Mireia Giménez-Barcons

Sequence Homology Required by Human Immunodeficiency Virus Type 1 To Escape from Short Interfering RNAs

ABSTRACT Short interfering RNAs (siRNAs) targeting viral or cellular genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, the emergence of mutations in the gene being targeted could lead to the rapid escape from the siRNA. Here, we simulate viral escape by systematically introducing single-nucleotide substitutions in all 19 HIV-1 residues targeted by an effective siRNA. We found that all mutant viruses that were tested replicated better in the presence of the siRNA than in the presence of the wild-type virus. The antiviral activity of the siRNA was completely abolished by single substitutions in 10 (positions 4 to 11, 14, and 15) out of 16 positions tested (substitution at 3 of the 19 positions explored rendered nonviable viruses). With the exception of the substitution observed at position 12, substitutions at either the 5′ end or the 3′ end (positions 1 to 3, 16, and 18) were better tolerated by the RNA interference machinery and only in part affected siRNA inhibition. Our results show that optimal HIV-1 gene silencing by siRNA requires a complete homology within most of the target sequence and that substitutions at only a few positions at the 5′ and 3′ ends are partially tolerated.

Infection with a novel human DNA virus (TTV) has no pathogenic significance in patients with liver diseases

BACKGROUND/AIMS A recently identified DNA virus, termed TT virus (TTV), has been associated with post-transfusional hepatitis, and a high prevalence of TTV infection in patients with acute or chronic liver disease of unknown etiology has been reported from Japan, but few data are available about TTV infection in other countries. METHODS Using hemi-nested-PCR amplification to detect TTV-DNA sequences in serum, we investigated TTV infection in blood donors and in patients with liver diseases of varied etiology. RESULTS The prevalence of TTV infection was 13.7% in blood donors (23/168), 18.6% in chronic hepatitis C (19/102), 28.6% in chronic hepatitis B (16/56), 29.9% in hepatocellular carcinoma (20/67), 9.1% in cryptogenic chronic liver disease (2/22) and 39.6% in fulminant hepatitis (19/48). The prevalence of TTV infection in patients with virus-induced or idiopathic fulminant hepatitis was similar. Comparison of TTV-infected and non-infected patients did not reveal significant differences concerning demographic, epidemiological or histopathological features. In patients with hepatitis C, response to interferon therapy was not related to TTV infection. Phylogenetic analysis of TTV isolates showed that at least three different types of TTV are present in Spain. CONCLUSIONS Our data suggest that TTV infection is frequent among blood donors and patients with acute liver disease. However, pathogenic effects associated with TTV infection were not observed.

Outbreak of Nosocomial Hepatitis C Virus Infection Resolved by Genetic Analysis of HCV RNA

ABSTRACT In July 2000, symptomatic acute hepatitis C was diagnosed in five patients who had attended the emergency room of a municipal hospital on the same day, about 6 weeks before. Investigation of the remaining 65 patients visited at the emergency room on that day disclosed that 8 patients had a positive anti-hepatitis C virus (anti-HCV) test and 4 of them had biochemical evidence of acute anicteric hepatitis. HCV RNA was detected in 12 of the 13 anti-HCV-positive patients. Phylogenetic analysis of the nonstructural 5A (NS5A) and E2 regions showed that 10 patients, including all 9 with acute hepatitis, were infected with a closely related HCV strain, while the remaining 2 patients harbored unrelated strains. Flushing of intravenous catheters with heparin retrieved from a multidose heparin solution in saline was carried out for all the patients involved in the hepatitis outbreak but in only 1 of 23 (4%) matched controls recruited among HCV-noninfected patients attending the emergency room on the same day, and this was the only significant difference concerning risk factors for HCV infection between patients and controls. Thus, accidental contamination of a multidose heparin solution with blood from an unrecognized HCV carrier was identified as the source of this nosocomial outbreak of hepatitis C.

Prevalence and route of transmission of infection with a novel DNA virus (TTV), hepatitis C virus, and hepatitis G virus in patients infected with HIV.

Objectives: To evaluate the prevalence, route of transmission and clinical significance that current co‐infection with TT virus (TTV), hepatitis C virus (HCV), and hepatitis G virus (HGV) have in HIV‐1‐infected patients. Design: Presence of TTV, HCV, and HGV was analyzed in plasma samples from 160 HIV‐1‐infected patients with parenteral (38 intravenous drug users [IVDUs] and 41 patients with hemophilia) or sexual (39 homosexuals and 42 heterosexuals) risk of exposure, and in 168 volunteer blood donors. Alanine aminotransferase (ALT) levels and CD4+ counts were also analyzed. Methods: HCV and HGV RNA were detected by specific reverse transcriptase (RT) nested polymerase chain reaction (PCR) and TTV DNA by specific heminested PCR. Results: TTV DNA was detected in 39% of the patients and in 14% of the volunteer blood donors. HCV and HGV infections were detected in 42% and in 14% of the patients, and in 0% and 3% of the blood donors, respectively. Prevalences of TTV and HCV infection were higher among patients with parenteral (62% and 68%) than in those with sexual (17% and 16%) risk of exposure. A higher prevalence of TTV infection (but not of HCV or HGV infection) was observed among patients with hemophilia (76%) than IVDUs (47%), and among homosexuals (26%) than among heterosexuals (10%). Abnormal ALT levels were related with the presence of HCV infection, independently of the detection of TTV DNA. TTV infection did not seem to alter the levels of CD4+ T cells. Conclusions: Prevalence of current TTV infection is high among HIV‐infected patients with parenteral risk of exposure, but TTV is also transmitted through sexual routes; detection of TTV does not seem to influence the clinical or immune status of HIV‐infected patients.

Prevalence and genotypes of GB virus C/hepatitis G virus (GBV-C/HGV) and hepatitis C virus among patients infected with human immunodeficiency virus : Evidence of GBV-C/HGV sexual transmission

The development of new antiretroviral agents may improve survival of HIV‐infected individuals, and therefore chronic viral hepatitis may become more relevant in these patients. The presence of GBV‐C/HGV and hepatitis C virus (HCV) RNA were investigated by reverse transcriptase‐nested polymerase chain reaction in plasma from 168 Spanish HIV‐infected patients belonging to four different risk groups: intravenous drug users (IVDUs), hemophiliacs, homosexuals, and heterosexuals. GBV‐C/HGV‐RNA and HCV‐RNA were detected in 18% and 43% of the patients, respectively. The prevalence of current infection with these viruses was notably high, 19% for GBV‐C/HGV and 69% for HCV, among individuals with parenteral risk of infection (intravenous drug abusers and hemophiliacs), but sexual transmission with GBV‐C/HGV was also suggested because 16.5% of patients with sexual risk, either homosexual or heterosexual, had GBV‐C/HGV‐RNA in plasma. Although investigation of GBV‐C/HGV‐RNA possibly underestimates the actual prevalence of infection with GBV‐C/HGV, the above data suggest that sexual contact may play a relevant role in the spread of this virus. Phylogenetic analysis showed no evidence for clustering of NS3 sequences into different genotypes or subtypes of GBV‐C/HGV. J. Med. Virol. 55:293–299, 1998.

Endoribonuclease-Prepared Short Interfering RNAs Induce Effective and Specific Inhibition of Human Immunodeficiency Virus Type 1 Replication

ABSTRACT Short interfering RNAs (siRNAs) targeting viral or cellular genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, optimal HIV-1 gene silencing by siRNA requires precise complementarity with most of the target sequence. The emergence of mutations in the targeted gene could lead to rapid viral escape from the siRNA. In the present study, Escherichia coli endoribonuclease III (RNase III) or mammalian Dicer was used to cleave double-stranded RNA into endoribonuclease-prepared siRNA (esiRNA). esiRNAs generate a variety of siRNAs which can efficiently and specifically target multiple sites in the cognate RNA. esiRNAs targeting the region encoding the HIV-1 reverse transcriptase (RT) reduced viral replication by 90%. The inhibition was dose dependent and sequence specific because several irrelevant esiRNAs did not inhibit HIV-1 replication. Importantly, esiRNAs obtained from the prototypic RT sequence of the HXB2 strain and from highly mutated RT sequences showed similar degrees of viral inhibition, suggesting that the heterogeneous population of esiRNAs could overcome individual mismatches in the RT sequence. Finally, esiRNAs generated by Dicer cleavage were five times more potent than those generated by bacterial RNase III digestion. These results show that esiRNAs are potent HIV-1 inhibitors. Moreover, sequence targets do not need to be highly conserved to reach a high level of viral replication inhibition.

Analysis of Chemokine and Cytokine Expression in Patients with HIV and GB Virus Type C Coinfection

BACKGROUND Plasma levels of several chemokines and cytokines were evaluated in a cohort of 161 human immunodeficiency virus (HIV)positive patients to shed light on a clinically relevant mechanism that would explain the putative beneficial effect of GB virus type C (GBV-C) coinfection. METHODS Markers for GBV-C infection were assessed in plasma samples. The syncitium-inducing (SI) capacity of isolated virus from each patient was determined in MT-2 cells. Plasma cytokine and chemokine levels were quantified with use of a commercial enzyme-linked immunosorbent assay. RESULTS GBV-C viremia was found in 44 (27%) of 161 patients, and anti-E2 antibodies were found in 18 (21%) of 87. In contrast to the findings of ex vivo analysis, no statistically significant differences were observed in levels of CCL5, stromal cell-derived factor 1, interleukin-7, and tumor necrosis factor-alpha in plasma of patients with or without GBV-C viremia. Seventy-two (45%) and 89 (55%) of our patients harbored SI and non-SI (NSI) strains, respectively. GBV-C viremia was less prevalent among patients with SI strains (13 [18%] of 72) than among patients with NSI strains (30 [34%] of 89; P = .6). Of interest, coinfected patients with SI strains had significantly higher CD4+ T cell values than did patients who were not coinfected. CONCLUSIONS Our results suggest that GBV-C infection does not appear to influence the expression of the cytokines and chemokines analyzed herein in a clinically relevant context. Alternative explanations for the elevated levels of HIV-inhibitory chemokines are needed to explain the putative beneficial effect of GBV-C.