Weilu Zhang

Analysis of recombination and natural selection in human enterovirus 71

The development of effective vaccines and antiviral prophylaxis against human enterovirus 71 (EV71) has been hampered by the extensive antigenic diversity of the virus. To gain new insights into the evolutionary processes that create this genetic diversity, the TreeOrder Scan Method and RDP program were employed to detect recombination events in the genome, and then parsimony-based and maximum-likelihood-based methods were used to detect natural selection effects on viral proteins. Recombination analysis provided strong evidence for recombination events in the majority of the sequences analyzed. Recombination events were found to be distributed nonrandomly with the highest frequency at the 3D region. Furthermore, positive selection was only detected at site 145 of VP1 by the maximum likelihood-based method. These results reveal that EV71 proteins are extensively influenced by stabilizing selection. We conclude that recombination may play a more important role than positive selection in the formation of genetic diversity.

Establishment and characterization of a spontaneously immortalized trophoblast cell line (HPT-8) and its hepatitis B virus-expressing clone

BACKGROUND Most trophoblast cell lines currently available to study vertical transmission of hepatitis B virus (HBV) are immortalized by viral transformation. Our goal was to establish and characterize a spontaneously immortalized human first-trimester trophoblast cell line and its HBV-expressing clone. METHODS Chorionic villi of Asian human first-trimester placentae were digested with trypsin and collagenase I to obtain the primary trophoblast cell culture. A spontaneously immortalized trophoblast cell line (HPT-8) was analyzed by scanning and transmission electron microscopy, cell cycle analysis, immunohistochemistry and immunofluorescence. HPT-8 cells were stably transfected with the adr subtype of HBV (HPT-8-HBV) and characterized by PCR and enzyme-linked immunosorbent assay. RESULTS We obtained a clonal derivative of a spontaneously immortalized primary cell clone (HPT-8). HPT-8 cells were epithelioid and polygonal, and formed multinucleate, giant cells. They exhibited microvilli, distinct desmosomes between adjacent cells, abundant endoplasm, lipid inclusions and glycogen granules, which are all characteristic of cytotrophoblasts. HPT-8 cells expressed cytokeratin 7, cytokeratin 18, vimentin, cluster of differentiation antigen 9, epidermal growth factor receptor, stromal cell-derived factor 1 and placental alkaline phosphatase. They secreted prolactin, estradiol, progesterone and hCG, and were positive for HLA-G, a marker of extravillous trophoblasts. HPT-8-HBV cells were positive for HBV relaxed-circular, covalently closed circular DNA and pre-S sequence. HPT-8-HBV cells also produced and secreted HBV surface antigen and HBV e antigen. CONCLUSIONS We established a trophoblast cell line, HPT-8 and its HBV-expressing clone which could be valuable in exploring the mechanism of HBV viral integration in human trophoblasts during intrauterine infection.

Recombination and Natural Selection in Hepatitis E Virus Genotypes

To gain new insights into the evolutionary processes that created the genetic diversity of the hepatitis E virus (HEV), the Recombination Detection Program (RDP) and SimPlot program were employed to detect recombination events in the genome, then the fixed‐effects likelihood (FEL) method was used to detect natural selection effects on viral proteins. Recombination analysis provided strong evidence for both intergenotype and intragenotype recombination events in the sequences analyzed. Recombination events were found to be distributed non‐randomly, with the highest frequency in the X domain and the helicase. Strain DQ450072 was identified as intergenotype‐recombinant. Natural selection analysis revealed that codons under both negative selection and positive selection were distributed non‐randomly. ORF1 and ORF2 have experienced strong purifying selection across genotypes. Furthermore, potentially important sites were also found under positive selection in the N‐terminal end of ORF2 and the C‐terminal end of ORF3. No significant difference was found among the selective pressures on different genotypes. J. Med. Virol. 84:1396–1407, 2012.

A description of the hepatitis B virus genomic background in a high-prevalence area in China

BackgroundHepatitis B (HB) is an important disease worldwide. Almost 350 million people are positive for Hepatitis B virus surface antigen (HBsAg), and one-third of them live in China. According to a nation-wide serosurvey in China in 2006, the prevalence of HBsAg was higher in Northwest China than in other areas. However, the epidemic HBV strains in this area are poorly studied.ResultsIn this study, 242 complete hepatitis B virus (HBV) genome sequences were obtained from HBV asymptomatic carriers in major cities of Northwest China. The 242 HBV sequences clustered into genotypes B, C and D. Through comparison of the genotype consensus sequences, 158 genotype-dependent positions were observed in P, S and X ORFs. Clinically relevant mutation screening in this study revealed that no HBV antiviral drug resistance mutations were observed and the vaccination failure mutations were heavily underrepresented.ConclusionsThe role of genotype D strains in HBV prevalence should not be ignored in Northwest China. Due to low prevalence of vaccination failure mutations, it can be inferred that the genotype B, C and D strains in Northwest China may have less likelihood of vaccine escape.

Maternal immunization promotes the immune response of neonates towards hepatitis B vaccine.

Infants infected with hepatitis B virus (HBV) face the risk of developing severe complications. Unfortunately, in spite of universal vaccination programmes, 5% or more of vaccinated newborns still do not achieve protective levels of anti‐hepatitis B virus surface antigen titres (anti‐HBs). The aim of this study was to use animal experiments and population‐based research to determine whether maternal vaccination against HBV affects the outcome of neonatal vaccination. Six sows and 53 newborn piglets were used for this study and randomly assigned to the vaccination group (three 20 μg doses of recombinant HBV vaccine). All the piglets were followed up to 10 weeks of age, and peripheral blood was withdrawn for measurement of anti‐HBs. A cross‐sectional study was also conducted on 449 mothers with infants. A structured questionnaire was used to collect demographic, medical and maternal data, and their peripheral blood was collected for measurement of anti‐HBs. The results of animal experiments demonstrated that nonvaccinated piglets born to vaccinated sows and nonvaccinated piglets born to nonvaccinated sows were negative for anti‐HBs. Repeated measures analysis of variance showed that the titres of anti‐HBs in vaccinated piglets born to vaccinated sows were significantly higher than in vaccinated piglets born to nonvaccinated sows (P < 0.05). In a population‐based study, a cumulative logistic regression analysis showed that the strongest influences on neonatal anti‐HBs titres were delay of the first vaccination dose [OR = 3.02(95% CI: 1.72–5.30)] and maternal anti‐HBs titres [OR = 2.48(95% CI: 2.03–3.04)]. In conclusion, high maternal anti‐HBs titres can enhance the response to HBV vaccination in infants.

A meta-analysis of HBsAg-positive rate among general Chinese populations aged 1--59 years.

Abstract Background: The third nationwide hepatitis B virus (HBV) sero-epidemiological survey conducted in China in 2006 showed a hepatitis B surface antigen (HBsAg)-positive rate of 7.18% and the number of carriers to be 93 million. To compensate for the lack of data on the national prevalence of hepatitis B from 2006 onwards, we performed a meta-analysis to investigate HBsAg-positive rates among the general Chinese population aged 1–59 years. Methods: We systemically reviewed published studies (January 2007 to September 2013) from full-text databases such as VIP, Chinese National Knowledge Infrastructure, and PubMed and assessed HBsAg-positive rates using a random effects regression model with comprehensive meta-analysis software. Results: After evaluation of the quality of the papers, 36 were finally included, with a total sample size of 314 103 individuals. The meta-analysis showed that the combined HBsAg-positive rate was 6.1% (95% confidence interval (CI), 5.4–6.8%) among general Chinese populations aged 1–59, with a higher HBsAg-positive rate in males (6.6%; 95% CI, 5.7–7.7%) than in females (5.1%; 95% CI, 4.4–5.9%). Our data also showed a higher HBsAg-positive rate of 6.9% in mid-western China (95% CI, 5.2–9.1%) against 5.6% in eastern China (95% CI, 4.9–6.4%). Conclusion: This meta-analysis suggested that the positive rates of HBsAg in China are reducing compared with previous 2006 sero-survey results, and China has changed from a high endemic area for HBV to an intermediate endemic area. However, owing to the large population, there remain an estimated 80 million HBsAg carriers in China. Thus, the prevention and control of hepatitis B represent a serious challenge, particularly in mid-western China.

Prevalence and natural course of occult hepatitis B virus infection in residents of two communities of Wuwei City, Gansu Province, China

Occult hepatitis B infection (OBI) is characterized by serum hepatitis B surface antigen (HBsAg) negative and hepatitis B virus (HBV) DNA positive (HBsAg‐/HBV DNA+). Occult hepatitis B infection in community‐based populations has been scarcely investigated, and OBI outcomes remain unclear, especially in Wuwei, a region located in Northwest China. This region is one of the areas in China that has the highest prevalence of chronic HBV infection. A prospective study was performed in the general population of 2 towns of Wuwei from June 2011 to May 2014. A questionnaire was used to collect demographic and medical data, and serum samples were collected from the participants and stored until analysis. DNA was detected using quantitative PCR (qPCR) or nested PCR, the HBV DNA from HBV DNA‐positive or possible positive (below the detection limit) subjects was extracted and amplified by nested PCR, and the PCR products were sequenced. Sequence analysis was performed using the Mega 6.0 program and CLC sequence viewer software. Hepatitis B virus DNA was detected in 90 of 3,080 HBsAg‐negative subjects, and the prevalence of OBI in the study population was 2.92% (90/3,080, 95% CI: 2.33%‐3.51%). Hepatitis B virus genomes in 51 of 80 objects (63.75%) contained mutations in the “a” determinant of HBsAg. After 2 years follow‐up, 42 of 90 HBV DNA of OBI subjects remained positive, and the natural clearance rate of OBI subjects was 53.3%. Occult hepatitis B infection prevalence in this cohort was much lower than chronic HBV infection in the same region. HBV DNA was cleared in most OBI subjects during the 2 year period. Our data suggest that some OBI may represent a late stage of resolving the HBV infection process.

Green Tea Liquid Consumption Alters the Human Intestinal and Oral Microbiome

Scope GTPs (green tea polyphenols) exert anti‐CRC (colorectal cancer) activity. The intestinal microbiota and intestinal colonization by bacteria of oral origin has been implicated in colorectal carcinogenesis. GT modulates the composition of mouse gut microbiota harmonious with anticancer activity. Therefore, the effect of green tea liquid (GTL) consumption on the gut and oral microbiome is investigated in healthy volunteers (n = 12). Methods and results 16S sequencing and phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis of both fecal and saliva samples (collected before intervention, after 2 weeks of GTL (400 mL per day) and after a washout period of one week) in healthy volunteers show changes in microbial diversity and core microbiota and difference in clear classification (partial least squares‐discriminant analysis [PLS‐DA]). An irreversible, increased FIR:BAC (Firmicutes to Bacteroidetes ratio), elevated SCFA producing genera, and reduction of bacterial LPS synthesis in feces are discovered in response to GTL. GTL alters the salivary microbiota and reduces the functional pathways abundance relevance to carcinogenesis. Similar bacterial networks in fecal and salivary microbiota datasets comprising putative oral bacteria are found and GTL reduces the fecal levels of Fusobacterium. Interestingly, both Lachnospiraceae and B/E (Bifidobacterium to Enterobacteriacea ratio—markers of colonization resistance [CR]) are negatively associated with the presence of oral‐like bacterial networks in the feces. Conclusion These results suggest that GTL consumption causes both oral and gut microbiome alterations.

Effects of vitamin C supplementation on blood pressure and hypertension control in response to ambient temperature changes in patients with essential hypertension

ABSTRACT Evidence for blood pressure-lowering effects of vitamin C (VC) supplementation in clinical trials is inconsistent and limited studies have examined the effect of VC supplementation on hypertension (HTN) control. In this study, eligible patients were cluster assigned to receive 300 mg VC per day or nothing for 6 months. During the 6-month follow-up period, a questionnaire survey was obtained and standardized blood pressure measurements were performed on all subjects. Oral administration of VC significantly decreased the diastolic blood pressure and pulse pressure with a significant increase in HTN control. After adjusting for confounding variables, treatment with VC was associated with ~ 0.5 risk reduction of uncontrolled HTN in subjects received anti-hypertensive medications, whereas lower indoor and outdoor and ground temperature were significantly associated with an increased risk of uncontrolled HTN in all patients. Our results warrant further studies investigating the mechanisms underlying the association between VC and HTN control.

Overt and occult hepatitis B virus infection among community children in Northwest China

Although a universal newborn hepatitis B (HB) immunization programme has been implemented in China, hepatitis B virus (HBV) breakthrough infection, including HB surface antigen (HBsAg)‐positive infection and occult HBV infection (OBI), still occurs during infancy or childhood. Obtaining the actual prevalence of HBV infection in general children is important for preventing and controlling the spread of HB. Accordingly, we investigated the prevalence of overt infection and OBI in community children and compared the serological and virological characteristics of OBI and HBsAg carrier children to clarify the mechanisms related to OBI.