Distribution of IL28B Polymorphism in a Cohort of Italians and Immigrants with HCV Infection: Association with Viraemia, Stage of Fibrosis and Response to Treatment.

Aims of the study are to investigate, in a cohort of patients affected by HCV chronic hepatitis with genotypes 1 and 4, the prevalence of interleukin 28B (IL28B) genotypes, the possible association between IL28B polymorphism and severity of liver damage, the role of IL28B CC as a predictor of outcome. 365 patients with HCV infection were observed between 2013 and 2014. Demographic, virological, biochemical, and genetic characteristics of each patient were investigated. Liver fibrosis was assessed by transient elastometry. Mean age of the patients (72.9 % males, 27.1 % females) is 50 years. 91.5 % % of patients are Caucasian, 8.5 % African. In the patients with HCV1 and HCV4 a higher frequency of IL28B CT is observed with a prevalence of 52.1 and 61.8 % respectively. As regards ethnic group, African people have a prevalence of 35.5 % for CC, while Caucasians have a prevalence of 23.8 % for CC. In our cohort, IL28B polymorphism does not show significant differences among ethnic groups and in HCV1 and HCV4 genotypes. As described in literature, IL28B CC genotype is confirmed as predictor of sustained virological response in both Caucasians and Africans. A significant correlation between liver fibrosis and IL28B polymorphism emerges.

HBV whole-genome mutation profile in HIV-1/HBV coinfected patients in a long-term follow-up study.

PURPOSE: Human immunodeficiency virus (HIV-1)-infected patients frequently harbour hepatitis B and C viruses (HBV and HCV, respectively). Possible modifications of the natural history of hepatitis B may occur. The aim of this study was to characterise HBV diversity and evolutionary and mutational viral genome profiles in HIV-1/HBV coinfections. METHODS: HIV-1 and HBV markers determinations (Roche, FRG; Abbott, USA) and HBV genome-length retrospective analysis were performed in follow-up isolates from patients who were either stably HBsAg-negative with a low level of HBV DNA (occult hepatitis B infection, OBI) or HBsAg-positive with a high level of HBV DNA. Phylogenetic analysis (maximum likelihood method, MEGA5), statistical analysis and evolutionary rates calculation (d S/d N) were applied. RESULTS: Positive selection pressures in the PreS/S region and a significantly higher number of mutations in this region including the major hydrophilic region (MHR) and the "a" determinant were shown in HBsAg-negative (possibly OBI) compared to stably HBsAg-positive HIV-1/HBV subgenotypes D3/A2 coinfected patients. Mutants previously described in HIV-1/HBV coinfected patients were found. Known mutants Y100C, P127T and P120A associated to Y134H and S143T and new S mutants, which may potentially affect HBsAg expression and secretion and anti-HBs binding, were detected in baseline sera persisting up to the end of 9 years follow-up. Known mutations of BCP, Pre-C, C and X regions were also characterised. Natural mutants strictly known as being involved in diagnostic failure were not detected; however, numerous corresponding sites showed amino acid variations. CONCLUSIONS: Evolutionary and genotypic differences observed, particularly in the PreS/S region, between HBsAg-negative (OBI) and HBsAg-positive HIV-1/HBV coinfected patients, may contribute, in association with mutations of other genomic regions, to the HBsAg-negative phenotype.

Virological and epitope evolution of HCV infection from acute hepatitis C to subsequent episodes of HCV-related acute liver cell necrosis.

AIM: To evaluate the virological and clinical events occurring during a 3-year follow-up in three patients who, after symptomatic acute hepatitis C (AHC), experienced subsequent episodes of HC virus (V)-related acute liver cell necrosis. PATIENTS AND METHODS: The three patients were investigated for viral variability in the core, E1/E2, and NS5b regions during different phases of infection, and a computer-assisted analysis of the variation of known predicted epitopes in the consensus sequence was performed. RESULTS: The first patient showed numerous genetic variations, which may be related to the maintenance of a chronic HCV infection state and to episodes of liver disease exacerbation. The second patient showed minimal viral variations associated with apparent resolution of the infection, but the same virus isolate, based on phylogenetic analysis, produced a second acute episode after the occult phase. The third patient, after the resolution of AHC, manifested a second episode of HCV infection by a different HCV sub-genotype. CONCLUSION: Episodes of HCV-related acute liver cell necrosis after AHC may be associated to different virological patterns, such as the establishment of a chronic HCV infection, a reactivation of an occult virus, or a reinfection by a different HCV genotype.

Kinetics of WHV-HDV replication in acute fatal course of woodchuck hepatitis.

The objective of this study was to evaluate, by developing one-step real-time PCR, the outcome of superinfection with hepatitis D virus (HDV) genotype I in woodchucks that were chronic carriers of woodchuck hepatitis virus (WHV) and did not show relevant signs of liver damage. Three woodchucks (Marmota monax) chronically infected with WHV were superinfected with a woodchuck HDV inoculum. The evolution of the WHV and HDV infections was monitored by quantifying HDV-RNA, WHV-DNA, and HDV-WHV antigens and antibodies. WHV and HDV sequencing was also performed and liver markers were evaluated. Liver damage was assessed using the Ishak method. All woodchucks showed a high HDV viral load, antigenemia and short survival after superinfection. Histopathological examination of autoptic liver samples showed massive liver necrosis compatible with an acute fatal course of hepatitis. The WHV sequencing showed that the virus population was not substituted by the WHV inoculum. The HDV sequencing performed during superinfection and at autopsy indicated amino acid changes in immune dominant regions of the HDV antigen. The strong correlation between acute infection with HDV genotype I and rapid and fatal liver failure indicates that HDV can be an important factor in the prognosis of HDV-WHV-superinfected woodchucks.

Nosocomial transmission in simultaneous outbreaks of hepatitis C and B virus infections in a hemodialysis center.

Reported here are details of a simultaneous outbreak of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections that occurred in a hemodialysis centre in northern Italy, with three patients seroconverting for HBsAg and four patients seroconverting for HCV antibodies. Phylogenetic analysis of the E2 region of the isolates from HCV-seroconverted patients showed the sequences were grouped in the same distinct branch as in a chronically HCV-infected patient, suggesting that the chronically infected patient was the index case. For the patients with HBV infection, phylogenetic analysis showed strong clustering among the sequences of the three patients who seroconverted to HBsAg and no relatedness between them and the sequences of patients chronically infected with HBV. For one of the patients who seroconverted to HBsAg, the last test with negative results for HBV markers had been performed 18 months prior to HBsAg seroconversion. This patient may have been previously infected with HBV and is presumed to be the source of the outbreak. This report emphasizes the importance of using universal precaution measures and HBV vaccination to prevent the transmission of viral hepatitis among chronic hemodialysis patients.

Molecular epidemiology of hepatitis C virus genotype 4 isolates in Egypt and analysis of the variability of envelope proteins E1 and E2 in patients with chronic hepatitis.

We analyzed hepatitis C virus (HCV) genotype 4 isolates circulating in the Alexandria District (Egypt) in terms of genetic divergence and the presence of different subtypes. Hypervariable region 1 (HVR1) and the NH2 region of the E2 protein were characterized, and the heterogeneity of subtype 4a isolates was evaluated by analyzing epitope frequencies, immunoproteasome prediction, and possible glycosylation patterns. The heterogeneity of the nucleotide sequences was greater than that found in previous studies, which reported only subtype 4a. Subtype 4a was most common (78% of cases), yet four new subtypes were found, with subtype 4m representing 11% of the cases and the other three subtypes representing another 11%. Substantial heterogeneity was also found when the intrasubtype 4a sequences were analyzed. Differences in the probability of glycosylation and in the positions of the different sites were also observed. The analysis of the predicted cytotoxic-T-lymphocyte epitopes showed differences in both the potential proteosome cleavage and the prediction score. The Egyptian isolates in our study also showed high variability in terms of the HVR1 neutralization epitope. Five of these isolates showed amino acid substitutions never previously observed (a total of six positions). Four of these residues (in four different isolates) were in positions involved in anchoring to the E2 glycoprotein core and in maintaining the HVR1 conformation. The results of this study indicate that HCV genotype 4 in Egypt is extremely variable, not only in terms of sequence, but also in terms of functional and immunological determinants. These data should be taken into account in planning the development of vaccine trials in Egypt.

Clinical and molecular characterization of chronic hepatitis B in Albania: a country that is still highly endemic for HBV infection.

Albania is a Mediterranean country, still with a high endemicity level of hepatitis B virus (HBV) infection. The chronic hepatitis B profile was characterized in this geographical area and used as a model to investigate the impact of endemicity level on the prevalence of the two major forms of chronic hepatitis B (HBeAg-positive and HBeAg-negative chronic hepatitis B). A cross-sectional study was conducted among 62 chronic hepatitis B patients consecutively admitted to the most important tertiary health care center for the diagnosis and treatment of liver disease in Albania. HBV-DNA was measured with an in-house PCR with a sensitivity of 10(4) copies/ml which uses primers encompassing the pre-core/core region. PCR products were subjected to sequencing and oligohybridization assay. Of the 62 patients, 75.8% had HBeAg-negative chronic hepatitis B. Genotype D was found in all 39 patients with detectable HBV viremia, for whom the heterogeneity of the region modulating HBeAg expression was assessed. Basic core promoter (BCP) mutations (1762/1764) were observed more often in anti-HBe-positive and older patients. In more than 90% of the HBeAg-negative chronic hepatitis B patients with detectable viremia, HBV that carries the G to A pre-core mutation at nucleotide 1896 was found. Patients with HBeAg-positive chronic hepatitis B were younger than HBeAg-negative chronic hepatitis B patients, and for symptomatic and asymptomatic liver-disease patients, the age of peak prevalence was at least 10 years lower for HBeAg-positive chronic hepatitis B patients. In conclusion, the virological and clinical pattern of chronic hepatitis B in Albania is similar to that observed in other Mediterranean countries; it seems to be independent of the HBV endemicity level.

Molecular characterisation of SENV and TTV infections in hepatopathic liver-transplant patients. Brief Report.

The presence of SENV and TTV infections among 50 patients who had undergone liver transplantation was evaluated. UTR amplification showed that 46 (92%) sera were positive. ORF-1 amplification showed that 25 (50%) patients were positive for either SENV (51.3%), TTV (10.8%), or both (37.8%) all confirmed by sequencing and phylogenetic analysis. SENV-D and SENV-H were the most prevalent viruses. The phylogenetic analysis of isolates showed that whereas SENV-D and SENV-G viruses showed sequence stability and strain persistence, SENV-H had cleared or mutated. Biological differences seem to exist among different genotypes in terms of viral replication and their persistence.

Different levels of variability in subtypes 1b and 4a of hepatitis C viruses.

We performed genetic and phenic analyses to evaluate nucleotide and amino-acid sequences of the amino-terminus of the E1 protein of HCV genotype 1b (extracted from databank) and 4a (characterised in this study). The non-synonymous (ka) mutation analysis demonstrated that the genome of genotype 1b was not saturated by variations, with a rate of transition/transversion (s/v) of 1.5, which is similar to the expected ratio (i.e., 2.0). The s/v ratio in genotype 4a isolates was lower (0.98), indicating saturation due long-term variability. Moreover, the genotype 1b sequences showed a higher number of ka mutations (s+v) (mean of 2.8 per sequence) than genotype 4a (mean of 1.5). The introduction of ka mutations resulted in a higher degree of amino acid variability in genotype 4a. In the genome of genotype 1b, each nucleotide mutation introduced new amino acids, with a Granthan distance of 3.35-42.5, whereas for genotype 4a the distances ranged from 48.8 to 102.1. The phenic analysis also indicated different and complex patterns of amino-acid substitution. Finally, diverse isoelectric points and hydrophobicity were predicted for the two genotypes, with a higher acidity for genotype 4a E1 proteins.

Scaffold attachment region located in a locus targeted by hepadnavirus integration in hepatocellular carcinomas.

The role of viral integration in HBV induced hepatocellular carcinoma (HCC) is still controversial. In the WHV/woodchuck animal model, WHV integration was found to activate the N-myc2 oncogene either by enhancer insertion in proximity of the gene, or by integration in a distantly located uncoding locus, win. In addition, we have reported that N-myc2 activation also results from WHV integration in the b3n locus, located several kilobases downstream of N-myc2. In this work we report the search for function(s) of the b3n locus that might be possibly affected by WHV integration and indirectly activate N-myc2. A 0.5 kb region of the sequence of this locus exhibited unusual features, typical of scaffold/matrix attachment regions (S/MAR). Standard in vitro binding assays are commonly used to assess if a DNA fragment is a S/MAR. DNA fragments cloned from the b3n locus were tested for in vitro binding affinity for both heterologous and autologous nuclear scaffold preparations. Only the fragment spanning the region rich of S/MAR motifs was found to bind specifically nuclear scaffolds, thus demonstrating that a S/MAR element is present in the b3n locus. Based on these findings, we speculate that WHV integration might deregulate the S/MAR element and indirectly affect the expression of the N-myc2 gene located upstream of the S/MAR. Our findings also suggest that the role of HBV integration should be reconsidered, because a similar mechanism has not been investigated to date in human HCC.

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.

Molecular evolution of HCV genotype 2c persistent infection following mother-to-infant transmission.

The molecular evolution of HCV 2c in a case of vertical transmission was studied by comparing the virus quasispecies in the sera from the mother and from the child in a two-year follow-up. The positivity of HCV-RNA since the delivery accounted for an in-utero infection. The Core-E1 genome region (nt 928-1225) was amplified by polymerase chain reaction (PCR) from serum samples collected at delivery and at 3, 9, 18 and 24 months after birth. The RIBA pattern was characterised by isolated anti-c22 positivity in the serum from mother and in sera from the child during the first 9 months. Additional presence of anti-c33 was observed afterwards. Genetic relatedness among isolates and with a mother minor variant serum (Mo1. 13) was found (mean variability ranged between 0.79% and 1.20%). From phylogenetic analysis this variant was identified as the origin of one of the two main lineages that included all isolates from child sera at 9, 18 and 24 months. The variability analysis has shown that high viral heterogeneity is present in the child serum collected at birth (3.16%). In this phase the dn/ds index (1.26%) indicates the presence of strong selective pressures. The development of child specific immune response at 9th month was concurrent with the disappearance of two mutants at positions 11 and 104 of E1. This rare case of in-utero mother-to-infant transmission can be considered as a model to elucidate the HCV quasispecies diversification during the first stage of infection.

Acute quadriplegia and loss of muscle myosin in patients treated with nondepolarizing neuromuscular blocking agents and corticosteroids: mechanisms at the cellular and molecular levels.

OBJECTIVE: Long-term treatment with nondepolarizing neuromuscular blocking agents and corticosteroids in the intensive care unit is not benign, and an increasing number of patients with acute quadriplegic myopathy have been reported with increased use of these drugs. The purpose of this study was to investigate the mechanisms underlying acute quadriplegic myopathy. DESIGN: Percutaneous muscle biopsy samples were obtained, and electrophysiologic examinations were performed during the acute phase and during recovery in patients with acute quadriplegic myopathy. Regulation of muscle contraction and myofibrillar protein synthesis was studied using cell physiologic techniques, ultrasensitive electrophoresis, in situ hybridization, and histopathologic techniques. SETTING: All patients were seen in the intensive care unit of different university hospitals. PATIENTS: All patients were critically ill with sepsis. They had been given massive doses of corticosteroids in combination with variable doses of neuromuscular blocking agents. All patients developed paralysis of spinal nerve-innervated muscles. On the other hand, cranial nerve-innervated muscle and sensory and cognitive functions were well maintained after discontinuation of treatment with neuromuscular blocking agents. INTERVENTION: Muscle biopsy samples were obtained and electrophysiologic examinations were performed in all patients. MEASUREMENTS AND MAIN RESULTS: The major observations in patients with acute quadriplegic myopathy were, as follows: a) a general decrease in myofibrillar protein content; b) specific but highly variable partial or complete loss of myosin and myosin-associated proteins; c) very low thick-filament/thin-filament protein ratios; d) absence of myosin messenger RNA; and e) a dramatically impaired muscle cell force-generating capacity in the acute phase of acute quadriplegic myopathy. During clinical improvement, normal expression of myosin messenger RNAs, reexpression of thick-filament proteins, and increased specific tension were observed. CONCLUSIONS: Acute quadriplegic myopathy is associated with a specific decrease in thick-filament proteins related to an altered transcription rate. Although the decreased content of thick-filament proteins is important for prolonged muscle weakness, it is not the primary cause of muscle paralysis in the acute stage, during which impaired muscle membrane excitability probably plays a more significant role. Several factors contribute to this condition, but the action of corticosteroids seems to be the predominant one, along with potentiation by neuromuscular blocking agents, immobilization, and probably also concurrent sepsis.

Molecular characterization of HCV 1b intra-familiar infection through three generations.

Vertical transmission is an uncommon route of hepatitis C virus (HCV) infection. Little is known about the way of virus spread between relatives. Furthermore, the nucleotide sequence variability studies that can be used for the definition of cases of HCV transmission still need accurate standardization. In this study, we analyzed the HCV positive sera from subjects belonging to one family. Five out of seven individuals were positive both for anti-HCV and HCV-RNA. The epidemiological data, in our knowledge, excluded the possible risk of parenteral exposure to HCV for the members of the family. The genetic relatedness of the viruses infecting the members of this family was demonstrated by the phylogenetic analysis of sequences from E1 genome region. The analysis included the calculation of the genetic divergence specific index, based on the ratio of synonymous/non-synonymous mutations. By the analysis of this genome region, we demonstrated the occurrence of HCV transmission among family members. In 2 cases out of 3, Mother-to-Infant transmission was demonstrated that involved three generations of the family. Transmission by sexual route was absent. A method of sequence analysis of E1 HCV genome region is proposed as molecular approach for the definition of transmission cases of HCV.

Activation of the N-myc2 oncogene by woodchuck hepatitis virus integration in the linked downstream b3n locus in woodchuck hepatocellular carcinoma.

In the woodchuck hepatitis virus (WHV)/woodchuck model for hepatitis B virus-induced hepatocellular carcinoma, frequent activation of N-myc oncogenes by WHV integration has been firmly established. N-myc2, the most frequently affected gene, was reported to be activated by WHV insertion either in the proximity of the gene or in a distant uncoding locus, win. We previously reported that a WHV integration cloned from a liver tumor was located in a chromosomal locus already described by others as the site of WHV integration in another hepatocellular carcinoma. On this basis, the locus, named b3n, was defined as a recurrent site of WHV integration. A scaffold or matrix attachment region (S/MAR) element was subsequently shown to be located in this locus approximately 1 kb from the WHV insertion sites. S/MARs are genetic elements involved both in structural and functional organization of chromosomal DNA and in stimulation of gene expression. In the present work, we investigated the possibility that an N-myc gene might be affected by integration in b3n. Analysis of a liver tumor harboring WHV integration in this locus showed N-myc2 overexpression. By restriction analysis, the b3n locus was shown to be located downstream of N-myc2, so the known sites of viral insertion in b3n were approximately 11 kb downstream of the N-myc2 promoter. Although these data support that WHV insertion in b3n activates N-myc2, the mechanisms previously described to be involved in N-myc2 activation do not appear to properly account for activation in this subset of WHV integrations. Available data suggest that activation of N-myc2 by WHV integration in b3n might be mediated by the S/MAR located near the WHV insertion.

Hepadnavirus evolution and molecular strategy of adaptation in a new host.

In order to elucidate the mechanisms of hepadnavirus evolution in vivo and to trace the fate of known quasispecies in a single animal during the acute phase of infection, a woodchuck (Marmota monax) was infected with the hepadnavirus woodchuck hepatitis B virus (WHV). Woodchuck 197 (W197) was injected intravenously with pooled sera collected from a chronic carrier that had been infected originally with a molecular clone of known genome sequence (WHV7). Viral genome variants from both the inoculum and the follow-up sera from W197 were characterized for the presence of quasispecies related to the WHV7 sequence. Interestingly, WHV7-related genomes were predominant 6 weeks post-infection (p.i.), whereas a highly heterogeneous virus population was present in the first viraemic serum (4 weeks p.i.). Using WHV7 as the prototype, the variability of the Pol and PreS/S regions in the first 11 weeks p.i. has been calculated. The sequence population in serum collected 6 weeks p.i. was highly homogeneous, with a mean variability of 0.36% in the region analysed. Mean variability values ranging from 0.82% to 1.61% were found in quasispecies from the other sera. The presence of possible selective pressure was analysed by means of the non-synonymous versus synonymous variation ratio (dn/d5). We found that the dn/d5 values were stable for the S ORF (ranging from 2.6 to 3.0), whereas a wider range was observed for the Pol ORF (from 1.4 to 3.0). Furthermore, from the analysis of the variability of the codon positions for the two overlapping ORFs it was found that, in most cases, non-synonymous mutations at position 1 of the Pol ORF (position 3 of the S ORF) corresponded to synonymous variation in the S (Pol) ORF, indicating independent evolution of the encoded proteins.

Age-related persistent clonal expansions of CD28(-) cells: phenotypic and molecular TCR analysis reveals both CD4(+) and CD4(+)CD8(+) cells with identical CDR3 sequences.

In a small group of subjects we had identified persistent expansions (range 6-72%) of CD4(+)CD8(+) double-positive (DP) peripheral blood (PB) cells which express the CD8 alpha/alpha homodimer. Here, DP cells present in a larger cohort were further investigated and found by FACS analysis to express a single or a dominant TCRBV family. In these subjects, with a mean age of about 64 years, expansions of CD4(+) cells with the same TCRBV family specificity as in the respective DP cells also were consistently detected. TCR heterogeneity of the dominant TCRBV family was specifically evaluated: The amplified CDR3 region was cloned and found to consist of one single or two largely dominant sequence patterns. Furthermore, cloning of the CDR3 region from FACS-sorted DP, CD4(+), or CD8(+) cells indicates that both DP and CD4(+), but not CD8(+) cells, isolated from the same individual possess a striking identity of the CDR3 regions. As indicated by FACS analysis, the clonally expanded cells occur in the CD4(+)CD28(-) cells. Taken together, these results suggest that expanded CD4(+)CD28(-) cells might also acquire CD8 alpha/alpha expression and become DP and imply that CD4 clonality is a more frequent phenomenon than previously suspected. In conclusion, the persistent expansions described in this report represent a novel group of age-related benign clonal expansions of still undefined significance of a rare CD28(-) T cell subset.

Identification of scaffold/matrix attachment region in recurrent site of woodchuck hepatitis virus integration.

Scaffold or matrix attachment regions (S/MARs) are noncoding genomic DNA sequences displaying in vitro selective binding affinity for nuclear scaffold. They have been reported to be involved in the physical attachment of genomic DNA to the nuclear scaffold, and thus in the organization of the chromatin in functional loops or domains, and in the regulation of gene expression. In this work, we report the identification of an S/MAR in a woodchuck chromosomal locus, named b3n, previously described as a recurrent site of woodchuck hepatitis virus (WHV) DNA integration in woodchuck hepatocellular carcinoma (HCC). The 4.3-kb sequence of this locus contains several Alu-like repeats and a gag-like coding region with frameshift mutations. Computer analysis revealed the presence of a region with unusually high AT content, typical of most S/MARs, and of specific motifs (A boxes, T boxes, topoisomerase II sites, and unwinding elements) overlapping or in proximity to the region with high AT content, predicting that b3n might contain an S/MAR. Fragments of the b3n locus were isolated by conventional and inverse PCR techniques. In in vitro binding experiments with both heterologous and autologous scaffold preparations, a 592-bp fragment spanning the region rich in S/MAR features showed marked scaffold affinity, which was specific when autologous scaffolds were used. The presence of an S/MAR at the b3n locus and its nature as a recurrent WHV integration site in HCC suggest the involvement of S/MAR elements in some of the mechanisms leading to liver oncogenesis.

Immunogenicity of hepatitis B vaccines among infant recipients of acellular and whole cell pertussis DTP vaccines.

The study was conducted to assess the immunogenicity of three doses of two recombinant hepatitis B virus (HBV) vaccines, administered simultaneously with a DT vaccine or one of three different pertussis vaccines combined with diphtheria and tetanus toxoids. The study population consisted of 1237 children selected from the cohort of 15,601 children enrolled in the Italian trial on pertussis vaccines. HBV vaccination was performed at 2, 4 and 12 months of age, with the first two doses concurrent with OPV and DTP vaccination. The DTP vaccines administered in the pertussis trial included one whole cell DTP, licensed in the USA, and two three-component acellular DTaPs, manufactured in Europe. Immunogenicity to HBV was evaluated on serum samples collected 9 months after the third dose of HBV vaccine. Antibodies against HBsAg were detected by ELISA and expressed in mlU/ml. In 13 children, the antibody response was below the protective level of 10 mlU/ml-1. No statistical difference was found among the various study groups with respect to the proportion of children showing protective response. Higher humoral response was observed in children receiving mixed HBV vaccines in each pertussis study groups.

Molecular heterogeneity and new subtypes of HCV genotype 4.

The subtype distribution of HCV genotype 4 was studied in two different African countries, Egypt and Tanzania. The HCV isolates were obtained from epidemiological studies involving, respectively, 135 hepatopatic patients and 1043 pregnant women and outpatients. Sequence comparison and phylogenetic analysis of the NS5b genome region (nt 8327-8499) were performed. Fourteen out of 18 isolates from Egypt, but only 3 out of 6 isolates from Tanzania clustered in the same branch of subtype 4a. Three new proposed subtypes have been identified. The first includes 1 isolate from Egypt (EGY15); the second, 2 isolates from Egypt (EGY193 and EGY44) and 2 isolates from Tanzania (D776, D61); and the third, 1 isolate from Egypt (EGY47) and 1 isolate from Tanzania (D70). These isolates cluster in branches different from any other, corresponding to a known subtype of genotype 4. In conclusion, remarkable genetic heterogeneity has been found among genotype 4 isolates simultaneously circulating in a restricted area. This was particularly observed in the study performed in Tanzania. Potential concern about the sensitivity of diagnostic assays and possible implications in the development of future vaccines have been stressed.