at -20 degrees C and -30 degrees C, respectively. When T-cell-tropic and macrophage-tropic laboratory strains of HIV-1 were kept at -10 degrees C, the virus infectivity decreased to approximately 1/100, and was completely lost at -20 degrees C and -30 degrees C. Four T-cell-tropic and four macrophage-tropic laboratory strains and clinical isolates of HIV-1 became completely inactivated at -30 degrees C. Treatment by FPGM at -20 degrees C to -30 degrees C reduced HIV-1 reverse transcriptase activity to approximately one tenth. In addition, treatment by FPGM at -20 degrees C was found to destroy the ability of HIV-1 to bind to CD4+ cells. In conclusion, this study showed that treatment by FPGM at -20 degrees C to -30 degrees C destroyed the infectivity of a wide range of HIV-1 strains, and suggested that the mechanisms of HIV-1 inactivation were the reduction in viral enzyme activity and the loss of the cell-binding ability of a viral envelope protein.

12-O-acetylphorbol-13-decanoate potently inhibits cytopathic effects of human immunodeficiency virus type 1 (HIV-1), without activation of protein kinase C.

Through bioactivity-guided fractionation, eight phorbol diesters, including five new ones (1-5), were isolated from the seeds of Croton tiglium collected in Egypt. 12-O-Acetylphorbol-13-decanoate (6) and 12-O-decanoylphorbol-13-(2-methylbutyrate) (4) potently inhibited the HIV-1-induced cytopathic effect on MT-4 cells (IC100 values of 7.6 ng/ml and 7.81 micrograms/ml, and CC0 values of 62.5 micrograms/ml and 31.3 micrograms/ml, respectively) without activating protein kinase C.

Design and racemic synthesis of conformationally restricted carbocyclic pyrimidine nucleoside analogs based on the structure of the L-nucleoside residue in heterochiral DNA.

Carbocyclic pyrimidine nucleoside analogs which have restricted glycosidic conformation at chi approximately 180 degrees were designed, based on the conformational features of the L-nucleotide residue in heterochiral DNA, and synthesized. The synthesis of (+/-)-carbocyclic 6,6'-O-cyclo-2'-deoxyuridine was achieved via bromination and subsequent intramolecular cyclization of carbocyclic 6'beta-hydroxy-2'-deoxyuridine. (+/-)-Carbocyclic 6,6'-O-cyclo-2'-deoxycytidine was synthesized from protected carbocyclic 6,6'-O-cyclo-2'-deoxyuridine via the 4-triazole intermediate.

Inhibition of octapeptide N-myristoylation by acyl amino acids and acyl alkanolamines.

Several acyl amino acids and acyl alkanolamines were prepared and screened for their inhibition of octapeptide N-myristoylation and HIV-1 replication in MT-4 cells. Of the 62 acyl derivatives tested, N-myristoyl-O-caproyl-L-serine, N-myristoyl-O-caproyl-D-serine and N-decanoyl-O-myristoyl-L-serine were found to be uncompetitive inhibitors of N-myristoylation, but did not prevent HIV-induced cytopathicity in MT-4 cells. However, other acyl derivatives such as N-3-hydroxymyristoyl ethanolamine, N-3-hydroxymyristoyl-D-serine and N-myristoyl-L-cysteine, which did not inhibit N-myristoylation, suppressed the cytopathicity in the infected cells. The acyl derivatives described here may serve as lead compounds for antiviral agents.

Inhibitory effects of Sudanese plant extracts on HIV-1 replication and HIV-1 protease.

Forty-eight methanol and aqueous extracts from Sudanese plants were screened for their inhibitory activity on viral replication. Nineteen extracts showed inhibitory effects on HIV-induced cytopathic effects (CPE) on MT-4 cells. The extracts were further screened against HIV-1 protease (PR) using an HPLC assay method. Of the tested extracts, the methanol extracts of Acacia nilotica (bark and pods), Euphorbia granulata (leaves), Maytenus senegalensis (stem-bark) and aqueous extracts of A. nilotica (pods) and M. senegalensis (stem-bark) showed considerable inhibitory effects against HIV-1 PR. Inhibitory principles were isolated from M. senegalensis and their activities were also discussed.

Boromycin, an anti-HIV antibiotic.

The polyether-macrolide antibiotic, boromycin, was isolated as a potent anti-human immunodeficiency virus (HIV) antibiotic from a fermentation broth of Streptomyces sp. A-3376. Boromycin was found to strongly inhibit the replication of the clinically isolated HIV-1 strain as well as the cultured strain in in vitro laboratory experiments. The mechanism for the anti-HIV activity of boromycin is suggested to involve blocking the later stage of HIV infection, and probably the maturity step for replication of the HIV molecule.

Anti-HIV-1 activity of herbs in Labiatae.

The anti-HIV-1 activity of aromatic herbs in Labiatae was evaluated in vitro. Forty five extract from among 51 samples obtained from 46 herb species showed significant inhibitory effects against HIV-1 induced cytopathogenicity in MT-4 cells. In particular, the aqueous extracts of Melissa officinalis, a family of Mentha x piperita "grapefruit mint," Mentha x piperita var. crispa, Ocimum basilicum cv "cinnamon," Perilla frutescens var. crispa f. viridis, Prunella vulgaris subsp. asiatica and Satureja montana showed potent anti-HIV-1 activity (with an ED of 16 microg/ml). The active components in the extract samples were found to be water-soluble polar substances, not nonpolar compounds such as essential oils. In addition, these aqueous extracts inhibited giant cell formation in co-culture of Molt-4 cells with and without HIV-1 infection and showed inhibitory activity against HIV-1 reverse transcriptase.