Umbertina Villano

Seroprevalence and anti‐HEV persistence in the general population of the Republic of San Marino

The prevalence of anti‐HEV was assessed in 2,233 subjects aged 20–79 years in the Republic of San Marino in the years 1990–1991. The sera were tested by ELISA and further confirmed by Western blot (WB) analysis. The overall anti‐HEV prevalence was 1.5%. A significant trend by age was observed. Anti‐HEV prevalence was 0.6% in subjects <30 years and 3.3% in those older than 70 years of age. Family size larger than four persons (OR = 3.8; 95% CI = 1.8–13.2) was the sole independent predictor of anti‐HEV positivity in the multivariate analysis. Anti‐HAV and anti‐HEV prevalences did not show a parallel trend by age. No association was found either between hepatitis E virus (HEV) or hepatitis C virus (HCV) infections. Follow‐up samples 5 years apart were available for 38 out of 54 (70%) anti‐HEV ELISA‐positive subjects. Eight out of 22 (37%) WB‐confirmed anti‐HEV–positive subjects were still anti‐HEV–positive after 5 years. However, anti‐HEV remained positive in all but two (75%) of the subjects with WB‐confirmed ELISA positivity value of S/CO ≥ 2 (cutoff 1.2), but in only 2 out of the 14 subjects (14%) with a WB‐con‐ firmed ELISA positivity value of S/CO < 2 (P < 0.005). None of the 16 subjects ELISA‐positive but not WB‐confirmed was anti‐HEV–positive 5 years apart. Therefore, only a relative proportion of subjects once infected with HEV maintain for at least 5 years anti‐HEV antibodies. J. Med. Virol. 58:49–53, 1999.

Molecular characterisation of HCV genotype 4 isolates circulating in Italy.

The characteristics of genotype 4 subtype variability of HCV isolates circulating in Italy were studied. The viral isolates were identified from 736 HCV‐RNA positive sera originated from seroepidemiological studies undertaken in 4 different regions of North, South Italy and Sardinia. 24 out of 28 genotype 4 isolates (86%) were classified by phylogenetic analysis of E1 genome region (915–1128) as belonging to subtype 4d (Neighbour Joining Method). Three isolates classified as subtype 4a were detected in haemophilic patients, possibly related to infections from blood products. One isolate classified as a new subtype derived from an Eritrean patient subjected to haemodialysis. Very high genome homogeneity (mean 4.3%) was shown by genetic comparisons (DNA dist programs Phylip Package) for all the 4d isolates relative to the studies performed in Veneto, Calabria and Sardinia and originated from subjects from the general population and outpatients (19 subtype 4d isolates out of 24). In the 3 studies different prevalence rates of HCV genotype 4 (3.1%, 1.3%, 14% respectively) were found. In contrast a considerable degree of heterogeneity, both intragroup and with the other groups (mean 8.2% and 8.7%, respectively) was observed among subtype 4d isolates identified in the patients of a haemodialysis centre in Apulia region. In conclusion the subtype 4d of genotype 4 was highly prevalent and endemic in Italy. An elevated level of viral heterogeneity was observed in one study carried out in a region of Southern Italy. This can be related to a longer period of past endemicity of this genotype and to a high level of exposure to reinfections in particular categories of patients such as haemodialysis patients. J. Med. Virol. 62:84–90, 2000.

Seroprevalence of hepatitis A virus and Helicobacter pylori infections in the general population of a developed European country (the San Marino study): evidence for similar pattern of spread.

Objective: To evaluate the role of faecal‐oral transmission in the spread of Helicobacter pylori. Design: A cross‐sectional comparison of the patterns of hepatitis A and H. pylori seropositivity. Methods: At interview, blood samples and questionnaire data were collected from a random sample of 1528 healthy subjects aged 20‐85 years from the Republic of San Marino. Serum samples from each subject were then tested for anti‐H. pylori and anti‐hepatitis A antibodies. Results: Overall, 529 of 670 H. pylori‐seropositive subjects (78.9%) and 460 of 858 H. pylori‐seronegative subjects (53.6%) were hepatitis A seropositive (P<0.01; odds ratio=3.2; confidence interval 95%=2.6‐4.1). This association remained after adjustment by a multiple logistic regression analysis for the confounding effect of age and length of schooling, as surrogate for socio‐economic status (OR=2.0; CI 95%= 1.3‐3.3). The agespecific prevalence curves for H. pylori and hepatitis A infections showed a parallel increase by age, although to a lesser extent for H. pylori. Conclusion: These findings provide evidence that in the community studied H. pylori may have spread in a manner similar to that of hepatitis A.

An integrated approach identifies IFN-regulated microRNAs and targeted mRNAs modulated by different HCV replicon clones

BackgroundInfections with hepatitis C virus (HCV) progress to chronic phase in 80% of patients. To date, the effect produced by HCV on the expression of microRNAs (miRs) involved in the interferon-β (IFN-β) antiviral pathway has not been explored in details. Thus, we compared the expression profile of 24 selected miRs in IFN-β-treated Huh-7 cells and in three different clones of Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system).MethodsThe expression profile of 24 selected miRs in IFN-β-treated Huh-7 cells and in HCV replicon 21-5 clone with respect to Huh-7 parental cells was analysed by real-time PCR. To exclude clone specific variations, the level of 16 out of 24 miRs, found to be modulated in 21-5 clone, was evaluated in two other HCV replicon clones, 22-6 and 21-7. Prediction of target genes of 3 miRs, confirmed in all HCV clones, was performed by means of miRGator program. The gene dataset obtained from microarray analysis of HCV clones was farther used to validate target prediction.ResultsThe expression profile revealed that 16 out of 24 miRs were modulated in HCV replicon clone 21-5. Analysis in HCV replicon clones 22-6 and 21-7 indicated that 3 out of 16 miRs, (miR-128a, miR-196a and miR-142-3p) were modulated in a concerted fashion in all three HCV clones. Microarray analysis revealed that 37 out of 1981 genes, predicted targets of the 3 miRs, showed an inverse expression relationship with the corresponding miR in HCV clones, as expected for true targets. Classification of the 37 genes by Panther System indicated that the dataset contains genes involved in biological processes that sustain HCV replication and/or in pathways potentially implicated in the control of antiviral response by HCV infection.ConclusionsThe present findings reveal that 3 IFN-β-regulated miRs and 37 genes, which are likely their functional targets, were commonly modulated by HCV in three replicon clones. The future use of miR inhibitors or mimics and/or siRNAs might be useful for the development of diagnostic and therapeutic strategies aimed at the recovering of protective innate responses in HCV infections.

microRNA levels in paraffin-embedded indolent B-cell non-Hodgkin lymphoma tissues from patients chronically infected with hepatitis B or C virus

BackgroundEpidemiological evidence links Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) to B-cell non-Hodgkin lymphoma (B-NHL). These B-NHLs, particularly those associated with HCV, may represent a distinct sub-group with peculiar molecular features, including peculiar expression of microRNAs (miRs).The aim of the present study was to search for miRs whose level in indolent B-NHL tissues could be associated with HBV or HCV infection.MethodsFourteen formalin fixed paraffin embedded (FFPE) tissues from HBV+, HCV+ and HBV-/HCV- indolent B-NHL patients were analyzed for levels of 34 selected miRs by quantitative Real-Time PCR. Reactive lymph nodes (RLNs) from HBV-/HCV- patients were included as non-tumor control. Statistical analysis of output data included Pearson and Spearman correlation and Mann-Whitney test and were carried out by the STATA software.ResultsMiR-92a was decreased exclusively in HBV-/HCV- B-NHLs, while miR-30b was increased in HBV+ and HCV+ samples, though only the HCV+ achieved full statistical significance. Analysis of a small subset of B-NHLs belonging to the same histological subtype (Nodal Marginal Zone Lymphoma) highlighted three miRs associated with HCV infection (miR-223, miR-29a and miR-29b) and confirmed decreased level of miR-92a in HBV-/HCV- samples also when considering this restricted B-NHL group.ConclusionsAlthough caution is needed due to the limited number of analyzed samples, overall the results suggest that differences at the miR expression level exist between indolent B-NHLs developed in patients with or without HBV or HCV infection. The identification of three further miRs associated with HCV by analyzing histologically homogeneous samples suggests that variations of miR levels possibly associated with HBV or HCV may be obscured by the tissue-specific variability of miR level associated with the different histological subtypes of B-NHL. Thus, the identification of further miRs will require, in addition to an increased sample size, the comparison of B-NHL tissues with the same histological classification.

Evolutionary dynamics of HBV‐D1 genotype epidemic in Turkey

Hepatitis B virus (HBV), is the leading cause of liver diseases infecting an estimated 240 million persons worldwide. The HBV prevalence rates are variables between different countries, with an high level of endemicity in the south‐eastern part of Europe. Seven main HBV‐D subgenotypes have been described until now (D1–D7). Turkey, seems to have played an important role in the penetration of HBV‐D1 in the Mediterranean area. The importance of Turkey in the European epidemiology of HBV is also suggested by the observation that the highest spread of HBV infection in the Continent are reported in Turkey with Romania, Bulgaria, Greece, Albania and some southern regions of Italy. In this paper the molecular epidemiology and the epidemiological history of HBV‐D in Turkey was studied, by characterizing 34 new Turkish isolates and performing a phylogeographic reconstruction. By using a phylodynamic and phylogeographic Bayesian approach, the analysis suggested that HBV‐D1 originated in Turkey about in the early 1940s. The large prevalence of D1 in comparison to the other subgenotypes in Turkey confirms the importance of this Country as epidemiological reservoir of HBV‐D1 dispersion. The phylogeny suggests that after each initial introduction of the virus in a specific population, separate transmission clusters have been evolving along independent phylogenetic lineages. Better characterization and continuous monitoring of such groups are going to be crucial to understand in detail the epidemiology of HBV‐D1 subgenotype in Turkey and to assess the efficacy of prevention, vaccination and therapy in controlling the epidemic. J. Med. Virol. 86:109–116, 2014.

A computational approach to identify point mutations associated with occult hepatitis B: significant mutations affect coding regions but not regulative elements of HBV.

BackgroundOccult Hepatitis B Infection (OBI) is characterized by absence of serum HBsAg and persistence of HBV-DNA in liver tissue, with low to undetectable serum HBV-DNA. The mechanisms underlying OBI remain to be clarified. To evaluate if specific point mutations of HBV genome may be associated with OBI, we applied an approach based on bioinformatics analysis of complete genome HBV sequences. In addition, the feasibility of bioinformatics prediction models to classify HBV infections into OBI and non-OBI by molecular data was evaluated.Methods41 OBI and 162 non-OBI complete genome sequences were retrieved from GenBank, aligned and subjected to univariable analysis including statistical evaluation. Their S coding region was analyzed for Stop codon mutations too, while S amino acid variability could be evaluated for genotype D only, due to the too small number of available complete genome OBI sequences from other genotypes.Prediction models were derived by multivariable analysis using Logistic Regression, Rule Induction and Random Forest approaches, with extra-sample error estimation by Multiple ten-fold Cross-Validation (MCV). Models were compared by t-test on the Area Under the Receiver Operating Characteristic curve (AUC) distributions obtained from the MCV runs for each model against the best-performing model.ResultsVariations in seven nucleotide positions were significantly associated with OBI, and occurred in 11 out of 41 OBI sequences (26.8%): likely, other mutations did not reach statistical significance due to the small size of OBI dataset. All variations affected at least one HBV coding region, but none of them mapped to regulative elements. All viral proteins, with the only exception of the X, were affected. Stop codons in the S, that might account for absence of serum HBsAg, were not significantly enriched in OBI sequences. In genotype D, amino acid variability in the S was higher in OBI than non-OBI, particularly in the immunodominant region. A Random Forest prediction model showed the best performance, but all models were not satisfactory in terms of specificity, due to the small sample size of OBIs; however results are promising in the perspective of a broader dataset of complete genome OBI sequences.ConclusionsData suggest that point mutations rarely occur in regulative elements of HBV, if ever, and contribute to OBI by affecting different viral proteins, suggesting heterogeneous mechanisms may be responsible for OBI, including, at least in genotype D, an escape mutation mechanism due to imperfect immune control. It appears possible to derive prediction models based on molecular data when a larger set of complete genome OBI sequences will become available.

Population-based survey of hepatitis A virus infection in the Republic of San Marino

In 1990–1991, the prevalence of antibodies to hepatitis A virus infection (anti-HAV) was assayed by the ELISA method among 1528 apparently healthy subjects, 20–85 years old in the Republic of San Marino. Subjects were selected from the list of residents by a random stratified sampling procedure with a proportional allocation by age, sex and district of residence. The overall anti-HAV prevalence was 64.7%; it increased from 28.6% in subjects 20–30 years old to 97% in those > 60 years (p < 0.01). No gender difference was observed. At the multivariate analysis age > 40 years (OR: 39.5; 95% CI: 12.4–126) and lowest level of schooling (OR: 1.8; 95% CI: 1.1–2.9), which is a good indirect indicator of socio-economic status, resulted both independent predictors of anti-HAV seroposi-tivity. These findings reflect the improved sanitation standards in this area and indicate that the proportion of non-immune adults is increasing with a higher risk of symptomatic infection in the near future.

Naturally Occurring Surface Antigen Variants of Hepatitis B Virus in Tunisian Patients

In Tunisia, the prevalence of naturally occurring surface (S) gene variants of hepatitis B virus (HBV) has not been determined. In the present study, the prevalence of these variants was examined in terms of the clinical and viral state in a series of 99 Tunisian patients with HBV infection. The S genes were amplified and directly sequenced. Genotype D was predominant (98%), 40.4% isolates belonged to subgenotypes D7 and 1 to subgenotype D2. The most common subtype was ayw2 (95.9%). In total, 60.6% of the studied strains harbored S mutations. Several novel mutation patterns were detected. Interestingly, the presence of S mutations was significantly correlated with the D7 subgenotype, low HBV DNA and advancing age (≥35 years), and tended to be higher in liver cirrhosis than in chronic infection. The global prevalence of the major hydrophilic region variants was 12.1%, with substitution S143L/T as the most frequent (4%). Only 33.9% of S substitutions produced amino acid changes in the polymerase gene. In conclusion, a high prevalence of naturally occurring HBsAg variants was observed among Tunisian HBV carriers. Natural viral variability in a geographical region and duration of infection are among the major factors associated with the occurrence of S mutations.

Evolutionary dynamics of HBV‐D7 subgenotype in Tunisia

Hepatitis B virus (HBV) is the main cause of diseases liver related infecting more than 200 milion persons worldwide. HBV infection shows high level of prevalence in South‐East Europe and in Mediterranean basin. In Tunisia, a country with an intermediate level endemicity, HbsAg prevalence ranges from 2 to 5%. Most of the HBV isolates from Tunisia were classified as subgenotype D7 whose circulation is restricted to a specific area of North Africa including Maghreb region. In this paper, the phylogeny of HBV‐D7 isolated from 38 Tunisian patients was investigated by analyzing the S gene region of HBV. A Bayesian coalescent‐based framework was used to estimate the origin of the HBV‐D7 in the country. The Tunisian D7 isolates were found to share a common ancestor whose origin was traced back to 1958. Population dynamics indicated that HBV‐D7 epidemic in Tunisia grew exponentially from 1960s to 1990s. After that, the curve reached a plateau around the years 2000 likely due to the implementation of the infant vaccination program in 1996. Epidemiological data suggested that the exponential growth phase was likely sustained by intra‐familial transmission events occurring during infancy. Further characterization of HBV‐D7 isolates should be performed to evaluate, in the post‐vaccination era, the emergence of new transmission routes, and to monitor the efficacy of the vaccination program. J. Med. Virol. 89:469–475, 2017.