Hui Zhuang

N-glycomic changes in hepatocellular carcinoma patients with liver cirrhosis induced by hepatitis B virus†

We evaluated the use of blood serum N‐glycan fingerprinting as a tool for the diagnosis of hepatocellular carcinoma (HCC) in patients with cirrhosis induced by hepatitis B virus (HBV). A group of 450 HBV‐infected patients with liver fibrosis or cirrhosis with or without HCC were studied. HCC was diagnosed by α‐fetoprotein (AFP) analysis, ultrasonography, and/or computed tomography and was studied histologically. N‐glycan profiles of serum proteins were determined with DNA sequencer–based carbohydrate analytical profiling technology. In this study, we found that a branch alpha(1,3)‐fucosylated triantennary glycan was more abundant in patients with HCC than in patients with cirrhosis, patients with fibrosis, and healthy blood donors, whereas a bisecting core alpha(1,6)‐fucosylated biantennary glycan was elevated in patients with cirrhosis. The concentration of these 2 glycans and the log ratio of peak 9 to peak 7 (renamed the GlycoHCCTest) were associated with the tumor stage. Moreover, for screening patients with HCC from patients with cirrhosis, the overall sensitivity and specificity of the GlycoHCCTest were very similar to those of AFP. Conclusion: This study indicates that a branch alpha(1,3)‐fucosylated glycan is associated with the development of HCC. The serum N‐glycan profile is a promising noninvasive method for detecting HCC in patients with cirrhosis and could be a valuable supplement to AFP in the diagnosis of HCC in HBV‐infected patients with liver cirrhosis. Its use for the screening, follow‐up, and management of patients with cirrhosis and HCC should be evaluated further. (HEPATOLOGY 2007.)

Aberrant expression of microRNA 155 may accelerate cell proliferation by targeting sex-determining region Y box 6 in hepatocellular carcinoma.

Recent research has suggested that the oncomir microRNA 155 (miR‐155) is up‐regulated in hepatocellular carcinoma (HCC). In this study, the authors investigated the tumorigenic mechanism of this oncomir in the development of HCC.

Transmission of hepatitis E virus from rabbits to cynomolgus macaques.

The recent discovery of hepatitis E virus (HEV) strains in rabbits in the People’s Republic of China and the United States revealed that rabbits are another noteworthy reservoir of HEV. However, whether HEV from rabbits can infect humans is unclear. To study the zoonotic potential for and pathogenesis of rabbit HEV, we infected 2 cynomolgus macaques and 2 rabbits with an HEV strain from rabbits in China. Typical hepatitis developed in both monkeys; they exhibited elevated liver enzymes, viremia, virus shedding in fecal specimens, and seroconversion. Comparison of the complete genome sequence of HEV passed in the macaques with that of the inoculum showed 99.8% nucleotide identity. Rabbit HEV RNA (positive- and negative-stranded) was detectable in various tissues from the experimentally infected rabbits, indicating that extrahepatic replication may be common. Thus, HEV is transmissible from rabbits to cynomolgus macaques, which suggests that rabbits may be a new source of human HEV infection.

Distribution and clinical correlates of viral and host genotypes in Chinese patients with chronic hepatitis C virus infection.

Chronic hepatitis C virus (HCV) infection is relatively frequent in China. This study investigated the clinical, demographic, and viral and host genetic characteristics that may influence disease manifestations and clinical management.

Phylogenetic analysis of 626 hepatitis E virus (HEV) isolates from humans and animals in China (1986-2011) showing genotype diversity and zoonotic transmission.

Hepatitis E is considered as a public health problem in China. To determine the overall molecular epidemiology of hepatitis E virus (HEV) and analyze the situation of cross-species transmission between humans and swine in China over the last 25 years (1986-2011), 626 HEV complete and partial sequences (89 isolates identified by our group) isolated from humans and animals in China were retrieved from GenBank and subjected to phylogenetic analysis. There were three genotypes and 11 sub-genotypes of HEV prevailing in China. Furthermore, rabbit HEVs, of which the genotype is controversial, are also widespread in China. Genotype 1 was the most isolated genotype prior to 2000 and mainly detected in Xinjiang, Beijing and East China. However, genotype 4, which was identified in most regions of China during the last 10 years, has overtaken genotype 1 in frequency of isolation nationwide. Genotype 3 HEV strains have been found only in eastern China and were thought to be imported from Japan. Both genotypes 3 and 4 were found in humans and swine and cross-species transmission from pigs to humans of the two genotypes may have occurred in Northeast, Northwest, North, East and South China. These results indicate that HEV strains with considerable genetic diversity are widespread and the zoonotic transmission between swine and humans appears ubiquitous in China.

Hepatitis B: overview of the burden of disease in the Asia‐Pacific region

Abstract: Hepatitis B virus (HBV) infection and its liver‐related complications are a substantial health concern in the Asia‐Pacific region. Over the last two decades, public health interventions and the implementation of universal vaccination programs have substantially reduced the incidence of HBV infections in many countries in this region. However, large proportions of individuals remain chronically infected and subject to an increased risk for serious sequelae, including cirrhosis, decompensated liver disease, and hepatocellular carcinoma. The management of HBV infection varies throughout the Asia‐Pacific region, with each country confronting different issues related to prevention, screening, and treatment. These issues include the availability of diagnostic testing and treatment, the cost of diagnosis and treatment, the availability of trained medical professionals and medical facilities, and disease awareness among primary care physicians and the public. This article reviews the epidemiology of HBV infection in the Asia‐Pacific region, explains factors influencing hepatitis B prevalence and prevention, and discusses barriers to prevention and treatment of chronic hepatitis B and its liver‐related complications.

Thermal stability and inactivation of hepatitis C virus grown in cell culture

BackgroundHepatitis C virus (HCV) is a blood-borne flavivirus that infects many millions of people worldwide. Relatively little is known, however, concerning the stability of HCV and reliable procedures for inactivating this virus.MethodsIn the current study, the thermostability of cell culture-derived HCV (HCVcc, JFH-1 strain) under different environmental temperatures (37°C, room temperature, and 4°C) and the ability of heat, UVC light irradiation, and aldehyde and detergent treatments to inactivate HCVcc were evaluated. The infectious titers of treated viral samples were determined by focus-forming unit (FFU) assay using an indirect immunofluorescence assay for HCV NS3 in hepatoma Huh7-25-CD81 cells highly permissive for HCVcc infection. MTT cytotoxicity assay was performed to determine the concentrations of aldehydes or detergents at which they were no longer cytotoxic.ResultsHCVcc in culture medium was found to survive 37°C and room temperature (RT, 25 ± 2°C) for 2 and 16 days, respectively, while the virus was relatively stable at 4°C without drastic loss of infectivity for at least 6 weeks. HCVcc in culture medium was sensitive to heat and could be inactivated in 8 and 4 min when incubated at 60°C and 65°C, respectively. However, at 56°C, 40 min were required to eliminate HCVcc infectivity. Addition of normal human serum to HCVcc did not significantly alter viral stability at RT or its susceptibility to heat. UVC light irradiation (wavelength = 253.7 nm) with an intensity of 450 μW/cm2 efficiently inactivated HCVcc within 2 min. Exposures to formaldehyde, glutaraldehyde, ionic or nonionic detergents all destroyed HCVcc infectivity effectively, regardless of whether the treatments were conducted in the presence of cell culture medium or human serum.ConclusionsThe results provide quantitative evidence for the potential use of a variety of approaches for inactivating HCV. The ability of HCVcc to survive ambient temperatures warrants precautions in handling and disposing of objects and materials that may have been contaminated with HCV.

Comparative epidemiology and virology of fatal and nonfatal cases of hand, foot and mouth disease in mainland China from 2008 to 2014.

This study aimed to analyze the epidemiology and virology of fatal and nonfatal hand, foot, and mouth disease (HFMD) cases in Mainland China. A total of 10 714 237 survivors and 3046 deaths were reported from 2008 to 2014 June, with a case fatality rate of 0.03%. The morbidity of the survivors increased from 37.6/100 000 in 2008 to 139.6/100 000 in 2013 and peaked in 2012 at 166.8/100 000. However, the mortality varied around 0.03–0.04/100 000 across the time. Most of the survivors were distributed in the southern and eastern China, predominantly in the Guangxi and Hainan Province, whereas deaths were dominant in southern (Guangxi) and southwestern (Guizhou) China. The two groups showed similar seasonal fluctuations from 2008 to 2014, peaking in spring and early summer. Of the total cases, 93.97% were children less than 5 years of age, with those ≤ 2 years old accounting for 60.08% versus 84.02% in the survivor and death groups, respectively. Boys were at higher risk of infection than girls in both groups. Five years of virological surveillance showed that 43.73%, 22.04%, and 34.22% of HFMD cases were due to EV71, CoxA16 and other enteroviruses, respectively. EV71 was encountered in most deaths, with no substantial effect of age, gender, month, and year on incidence. Subgenotype C4a was the prevalent EV71 strain in Mainland China, with no significant difference in the VP1 gene related to virulence between the two groups. In conclusion, based on the largest population study, fatal and nonfatal HFMD cases, mainly caused by C4a of EV71, are circulating in Mainland China with a low‐cause fatality rate. Copyright

Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication.

AIM To screen and investigate the effective gRNAs against hepatitis B virus (HBV) of genotypes A-D. METHODS A total of 15 gRNAs against HBV of genotypes A-D were designed. Eleven combinations of two above gRNAs (dual-gRNAs) covering the regulatory region of HBV were chosen. The efficiency of each gRNA and 11 dual-gRNAs on the suppression of HBV (genotypes A-D) replication was examined by the measurement of HBV surface antigen (HBsAg) or e antigen (HBeAg) in the culture supernatant. The destruction of HBV-expressing vector was examined in HuH7 cells co-transfected with dual-gRNAs and HBV-expressing vector using polymerase chain reaction (PCR) and sequencing method, and the destruction of cccDNA was examined in HepAD38 cells using KCl precipitation, plasmid-safe ATP-dependent DNase (PSAD) digestion, rolling circle amplification and quantitative PCR combined method. The cytotoxicity of these gRNAs was assessed by a mitochondrial tetrazolium assay. RESULTS All of gRNAs could significantly reduce HBsAg or HBeAg production in the culture supernatant, which was dependent on the region in which gRNA against. All of dual gRNAs could efficiently suppress HBsAg and/or HBeAg production for HBV of genotypes A-D, and the efficacy of dual gRNAs in suppressing HBsAg and/or HBeAg production was significantly increased when compared to the single gRNA used alone. Furthermore, by PCR direct sequencing we confirmed that these dual gRNAs could specifically destroy HBV expressing template by removing the fragment between the cleavage sites of the two used gRNAs. Most importantly, gRNA-5 and gRNA-12 combination not only could efficiently suppressing HBsAg and/or HBeAg production, but also destroy the cccDNA reservoirs in HepAD38 cells. CONCLUSION These results suggested that CRISPR/Cas9 system could efficiently destroy HBV expressing templates (genotypes A-D) without apparent cytotoxicity. It may be a potential approach for eradication of persistent HBV cccDNA in chronic HBV infection patients.

Study on prevalence and genotype of hepatitis E virus isolated from Rex Rabbits in Beijing, China.

Summary.  A novel genotype of hepatitis E virus (HEV) isolated from rabbits is reported. The aim of this study was to confirm and further investigate the prevalence of the novel HEV genotype in rabbits in China. Sera and faecal samples were collected from farmed rex rabbits in Beijing, China. All serum samples were tested for anti‐HEV antibody by EIA. Both the serum and the faecal samples were evaluated for detection of HEV RNA using a nested RT‐PCR assay. The nucleotide sequences of rabbit HEV were then analysed, and sequence homology of rabbit HEV compared against human HEV genotypes 1–4, and avian HEV. Results: The prevalence of positive serum anti‐HEV from rex rabbits was 54.62% (65/119). The detection rate of HEV RNA using ORF2 primers was 6.96% (8/115) amongst rabbit faecal samples. All eight amplicons shared 98.3–100% nucleotide homology with each other and had identities of 75.8–78.6%, 73.9–75.0%, 77.5–81.0%, 74.2–78.6% and 54.8–57.6% with the corresponding regions of genotypes 1–4 and avian HEV, respectively. Phylogenetic analysis showed that the eight sequences formed one individual branch and were on the same branch with GDC9 and GDC46, both of which were reported to be a novel genotype of HEV isolated from rabbits. The conclusion is that this study provides further information about HEV infecting rabbits, which may be a new animal host of HEV, as well as genetical evidence of a new mammalian genotype of HEV.