Role of p53, apoptosis, and cell proliferation in early stage Epstein-Barr virus positive and negative gastric carcinomas.

AIMS: Mechanisms of Epstein-Barr virus (EBV) associated gastric tumour development are incompletely understood. The interrelations between EBV infection, apoptosis, cell proliferation, and the expression of the tumour suppressor gene p53 was investigated in 133 early stage gastric carcinomas. METHODS: Tumour tissue was compared with paired non-tumour tissue. EBV encoded small RNAs (EBERs) determined EBV status. The apoptotic index (AI) was determined by morphology and verified biochemically. p53 and Ki-67 expression (cell proliferation) was assessed using immunohistochemistry. RESULTS: EBV was detected in 14.3% of the cases. Cell proliferation did not differ significantly between EBV positive and negative cancers. However, within both these groups, the p53 positive and negative subsets differed significantly (EBV positive group: 76.8% and 55.3% were p53 positive or negative cancers, respectively; p<0.05; EBV negative group: 65.2% and 51.7% were p53 positive or negative, respectively; p<0.005). The numbers of p53 expressing EBV positive and negative cases were significantly different (57.9% and 82.5%, respectively; p<0.05). Compared with cell proliferation, apoptosis was significantly lower in EBV positive versus negative cancers (AI of 4.36 and 6.50, respectively; p<0.01). The p53 positive and negative subsets also differed significantly in AI (EBV positive group: AI of 5.13 and 3.30 for p53 positive and negative cancers, respectively; p<0.05: EBV negative group: AI of 6.84 and 4.90 for p53 positive and negative cancers, respectively; p<0.05). CONCLUSIONS: These factors probably combine to promote development and progression of early stage gastric carcinomas and, at the same time, ensure the survival of EBV itself.

A phylogenetic-tree analysis elucidating nosocomial transmission of hepatitis C virus in a haemodialysis unit.

Nosocomial transmission of hepatitis C virus (HCV) subtype 1b involving 11 haemodialysis patients occurred in a haemodialysis unit in Japan in March 2000. Sequencing of the HCV-E1 region (411-bp) and phylogenetic-tree analysis showed near identity between HCV isolates derived from these patients and a haemodialysis patient who was known to be HCV-positive. The mode of transmission could not be conclusively established, but retrospective analysis suggested that the sharing of contaminated multidose vials of heparin-saline solutions, which were prepared in the Haemodialysis Center using accidentally contaminated instruments such as needles, may have been responsible for the outbreak. To prevent transmission of HCV in a haemodialysis unit, it may be important to observe strictly standard precautions and to prepare all medications in the Pharmacy. After these measures were taken, no new seroconversions and no new nosocomial transmissions of HCV have been observed in our haemodialysis unit.

Novel deletion of HIV type 1 reverse transcriptase residue 69 conferring selective high-level resistance to nevirapine.

A novel deletion of residue 69 of the HIV-1 reverse transcriptase (RT) gene was detected in combination with mutations V75I/V and F77L/F in a patient with partial virological response to several antiretroviral drug regimens, including stavudine (D4T), didanosine (DDI), lamivudine (3TC), saquinavir (SQV), and nevirapine (NVP). Longitudinal analysis of samples revealed that this deletion emerged upon reinitiation DDI/D4T therapy following a toxicity-induced short discontinuation of all antiretrovirals. Analysis of the resistance phenotype showed a greater than 62-fold increase of the IC50 of NVP, but no significant change in sensitivity to other single nonnucleoside reverse transcriptase inhibitors (NNRTIs). The mutated virus showed only a moderately reduced sensitivity to DDI (6.7-fold) and D4T (4.8 fold). In a subsequent sample 3 months later additional RT mutations were found, including A62V, Y188L, and Q151M, conferring high-level cross-resistance to multiple nucleoside analogs. Our findings provide evidence that the deletion of RT residue 69 selectively confers high-level NVP resistance.

Impact of HIV type 1 protease, reverse transcriptase, cleavage site, and p6 mutations on the virological response to quadruple therapy with saquinavir, ritonavir, and two nucleoside analogs.

Genotype alterations of HIV-1 protease, reverse transcriptase, cleavage sites p7/p1 and p1/p6, as well as p6(gag) and transframe protein p6* were studied in an observational cohort of 42 individuals who received antiretroviral therapy consisting of saquinavir, ritonavir, and two nucleoside analogs. In a multivariate logistic regression analysis, the prior protease inhibitor experience (odds ratio, 6.20; 95% CI, 1.22-31.38) and the presence of primary protease mutations (odds ratio, 9.99; 95% CI, 1.05-94.72) were independently associated with virological failure. Moreover, a trend was observed in that individuals with N-terminal amino acid insertions in the proline-rich motif of the p6(gag) protein were less likely to experience virological failure (OR, 0.17; 95% CI, 0.02-1.35; p = 0.09). In contrast, the presence of secondary protease, reverse transcriptase, or cleavage site mutations was not independently associated with treatment failure. However, mutations at cleavage site p7/p1 (p = 0.01) and C-terminal p6* mutations (p = 0.02) were both associated with primary protease mutations. In conclusion, the presence of primary protease mutations was the most important predictor of the subsequent virological response. Moreover, there is some evidence that insertions in the proline-rich area of the p6(gag) protein may affect the virological response. The relationship between mutations of cleavage sites or C-terminal p6* residues and protease mutations suggests that these alterations may serve a compensatory role, increasing viral fitness.

Hepatitis C virus core mutations reduce the sensitivity of a fluorescence enzyme immunoassay.

Four of 107 samples obtained from hepatitis C virus (HCV) carriers showed lower HCV core antigen levels in a fluorescence enzyme immunoassay (FEIA) than expected from corresponding HCV RNA levels. Nucleotide sequencing revealed a mutation in the HCV core region (Thr49Pro) that appears to have reduced the FEIA sensitivity.

Identification of a novel 23kDa protein encoded by putative open reading frame 2 of TT virus (TTV) genotype 1 different from the other genotypes.

We report the entire open reading frames (ORFs) sequences of four TT virus (TTV) isolates, one genotype 2 (G2) and three G4 isolates. Despite a DNA virus, TTV possesses high rate of amino acid (aa) substitution: the aa sequence homology of ORF1 and 2 is lower than the nucleotide homology. The partial 'N22' region of ORF1 is suitable for genotyping of 'prototype TTV' isolates, because the phylogenetic tree from partial 'N22' sequence is consistent with that from the entire ORF1. Based on our sequence data, ORF2 from most isolates excluding G1 encode truncated 49 aa (pORF2a) because of an in-frame stop codon, although ORF2s from most G1 isolates encode 202 aa (pORF2ab). Just downstream the stop codon, another ORF encoding a protein of approximately 150 aa (pORF2b) is found, whose homology is quite low among these genotypes. Our in vitro transcription/translation study supports that all G1a and a part of G b without an in-frame stop codon dominantly encode pORF2ab, a novel 23 kDa protein, whereas the other genotypes with an in-frame stop codon encode pORF2b (17 kDa). Our data indicate TTV G1a and a part of G1b should have different characteristics from the other genotypes.

Lack of association between TTV viral load and aminotransferase levels in patients with hepatitis C or non-B-C.

TT virus (TTV) is a newly identified un-enveloped single-stranded DNA virus. Although TTV was initially thought to be a new hepatitis virus, it is still unclear whether it causes hepatitis. To clarify the natural history and pathogenesis of TTV infection, serial serum samples from patients with chronic hepatitis were analysed. TTV DNA was quantified by real-time detection polymerase chain reaction assay (RTD-PCR), which was adapted for TTV. Five patients with chronic hepatitis, 4 with hepatitis C and 1 with non-B-C, were studied. The study period ranged from 9 to 50 months. In 3 patients there were frequent increases in TTV DNA titres, but no concomitant elevation of the aminotransferase (ALT) levels. In 2 patients who were treated with interferon, the changes in TTV titres were not synchronized with those of the ALT levels. Thus, in cases of chronic hepatitis, no correlation was observed between the serum TTV DNA titres and the ALT levels.

GB virus C/hepatitis G viremia and antibody response to the E2 protein of hepatitis G virus in hemodialysis patients.

The aim of this study was to assess the relationship between the prevalence of GB virus C/hepatitis G virus (GBV-C/HGV) RNA and that of antibody to the putative E2 protein (anti-E2) in hemodialysis patients. GBV-C/HGV RNA in serum was detected by a reverse transcription polymerase chain reaction (RT-PCR) assay, and anti-E2 was measured in 244 hemodialysis patients by enzyme-linked immunosorbent assay using recombinant E2 protein. The GBV-C/HGV RNA level was determined by competitive RT-PCR with an interval of 1 year. GBV-C/HGV RNA, anti-E2. and both together were detected in 11 (4.5%), in 19 (7.8%), and in 3 patients (1.2%), respectively. Comparison of clinical characteristics between GBV-C/HGV RNA-positive patients and negative patients revealed the longer duration of hemodialysis (9.8 years vs. 6.0 years; p < 0.05), and the greater frequency of anti-hepatitis C virus (HCV) (63.6% vs. 20.3%; p < 0.05) and HCV RNA (36.4% vs. 12.9%; p < 0.05) in GBV-C/HGV RNA-positive patients. The GBV-C/HGV RNA levels of patients who were positive for anti-E2 remained under detection limit (< 10(2) copies/mL), whereas only one of eight patients who were negative for anti-E2 showed a GBV-C/HGV RNA level under detection limit (p < 0.05). The presence of anti-E2 in serum was associated with loss of detectable GBV-C/HGV RNA or with a very small amount of HCV RNA in hemodialysis patients.

Iatrogenic GB virus C/hepatitis G virus infection in an area endemic for hepatitis C virus.

GB virus C/hepatitis G virus (GBV-C/HGV) is reported to be transmitted by blood products. This study reports infection with GBV-C/HGV from Area-O of town T, an area of high prevalence of antibody to hepatitis C virus (anti-HCV). Four hundred and thirty-five inhabitants of Area-O in town T were examined. Three hundred and forty-three inhabitants of Area-H in town T (where differences of age or sex are not markedly different to Area-O) were studied as controls. We investigated the virus markers and conducted a survey of life history in both areas. The seroprevalence of anti-HCV and GBV-C/HGV markers in Area-O was 17.7% and 11.7%, significantly higher than in Area-H (1.5% and 4.4%). The prevalence of GBV-C/HGV markers was significantly higher in the anti-HCV-positive group than in the sero-negative group. Anti-HCV- or GBV-C/HGV positive subjects tended to have a history of intravenous medications at hospital C in town T, suggesting iatrogenic infection through insufficient sterilization of needles and/or syringes.

Development of a TT virus DNA quantification system using real-time detection PCR.

Although TT virus (TTV) was isolated from a cryptogenic posttransfusion hepatitis patient, its pathogenic role remains unclear. It has been reported that the majority of the healthy population is infected with TTV. To elucidate the differences between TTV infection in patients with liver diseases and TTV infection in the healthy population, a quantification system was developed. TTV DNA was quantified by a real-time detection PCR (RTD-PCR) assay on an ABI Prism 7700 sequence detector. With this system, TTV DNA was quantified in 78 hepatitis C virus (HCV)-infected patients (63 with elevated serum alanine aminotransferase [ALT] levels and 15 with normal ALT levels) and in 70 voluntary blood donors (BDs). The quantification range was 2.08 to 7.35 log copies/ml. The intra-assay and interassay coefficients of variation were 0.37 to 6.33% and 0.60 to 7.07%, respectively. The mean serum TTV DNA levels in the HCV-infected patients with both elevated and normal ALT levels and BDs were 3.69 +/- 0.89, 3.45 +/- 0.76, and 3.45 +/- 0.67 log copies/ml, respectively. Comparison of the serum TTV DNA levels among the HCV-infected patients revealed that they were not related to the serum ALT and HCV core protein levels or to the histopathological score on liver biopsy. This study showed that (i) the RTD-PCR assay for the detection of TTV was accurate and had a high degree of sensitivity, (ii) the mean serum TTV DNA level was similar among HCV-infected patients, irrespective of their ALT level, and also among BDs, and (iii) a high serum TTV DNA level does not affect the serum ALT and HCV levels or liver damage in HCV-infected patients.

Analysis of HBs antigen negative variant of hepatitis B virus: unique substitutions, Glu129 to Asp and Gly145 to Ala in the surface antigen gene.

We analyzed the surface gene (S gene) of a hepatitis B virus (HBV) isolate with mutations of envelope protein that rendered it undetectable by both a monoclonal hepatitis B surface antigen (HBsAg) enzyme-linked immunosorbent assay (ELISA) and polyclonal HBsAg radioimmunoassay (RIA). Sequencing of independently cloned products of HBV polymerase chain reaction revealed several point mutations within the S gene. Rare substitution was identified both at positions 129 (glutamine to asparagine) and at position 145 (glycine to alanine) in the 'a' determinant region, which is considered to be within a larger antigenic area known as the major hydrophilic region (MHR). A computer-assisted analysis of protein secondary structure could not find any significant difference between this mutant and wild-type HBsAg. However, the substitution of substitution glycine to alanine at position 129 introduce a putative glycosylation site (Asn-Gly-Thr), which may interfere with the antigenicity of HbsAg. Also, HBV variant with substitution at position 145 (Gly to Ala) has been recently reported to be antigenically altered and to show impaired recognition by polyclonal hepatitis B hyperimmune globulin in vitro. These genetic mutations in the S gene inside MHR may allow to escape detection by standard HBsAg assays.

High prevalance of TT virus infection in Japanese patients with liver diseases and in blood donors.

BACKGROUND/AIMS: Although a novel DNA virus, TT virus (TTV), has been isolated from a patient with cryptogenic post-transfusion hepatitis, its pathogenic role remains unclear. To elucidate its prevalence and clinical impact in patients with liver diseases, the presence of TTV DNA was assessed in patients with liver diseases and blood donors (BDs) in Japan using two primer sets, one conventional and the other new and highly sensitive. METHODS: We studied 261 samples, 72 with chronic hepatitis associated hepatitis C virus (HCV-CH), 57 with hepatocellular carcinoma associated HCV (HCV-HCC), 12 with HCC without either HCV or hepatitis B virus (NBNC-HCC), and 120 of BDs. RESULTS: Using two primer sets, TTV DNA was detected in 68 (94.4%), 53 (93.0%), 12 (100%), and 98 (81.7%) HCV-CH, HCV-HCC, NBNC-HCC, and BDs, respectively. The prevalence was not significantly different between HCV-CH and HCV-HCC, or between HCV-HCC and NBNC-HCC. Comparison between patients with and without TTV revealed no significant differences in backgrounds or biochemical findings. Histopathological findings in patients with HCV-CH, and number, maximum diameter, and histological differentiation of HCC also did not demonstrate any relation to TTV infection. TTV strains can be divided into five groups using phylogenetic analysis, but no disease-specific group appears to exist. CONCLUSIONS: Our data suggest that: 1) TTV is very prevalent among patients with liver diseases and even among BDs in Japan, 2) TTV infection does not impact on liver damage with HCV infection, and 3) TTV infection also does not affect the development or progression of HCC.

Hepatitis B virus genotype assignment using restriction fragment length polymorphism patterns.

Hepatitis B virus (HBV) is classified into genotypes A-F, which is important for clinical and etiological investigations. To establish a simple genotyping method, 68 full-genomic sequences and 106 S gene sequences were analyzed by the molecular evolutionary method. HBV genotyping with the S gene sequence is consistent with genetic analysis using the full-genomic sequence. After alignment of the S sequences, genotype specific regions are identified and digested by the restriction enzymes, HphI, NciI, AlwI, EarI, and NlaIV. This HBV genotyping system using restriction fragment length polymorphism (RFLP) was confirmed to be correct when the PCR products of the S gene in 23 isolates collected from various countries were digested with this method. A restriction site for EarI in genotype B was absent in spite of its presence in all the other genotypes and genotype C has no restriction site for AlwI. Only genotype E is digested with NciI, while only genotype F has a restriction site for HphI. Genotype A can be distinguished by a single restriction enzyme site for NlaIV, while genotype D digestion with this enzyme results in two products that migrates at 265 and 186 bp. This simple and accurate HBV genotyping system using RFLP is considered to be useful for research on HBV.

Amino acid substitutions in NS5A region of GB virus C and response to interferon therapy.

GB virus C (GBV-C) is related to hepatitis C virus (HCV) and has a similar genomic structure. Some predictors for the efficacy of interferon (IFN) therapy on HCV have been reported: genotype, viral load, IFN dose, and the amino acid substitutions in the NS5A region, designated as the interferon sensitivity determining region (ISDR). To evaluate the correlation between the amino acid substitutions in the GBV-C NS5A region and the response to IFN therapy, single-strand conformation polymorphism (SSCP) analysis was performed in the 12 concomitantly GBV-C-and HCV-infected patients who received IFN therapy at three time points: before, end-point, and after the IFN therapy. The region in the GBV-C NS5A studied includes the amino acids that exhibit some homology to the ISDR and the various substitutions. By SSCP analysis, amplicons were separated into 1-4 bands, which indicated the existence of heterogeneity in each host. However, the deduced amino acid sequences in these bands exhibited no characteristic differences among these strains irrespective of response to IFN therapy. Of the 32 strains separated by SSCP, 7 strains were responders, and 25 were nonresponders. The mean amino acid substitution, compared with the consensus sequence of nonresponders, was 1.00+/-0.93 among responders, and 1.40+/-0.85 among non-responders (P= NS). No correlation between the amino acid sequence in the GBV-C NS5A region and response to IFN therapy was found, indicating that the GBV-C NS5A region dose not act as the ISDR.

GB virus C infection: clinical significance.

GB virus C (GBV-C) RNA positivity rates were examined in serum specimens from 231 patients with liver disease (23 patients with hepatitis B, 175 patients with hepatitis C, five patients with hepatitis B virus plus hepatitis C virus coinfection, and 28 patients with non-A, non-B, non-C hepatitis) to clarify the clinical significance of this virus. GBV-C RNA was detected in none of 12 patients with fulminant hepatitis, one of two patients with acute hepatitis positive for hepatitis B surface antigen and one of four patients with acute non-A, non-B, non-C hepatitis. Pathogenetic involvement of GBV-C was suspected in some patients in the latter group. Among patients with the non-B, non-C type of chronic disease, one of seven with cirrhosis (14%) and none with chronic hepatitis or hepatocellular carcinoma were GBV-C-positive. In chronic hepatitis C patients who had received interferon treatment, no difference was found in clinical findings, alanine aminotransferase (ALT) concentrations, histology or response to interferon between 11 patients who were GBV-C RNA-positive and 101 patients who were GBV-C RNA-negative. Moreover, changes in ALT after interferon therapy showed no relation to positivity for GBV-C RNA. On the basis of these findings, GBV-C appears to be an unlikely cause of initiation or progression of chronic hepatic diseases.

New genotypes of TT virus (TTV) and a genotyping assay based on restriction fragment length polymorphism.

A phylogenetic analysis, using the open reading frame I sequence of 93 TT viruses (TTV) obtained from various geographical areas, indicated that the virus could be classified into six different genotypes including three hitherto unreported genotypes. The high reliability of the six clusters was confirmed by bootstrap analysis. On the basis of these sequence data, a new simple genotyping assay based on a restriction fragment length polymorphism of TTV was developed. Using the enzymes NdeI and PstI, followed by cleavage with NlaIII or MseI, it was possible to distinguish between the six TTV genotypes. This system will provide the framework for future detailed epidemiological and clinical investigations.

Heterogeneity in E2 region of GBV-C/hepatitis G virus and hepatitis C virus.

GB virus C/hepatitis G virus (GBV-C/HGV) is related distantly to hepatitis C virus (HCV). HCV has a hypervariable region (HVR), and exists as quasispecies in vivo. Although GBV-C/HGV also has replaceable amino acids in the presumed antigenic region, the existence and fluctuation of population of heterogeneous virus have not been evaluated. In this study, the heterogeneity of GBV-C/HGV and HCV was investigated by the single-strand conformation polymorphism (SSCP) analysis in six concomitantly infected patients. Two patients were observed for 4 years without any treatment, and four were treated with interferon-alpha (IFN). By SSCP analysis, amplicons of GBV-C/HGV RNA were separated into 1-5 bands on gels for each patient. The amplicons had different nucleotide but the same amino acid sequences in the presumed antigenic region. The amplicons of HCV RNA, separated into 1-4 bands, had different nucleotide and amino acid sequences in the HVR. In the two patients without treatment, the predominant strain of GBV-C/HGV was unchanged for the 4 years. In the four patients administered IFN, some strains of GBV-C/HGV disappeared after IFN therapy, whereas other strains persisted. The mean genetic distance among GBV-C/HGV strains represented by SSCP analysis was significantly lower than that of HCV (P < 0.05). The data indicate that: 1) GBV-C/HGV can be devoid of antigenic drift unlike HCV; 2) GBV-C/HGV has no HVR as seen in HCV in the presumed antigenic region; and 3) the sensitivity to IFN differs among GBV-C/HGV strains in the same hosts, as with HCV.

Interferon-alpha therapy in patients dually infected with hepatitis C virus and GB virus C/hepatitis G virus--virological response of HGV and pretreatment HGV viremia level.

BACKGROUND/AIMS: The response to interferon-alpha (IFN) therapy of recently isolated GB virus C and hepatitis G virus (HGV) is still unclear. To investigate the biochemical and virological response to IFN therapy in patients with chronic hepatitis C virus (HCV) infection concomitantly infected with HGV, 196 patients with HCV who had received IFN therapy were retrospectively studied. METHODS: HGV and HCV RNA were detected by reverse transcription nested polymerase chain reaction (RT-PCR). Serum HGV RNA levels were quantified by competitive RT-PCR. The HGV genotype was detected by restriction fragment length polymorphism analysis using the PCR products. RESULTS: Of 196 patients, 16 (8.2%) were positive for both HCV and HGV RNA before IFN therapy. There were no significant clinical and virological differences between the patients with dual infection and those with only HCV infection. During the therapy, a decrease or loss of serum HGV RNA level was observed in these patients. Six months after cessation of the therapy, five of 16 patients became negative for HGV RNA by RT-PCR. The pretreatment HGV RNA level of the patients who lost HGV RNA after cessation of IFN was low (median=10(3) copies/ml), compared to the level (median=10(7) copies/ml, p<0.01) in the patients with positive HGV RNA after the therapy. The HGV genotype of these 16 patients was the same type. CONCLUSIONS: These data suggest that: 1) there is no significant difference in response to IFN therapy between patients with dual and single infection; 2) HGV shows sensitivity to IFN therapy; and 3) in the patients who show a low pretreatment HGV RNA level, serum HGV RNA becomes undetectable by RT-PCR after cessation of IFN therapy.

Prevalence and molecular epidemiology of GB virus C/hepatitis G virus infection in Mongolia.

We studied the prevalence of GB virus C/hepatitis G virus (GBV-C/HGV) infection among 112 patients with liver disease and 121 blood donors in Ulaanbaatar, Mongolia. Reverse transcription and polymerase chain reaction were employed to detect GBV-C/HGV RNA using the specific primers derived from the 5'-untranslated region (5'-UTR) of the GBV-C/HGV genome. Nucleotide sequences of all positive samples for GBV-C/HGV RNA were determined. The sequences were analyzed by a molecular evolutionary method. Twenty-five (10.7%) of 233 people were positive for GBV-C/HGV RNA. Eight (6.6%), 11 (9.1%), and 30 (24.8%) blood donors were positive for GBV-C/HGV RNA, HBsAg, and anti-HCV, respectively, although 17 (15.2%), 65 (58.0%), and 64 (54.5%) patients with liver disease were positive for each viral marker. The prevalences of GBV-C/HGV RNA, HBV, and HCV in the patients were significantly higher than those in blood donors (P < 0.05). There was no significant difference in the prevalence of anti-HCV among people with and without GBV-C/HGV RNA, while the prevalence of HBsAg among people with GBV-C/HGV RNA was significantly higher than among those without GBV-C/HGV RNA (P < 0.05). The molecular evolutionary tree showed that GBV-C/HGV was a heterogeneous virus and all strains could be divided into 2 types. One is the same phylogenetic type as HGV, and the other is a new type that is different from GBV-C and HGV.