Hepatitis B virus subgenotype C2 is the most prevalent subgenotype in northeast China

The geographical distribution of hepatitis B virus (HBV) subgenotypes and their clinical implications in patients with acute and chronic hepatitis B in the Heilung-kiang province of northeast China were investigated. Nested PCR and multiplex PCR were performed with genotype-specific primers and with subgenotype-specific primers to identify genotypes and subgenotypes from serum samples of 412 HBV infections including 69 with acute self-limited hepatitis (ASH) and 343 with chronic hepatitis (CH). A total of 361 samples were genotyped and 304 were further subgenotyped. The most common HBV genotype was C (93.63%, 338/361), with subgenotype group C2 (83.73%, 283/338) predominating. Genotype B was also found and subgenotype B2 predominated within this genotype. Out of 69 infected patients with ASH, 48 were identified as genotype C and all belonged to subgenotype C2. Of 343 infected patients with CH, 313 were genotyped and 256 were subgenotyped; amongst these, C2 (91.80%, 235/256), B2 (7.42%, 19/256) and mixed subgenotypes B2 and C2 (0.78%, 2/256) were found. In HBV subgenotype C2 infections, ASH had a higher ratio of women than CH patients. These results show that HBV subgenotypes C2 and B2 were found in Heilung-kiang province of northeast China. In ASH and CH groups, the distributions of subgenotypes were coincident with C2, the predominant subgenotype. Analysis of the association between subgenotype and the outcomes of HBV infection was inconclusive in our study.     

Identification of hepatitis B virus subgenotype A3 in rural Gabon

An hepatitis B virus (HBV) molecular survey was conducted in five remote villages in the equatorial forest in Gabon, Central Africa. Two hundred seventy out of 311 inhabitants (86.8%) were HBV-infected or had evidence of past HBV infection. Chronic hepatitis corresponding to hepatitis B surface antigen (HBsAg) positivity was suspected in 27 (8.6%) of the HBV-infected subjects. High HBV viral loads were detected mainly in children aged 4-7 years. The pre-S/S domains were sequenced in 13 cases and 12 strains belonged to HBV-A genotype. In one case we found evidence for recombination between genotypes A and E. Phylogenetic analysis revealed that Gabonese HBV strains were distinct from HBV-A subgenotypes (A1 and A2). These new HBV strains from Gabon clustered with previously reported HBV-A3 subgenotype strains from Cameroon and Democratic Republic of Congo. The analysis of the pre-S2 domain allowed us to determine two amino acid substitutions (N/152/S and N/174/T) specific to the Central African HBV-A3 subgenotype strains and one amino acid substitution (P/155/Q) unique to these new Gabonese HBV-A3 subgenotype isolates. Two full genome sequences of two new Gabonese HBV isolates are also presented and confirm the distinctive HBV-Gab-A3 cluster.     

Analysis of the entire nucleotide sequence of hepatitis B causing consecutive cases of fatal fulminant hepatitis in Miyagi Prefecture Japan

We encountered five consecutive patients with fulminant hepatitis induced by acute hepatitis B virus (HBV) infection in 2000--2001 in Japan. They had not had previous contact each other, and were referred to us from different hospitals. Although a 69-year-old woman could be rescued by intensive internal treatment, the four patients died. We analyzed the partial (nt 278-646) and entire nucleotide sequences of the HBV obtained from them, and their divergences were 0-0.3% and 0-0.2%, respectively. The results suggested that they had been infected with the same HBV isolates. The isolates belonged to genotype B and subgenotype B2 on the phylogenetic tree analysis (AB302942-AB302946). As for the nucleotides sequences of them, previously reported mutations of G1896A, A1762T, and G1764A were present. Amino acid analysis revealed that previously reported Ile97Leu and Pro130Non-Pro in the core region and Trp28Stop in the precore region were present. As for the entire nucleotide sequences among B2, AB302942 showed low divergences with AF121245 and AB073834 (1.7%), and X97850 from patients with fulminant hepatitis (3.2%). We compared the two consensus nucleotides derived from AB302942 and X97850 (fulminant hepatitis) versus AY121245 and AB073834 (non-fulminant hepatitis), which revealed a difference in nt 1,504 located in the P and X region. Nucleotide 1,504 was C for isolates from fulminant hepatitis and G for non-fulminant hepatitis, and it was recognized among most of the isolates belonging to B2 registered on GenBank. Further studies could disclose the mechanism of severe inflammation of liver that finally leads to fulminant hepatitis.     

Another novel subgenotype of hepatitis B virus genotype C from papuans of Highland origin

Hepatitis B virus (HBV) genotypes and subtypes have been identified worldwide. As HBV genotypes/subtypes, the HBV subgenotypes seem to be associated with their geographical distribution and ethnic origin. A previous study showed the novel HBV subgenotype C6 based on the complete genome sequences of isolates in Papua, Indonesia. In the present study, further characterization of HBV in Jayapura (capital of Papua Province), particularly from native people of Papua originating from the highland (highland Papuans) and those from the lowland (lowland Papuans) were examined. Of 32 HBV isolates from both highland and lowland Papuan blood donors with HBsAg positive, part of the S gene and the core gene sequences were analyzed. Analyses of some isolates from highland Papuans were confirmed by the complete genome sequences. Most HBV isolates were classified into genotype C (78.1%), followed by genotype B (18.8%), and genotype D (3.1%). The subtype adr was predominant (71.9%), followed by adw2 (25.1%), and ayw2 (3.1%). As with previous findings, phylogenetic analyses revealed that most HBV isolates from Papuans, C/adr, belonged to subgenotype C6. Interestingly, some C/adr isolates from highland Papuans formed a distinct cluster from all reported subgenotypes of HBV/C, and they differed from HBV/C1-C10 by 4.2-7.2% over the complete genome. SimPlot analysis showed no evidence of recombination with HBV/C1-C10. The isolated life and closed social systems of highland Papuans, even though some have been moving to Jayapura, likely contribute to the formation of this unique cluster of infection with a novel subgenotype of HBV, named C11.     

Long-term follow-up of hepatitis B virus and hepatitis C virus replicative levels in chronic hepatitis patients coinfected with both viruses

Dual infection with hepatitis B and C viruses is often encountered in endemic areas of both viruses. However, understanding of the clinical and virological implications is limited. The aim of this study was to investigate the role of each virus in liver injury and the interaction between the two viruses in dual infection with hepatitis B and C viruses. Three patients who had chronic infection with both hepatitis B and C viruses were examined, and a longitudinal study of both serum hepatitis B virus DNA and hepatitis C virus RNA levels over 4 years was undertaken. The results were correlated with serum alanine aminotransferase levels. Serum alanine aminotransferase values showed a relationship with hepatitis B virus replicative levels, but not with hepatitis C virus replicative levels in all 3 patients. Serial changes of replicative levels of both viruses were studied, and it was found that hepatitis C virus replicative levels were enhanced after the decline of hepatitis B virus replication in 1 of the 3 patients. In the remaining 2 patients, a transient rise of hepatitis C virus replicative levels in association with a decrease of hepatitis B virus replication was also observed during part of the follow-up period. These findings indicate that hepatitis B virus may play a dominant etiological role in liver injury, and that a suppressive action between hepatitis B and C viruses may occur in dual infection with both viruses. © 1995 Wiley-Liss, Inc.     

Characterization of hepatitis B virus genome variability in Iranian patients with chronic infection, a nationwide study

Hepatitis B virus (HBV) isolates from Iranian patients around the country were characterized. Eighty-one complete genomes from HBV isolates were sequenced and analyzed. The studied population was grouped into three categories including inactive carriers, patients with chronic hepatitis, and patients with liver cirrhosis. Molecular and phylogenetic analyses revealed that Iranian patients were infected with HBV genotype D and subgenotype D1. The most common subtype was ayw2, followed by ayw3 and ayw4. Several deletions and insertions that had no correlation with disease outcome were observed in the HBV genomes. The most frequent mutation in the major hydrophilic region (MHR) of HBV surface antigen (HBsAg) was sP120S. Almost half of the patients studied carried precore (PC) mutant variants and one-third of the studied population was infected with variants carrying basal core promoter (BCP) mutations. PC and BCP mutations were observed in older patients, especially in those with chronic liver disease. Sixty-seven patients (82.7%) were HBeAg negative, and the prevalence of precore mutant isolates (G1896A) was higher in this group than in HBeAg-positive patients. Lamivudine drug resistance mutations were detected after 1 year of treatment in about 30% of lamivudine-treated patients. In conclusion, these results demonstrate that HBV subgenotype D1 is the only subgenotype circulating in Iran, and there is no evidence of any exotic genotype in the region. The HBV PC (G1896A) mutation may play an important role in the clinical outcome of the disease by increasing the risk of progressive liver disease among Iranian patients infected with HBV.     

Concurrent hepatitis C virus and hepatitis delta virus superinfection in patients with chronic hepatitis B virus infection.

Since hepatitis C virus (HCV) and hepatitis delta virus (HDV) are transmitted by the same routes as hepatitis B virus (HBV), simultaneous or concurrent HCV and HDV infection in patients with chronic HBV infection may occur. To test this hypothesis and to examine the clinicohistological and immunopathological presentations of such multiple hepatitis virus infections, acute and/or convalescent serum specimens from 86 patients with acute HDV superinfection were tested by enzyme immunoassay for antibodies to HCV. Of the 86 patients, 18 (20.9%) were associated with HCV infection. Although patients with early mortality cannot be evaluated by the HCV markers used in this study, the results showed that the clinical and histologic features were similar except that patients with HCV infection were older than those without HCV infection (P less than 0.01). Immunopathological studies carried out within 2 months after the onset of acute HDV superinfection demonstrated that hepatitis B core antigen (HBcAg) was not detected in any patient and HDV antigen was detected in 18.2% of the patients with HCV infection whereas HBcAg and HDAg were found in 7% and 65.1%, respectively, of those without HCV coinfection (P less than 0.02). It is concluded that concurrent HCV and HDV superinfections can and do occur in patients with chronic HBV infection. In these triple viral infections, HCV may even transiently suppress HDV and HBV.     

Analysis of the full-length genomes of novel hepatitis B virus subgenotypes C11 and C12 in Papua, Indonesia

Two novel subgenotypes (C6 and D6) of hepatitis B virus (HBV) were identified recently in Papua, a multiethnic area of Indonesia. To characterize further the HBV strains in Papua, serum samples collected from 59 viremic subjects (44 males and 15 females; mean age: 30.0 ± 15.5 years) among indigenous inhabitants in Papua, were subjected to phylogenetic analysis of an 1.6-kb partial sequence. Forty-five samples (76%) had genotype C HBV (HBV/C) [C5 (n = 1), C6 (n = 40), and unclassifiable (n = 4)], while seven samples (12%) were HBV/D [D1 (n = 1) and D6 (n = 6)] and six samples (10%) were HBV/B [B2 (n = 1), B3 (n = 3), B7 (n = 1), and B8 (n = 1)]; the remaining sample possessed B3 and C6. An analysis of the full-length sequence of the four HBV/C isolates (NMB09122, NMB09124, NMB09075, and MRK89073) that were unclassifiable into any of the 10 known HBV/C subgenotypes (C1-C10) showed no significant evidence of recombination. Over the entire genome, the NMB09122 and NMB09124 isolates shared 99.8% identity and segregated into a cluster with a bootstrap value of 100%, differing from HBV/C1-HBV/C10 by 3.8-6.9% (mean, ≥4.0%), indicating that NMB09122 and NMB09124 can be classified into a novel subgenotype within genotype C (tentatively designated C11). The NMB09075 and MRK89073 isolates were 97.4% identical to each other and differed from known HBV/C isolates, including the C11 strains, by 4.0-7.2% (mean, ≥4.5%) over the entire genome, indicating that NMB09075 and MRK89703 can be classified into another novel HBV/C subgenotype (C12). The distribution of C11 and C12 seemed to be associated with particular language speakers in Papua.     

Tattooing as a risk of hepatitis C virus infection.

The association of hepatitis C virus (HCV) infection and tattooing was studied in 87 tattooed and 126 tattoo free healthy young men who did not engage in intravenous drug use or multiple sexual activity. Antibody against HCV (anti-HCV) was tested in serum specimens by enzyme immunoassay with C100-3, NS3, and core antigens; 11 of the 87 (12.6%) tattooed and 3 of the 126 (2.4%) tattoo free subjects were positive for anti-HCV (odds ratio = 5.9, 95% CI = 1.6-22.0). A relationship was demonstrated by an increased risk for HCV infection with an increasing number of tattooed site (P(trend) = 0.002). All but one of the 87 tattooed subjects had been infected by hepatitis B virus (HBV) and 25 were carriers of hepatitis B surface antigen (HBsAg). None of the 25 HBsAg carriers was positive for anti-HCV whereas 11 of the 62 HBsAg non-carriers had anti-HCV, suggesting a negative association between the HBsAg carriage and the long lasting anti-HCV (P = 0.02, Fisher's exact). The status of the tattooer was also an important determinant for HCV infection; the risk was higher if tattooing was done by a non-professional friend than by a professional tattooist. Tattooing, probably with improperly sterilized needles, can clearly pose an increased risk for HCV infection in Taiwan. This study indicates the need for legal standards for hygienic tattooing as part of preventive measures for the control of parenterally transmitted infections.     

Association of concurrent hepatitis B surface antigen and antibody to hepatitis B surface antigen with hepatocellular carcinoma in chronic hepatitis B virus infection

Antibody to hepatitis B surface antigen (HBsAg) (anti‐HBs) can exist in patients with chronic hepatitis B virus (HBV) infection. To date, little is known about the association of concurrent HBsAg and anti‐HBs (concurrent HBsAg/ anti‐HBs) with hepatocellular carcinoma (HCC). The aim of this study was to investigate the clinical relevance of concurrent HBsAg/anti‐HBs with preS deletion mutations and HCC in chronic HBV infection. A total of 755 patients with chronic HBV infection were included consecutively at a tertiary center. Logistic regression analysis was used to identify risk factors for HCC, and serum HBV DNA was amplified, followed by direct sequencing to detect preS deletions. The prevalence of concurrent HBsAg/anti‐HBs was 6.4% (48/755) and all HBVs tested were genotype C. HCC occurred more frequently in the concurrent HBsAg/anti‐HBs group than in the HBsAg only group [22.9% (11/48) vs. 7.9% (56/707), P = 0.002]. In multivariate analyses, age >40 years [odds ratio (OR), 14.712; 95% confidence interval (CI), 4.365–49.579; P < 0.001], male gender (OR 2.431; 95% CI, 1.226–4.820; P = 0.011), decompensated cirrhosis (OR, 3.642; 95% CI, 1.788–7.421; P < 0.001) and concurrent HBsAg/anti‐HBs (OR, 4.336; 95% CI, 1.956–9.613; P < 0.001) were associated independently with HCC. In molecular analysis, preS deletion mutations were more frequent in the concurrent HBsAg/anti‐HBs and HCC groups than in the HBsAg without HCC group (42.3% and 32.5% vs. 11.3%; P = 0.002 and 0.012, respectively). In conclusion, concurrent HBsAg/anti‐HBs is associated with preS deletion mutations and may be one of the risk factors for HCC in chronic HBV infection with genotype C. J. Med. Virol. 81:1531–1538, 2009. © 2009 Wiley‐Liss, Inc.     

Hepatitis B virus genotypes and spontaneous hepatitis B e antigen seroconversion in Taiwanese hepatitis B carriers

Hepatitis B virus (HBV) is classified into eight genotypes (A-H), and genotype C is associated with more aggressive liver disease compared to genotype B. However, the mechanisms responsible for the clinical differences remain unclear. To test whether genotype C patients had with lower rates of spontaneous hepatitis B ge antigen (HBeAg) seroconversion than genotype B patients, stored serum samples from 146 Taiwanese adult HBeAg-positive hepatitis B carriers followed-up for a mean of 52 months (range, 12-120 months) were tested for HBV genotype by a molecular method. Genotype C patients were significantly older than genotype B patients (mean age, 37 +/- 12 vs. 29 +/- 10 years, P < 0.001). During the follow-up period, genotype C patients had a significantly lower rate of spontaneous HBeAg seroconversion than genotype B patients (27 vs. 47%, P < 0.025). Spontaneous HBeAg seroconversion occurred one decade later in genotype C patients compared with genotype B patients. Multivariate analyses identified age < or =35 years (odds ratio: 2.08; 95% confidence interval [CI], 1.07-4.0; P < 0.05), high baseline serum alanine aminotransferase level (odds ratio: 2.34; 95%CI, 1.39-4.09; P < 0.005), and HBV genotype B (odds ratio: 1.94; 95%CI, 1.03-3.63; P < 0.05) as independent factors associated with spontaneous HBeAg seroconversion. In conclusion, genotype C patients, compared to genotype B patients, have a delayed HBeAg seroconversion in the immune clearance phase of chronic HBV infection, which may contribute to a more progressive liver disease and more refractory to antiviral therapy.     

Analysis of the full-length genome of a subgenotype IIIB hepatitis A virus isolate: primers for broadly reactive PCR and genotypic analysis

Among six known subgenotypes (IA, IB, IIA, IIB, IIIA, and IIIB) of human hepatitis A virus (HAV), the complete genomic sequence has not been determined for IIIB. In this study, the full-length genomic sequence of a IIIB HAV isolate (HA-JNG06-90F) recovered from a Japanese patient who contracted sporadic hepatitis A in 1990, was determined. The HA-JNG06-90F genome, which comprised 7462 nt excluding the poly(A) tail, was related most closely to NOR-21 of subgenotype IIIA with an identity of 89.1%, and was only 82.6-83.4% similar to human HAV isolates of genotypes I and II over the entire genome. Comparison of full-length genomic sequences of 20 reported isolates and HA-JNG06-90F generated optimal results for separation of different levels: the nucleotide identities were 80.7-86.6% at the genotype level, 89.1-91.9% at the subgenotype level, and 94.6-99.7% at the isolate level. Similar ranges of nucleotide identity were observed when comparing partial nucleotide sequences of the VP1-2B (481 nt; primer sequences at both ends excluded) and 3C/3D (590 nt) regions, which were amplifiable by PCR with primers designed from well-conserved areas of the HAV genome. All 66 samples with IgM-class HAV antibodies tested positive for HAV RNA by both VP1-2B (481 nt)-PCR and 3C/3D (590 nt)-PCR: subgenotype assignment was concordant in all samples tested (IA [n = 61], IB [n = 1], IIIA [n = 2] and IIIB [n = 2]). These results suggest that two broadly reactive PCRs using primers derived from the VP1-2B and 3C/3D regions, respectively, may be applicable to universal detection and phylogenetic analysis of various HAV strains.     

Prevalence and association of human parvovirus B19V with hepatitis B and C viruses in Nigeria

Co-infection of parvovirus B19 with hepatitis B virus has been found in patients with acute and chronic hepatitis. The clinical significance of parvovirus B19 in hepatitis B co-infected patients is still controversial. In this study parvovirus B19 antibodies and DNA were investigated in serum samples from 76 patients with HBV infection, 17 with HBV/HCV co-infection and 44 healthy controls. In the sera from patients with HBV infection, anti-B19V IgM and IgG antibodies were detected in 24/76 (32%) and 25/76 (33%), in 6/17 (35%) and 8/17 (47%) of HBV/HCV co-infected patients, and in 14/44 (32%) and 12/44 (12%) of a non-hepatitis healthy controls, respectively. B19V DNA was detected in 8/76 (11%) of patients with HBV infection and in 3/17 (18%) of patients with a HBV/HCV co-infection, and in 4/44 (9%) healthy controls. The occurrence of parvovirus B19 DNA was significantly higher in patients with symptomatic HBV 4/20 (20%) compared to asymptomatic HBV carrier 4/56 (7%) (P<0.05). Ten of the positive B19V DNA sequences belonged to B19V genotype 1 while two belonged to genotype 3. The results of this study showed a significant difference in the prevalence of parvovirus B19 DNA in symptomatic HBsAg positive as compared to asymptomatic HBsAg positive individuals; however, the conclusion that parvovirus B19 infection increased the frequency of liver disease was not supported. Long-term longitudinal studies are, however, required to determine the synergistic effect of parvovirus B19 infection in HBV or HBV and HCV co-infected persons.     

Changing seroepidemiology of hepatitis B, C, and D virus infections in high-risk populations

Needle-sharing and sexual contact are important transmission routes of hepatitis B, C, and D virus (HBV, HCV, HDV) infection. This study aimed to investigate the current status of these viral infections among high-risk populations including prostitutes and intravenous (i.v.) drug users, compared with the prevalence rate reported previously to examine the changing seroepidemiology. Of the 916 female prostitutes, 79 (9%) were positive for antibody to HCV (anti-HCV), 111 (12%) were positive for HBV surface antigen (HBsAg), and 5 (5%) had antibody to HDV (anti-HDV). The prevalence rate was significantly lower compared to that in 1989-1991 (12%, P = 0.037) for HCV infection, and to that in 1988 (59%) and 1996 (40%) (P < 0.0001) for HDV infection. Of the 494 i.v. drug users, 87 (18%) patients were HBsAg carriers and 12 (14%) were anti-HDV-positive. The prevalence rate of HDV infection was significantly lower than that reported in 1985 (79%, P < 0.0001). Among the 443 tested i.v. drug users, 182 (41%) were anti-HCV-positive, significantly lower than that in 1985 (53%, P = 0.026). Of the 263 male prostitutes, 11 (4%) were anti-HCV-positive, 45 (17%) were HBsAg-positive, and 7 (16%) were anti-HDV-positive. Of the 129 illegal immigrant prostitutes, 7 (5%) were anti-HCV-positive, 15 (12%) were HBsAg-positive and none were positive for anti-HDV. In conclusion, the findings indicate a declining prevalence of HCV and HDV infections among drug users and prostitutes over the past 16 years. Male prostitutes and immigrant prostitutes are new "high-risk" populations and may become a reservoir for disease transmission.     

Mutations within enhancer II and BCP regions of hepatitis B virus in relation to advanced liver diseases in patients infected with subgenotype B3 in Indonesia

Studies on the characteristics of mutations within the hepatitis B virus (HBV) genome, their roles in the pathogenesis of advanced liver diseases, and the involvement of host properties of HBV‐infected individuals have not been conducted in subgenotype B3‐infected populations. For addressing this issue, 40 cases with HBV surface antigen (HBsAg)—positive advanced liver diseases, including advanced liver cancer and cirrhosis (male 31, female 9, age 54.4 ± 11.6‐year‐old), were collected and compared with 109 cases with chronic hepatitis B (male 71, female 38, age 38.0 ± 13.4‐year‐old). Mutations in enhancer II (Enh II) and basal core promoter (BCP)/precore regions were analyzed by PCR‐direct sequencing method. HBV viral load was examined by real‐time PCR. For all examined regions, the prevalence of mutation was significantly higher in cases with advanced liver diseases. Multivariate analysis showed that, in patients older than 45 years, C1638T and T1753V mutations constituted independent risk factors for the advancement of liver diseases. The presence of C1638T and T1753V mutations may serve as predictive markers for the progression of liver diseases in Indonesia and other countries, where subgenotype B3 infection is prevalent. J. Med. Virol. 84:44–51, 2011. © 2011 Wiley Periodicals, Inc.     

Subgenotype diversity of hepatitis B virus American genotype F in Amerindians from Venezuela and the general population of Colombia

The objective of this study was the evaluation of the genetic diversity found in HBV circulating among Venezuelan Amerindians and the general population in Colombia. Phylogenetic analysis of the S region in 194 isolates showed that genotype F is highly predominant in Colombia and Venezuela. This might be related to the genetic background of the population. F3 is the main subgenotype which circulates in both countries. Phylogenetic analysis of 61 complete genome sequences of HBV American genotypes confirms the presence of two genotypes F and H, and 4 F subgenotypes. In Venezuela, subgenotypes F1, F2, and F3 circulate in East and West Amerindians, while only F3 was found among South Amerindians. Japreira community derived from Yucpa Amerindians around 150 years ago. However, several Japreira HBV sequences were forming a clade that can be classified as subgenotype 2b, differing from Yucpa sequences that belong mainly to subgenotype F3. The apparent absence of correlation between the phylogenetic groupings of HBV isolates with the ethnical origin in aboriginal populations might be suggesting a recent origin of HBV American subgenotypes, or a genetic drift effect.