Impact of occult hepatitis B on the clinical outcomes of patients with chronic hepatitis C virus infection: A 10-year follow-up.

BACKGROUND/PURPOSE: Occult hepatitis B infection (OHB) is not rare in countries that are endemic for hepatitis B virus (HBV) and in patients with chronic hepatitis C virus (HCV) infection. Notably, OHB has been shown to play a role in the progression of liver diseases, including the development of hepatocellular carcinoma (HCC); however, the data is inconsistent. We aim to clarify the contribution of concurrent OHB to the progression of liver diseases in a long-term cohort of patients with HCV infection and to investigate the value of total anti-hepatitis B core (anti-HBc) antibody as a surrogate OHB biomarker. METHODS: We included 250 chronic anti-HCV-positive patients who had resolved HBV infection (anti-HBc positive and hepatitis B surface antigen negative). OHB was then detected using a sensitive commercial assay for serum HBV DNA with a low limit of detection of 6 IU/mL. Clinical outcomes, including the development of liver cirrhosis, HCC, and all-cause deaths, were compared between OHB-positive and OHB-negative patients. RESULTS: At baseline, only 183 (73.20%) patients had positive HCV ribonucleic acid, and 56 (30.60%) of these 183 patients with active HCV infection had OHB. The presence of OHB did not correlate with any adverse clinical outcome in multivariate analyses. In addition, chronic hepatitis C patients with OHB did not have a higher level of serum total anti-HBc. CONCLUSION: OHB infection may not contribute to the development of adverse liver outcomes in patients with chronic HCV.

Micro-evolution of the hepatitis B virus genome in hepatitis B e-antigen-positive carriers: comparison of genotypes B and C at various immune stages.

BACKGROUND AND AIM: Patients with hepatitis B virus (HBV) genotype B infection experience hepatitis B e-antigen (HBeAg) seroconversion at an earlier stage than do patients with genotype C infection. Therefore, this study investigated whether the differential phenotypes are related to HBV genomic evolution. METHODS: Thirty-three HBeAg-positive patients with a mean follow-up of 3.1 years were enrolled: 16 at the immune tolerance stage (group I) and 17 at the immune clearance stage (group II). The evolution rates of paired viral genomes at enrollment and at the final follow-up in the full-length genome (µf), nonoverlapping regions (synonymous [µs] and nonsynonymous [µa]), and overlapping regions (µ) were calculated. The evolution rates were then compared according to serum alanine aminotransferase (ALT) levels and HBV genotype. RESULTS: The overall µf evolution rate was lower in group I than in group II (1.4 × 10(-5) ± 3.3 × 10(-5) vs 1.2 × 10(-3) ± 1.2 × 10(-3) nucleotide substitution/site/year, P < 0.001). We observed similar results for the µs, µa, and µ evolution rates. All evolution parameters were comparable between genotypes B and C. We determined a positive correlation between µa/y and the area under the average ALT time curve in genotype B (R(2) = 0.6935, P < 0.0001), but not in genotype C (R(2) = 0.1606, P = 0.124). CONCLUSION: The evolution rate of the HBV genome is higher at the immune clearance stage than at the immune tolerance stage. Host immune selection might play a role in triggering evolution of genotype B.

Clinical and virological features of occult hepatitis B in patients with HBsAg seroclearance post-treatment or spontaneously.

BACKGROUND: Occult hepatitis B virus (HBV) infection (OHB) may exist in patients experiencing hepatitis B surface antigen (HBsAg) seroclearance. AIMS: We examined the clinical and virological features of OHB in patients who lost HBsAg post-treatment or spontaneously. METHODS: We collected 44 patients with HBsAg seroclearance: 15 patients with dual HBV/hepatitis C virus (HCV) infection who lost HBsAg after peginterferon alfa-2a (PEG-IFN) plus ribavirin therapy; 13 HBV mono-infected patients who lost HBsAg after various oral antiviral therapies; and 16 patients who lost HBsAg spontaneously. OHB was defined as detectable serum HBV DNA in the absence of HBsAg. Viral mutations associated with OHB were identified by comparison with matched controls that remained positive for HBsAg, and further characterized in vitro. RESULTS: The prevalence of OHB was 34.1% (15/44) in all patients, which was not significantly different among three groups. One mutation in surface promoter/polymerase region, C3050T (preS1T68I), was identified to be associated with the seroclearance of HBsAg in six cases. This mutation does not change the amino acid sequence of the polymerase protein. The S promoter activity was significantly lower in the construct containing C3050T mutation as compared with the wild-type (P = 0.0008). However, this mutation did not affect HBV replication, transcription and translation in the context of the full-length HBV genome. OHB was not rare in patients with HBsAg seroclearance. CONCLUSIONS: One mutation, C3050T (preS1T68I), decreased S promoter activity; nevertheless, other factors may play more important role in the clearance of HBsAg in these OHB patients.

Value of interleukin-28B genetic polymorphism on retreatment outcomes of chronic hepatitis C genotype 1 relapsers by peginterferon alfa plus ribavirin.

BACKGROUND AND AIM: Chronic hepatitis C (CHC) infection is a leading cause of cirrhosis and hepatocellular carcinoma worldwide. Pegylated interferon (PEG-IFN) plus ribavirin (RBV) combination therapy remains the standard of care for CHC genotype 1 in many Asian countries, and single nucleotide polymorphism or genotype of the interleukin-28B (IL28B) gene is associated with the development of sustained virologic response (SVR). The predictive value of IL28B genotype for retreatment outcomes of patients with CHC was only partly clarified and deserves further investigation. METHODS: A total of 75 CHC genotype 1 Taiwanese patients who relapsed after 24-week PEG-IFN/RBV combination therapy and received retreatment with a 48-week PEG-IFN/RBV therapy were consecutively enrolled since November 2009. The associations among IL28B rs8099917 genotype, virologic kinetics, and treatment outcomes were evaluated. RESULTS: Rapid virologic response (RVR) at week 4, end-of-treatment virologic response (EOT-VR) and SVR was 37%, 73%, and 52%, respectively. Relapse rate was 29%. None of patients had rs8099917 GG genotype. Patients with TT genotype (n = 54, 72%) had higher rates of RVR (50% vs 5%, P = 0.0002), end-of-treatment virologic response (85% vs 43%, P = 0.0001), and SVR (67% vs 14%, P = 0.0001) than those with GT genotype (n = 21, 28%). Combination of IL28B TT genotype and achieving RVR had 85% positive and 90% negative predictive values of SVR. CONCLUSIONS: About half of the Taiwanese CHC relapsers to a previous 24-week combination therapy achieve SVR after retreatment for 48 weeks. IL28B genotype influences on-treatment viral kinetics and SVR rate in these retreated patients. Baseline IL28B genotype and RVR can serve as early predictors for treatment success.

Serum cytokine/chemokine profiles in acute exacerbation of chronic hepatitis B: clinical and mechanistic implications.

BACKGROUND AND AIMS: Acute exacerbation (AE) of chronic hepatitis B virus (HBV) infection is common and negatively impacts the clinical outcome. Although upsurge of viral load always precedes or coincides with AE, the underlying immunological mechanisms remain unclear and were investigated. METHODS: We prospectively followed the serum cytokine/chemokine profiles, viral load, and alanine aminotransferase (ALT) levels in 250 patients and identified 44 consecutive patients (male: 72.7%; age: 40.4 ± 9.7 years; hepatitis B e antigen [HBeAg] positivity: 63.6%; genotype B/C: 75%/25%) who developed AE during the follow-up in a medical center. The impact of clinical characteristics (age, gender, HBeAg, ALT, HBV genotype), cytokines (tumor necrosis factor-alpha, interferon gamma, interleukin [IL]-2, IL-4, IL-6, and IL-10), and chemokines (CXCL10/interferon gamma-induced protein [IP]-10, CCL2/MCP-1, CXCL9/MIG, CCL5/RANTES, and CXCL8/IL-8) on the serum HBV DNA dynamics at different time points (baseline, peak of serum HBV DNA level, peak of serum ALT level, and after AE) were analyzed. RESULTS: Of 44 patients, serum HBV DNA level surged before the peak of serum ALT level in 23 (52.3%), and coincided with the peak of ALT in 21 (47.7%). The upsurge of serum viral load significantly correlated with the increase of IL-10 (P = 0.0037) and CXCL10/IP-10 (P = 0.0044). Upsurge of serum viral load was preceded by an increase in serum IL-4 (P < 0.05), IL-6 (P < 0.05), and IL-10 (P < 0.05). Combination of HBV genotype, IL-6 level at baseline, and ALT level at the peak of serum HBV DNA reliably predicted subsequent AE pattern (P = 0.0116). CONCLUSIONS: Enhanced Th2 activity is likely involved in the surge of HBV DNA level before hepatitis exacerbation.

Emerging lethal infectious bronchitis coronavirus variants with multiorgan tropism.

Infectious bronchitis virus (IBV) causes respiratory diseases in chickens and poses an economic threat to the poultry industry worldwide. Despite vaccine use, there have been field outbreaks of IBV in Taiwan. This study aimed to characterize the emerging IBV variants circulating in Taiwan. The analysis of the structural protein genes showed that these variants emerged through frequent recombination events among Taiwan strains, China strains, Japan strains and vaccine strains. Cross-neutralization tests revealed that two of the variants exhibited novel serotypes. Clinicopathological assessment showed that two of the variants caused high fatality rates of 67% and 20% in one-day-old SPF chicks, and all the variants possessed multiorgan tropisms, including trachea, proventriculus and urogenital tissues. Furthermore, the commercial live-attenuated Mass-type vaccine conferred poor protection against these variants. This study identified novel genotypes, serotypes and pathotypes of emerging IBV variants circulating in Taiwan. There is an urgent need for effective countermeasures against these variant strains.

Anti-Influenza Protective Efficacy of a H6 Virus-Like Particle in Chickens.

H6 avian influenza viruses (AIVs) have a worldwide distribution, and they pose a potential concern for public health. In Taiwan, H6 AIVs have circulated in domestic chickens for more than 40 years, and certain strains have crossed the species barrier to infect mammals. With the goal of containing the disease, there is a pressing need to develop a safe and effective vaccine for pandemic preparedness. In this study, we prepared a virus-like particle (VLP) that consisted of the hemagglutinin (HA) and matrix protein 1 (M1) derived from a H6 AIV as a vaccine antigen, and we examined the immunogenicity and protective efficacy when combined with an adjuvant in a chicken model. Full-length HA and M1 protein genes were cloned and expressed using a baculovirus expression system, and VLPs were purified from the supernatant of insect cell cultures. We performed nanoparticle-tracking analysis and transmission electron microscopy to validate that the particle structure and properties resembled the native virions. In animal experiments, specific-pathogen-free chickens that received the H6 VLPs in combination with an adjuvant showed superior H6N1 virus-specific serum IgG and hemagglutination-inhibition antibody responses, which lasted more than 112 days. Following the H6N1 viral challenge, the vaccinated chickens showed reduced viral replication in the lungs, kidneys and conjunctival/cloacal shedding. The antibodies induced in the chickens by the vaccine were able to cross-react with the H6N1 human isolate and drifted avian H6N1 isolates. In summary, the H6 VLP vaccine elicited superb immunogenicity in vivo, and the use of an adjuvant further enhanced the antiviral protective efficacy. This vaccine formulation could potentially be used to manage H6 influenza virus infections in chickens.

Identification of an infectious bronchitis coronavirus strain exhibiting a classical genotype but altered antigenicity, pathogenicity, and innate immunity profile.

Avian coronavirus infectious bronchitis virus (IBV) poses economic threat to the poultry industry worldwide. Pathogenic IBV 3575/08 was isolated from broilers vaccinated with the attenuated viral vaccine derived from a Taiwan strain 2575/98. In this study, extensive investigations were conducted on the genome sequences, antigenicity, pathogenicity, and host immune responses of several IBV strains in specific-pathogen-free chickens. Sequence analyses revealed that 3575/08 and 2575/98 shared high homology in their structural genes, but not in non-structural accessory proteins such as 3a, 3b and 5b. Despite a high degree of homology in their spike protein genes, cross neutralization test showed low cross protection between 3575/08 and 2575/98, suggesting distinct antigenicity for the two strains. Animal challenge experiments exhibited strong respiratory and renal pathogenicity for 3575/08. In addition, early and prolonged viral shedding and rapid viral dissemination were observed. Immune gene expression profiling by PCR array showed chickens infected with 3575/08 had delayed expression of a subset of early innate immune genes, whereas chickens infected with the wild-type or attenuated-type 2575/08 revealed quick gene induction and efficient virus control. In summary, this study reveals a new IBV strain, which harbors a known local genotype but displays remarkably altered antigenicity, pathogenicity and host defenses.

Lack of interferon sensitivity-determining region in the genome of hepatitis B virus genotype Ba.

BACKGROUND/AIMS: In chronic hepatitis B, both host and viral factors may predict the response to interferon (IFN) treatment. Whether IFN sensitivity-determining regions exist within the hepatitis B virus (HBV) genomic background remains largely unknown. We therefore performed full-length viral genomic comparison between HBVs obtained from IFN responders and non-responders. METHODS: We enrolled 18 HBV genotype Ba patients who had received 24-week IFN 5 MU three times weekly and were followed monthly for 12 months post-treatment. There were 10 responders and eight non-responders. Pretreatment full-length viral nucleotide consensus sequence was obtained. In six non-responders and four responders, post-treatment viral nucleotide sequence was further compared with their corresponding pre-treatment specimens. In addition, the average number of nucleotide substitutions of the HBV quasispecies was compared between three responders and three non-responders. RESULTS: HBV nucleotide consensus sequence was identical between responders and non-responders. We found 0-15 (mean 7.7) nucleotide substitutions in the post-treatment HBV genome in the six non-responders and 0-14 (mean 3.8) nucleotide substitutions in the four responders, respectively. Genetic complexity of HBV quasispecies was comparable between responders and non-responders. CONCLUSIONS: Our results suggest that an IFN sensitivity-determining region might not exist within the genome of HBV genotype Ba. Host factors and virus-host interactions may be more important in determining the response to IFN treatment.

Origin of serum hepatitis B virus in acute exacerbation: comparison with HBV in the liver and from other exacerbation.

Acute exacerbation (AE) of chronic hepatitis B is usually preceded by reemergence or increase of hepatitis B virus (HBV) in the serum. To investigate the origin of the reemergence or increase, we compared the identity of the serum viral genome to that in the liver and in previous AE by full-length sequencing. The full-length viral genome and extent of quasispecies were obtained from serum and liver biopsy specimens at the same time from 9 subjects with hepatitis B exacerbation (group I). Composition of viral quasispecies was compared by the genetic diversity and the average number of nucleotide substitutions within and between different viral sources. Another 2 patients with repeated AEs (group II) were also enrolled, and their serial serum alanine aminotransferase, HBV DNA levels and full-length sequences were determined. In all group I patients, serum viral genome was identical to that in the liver. The genetic diversity and the average number of nucleotide difference were also comparable between serum and liver tissue. In 2 group II patients, the viral variant that emerged after previous AE was not identical to that caused by the subsequent AE. Dominant viral strains for serial AEs in a single patient did not show a sequential evolution, but presented as a horizontal selection of a minor population from the original viral pool. In conclusion, the findings suggest that viral strain in serum reflects the intrahepatic strain of the AE. Random reactivation of the original HBV pool, rather than a sequential evolution of one strain, also contributes to the onset of repeated AE. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).