Longer Poly(U) Stretches in the 3'UTR Are Essential for Replication of the Hepatitis C Virus Genotype 4a Clone in in vitro and in vivo.

The 3' untranslated region (UTR) of the hepatitis C virus (HCV) genome plays a significant role in replication including the poly(U) tract (You and Rice, 2008). Here we established an HCV clone that is infectious in vitro and in vivo, from an Egyptian patient with chronic HCV infection and hepatocellular carcinoma (HCC). First, we inoculated the patient plasma into a humanized chimeric mouse and passaged. We observed HCV genotype 4a propagation in the chimeric mouse sera at 1.7 × 107 copies/mL after 6 weeks. Next, we cloned the entire HCV sequence from the HCV-infected chimeric mouse sera using RT-PCR, and 5' and 3' RACE methodologies. We obtained first a shorter clone (HCV-G4 KM short, GenBank: AB795432.1), which contained 9,545 nucleotides with 341 nucleotides of the 5'UTR and 177 nucleotides of the 3'UTR, and this was frequently obtained for unknown reasons. We also obtained a longer clone by dividing the HCV genome into three fragments and the poly (U) sequences. We obtained a longer 3'UTR sequence than that of the HCV-G4 KM short clone, which contained 9,617 nucleotides. This longer clone possessed a 3'-UTR of 249 nucleotides (HCV-G4 KM long, GenBank: AB795432.2), because of a 71-nucleotide longer poly (U) stretch. The HCV-G4-KM long clone, but not the HCV-G4-KM short clone, could establish infection in human hepatoma HuH-7 cells. HCV RNAs carrying a nanoluciferase (NL) reporter were also constructed and higher replication activity was observed with G4-KM long-NL in vitro. Next, both short and long RNAs were intra-hepatically injected into humanized chimeric mice. Viral propagation was only observed for the chimeric mouse injected with the HCV-G4 KM long RNA in the sera after 21 days (1.64 × 106 copies/mL) and continued until 10 weeks post inoculation (wpi; 1.45-4.74 × 107 copies/mL). Moreover, sequencing of the HCV genome in mouse sera at 6 wpi revealed the sequence of the HCV-G4-KM long clone. Thus, the in vitro and in vivo results of this study indicate that the sequence of the HCV-G4-KM long RNA is that of an infectious clone.

Serum and ascitic D-dimer in cirrhotic patients with spontaneous bacterial peritonitis.

AIM OF THE STUDY: The study aimed to investigate serum and ascitic fluid D-dimer level in patients with liver cirrhosis with and without ascites and to evaluate the impact of spontaneous bacterial peritonitis (SBP) on circulating serum and ascitic fluid D-dimer levels. MATERIAL AND METHODS: This study was conducted on 60 subjects who were further subdivided into group I comprising 15 patients with liver cirrhosis and no ascites, group II comprising 15 cirrhotic patients with ascites, group III comprising 15 cirrhotic patients with ascites and SBP, and group IV comprising 15 healthy controls. All patients were subjected to full history taking, physical examination, laboratory investigations, and measurement of serum D-dimer in all groups and ascitic fluid D-dimer in groups II and III. The diagnostic performance of serum D-dimer was tested to detect SBP. RESULTS: Serum D-dimer differed significantly between groups III and IV, whilst no significant differences were detected between the other groups and group IV. Moreover, group III showed a significantly higher level of serum D-dimer. Ascitic fluid D-dimer mean levels showed no statistically significant differences. A statistically significant positive correlation was found between serum D-dimer level and ascitic fluid D-dimer in group III, r = 0.682. Using a sensitivity and specificity level of 80%, a cut-off value (COV) of > 323.2 ng/ml could differentiate between patients with SBP and patients with ascites only. CONCLUSIONS: Serum D-dimer significantly correlated with ascitic fluid D-dimer in patients with SBP, whereas no significant correlation was found in patients with cirrhotic ascites without bacterial infection. D-dimer showed good diagnostic performance for SBP among patients with liver cirrhosis.

Serum and Urinary Interferon-Gamma-Inducible Protein 10 in Lupus Nephritis.

BACKGROUND: Lupus nephritis is a serious manifestation of systemic lupus erythematosus (SLE). The objective of this study was to identify the sensitivity, specificity, and cut-off values of IP-10 in the serum and urine of patients with lupus nephritis compared to renal biopsy, albumin/creatinine ratio, and serum anti-dsDNA. METHODS: Thirty female SLE patients were included. SLEDAI was calculated and blood and urine samples were collected. Patients were divided into 10 SLE patients with renal involvement (six active and four inactive), 10 active SLE, and 10 inactive SLE patients. Ten age-matched healthy (control) were included. Serum and urinary levels of IP-10 were measured by ELISA. Anti-dsDNA, urine albumin/creatinine ratio were performed. RESULTS: Serum and urinary IP-10 in active SLE patients had significantly increased levels as compared to inactive SLE patients (P = 0.015, P = 0.033, respectively). However, there was no difference in serum and urinary levels between active renal and active non-renal patients. Albumin/creatinine ratio is a better marker in differentiating between lupus nephritis and SLE with no renal involvement. Any of serum and urinary IP-10, albumin/creatinine ratio, and anti-dsDNA did not correlate with the class of lupus nephritis in renal biopsy. CONCLUSION: Urinary and serum IP-10 are useful markers of lupus activity, but not indicative of renal activity. Albumin/creatinine ratio is superior in identifying lupus nephritis and renal activity.

A serine palmitoyltransferase inhibitor blocks hepatitis C virus replication in human hepatocytes.

BACKGROUND & AIMS: Host cell lipid rafts form a scaffold required for replication of hepatitis C virus (HCV). Serine palmitoyltransferases (SPTs) produce sphingolipids, which are essential components of the lipid rafts that associate with HCV nonstructural proteins. Prevention of the de novo synthesis of sphingolipids by an SPT inhibitor disrupts the HCV replication complex and thereby inhibits HCV replication. We investigated the ability of the SPT inhibitor NA808 to prevent HCV replication in cells and mice. METHODS: We tested the ability of NA808 to inhibit SPT's enzymatic activity in FLR3-1 replicon cells. We used a replicon system to select for HCV variants that became resistant to NA808 at concentrations 4- to 6-fold the 50% inhibitory concentration, after 14 rounds of cell passage. We assessed the ability of NA808 or telaprevir to inhibit replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in mice with humanized livers (transplanted with human hepatocytes). NA808 was injected intravenously, with or without pegylated interferon alfa-2a and HCV polymerase and/or protease inhibitors. RESULTS: NA808 prevented HCV replication via noncompetitive inhibition of SPT; no resistance mutations developed. NA808 prevented replication of all HCV genotypes tested in mice with humanized livers. Intravenous NA808 significantly reduced viral load in the mice and had synergistic effects with pegylated interferon alfa-2a and HCV polymerase and protease inhibitors. CONCLUSIONS: The SPT inhibitor NA808 prevents replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It might be developed for treatment of HCV infection or used in combination with pegylated interferon alfa-2a or HCV polymerase or protease inhibitors.

Genomic polymorphisms in 3ß-hydroxysterol Δ24-reductase promoter sequences.

It was recently reported by the present team that 3ß-hydroxysterol Δ24-reductase (DHCR24) is induced by hepatitis C virus (HCV) infection. In addition, upregulation of DHCR24 impairs p53 activity. In human hepatoma HuH-7 cells, the degree of DHCR24 expression is higher than in normal hepatic cell lines (WRL68) at the transcriptional level. The genomic promoter sequence of DHCR24 was characterized and nucleotide substitutions were observed in HuH-7 cells at nucleotide numbers -1453 (G to A), -1420 (G to T), -488 (A to C) and -200 (G to C). The mutations of these sequences from HuH-7 cell types to WRL68 cell types suppressed DHCR24 gene promoter activity. The sequences were further characterized in hepatocytes from patient tissues. Four tissues from HCV-positive patients with cirrhosis or hepatocellular carcinoma (#1, 2, 3, 5) possessed HuH-7 cell type sequences. Interestingly, one patient with liver cirrhosis (#4) possessed WRL68 cell-type sequences; this patient had been infected with HCV and was HCV negative for 17 years after interferon therapy. Next, the effect of HCV infection on these polymorphisms was examined in humanized chimeric mouse liver and HuH-7 cells. The human hepatocytes possess WRL68 cell type and did not show the nucleotide substitution after HCV infection. The HCV-replicon was removed by interferon treatment and established the cured K4 cells. These cells possess HuH-7 cell type sequences. Thus, this study showed the genomic polymorphism in DHCR24 promoter is not directly influenced by HCV infection.

Alterations in neutrophil surface expression of L-selectin (CD62L) and soluble L-selectin (sL-selectin) in hemodialysis patients: relation to HCV.

High levels of L-selectin (CD62L) are a strong indicator of endothelial dysfunction, and atherosclerosis. Atherosclerosis is the leading cause of mortality and morbidity in hemodialysis (HD) patients. Whether HCV infection (highly prevalent in HD patients and also associated with alterations in adhesion molecules) would affect the leukocytic expression and/or the soluble form of L-selectin in hemodialysis patients is unknown. Seventy-two HD patients, HCV-positive (n=48) and HCV-negative (n=24) and 10 normal control were studied. Blood samples were obtained just prior to the start of the dialysis session (predialysis) and at the end of 15 min. of dialysis (intradialysis). The following tests were performed on all patients: HCV-RNA by RT-nested PCR, quantitative determination of sL-selectin by ELISA and neutrophil surface expression of L-selectin (CD62L) by flowcytometry. Both CD62L and sL-selectin were found to be significantly higher in HD patients as compared to normal controls irrespective to HCV. Fifteen minutes after start of the dialysis session both CD62L and absolute neutrophil count decreased significantly [CD62L, p < 0.0001 (HCV-positive), p= 0.03 in (HCV-negative), [neutrophil count, p < 0.0001 each], while sL-selectin showed a significant increase [p = 0.004 (HCV-positive), p = 0.006 (HCV-negative)]. These changes were unrelated to HCV status. A significant increase in CD62L in HCV-positive patients compared to HCV-negative ones in both pre and intradialysis samples was noted (p = 0.007, p = 0.02 respectively). However, no difference was observed in either sL-selectin or absolute neutrophil count between the two groups in the two tested time points. In conclusions, the increased levels of neutrophil-expressed and soluble forms of L-selectin in HD patients, and the intradialysis increase in sL-selectin and decrease in CD62L and neutrophil count are unrelated to HCV viremia. The association between HCV positivity and neutrophil expression of pre andintradialysis L-selectin point to a possible role of HCV that needs further studies.

Molecular epidemiology of hepatitis B virus in the United Republic of Tanzania.

In the United Republic of Tanzania, 457 voluntary blood donors were enrolled in hepatitis B virus (HBV) serological screening; 4.8% (22/457) carried HBsAg, 13.6% (3/22) of whom were HBeAg-positive. The mean age among HBeAg-negative carriers was 31 years. HBV DNA was detectable in 81.8% (18/22), the mean level was 3.67 (+/-1.77) log copies/ml. Genotype A was determined in 90.9% (20/22) and 18/20 were classified into subgenotype Aa (Asia/Africa). The basal core promoter, precore and partial core nucleotide sequences were analyzed in the 18 strains; T1809/T1812 ("Kozak" sequence) and A/T1888 (encapsidation signal) variants were identified in 100% and 78%, respectively. The complete genome sequencing for one of the Tanzanian strains revealed no recombination. In conclusion, HBV seroprevalence is high among general population in Tanzania, and the HBV/Aa-infection is predominant. The indicated tendency to early HBeAg seroconversion and declining of the viral load should be confirmed further in case-control studies.

Distribution of HBV genotypes among HBV carriers in Benin:phylogenetic analysis and virological characteristics of HBV genotype E.

AIM: To determine the distribution of Hepatitis B virus (HBV) genotypes in Benin, and to clarify the virological characteristics of the dominant genotype. METHODS: Among 500 blood donors in Benin, 21 HBsAg-positive donors were enrolled in the study. HBV genotypes were determined by enzyme immunoassay and restriction fragment length polymorphism. Complete genome sequences were determined by PCR and direct sequencing. RESULTS: HBV genotype E (HBV/E) was detected in 20/21 (95.2%), and HBV/A in 1/21 (4.8%). From the age-specific prevalence of HBeAg to anti-HBe seroconversion (SC) in 19 HBV/E subjects, SC was estimated to occur frequently in late teens in HBV/E. The comparison of four complete HBV/E genomes from HBeAg-positive subjects in this study and five HBV/E sequences recruited from the database revealed that HBV/E was distributed throughout West Africa with very low genetic diversity (nucleotide homology 96.7-99.2%). Based on the sequences in the basic core promoter (BCP) to precore region of the nine HBV/E isolates compared to those of the other genotypes, a nucleotide substitution in the BCP, G1757A, was observed in HBV/E. CONCLUSION: HBV/E is predominant in the Republic of Benin,and SC is estimated to occur in late teens in HBV/E. The specific nucleotide substitution G1757A in BCP,which might influence the virological characteristics, is observed in HBV/E.

Automation of semen analysis using flow cytometer in comparison with manual methods.

In order to standardize techniques and limit the effect of human factors on the results of analyses of biological fluids, automation seems to be mandatory. In an attempt to automate semen analysis, computer assisted sperm analysis (CASA) system has been developed, however its use is still limited and its practical applications have many criticisms. In a trial to automate semen analysis, this study aimed to evaluate the usefulness of flow cytometer in the detection of some seminal parameters in comparison with the traditional manual methods. Isolated spermatogenic cells and isolated sperms from semen and EDTA blood of volunteers were analyzed by flow cytometer in order to define their respective regions. Ejaculates of 28 male patients were subjected to routine semen analyses, leucocytes detection by peroxidase test and monoclonal antibody CD53 using flow cytometer after preparation of the patients' semen samples for flow cytometeric analysis. A highly significant correlation (r=0.96, p= 0.001) of absolute neutrophils (pus cells) detected by peroxidase versus flow cytometer using CD53 monoclonal antibody. A poor correlation (r=0.39, p=0.035) of sperm counts assessed by manual technique and flow cytometer and a spurious sperm counts of 1.08 million/ml detected by flow cytometery in azoospermic patients. Flow cytometer could be used for the assessment of pus cells in semen but seems to be non reliable for the assessment of sperm count if gating depend on sperm size and granularity alone.

Role of a sentinel surveillance system in the context of global surveillance of infectious diseases.

In some nation states, sustained integrated global epidemiological surveillance has been weakened as a result of political unrest, disinterest, and a poorly developed infrastructure due to rapidly increasing global inequality. The emergence of severe acute respiratory syndrome has shown vividly the importance of sensitive worldwide surveillance. The Agency for Cooperation in International Health, a Japanese non-governmental organisation, has developed on a voluntary basis a sentinel surveillance system for selected target infectious diseases, covering South America, Africa, and Asia. The system has uncovered unreported infectious diseases of international importance including cholera, plague, and influenza; current trends of acute flaccid paralysis surveillance in polio eradication; and prevalence of HIV, syphilis, hepatitis B, and hepatitis C in individual areas covered by the sentinels. Despite a limited geographical coverage, the system seems to supplement disease information being obtained by global surveillance. Further development of this sentinel surveillance system would be desirable to contribute to current global surveillance efforts, for which, needless to say, national surveillance and alert system takes principal responsibility.

Soluble ICAM-1 in patients with chronic hepatitis C infection: a prognostic marker of disease activity.

Soluble intercellular adhesion molecule-I (sICAM-1) is an important early marker of response to inflammatory mediators and immune activation released from a variety of cells including hepatocytes. At present, the most reliable determination of severity and prognosis in chronic viral hepatitis is the histological staging of the disease which is an invasive procedure and is often not well accepted by patients. The search for alternative non-invasive methods is mandatory especially in follow ups after initial assessment by biopsy. Serum sICAM-1 level was measured in 19 patients with chronic HCV, 19 patients with non-B, non-C chronic liver diseases (NBNC-CLD) and in 19 healthy control subjects using ELISA. Serum sICAM-1 levels were significantly higher in patients with chronic HCV and in NBNC-CLD patients compared to normal subjects (mean +/- SD, [1003 +/- 453 vs. 232 +/- 177, p<0.001], and [881 +/- 328 vs. 232 +/- 177, p<0.001]), respectively. Furthermore, serum levels of sICAM-1 were significantly higher in HCV-RNA positive patients than in HCV-RNA negative patients (p<0.001). Positive correlations were detected between serum levels of sICAM-1 and serum alanine aminotranseferase (ALT) (p<0.001), aspartate aminotranseferase (AST) (p<0.001), prothrompin time (p<0.001), and alkaline phosphatase (p<0.001), while, a negative correlation with albumin was found (p<0.001). Also, there was a significant correlation between clinical, ultrasonic findings and the level of sICAM-1 in chronic HCV patients as regards hepatomegaly, splenomegaly and normal liver echogenecity. High knodell score was significantly associated with high sICAM-1 level (p<0.001) in both patient groups. while no association between sICAM-1 and fibrosis was found. In conclusion, the measurement of sICAM-1 serum levels in chronic hepatitis C and NBNC-CLD patients is a useful non-invasive marker for monitoring liver disease activity that could replace follow up liver biopsies that are mostly not welcomed by the patients.

Factors predisposing to the occurrence of cryoglobulinemia in two cohorts of Egyptian and Japanese patients with chronic hepatitis C infection: ethnic and genotypic influence.

The association between cryoglobulinemia and hepatitis C virus (HCV) infection has been reported. However, the factors underlying its wide variation of occurrence have not yet been well identified. To investigate this, cryoglobulinemia was studied in four cohorts of Egyptian and Japanese patients. Fifty Egyptian patients with chronic hepatitis C, infected with genotype 4 (the predominant HCV genotype in Egypt), were compared with 50 age- and sex-matched Japanese patients, infected with HCV genotype 1b (the predominant HCV genotype in Japan). Thirty-two Egyptian and 30 age- and sex-matched Japanese patients with chronic hepatitis B were included as controls. All patients were noncirrhotic. Antinuclear antibody (ANA), immunoglobulins (Ig), and cryoglubulins were assessed. Results showed a significantly higher prevalence of cryoglobulinemia in chronic hepatitis C Japanese genotype 1b (40%) as compared with Egyptian genotype 4 (14%), P = 0.003, while no difference was found between Japanese (17%) and Egyptian chronic hepatitis B controls (13%). Symptomatic cryoglobulinemia was more prevalent in the Japanese than in the Egyptian chronic hepatitis C group (10% vs. 4%), but the difference was not statistically significant. Univariate analysis showed no association between cryoglobulinemia and either age, sex, alanine aminotransferase level, or HCV viral load in Japanese or Egyptian patients, while the mean IgM level was significantly higher in the cryoglobulin-positive than in the cryoglobulin-negative chronic hepatitis C patients in each group (P = 0.003 and 0.017, respectively). Cryoglobulinemia was found to be significantly associated with both high IgG level (P = 0.020), and positive ANA (P < 0.001) in Japanese patients with chronic hepatitis C, genotype 1b but not in Egyptians with genotype 4. Multivariate analysis showed that the only factors predisposing to cryoglobulinemia were Japanese ethnicity with HCV genotype1b (P = 0.002, OR = 2.56), high IgM level of >245 mg/dl (P = 0.018, OR = 2.05) and female gender (P = 0.040, OR = 1/0.66). In conclusion, cryoglobulinemia is prevalent in Japanese patients with chronic hepatitis C infected with genotype 1b, but cryoglobulinemia is not common in Egyptians with HCV genotype 4. Although it was not possible to evaluate ethnicity and HCV genotype separately in this study, HCV genotype 1b appears to predispose more to cryoglobulinemia than does genotype 4. Female gender and high serum IgM level were also related.

Distribution of hepatitis B virus (HBV) genotypes among HBV carriers in the Cote d'Ivoire: complete genome sequence and phylogenetic relatedness of HBV genotype E.

The characteristics of hepatitis B virus (HBV) genotype E are not well known because only a few studies have been carried out by complete genome analysis. The aim of this study was to elucidate the distribution of HBV genotypes in Cote d'Ivoire, and to clarify the genotype-related characteristics of genotype E. The distribution of HBV genotypes among 48 HBV carriers in Cote d'Ivoire was determined using serological and genetic methods. The characteristics of genotype E were evaluated by complete genome sequences, and further investigations of small S gene, basic core promoter (BCP) mutation, and precore mutation were undertaken. HBV genotype distribution among the 48 carriers was 6.3% for genotype A, 6.3% for genotype D, and 87.4% for genotype E. Complete genomes of two genotype E strains were sequenced, and found to have 98.2% to 99.2% homology at the nucleotide level when compared with genotype E strains reported previously. In 24 genotype E carriers, the precore mutation was detected in 75% of the patients without HBeAg, in contrast to only 25% of the patients with HBeAg (P < 0.05). All 24 strains have T at nucleotide 1858 in the precore region. In contrast, BCP double mutation was detected in 17% of the patients with HBeAg, and 33% of the patients without HBeAg. These results indicated as the following: (1) genotypes A, D, and E of HBV exist in Cote d'Ivoire and genotype E is the most prevalent; (2) genotype E spread with low genetic diversity over the complete genome in West Africa; (3) HBV precore and/or BCP double variants were common among the patients with genotype E infections.