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.

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.

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.

GB virus C/hepatitis G virus exposure in Italian pediatric and young adult thalassemic patients.

BACKGROUND: The aim was to estimate the prevalence and the persistence of GB virus C/hepatitis G virus (GBV-C/HGV) exposure markers in a group at high risk for transfusion-transmitted agents. PATIENTS AND METHODS: Serum samples from 37 thalassemic patients were screened for GBV-C/HGV RNA by reverse transcription PCR (RT-PCR) and for antibodies to the envelope protein E2 of GBV-C/HGV (anti-E2). RESULTS AND DISCUSSION: GBV-C/HGV RNA and anti-E2 were detected in 13 (35%) and 12 (32%) sera, respectively. Contemporary presence of both markers was found in one patient. GBV-C/HGV exposure was found in 24 patients (64.8%). Mean levels of liver enzymes were similar in both exposed and unexposed GBV-C/HGV groups. 33 out of 35 patients showed no change in GBV-C/HGV RNA and anti-E2 status in sera taken 6 months apart. The rate of persistent infection was 92.3% and the anti-E2 seroconversion rate was 23% for sera taken at least 6 months apart. The temporal overlap between anti-E2 seroconversion and loss of detectable GBV-C/HGV RNA may last more than 6 months.

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.

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.

Genotyping HCV isolates from Italy by type-specific PCR assay in the core region.

A revision of the polymerase chain reaction (PCR) core procedure was performed for genotyping hepatitis C virus (HCV) in 139 patients from Italy. This procedure, developed prior to the identification of new genotypes, may be inadequate in several geographical areas. We proposed a new typing mixture in which primers for types 2c and 4, that are reported to be circulating in Italy, were added and a primer for type 2b was substituted. Using the modified procedure, 139 HCV-positive patients were analysed. The HCV genotype was identified in 96.4% of the cases. We observed double infections and unclassified genotypes in 5 (3.6%) and 5 (3.6%) patients, respectively. The classification of isolates into genotypes and subtypes 2b, 2c and 4 was confirmed by sequence analysis. Furthermore, the efficiency and accuracy of the modified core procedure were evaluated by parallel testing of 107 out of 139 samples using the line probe assay, and demonstrated a 98.9% degree of concordance. The results demonstrated the specificity of the selected primers for type 2c, 2b and 4 and confirmed the circulation of types 2c and 4 in Italy. In conclusion, the proposed modified PCR procedure is the only primer-specific PCR genotyping method available for identification of the 2c and 4 genotypes reported to be circulated in Italy and other European countries.

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.

Hepatitis C virus infection in Italian kidney graft recipients. Changing risk factors and hepatitis C virus genotypes.

BACKGROUND/AIMS: The risk for hepatitis C virus infection in kidney transplant recipients has been reduced by the introduction of accurate diagnostic tests. Little is known, however, of the current risk factors and the molecular genetics of hepatitis C virus infection in Italy. METHODS: We studied 101 Italian kidney allograft recipients, transplanted between 1975 and 1995, in Italy or abroad. Sera were assayed for biochemistry, presence of HBsAg, anti-hepatitis C virus antibodies, hepatitis C virus-RNA (by reverse transcription nested PCR) and hepatitis C virus genotyping. RESULTS: HBsAg was found in 4 sera and anti-Hepatitis C Virus antibodies in 33 (33%). The duration of pre-transplant dialysis was longer in anti-hepatitis C virus positive than in anti-hepatitis C virus negative patients (5.9 +/- 4.3 vs 2.8 +/- 1.9 years, p = 0.0004). Anti-hepatitis C virus seropositivity was more frequent among patients grafted before than after 1990 (50% vs 27%, p = 0.04) and varied depending on the country of transplantation (25% in Italy; 56% in other European countries; and 40% in non-European developing countries). Twenty-seven sera were hepatitis C virus-RNA positive, including 5 without anti-hepatitis C virus antibodies. Hepatitis C virus genotype 1b was found in 13 (48%) patients, the remainder being infected with genotypes 1a (6 cases), 2a, 2c, 3a and 4. Genotype 1b was largely predominant among patients grafted in Europe but never found in those transplanted in developing countries. All but one patient without a sustained antibody response were infected by non-1b genotypes. Hepatitis C virus-RNA seropositivity was associated (p = 0.03) with a higher dose of prednisone (p = 0.03) and a lower dose of cyclosporine (p = 0.05) used as immunosuppressants. CONCLUSIONS: Current risk factors for hepatitis C virus infection in Italian kidney graft recipients include the duration of haemodialysis, transplantation in developing countries and the level of post-transplant immunosuppression. The pattern of hepatitis C virus genotypes is changing from predominantly 1b to non-1b genotypes and the latter infection often occurs without a sustained antibody response. Few patients develop clinical liver disease.

Heterogeneity of hepatitis C virus genotype 2 variants in West Central Africa (Guinea Conakry).

An overall anti-hepatitis C virus (HCV) prevalence of 6.7% was found in a sero-epidemiological study carried out in the town of Conakry (Guinea Conakry, West Central Africa) on 1421 subjects who were either blood donors, pregnant women or in- and outpatients receiving treatment for conditions other than liver disease. Seven HCV isolates from a subsample of 73 sterile sera from this population were studied for genetic characterization and classification. The 5'NCR was analysed by the Line Probe Assay. This method assigned the isolates to genotype 2. Analysis of the 5'NCR sequences alone was unable to give a more accurate classification. Comparison of NS5b region sequences (nucleotides 7575-8196), from Guinea isolates and genotype 2 database sequences, showed evolutionary distances in the range 0.15-0.26. There was a high level of subtype heterogeneity among the genotype 2 Guinea HCV isolates. Four of the subtypes were possibly new.

Active and inactive renin after a single dose of captopril in hypertensive patients.

In 25 hypertensive patients (15 with renal artery stenosis and 10 with essential hypertension), captopril, in a single 12.5 mg dose, caused a prompt decrease in arterial pressure without changing the heart rate. Plasma active and trypsin-activated renin significantly increases, whereas inactive renin and plasma aldosterone decreased. The plasma active/inactive renin ratio was also increased, suggesting that captopril, together with a release of active renin, may induce an in vivo activation of inactive renin. No correlations were found between blood pressure changes and both pretreatment and captopril-induced variations of active, inactive and trypsin-activated renin or the active/inactive ratio. However, the percent decrease in mean arterial pressure was significantly related to the increase in the active/inactive renin ratio in a group of patients whose blood pressure was brought to normal (r = -0.78; p less than 0.001). This finding suggests the possibility that vasodilating substances, in addition to inhibiting angiotensin II formation, might play some role both in exerting a full effect of captopril on blood pressure and in triggering the in vivo mechanisms of inactive renin activation.

In-vivo activation of circulating inactive renin by the ischemic kidney in man.

The role of the kidney as a possible source or as activator of inactive renin was studied in 22 patients with Essential Hypertension (EH) and in 20 patients with Unilateral Renal Artery Stenosis (RAS). Active and inactive renin (trypsin activation) were measured in blood samples taken simultaneously from both renal veins and from a peripheral artery during acute diuretic stimulation induced by furosemide 40 mg i.v. In EH pts active and trypsin-activated renin were significantly higher in both renal veins than in arterial blood (P less than 0.001 and P less than 0.02 respectively) whereas no difference was seen as far as inactive renin is concerned. In unilateral RAS trypsin-activated and active renin from the ischemic kidney were significantly higher (P less than 0.01 and P less than 0.005 respectively) while inactive renin was significantly lower (P less than 0.005) than in arterial blood. No significant difference was seen between arterial and renal venous blood from the contralateral kidney as far as active and inactive renin are concerned. When comparing the V-A differences for active renin to the corresponding V-A differences for inactive renin from the ischemic kidney a significant negative correlation appeared (r = -0.49 p less than 0.05) whereas no correlation was found from the contralateral kidney (r = -0.26 n.s.). These data demonstrate that the ischemic kidney, in addition to its ability to release active renin, can also activate circulating inactive renin.

[Role of renin profile and age in the choice of the therapeutic approach of arterial hypertension (author's transl)]. / Ruolo del profilo reninico e dell'età nella scelta dell'approccio terapeutico dell'ipertensione arteriosa.

A group of 116 patients with grade I-II (W.H.O.) essential hypertension was studied. A diuretic treatment was instituted as a first step of therapy in 50 patients while the remaining 66 were firstly treated with beta-blockers. After 6 weeks of treatment the drugs were combined in those patients who did not achieve blood pressure control; this combination therapy was again maintained for 6 weeks and, if necessary, a vasodilating agent was added thereafter. The results show that with diuretic treatment alone diastolic blood pressure was brought to normal values (less than 95 mmHg) in 3/4 of our low renin patients, in 2/3 of normal renin and in 1/3 of high renin hypertensives. On the contrary among the group of patients treated with a beta-blocker alone more than half of the high and normal renin patients were controlled whereas this goal was achieved in only 1/4 of the low renin patients. Similar behaviour was observed subdividing the subjects in young (less than 35 yrs), middle-aged (36-49 yrs) and older (greater than 50 yrs) patients. In conclusion our data indicate that age must be taken into account in classifying hypertensive patients in renin subgroups. In addition if a renin-sodium profile is available, it can be useful in choosing between diuretics or beta-blocker as a first step treatment. Alternatively, younger patients are more likely to have better hypotensive responses with beta-blockers whereas in older patients antihypertensive treatment should preferentially be started with diuretics alone.