Zhuang H

Amino-terminal epitopes are exposed when full-length open reading frame 2 of hepatitis E virus is expressed in Escherichia coli, but carboxy-terminal epitopes are masked

We constructed a panel of overlapping and non‐overlapping fragments of cDNA derived from open reading frame 2 (ORF2) of hepatitis E virus (HEV) and fused to the gene encoding glutathione S‐transferase (GST), from which proteins were expressed in Eschericia coli. IgG‐specific immunoreactivity against each protein was measured by Western immunoblotting using sera from experimentally infected Rhesus macaques (Macaca mulatta) or from HEV‐infected patients. Under these conditions, full‐length ORF2 protein (GST‐ORF2) was strongly reactive with acute‐phase sera from either macaques or patients, but was poorly reactive with convalescent sera. Recombinant protein GST‐ORF2.3, representing amino acids 1–110 of the 660 encoded by ORF2, demonstrated a pattern of reactivity largely indistinguishable from the full‐length protein. Conversely, GST‐ORF2.1, representing amino acids 394–660 of the ORF2 protein was strongly reactive with both acute‐ and convalescent‐phase sera. Extension of GST‐ORF2.1 towards the N‐terminus led to a progressive loss of convalescent‐phase reactivity, apparent with as few as 20 additional HEV‐specific amino acids. Deletion of 40 or more amino acids from the N‐terminus of ORF2.1 also led to reduced convalescent‐phase reactivity, however a protein representing this “reactive” region, containing amino acids 394–473, was poorly reactive, suggesting that the convalescent‐reactive epitopes are conformational. Expression of full‐length ORF2 protein in E. coli therefore masks the convalescent‐reactive epitopes within the C‐terminal part of the protein, without affecting N‐terminal, acute‐reactive epitopes. J. Med. Virol. 52:289–300, 1997.

Epidemiology and prevention of hepatitis B virus infection in China

Summary.u2002 The concept of a hepatitis B vaccine was first introduced into China in 1978. China has been one of the first two developing countries to enact the universal hepatitis B vaccination programme for newborn babies in 1992, and has made tremendous achievements in the control of hepatitis B virus (HBV) infection since then. China now has both low and high endemic regions regarding HBV prevalence co‐existing. Although China’s drive to stop HBV spread has resulted in changes in HBV epidemic patterns, for the eventual elimination HBV infection in China, it is important to understand the current status of its epidemiology and the aspects of HBV transmission in different regions. More efforts are needed to improve and develop strategies for the control of HBV infection in China, particularly after implementing the policy of universal HBV immunization for all newborns.

Experimental infection of pregnant rabbits with hepatitis E virus demonstrating high mortality and vertical transmission

A high mortality rate of approximately 20% in pregnant women with hepatitis E has been reported in previous studies. However, other studies showed no difference between pregnant and nonpregnant women in the severity of hepatitis E. To determine the effects of HEV infection on pregnancy, we successfully established HEV infection in six pregnant rabbits (PR) and six nonpregnant rabbits (NPR) with a rabbit HEV isolate, taking three PR and one NPR without HEV infection as controls. Tests for HEV RNA by RT‐PCR, anti‐HEV antibodies by ELISA and HEV antigen via immunohistochemistry and histopathology were carried out. Two of six infected PR miscarried and three of the remaining four PR died which may be attributed to severe liver necrosis caused by HEV infection. Moreover, vertical transmission was found to be associated with the replication of HEV in placenta, indicated by the presence of HEV RNA and antigen in placenta from the infected PR. Our findings strongly suggest that HEV infection could lead to adverse outcomes in pregnancy and vertical transmission, suggesting the necessity for pregnant women at risk of HEV infection to be vaccinated.

Serum Immunoglobulin Levels in Acute A, B, and Non-A, Non-B Hepatitis

Immunoglobulin M, G, and A concentrations were determined by radial immunodiffusion in sera collected from 117 patients with acute hepatitis A, hepatitis B, or non-A, non-B hepatitis. Statistically significant differences in IgG and IgM levels were detected between the three groups. In particular, elevated IgG and almost-normal IgM levels were regularly detected in patients with non-A, non-B hepatitis while the opposite pattern was seen in patients with hepatitis A. Calculation of a serum IgG/IgM ratio enabled discrimination between most patients with non-B hepatitis. In 92% of patients with hepatitis A, the ratio was less than 6, whereas 82% of patients with non-A, non-B hepatitis had ratios of greater than 6. The IgG/IgM ratio may be of value in distinguishing between subjects with the two forms of the disease when specific serologic tests are unavailable.

An integrated analysis of SOCS1 down‐regulation in HBV infection‐related hepatocellular carcinoma

Persistent inflammation together with genetic/epigenetic aberrations is strongly associated with chronic Hepatitis B virus (HBV) infection‐related hepatocarcinogenesis. Here, we investigated the alterations of the suppressor of cytokine signalling (SOCS) family genes in HBV‐related hepatocellular carcinoma (HCC). A total of 116 patients with HCC were enrolled in this study. The methylation statuses of SOCS1‐7 and CISH genes were quantitatively measured and clinicopathological significance of SOCS1 methylation was statistically analysed. The gene copy number variation was assayed by aCGH. Luciferase reporter assay and Western blot were used to detect the involvement of SOCS1 in p53 signalling. We found high frequencies of SOCS1 gene hypermethylation in both tumour (56.03%) and adjacent nontumour tissues (54.31%), but tumour tissues exhibited increased methylation intensity (24.01% vs 13.11%, P < 0.0001), particularly in patients with larger tumour size or cirrhosis background (P < 0.0001). In addition, the frequency and intensity of SOCS1 hypermethylation in tumour tissues were both significantly higher than those in nontumour tissues in male gender patients and in patients ≥45 years old (P = 0.0214 and P < 0.0001, P = 0.0232 and P < 0.0001, respectively). SOCS1 gene deletion was found in 8 of 25 aCGH assayed tumour specimens, which was associated with lower SOCS1 mRNA expression (P = 0.0448). Furthermore, ectopic SOCS1 overexpression could activate the p53 signalling pathway in HCC cell lines. Hypermethylation of SOCS2‐7 and CISH genes was seldom found in HCC. Our results suggested that the gene loss and epigenetic silencing of SOCS1 were strongly associated with HBV‐related HCC.

Kinetics of Severe Acute Respiratory Syndrome (SARS) Coronavirus-Specific Antibodies in 271 Laboratory-Confirmed Cases of SARS

ABSTRACT The sensitivities and specificities of an immunofluorescence assay and an enzyme immunoassay for detection of antibodies specific for severe acute respiratory syndrome coronavirus (SARS-CoV) were compared for 148 laboratory-confirmed SARS cases. The appearance and persistence of SARS-CoV-specific antibodies were assessed, with immunoglobulin G detected in 59% of samples collected within 14 days and persisting for 60 to 95 days after the onset of illness.

Hepatitis B virus genotype C encoding resistance mutations that emerge during adefovir dipivoxil therapy: in vitro replication phenotype

IntroductionHepatitis B virus (HBV) can be classified into ten genotypes (A–J), with genotypes B and C being the most common in Asia. Recent data suggest that the HBV genotype can influence disease progression, and genotype C has been associated with more aggressive liver disease than that of other genotypes. Although there is a preventative vaccine, chronic infection remains a public health problem with oral nucleos(t)ide analog therapy being the most common treatment. The HBV genome is composed of four partially overlapping reading frames, meaning that substitutions in the HBV polymerase selected during NA therapy may also alter the overlapping HBV surface antigen (HBsAg). We have recently shown that for HBV genotype D, the rtA181T/sW172stop substitution conferring resistance to adefovir dipivoxil (ADV) alters secretion of HBsAg and exerts a dominant-negative effect on wild-type virion secretion. However, the effect of this and other ADV-resistance-associated mutations on HBV replication and HBsAg secretion for the HBV genotype C, the genotype with the most severe clinical prognosis, is unknown.Methods/ResultsWe constructed 1.2-mer infectious cDNA clones of HBV genotype C encoding mutations associated with ADV resistance and established an in vitro replication assay in Huh7 cells. Decreased levels of HBV DNA and HBsAg were detected for all ADV variants relative to the 1.2-mer wild-type polymerase control plasmid. Importantly, less HBsAg was detected in the cells transfected with the rtA181T resistance mutants, and the overlapping sW172stop mutation ablated secretion of HBsAg into cell culture supernatants.ConclusionsThe identification of secretion-defective HBV in the setting of ADV therapy for HBV genotype C, and to a lesser extent HBV genotype B, has major implications for the diagnosis and treatment of HBV in the Asia-Pacific region, as it is likely that quantitative HBsAg and viral load testing of serum from patients infected with HBV encoding rtA181T and rtN236T substitutions may not accurately reflect the level of replication within hepatocytes.

Composition and Interactions of Hepatitis B Virus Quasispecies Defined the Virological Response During Telbivudine Therapy.

Reverse transcriptase (RT) mutations contribute to hepatitis B virus resistance during antiviral therapy with nucleos(t)ide analogs. However, the composition of the RT quasispecies and their interactions during antiviral treatment have not yet been thoroughly defined. In this report, 10 patients from each of 3 different virological response groups, i.e., complete virological response, partial virological response and virological breakthrough, were selected from a multicenter trial of Telbivudine treatment. Variations in the drug resistance-related critical RT regions in 107 serial serum samples from the 30 patients were examined by ultra-deep sequencing. A total of 496,577 sequence reads were obtained, with an average sequencing coverage of 4,641X per sample. The phylogenies of the quasispecies revealed the independent origins of two critical quasispecies, i.e., the rtA181T and rtM204I mutants. Data analyses and theoretical modeling showed a cooperative-competitive interplay among the quasispecies. In particular, rtM204I mutants compete against other quasispecies, which eventually leads to virological breakthrough. However, in the absence of rtM204I mutants, synergistic growth of the drug-resistant rtA181T mutants with the wild-type quasispecies could drive the composition of the viral population into a state of partial virological response. Furthermore, we demonstrated that the frequency of drug-resistant mutations in the early phase of treatment is important for predicting the virological response to antiviral therapy.

Female gender lost protective effect against disease progression in elderly patients with chronic hepatitis B

Female gender and younger age are protective factors against disease progression in chronic hepatitis B (CHB). However, it is not clear whether the disease progression still remains slow in elderly females. This study investigated the interaction of female gender and older age on the development of cirrhosis in patients recorded in China Registry of Hepatitis B. A total of 17,809 CHB patients were enrolled in this multi-center cross-sectional study. The prevalence of cirrhosis in female CHB patients increased faster than that in male CHB patients over 50 years old. Multivariate analysis showed that the increase of adjusted ORs for developing cirrhosis in females started to accelerate after 50 years old: 11.19 (95% CI: 5.93–21.11) in women versus 14.75 (95% CI: 8.35–26.07) in men at ages of 50–59 years, 21.67 (95% CI: 11.05–42.47) versus 24.4 (95% CI: 13.00–45.80) at ages 60–69 years, and 18.78 (95% CI: 6.61–53.36) versus 12.09 (95% CI: 4.35–33.61) in those over 70 years. In conclusion, the protective effect of female gender against cirrhosis gradually lost with increasing age, therefore disease progression should be monitored more closely in elderly women with CHB.

Establishment of a reference panel for the detection of anti-SARS-CoV antibodies

n Abstractn n The immunological assays for detection of antibodies against SARS-CoV were developed in-house and some of them are available commercially. However, the antigens used in these assays differed. In order to validate the reliability of these assays, the standard panel should be established. In this study, we have expressed and purified severe acute respiratory syndrome (SARS) structural proteins and their fragments and developed indirect enzyme-linked immunosorbent assays (ELISAs) that detect antibodies against the SARS N, N1, N2, S1, SC, S2, and M proteins as well as the human coronavirus OC43 and 229E N proteins. These assays were used to screen 58 samples from SARS convalescent patients, 40 serial serum specimens from patients at different phases of SARS infection, and 88 plasma specimens from normal blood donors. The samples from normal blood donors were also tested for antibodies against other respiratory virus. The representative samples were chosen to comprise a reference panel of SARS antibodies that may be used for the detection of SARS. The panel is composed of 25 positive samples, 25 negative samples, 7 diluted samples for anti-N antibody, 6 diluted samples for anti-S antibody, and one sample for validating precision. Comparison of detection results with different SARS antibody assays indicated that our panel should differentiate the specificity and sensitivity of different assays.n n