Molecular epidemiology of hepatitis B and hepatitis delta viruses circulating in the Western Amazon region, North Brazil.

BACKGROUND: Hepatitis B virus (HBV) and hepatitis D virus (HDV) represent important public health problems in the Western Amazon region with reported cases of fulminant hepatitis. This cross sectional study describes HBV and HDV genotypes circulating in the Brazilian Amazon region. METHODS: HBsAg positive individuals (n = 224) were recruited in Manaus/Amazonas State (130 blood donors from the Hematology and Hemotherapy Foundation from Amazonas/HEMOAM; 60 subjects from outpatient clinic) and in Eirunepe city (n = 34) from 2003-2009. Most participants (n = 153) lived in Manaus, 63 were from 20 remote isolated municipalities, 8 lived outside Amazonas State. Genotyping was based on PCR products: HBV genotype A-F specific primers, restricted length polymorphism for HDV. HDV isolates were directly sequenced (delta antigen 405 nucleotide fragment) and phylogenetic analysis performed (MEGA; neighbor-joining, Kimura's two parameter). RESULTS: Most participants were young adult males and HBV mono-infection predominated (70.5%, 158/224). Among blood donors, outpatient subjects and individuals from Eirunepe, HBV/A prevailed followed by HBV/D and F (p > 0.05). HBV/A was more frequent in blood donors (p < 0.05). HBV-HDV coinfection rate was 8.5% in blood donors (11/130), 65.0% (39/60) in outpatient subjects and 47.0% (16/34) in individuals from Eirunepe. Compared to blood donors, coinfection was higher in outpatient subjects (65.0% versus 8.5%; RR = 5.0; CI 3.4-7.9; p < 0.0001) and in subjects from Eirunepe (47.0% versus 8.5%; RR = 5.5; CI 3.0-9.9; p < 0.0001). HBV-HDV coinfection rates were higher in patients from highly endemic remote cities. Only HDV genotype 3 was detected, HBV/F-HDV/3 predominated (20/38; 52.7%), followed by HBV/A-HDV/3 (31.6%; 12/38) and HBV/D-HDV/3 (15.8%; 6/38). CONCLUSIONS: The description of HBV and HDV genotypes circulating in the western Amazon can contribute to a better understanding of their relevance on the regional epidemics. These infections are highly endemic in the Amazon where their control is challenged by its vast territorial dimension with small, hard-to-reach municipalities dispersed into the jungle and populated by diverse ethnic groups.

[Hepatitis D virus superinfection--a rare cause of occupational disease]. / Nadkazenie wirusem zapalenia watroby typu D jako rzadka przyczyna choroby zawodowej.

The authors present a case of occupational HDV infection in a 38-year-old nurse, HBsAg carrier, injured by a needle contaminated with blood of a drug user infected with HIV, HBV and HCV. After 2 months she developed acute viral hepatitis. HBV, HCV, HIV, CMV, EBV and other non-viral liver diseases were excluded. Finally, based on the source of exposure with high probability of HDV infection, the patient's positive serological test for HDV, and the result of histological examination of the liver, the diagnosis of viral hepatitis type D was established. Our case report suggests the need to consider possible occupational HDV infection in certain circumstances as described above.

[Interferon therapy in chronic active viral hepatitis]. / Interferon kezelés vírus okozta krónikus aktív hepatitisben.

The authors give a short review of the recent data about the types of interferons and their biological activity. The role of interferons in the therapy of B, C and D chronic viral hepatitis is discussed. Interferon treatment means a substantial progress in the therapy of chronic viral hepatitis, however it represents a final recovery from chronic B or C hepatitis only in 25-40 percent or 40-45 percent of the cases, respectively. The authors refer to the combination therapy which seems to be promising in the future.

El espectro creciente de las hepatitis virales / The growing spectrum of viral hepatitis

El conocimiento de las hepatitis se remonta por lo menos al siglo VIII, pero éste ha avanzado vertiginosamente en los últimos 30 años gracias a factores como la aplicación de técnicas de inmunoquímica, microscopía electrónica, estudios epidemiológicos tanto clínicos como experimentales, técnicas de ingeniería genética, etc. Este trabajo revisa aspectos históricos de estas afecciones, las lateraciones histológicas hepáticas y manifestaciones clínicas que desencadenan. También describe las características principales de los virus A, B, C y E, la respuesta inmune que produce cada uno de ellos, su forma de infectar y la evolución, complicaciones y posibilidades de prevención de la enfermedad que desencadenan. Se comentan los resultados de los tratamientos con antivirales e interferón para las infecciones crónicas por virus B y C

Long-term follow-up of hepatitis B virus-infected recipients after orthotopic liver transplantation.

The outcome after OLT was studied in 53 patients with chronic hepatitis B virus (HBV)* infection, 15 of whom had, in addition, evidence of hepatitis delta virus (HDV) superinfection. Nine of 53 patients received short-term immunoprophylaxis with anti-hepatitis B surface (HBs) hyperimmunoglobulin up to 1 week after OLT and 44 of 53 patients received long-term unlimited immunoprophylaxis. Eight of 9 (89%) patients with short-term immunoprophylaxis showed reactivation of replication with HBV DNA in serum > 10 pg/ml independently of the preoperative HBV DNA level and HBsAg reappeared in all cases. Four (44%) patients in this group lost their graft because of fulminant hepatitis or cirrhosis and required retransplantation, and 2 patients (22%) died after reinfection in the second graft. Nineteen of 44 (43%) patients with long-term immunoprophylaxis developed HBV values > 10 pg/ml after transplant and 12 of 44 (27%) became HBsAg+ again. Most of them had quantifiable HBV DNA levels before OLT. Retransplantation was required in 5 of 44 (11%) patients and 4 of them died after HBV recurrence. The frequency of HBV reactivation and the development of viral hepatitis after OLT were associated with the preoperative presence of HBV, as determined by the molecular hybridization assay. With nested polymerase chain reaction, all 53 patients were HBV-DNA+ in the serum before and after OLT. with just one exception, none of the patients with HDV superinfection died, in spite of increased HDV replication after OLT. The data indicate that long-term immunoprophylaxis with anti-HBs hyperimmunoglobulin after OLT improves the prognosis in HBV-infected patients. The preoperative detection of HBV DNA in serum by molecular hybridization assay is correlated with graft infection and represents a prognostic parameter. The presence of HDV may have a protective effect after OLT.

Cis- and trans-acting ribozymes from a human pathogen, hepatitis delta virus.

Hepatitis delta virus (HDV) contains two self-cleaving RNA sequences (ribozymes) that may naturally function as such in human cells. A pseudo-knot-containing structural motif, which is distinct from the well-characterized secondary structures of self-cleaving RNAs common to the plant pathogenic RNAs, is shared by the cis-acting HDV ribozymes. Definition of the sequences and secondary structures of the HDV ribozymes has facilitated the design of novel catalytic molecules, such as small RNA circles, capable of site-specific cleavage of RNA in trans.

The middle hepatitis B virus envelope protein is not necessary for infectivity of hepatitis delta virus.

The hepatitis delta virus (HDV) envelope contains the large (L), middle (M), and small (S) surface proteins encoded by coinfecting hepatitis B virus. Although HDV-like particles can be assembled with only the S protein in the envelope, the L protein is essential for infectivity in vitro (C. Sureau, B. Guerra, and R. Lanford, J. Virol. 67:366-372, 1993). Here, we demonstrate that the M protein, previously described as carrying a site for binding to polymerized human albumin, is not necessary for infectivity. HDV-like particles coated with the S plus L or the S plus M plus L proteins are infectious in primary cultures of chimpanzee hepatocytes. We conclude that the S and L proteins serve two essential functions in the HDV replication cycle; the S protein ensures the export of the HDV genome from an infected cell by forming a particle, and the L protein ensures its import into a human hepatocyte.

Treatment of chronic hepatitis D virus infection with low and high doses of interferon-alpha 2a: utility of polymerase chain reaction in monitoring antiviral response.

We examined the efficacy of decreasing high doses (beginning at 18 MU/day) of interferon-alpha 2a vs. that of daily low doses (3 MU) in the treatment of chronic hepatitis delta virus infection. Patients treated with 18 MU had a somewhat higher frequency of normalization of serum ALT levels than patients treated with low doses (31% and 12%, respectively, on an intention-to-treat basis). A decrease in the percentage of hepatitis D virus RNA positivity was observed in both groups at the end of treatment. Thus, whereas in baseline samples 10 (62%) of the patients in each group were positive for hepatitis D virus RNA in serum on slot-blot hybridization, these numbers decreased to 5 (31%) and 4 (25%) patients in groups 1 and 2, respectively, at the end of therapy. However, hepatitis D virus RNA, detected by means of nested polymerase chain reaction, remained in all but two (one in each group) patients who completed the treatment. Finally, during posttreatment follow-up, hepatitis D virus RNA levels returned to baseline values, and only one patient remained negative for this marker. The beneficial effect of interferon-alpha was only transient. Only two patients (one from each treatment group) had persistently normal serum ALT levels after 18 mo of follow-up. Finally, the presence of serum hepatitis D virus RNA at the end of therapy, detected with nested polymerase chain reaction, might be a good marker for the prediction of viral replication relapse.

Chemiluminescent hybridization assay for hepatitis delta virus RNA in serum.

A nonradioactive hybridization assay for the detection of hepatitis delta virus RNA in serum is described. This assay utilizes a digoxigenin-labeled RNA hybridization probe and chemiluminescent immunodetection. The probe can detect as little as 0.4 pg of cDNA with a 60-min exposure. Results obtained are in agreement with serological indicators of hepatitis delta virus infection and are comparable to those obtained by hybridization assays employing radioactively labeled RNA.

Detection of small and large genomes of hepatitis D virus in serum of patients with hepatitis D.

By combining the polymerase chain reaction (PCR) with restriction enzyme digestion technologies, we characterized the genomes for the small and large delta proteins of HDV in retrospective analysis of sera from 10 patients with varied clinical outcomes. Both small and large genomes of HDV were present in all 13 serum samples from the 6 acute and 4 chronic cases studied, while the specific HDV proteins (P24 and P27) could be detected by immunoblot analysis in only 4 of them. The relative amounts and ratios of the genomes for the large and the small proteins of HDV were different for each individual. The molecular ratio of large to small HDV genomes in serum correlated with viral replication. When the replication of HDV RNA increased, the ratio decreased and vice-versa. No specific correlation, however, was found between the ratio of both molecular forms and the clinical outcome.

Factors associated with viremia and elevated transaminase levels in asymptomatic hepatitis D virus-infected risk groups.

To detect hepatitis D virus (HDV) RNA in asymptomatic HDV-infected risk groups, Northern blot hybridization was carried out using a strand-specific riboprobe. Univariate and multivariate analyses were carried out to evaluate factors associated with HDV viremia and elevated transaminase levels in these subjects. Two (15%) of 13 antibody to HDV (anti-HDV) -positive intravenous drug addicts, 15 (33%) of 45 anti-HDV positive prostitutes, and 6 (40%) of 15 anti-HDV positive brothel goers had detectable serum HDV RNA. Older age (> 31 years old) was negatively associated with HDV RNA (P < .04), while hepatitis B e antigen (HBeAg) was positively associated with it (P < .002) in univariate analysis. Only HBeAg was still significant in multivariate analysis (P < .05). Of the 76 asymptomatic anti-HDV positive case, 28 (37%) had mildly elevated serum ALT levels and only 5 (7%) had ALT levels more than twice normal (> 80 U/L). HBeAg (P < .05) and HDV RNA (P < .02) were two factors associated with ALT elevation in univariate analysis, and HDV RNA was the only significant factor in multivariate analysis (P < .005). In summary, active replication of HBV seemed to be of help for the assembly of HDV and viremia. However, active replication of HDV was associated mostly with mildly elevated ALT levels in these subjects. These cases may represent a particular group in the disease spectrum of HDV infection.

A genotype of hepatitis D virus that occurs in northern South America.

Hepatitis D virus (HDV) is the cause of an unusually severe form of liver disease with distinct histologic features (morula cell) that occurs throughout northern South America and certain other areas of the world. Clinical studies of HDV disease worldwide indicate that there is, in fact, a wide variation in pathogenesis, and the reasons for these differences are presently unknown. One possible explanation is that factors associated with the viral genotype are determinants of HDV pathogenesis. In this study, nucleic acid sequences were determined for three different northern South American HDV isolates which were obtained from individuals with severe disease or a family history of severe disease, in areas that are hyperendemic for this disease pattern. The sequences of these three isolates are very similar to one another but only distantly related to other published HDV sequences. Comparison of the sequence of a semiconserved region from a total of 14 isolates indicates that there are at least three HDV genotypes. Most published HDV sequences, including those from North America, Europe, the Middle East, the South Pacific, and Asia, belong to a single genotype which may have some geographically based subtypes. A single Japanese isolate is the sole representative of a second HDV genotype. The South American sequences reported here constitute a third genotype. The association of a particular genotype with the severe form of type D hepatitis that occurs in northern South America supports the hypothesis that HDV genetic factors are important determinants in the pathogenesis of type D hepatitis.

Delta hepatitis: molecular biology and clinical and epidemiological features.

Hepatitis delta virus, discovered in 1977, requires the help of hepatitis B virus to replicate in hepatocytes and is an important cause of acute, fulminant, and chronic liver disease in many regions of the world. Because of the helper function of hepatitis delta virus, infection with it occurs either as a coinfection with hepatitis B or as a superinfection of a carrier of hepatitis B surface antigen. Although the mechanisms of transmission are similar to those of hepatitis B virus, the patterns of transmission of delta virus vary widely around the world. In regions of the world in which hepatitis delta virus infection is not endemic, the disease is confined to groups at high risk of acquiring hepatitis B infection and high-risk hepatitis B carriers. Because of the propensity of this viral infection to cause fulminant as well as chronic liver disease, continued incursion of hepatitis delta virus into areas of the world where persistent hepatitis B infection is endemic will have serious implications. Prevention depends on the widespread use of hepatitis B vaccine. This review focuses on the molecular biology and the clinical and epidemiologic features of this important viral infection.

Polymerase chain reaction-based detection of hepatitis D virus genome in patients infected with human immunodeficiency virus.

The polymerase chain reaction (PCR) was used to detect hepatitis D (HD) viremia in patients infected with the human immunodeficiency virus (HIV). Nineteen (9%) of 206 such patients, unselected for liver disease or HBV infection, were found prospectively to be infected by HDV. Thirty-one anti-HIV-positive patients were studied by means of PCR, and the results were analyzed according to HDV and hepatitis B virus (HBV) serological status. HDV-PCR was positive in 5 patients. Two had detectable serum HDV antigen. Four patients had anti-HD IgM and IgG antibodies. All these patients were HBs antigen-positive, and 3 were HBV-DNA-positive. All the other patients were HDV-PCR-negative. Statistical analysis suggested more extensive liver damage and immunological impairment in HDV-PCR-positive patients. In this unselected HIV-infected population, HDV-RNA detection by PCR was restricted to HDV infected patients in whom 5/19 were positive. This test permitted direct diagnosis of HDV viremia and will be useful for monitoring HDV infection.

[Heterogeneity of delta virus RNA sequences in Japan].

Hepatitis delta virus RNA sequences were determined in isolates from two Japanese patients, M and S, by polymerase chain reaction and direct nucleotide sequencing and compared with three isolates from Italy, USA and Taiwan. The sequence obtained for hepatitis delta virus RNA from patient M was 92-96% identical to the sequences obtained for three other strains of hepatitis delta virus, whereas the sequence of hepatitis delta virus RNA obtained from patient S was approximately 80% identical to the other sequenced strains. This suggests that the delta agent in Japan has a heterogeneous origin and the delta virus RNA sequence from Japanese patient S is the most divergent delta virus isolate yet analyzed.

Occurrence of hepatitis delta virus infection in hepatitis B virus-infected patients with different serum patterns of preS gene-encoded proteins.

The occurrence of delta superinfection among viremic and nonviremic HbsAg-positive carriers with different serum patterns and levels of preS1 and preS2 antigens was investigated. PreS1 and preS2 antigens in serum, as well as their levels, were found to be independent of hepatitis B virus (HBV) replicative activity. Serological evidence of hepatitis delta virus (HDV) superinfection was found in 34 out of 233 (14.6%) HBsAg-positive carriers; all these 34 patients resulted positive for antibody to hepatitis B 'e' antigen, and 33 of them were negative for circulating HBV-DNA. Delta superinfection occurred only among HBsAg-positive carriers whose sera were reactive for both preS1 and preS2 antigens (30 out of 142; 21.1%) or at least for preS1 alone (4 out of 63; 6.3%), but not among the patients with undetectable levels of both these antigens. Serum levels of both preS1 (p < 0.005) and preS2 (p < 0.001) antigens were found to be significantly higher in delta-positive HBsAg-positive carriers than in patients with HBV infection uncomplicated by HDV. In addition to confirm previous observations that the detection of both preS antigens in HBsAg-positive sera is independent of HBV replication, these findings clearly show that HDV infection requires not only the presence of HBsAg, but also the presence of preS peptides, and seem to suggest a low susceptibility of HBsAg-positive carriers with low or undetectable synthesis and secretion of HBV surface proteins to delta superinfection.

Pathobiology of hepatitis delta virus.

Early observations in humans and infectivity studies in the chimpanzee suggested a direct cytotoxic effect as the major pathogenetic factor in delta hepatitis. Data acquired in liver transplant patients, as well as additional experimental and clinical evidence, have modified this view and indicate instead that liver damage depends on several factors. To explain the array of different clinical presentations, the replication strategy of hepatitis delta virus, the helper function and genetic heterogeneity of the co-infecting Hepadnavirus, and the response of the host immune system should all be taken into consideration.

Significance of low HDV replication levels during the natural history of HDV infection. Long-term follow-up.

The use of genetic amplification of the hepatitis delta virus (HDV) genome reveals the existence of different HDV replicative behaviours during the natural history of chronic HDV infection. While some of the patients (8/19, 42%) presented high and long-term maintained levels of HDV replication, as detected by slot-blot hybridization, others showed fluctuations from positive to negative, and in 5/7 (71%) polymerase chain reaction (PCR) demonstrated the presence of the HDV genome. Finally, 4 patients were persistently slot-blot-negative and in 3 of them HDV-RNA was detected by PCR in all samples tested. The correlation observed between the low levels of HDV replication and the ALT values, as well as the reactivation observed in one of the patients, suggests that PCR is useful in the virological surveillance of HDV infection, and indicates its usefulness in evaluating the effectiveness of antiviral therapy for chronic hepatitis D.