Chengyao Li

Characterization of Occult Hepatitis B Virus Infection from Blood Donors in China

ABSTRACT Prevalence and characteristics of occult hepatitis B virus (HBV) infection (OBI) of genotypes B and C prevalent in China have not been extensively explored. Characterization of OBI strains obtained from Chinese blood donors was based on clinical and serological analyses, follow-up testing, and sequence analyses. Twenty-eight samples from 165,371 HBV surface antigen (HBsAg)-negative plasmas were confirmed HBsAg negative and DNA positive(HBsAg−/DNA+), of which 22 were classified as OBIs and 6 as window period infections. The OBI incidence was 1:7,517 in blood donors, whose ages ranged between 20 and 45 years (median, 28 years). OBI donors had normal alanine aminotransferase (ALT) levels and low viral loads ranging between unquantifiable amounts and 178 IU/ml (median, 14 IU/ml). Sequences from 21 basic core promoter/precore (BCP/PC) regions, five whole genomes, and two additional pre-S/S regions from OBI strains were compared to genotypes B and C HBsAg+ reference strains. Eighty-six percent (6/7) of OBI strains were genotype C. Deletions, insertions, stop codons, and substitutions were detected in 15/21 (71%) core regulatory elements of OBI strains. Critical mutations were found in the core proteins of 5/5 OBI strains in parallel with random substitutions in pre-S/S proteins from 6/7 (86%) OBI strains. Critical mutations in core regulatory elements and core proteins might affect OBI genotype B and C strain replication. That there were few S protein substitutions suggests a minor role of the host immune defenses in OBI occurrence.

Prevalence of persistent and latent viruses in untreated patients infected with HIV-1 from Ghana, West Africa.

Only limited epidemiological data, pertaining to the prevalence of common persistent viruses has been reported in Ghana. This study was conducted to determine the prevalence of persistent viruses in individuals with untreated HIV‐1 infection and uninfected blood donors. Paired plasma and cellular samples from HIV‐negative blood donors, asymptomatic HIV and symptomatic/AIDS cohorts were screened by multiplex PCR then qPCR for parvovirus B19 (B19V), hepatitis B virus (HBV), GB virus‐C (GBV‐C), cytomegalovirus (CMV), Epstein–Barr virus (EBV), human herpesvirus‐8 (HHV‐8) and varicella‐zoster virus (VZV). IgG antibodies specific to each target virus were tested to determine exposure rates. No evidence of viraemia was found for B19V and VZV in any group. Prevalence of GBV‐C plasma viraemia was significantly higher in asymptomatic and symptomatic HIV infection (16.7%) and (16.2%) than in blood donors (4%) P < 0.005. Occult HBV infection was significantly more frequent in symptomatic HIV infection (10.9%) compared to asymptomatic HIV (3.6%) and blood donors (1.6%) P < 0.005. Although there was a high background of EBV viraemia in cellular fractions of blood donors (8.3%), it was significantly higher in asymptomatic (44.6%) and symptomatic HIV (14.6%) P < 0.0001. For CMV, the significantly increased prevalence of viraemia was only observed in the plasma fraction of the symptomatic HIV‐1/AIDS patients (7.6%) compared to asymptomatic individuals (1.8%) and blood donors (0.8%) P ≤ 0.001. The background seroprevalence in blood donors was high for B19V (≥64%), HBV (≥70%), CMV and EBV (≥90%) and was significantly increased in HIV infections for HBV, CMV, VZV (symptomatic HIV), and HHV‐8 (asymptomatic and symptomatic HIV). J. Med. Virol. 81:1860–1868, 2009.

Multiplex real-time PCR for the detection and quantification of latent and persistent viral genomes in cellular or plasma blood fractions.

In common with latent viruses such as herpesviruses, parvovirus B19, HBV and GBV-C are contained successfully by the immune response and persist in the host. When immune control breaks down, reactivation of both latent and persistent viruses occurs. Two multiplex assays were developed (B19, HBV, HHV-8), (EBV, CMV, VZV) for blood screening, and tested on blood donor samples from Ghana to determine baseline prevalence of viraemia in immunocompetent persons. Single-virus real-time quantitative PCR (qPCR) assays were optimised for viral load determination of positive initial screening. The qPCR method utilised was absolute quantification with external standards. Multiplex and single-virus qPCR assays had similar sensitivity, except for the B19 assay in which sensitivity was 100-fold lower. Assays were optimised for reproducibility and repeatability, with R(2) of 0.9 being obtained for most assays. With the exception of B19 and CMV, assays had 100% detection limit ranging between 10(1) and 10(2) copies, IU or arbitrary units under single-virus and multiplex assay conditions. The prevalence of viraemia was 1.6% HBV (0.8% DNA+/HBsAg-, 0.8% DNA+/HBsAg+), 0.8% parvovirus B19, and 3.3% GBV-C viraemia in the plasma fraction. The prevalence of four herpesviruses was 1.0% HHV-8, 0.85% CMV, and 8.3% EBV, and no detectable VZV viraemia.

Increasing threat of brucellosis to low-risk persons in urban settings, China.

Cases of brucellosis were diagnosed in 3-month-old twins and their mother. An epidemiologic survey suggested that raw sheep or goat meat might be the source of Brucella melitensis infection. This finding implies that the increasing threat of brucellosis might affect low-risk persons in urban settings in China.

Characterization of Periplasmic Protein BP26 Epitopes of Brucella melitensis Reacting with Murine Monoclonal and Sheep Antibodies

More than 35,000 new cases of human brucellosis were reported in 2010 by the Chinese Center for Disease Control and Prevention. An attenuated B. melitensis vaccine M5-90 is currently used for vaccination of sheep and goats in China. In the study, a periplasmic protein BP26 from M5-90 was characterized for its epitope reactivity with mouse monoclonal and sheep antibodies. A total of 29 monoclonal antibodies (mAbs) against recombinant BP26 (rBP26) were produced, which were tested for reactivity with a panel of BP26 peptides, three truncated rBP26 and native BP26 containing membrane protein extracts (NMP) of B. melitensis M5-90 in ELISA and Western-Blot. The linear, semi-conformational and conformational epitopes from native BP26 were identified. Two linear epitopes recognized by mAbs were revealed by 28 of 16mer overlapping peptides, which were accurately mapped as the core motif of amino acid residues 93DRDLQTGGI101 (position 93 to 101) or residues 104QPIYVYPD111, respectively. The reactivity of linear epitope peptides, rBP26 and NMP was tested with 137 sheep sera by ELISAs, of which the two linear epitopes had 65–70% reactivity and NMP 90% consistent with the results of a combination of two standard serological tests. The results were helpful for evaluating the reactivity of BP26 antigen in M5-90.

Evaluation of humoral and cellular immune responses to BP26 and OMP31 epitopes in the attenuated Brucella melitensis vaccinated sheep.

In recent years, the number of cases of human brucellosis has been increasing by approximately 10% per year in China. Most cases were caused by Brucella melitensis through contacts with infected sheep, goats or their products. An attenuated B. melitensis vaccine M5-90 is currently used to vaccinate both animals in China. This vaccine has not been investigated for critical parameters such as immune response and its association with protective efficacy. In this study, humoral and cellular immune response to the periplasmic protein BP26 and the outer membrane protein OMP31 were evaluated in M5-90 vaccinated Chinese merino and Kazak sheep. Antibodies to BP26 or OMP31 were detected at low levels, and specific IFN-γ response was quantified. Strongly reactive peptides derived from BP26 and OMP31 identified five T-cell epitopes (BP26-6, -8, -11, -12 and OMP31-23) common to both sheep species, five species-specific epitopes (BP26-10, -18, -21 and -22 and OMP31-12) and four animal-specific epitopes (BP26-15, -23, OMP31-6 and -21), which stimulated specific IFN-γ response in vaccinated sheep. Among those T-cell epitopes, reactivity to BP26-18 and -21 epitopes was significantly associated with MHC-I B allele (P=0.024). However, a specific T-cell response induced by the M5-90 vaccine was relatively week and did not sustain long enough, which might be suppressed by rapid activation of T-regulatory (Treg) cells following vaccination. These findings provide an insight in designing a safer and more effective vaccine for use in animals and in humans.

GB virus C and HIV-1 RNA load in single virus and co-infected West African individuals.

Background:Investigations on the impact of GB virus C (GBV-C) co-infection on HIV disease progression relied essentially on clinical follow-up but not on virologic parameters. Objectives:To detect and quantify GBV-C RNA in West African populations co-infected or not with HIV-1 and to correlate the RNA load of HIV-1 and GBV-C in co-replicating patients with different clinical conditions. Methods:Three Ghanaian populations (blood donors, pregnant women and HIV-infected patients) were subdivided into six groups according to HIV-1 and clinical status and GBV-C and HIV-1 RNA load was tested by quantitative real time reverse transcriptase-polymerase chain reaction. In one population with HIV-1 disease, CD4+ cell count was also measured. Results:Prevalence of GBV-C markers in HIV-1-infected groups and HIV-1 non-infected pregnant women were significantly higher than in healthy blood donors. Similar levels and distribution of GBV-C RNA load were found in each population irrespective of HIV-1 status except for a lower GBV-C RNA load in AIDS patients. There was a significant shift of HIV-1 load towards lower value when GBV-C RNA was present and a trend towards an inverse correlation between HIV-1 and GBV-C RNA load. A positive correlation between CD4+ cell count and GBV-C RNA load in symptomatic HIV-1-infected patients was observed. Conclusions:The moderate impact of GBV-C on HIV-1 viremia is unlikely to entirely account for a favourable clinical outcome of replicating co-infections.

Infection of common marmosets with hepatitis C virus/GB virus‐B chimeras

The development of vaccination and novel therapy for hepatitis C virus (HCV) has been hampered by the lack of suitable small‐animal models. GB virus B (GBV‐B), closely related to HCV, causes viral hepatitis in common marmosets (Callithrix jacchue jacchus) and might represent an attractive surrogate model for HCV infection. However, differences exist between GBV‐B and HCV in spite of a short genetic distance between the two viruses. Here we report common marmosets infected with two HCV/GBV‐B chimeras containing HCV structural genes coding for either whole core and envelope proteins (CE1E2p7) or full envelope proteins (E1E2p7) substituted for the counterpart elements of GBV‐B. Naïve animals intrahepatically injected with chimeric RNA transcripts or intravenously injected with sera from primary infected animals produced high levels of circulating infectious chimeric viruses and they developed chronic infection. Tacrolimus‐treated marmosets inoculated with a CE1E2p7 chimera had higher viral loads and long‐term persistent infection. A moderate elevation of serum aspartate aminotransferase (AST) levels was observed in parallel with viral replication. Chimeras recovered from liver samples revealed 1/958 adaptive viral mutations. Histopathological changes typical of viral hepatitis were observed in liver tissues from all types of HCV chimeras‐infected marmosets. HCV core and E2 proteins were detected in liver tissues from infected animals by immunohistochemical staining. Fluctuations of chimeric virus replication in marmosets with spontaneous and sporadic viral clearance might be related to specific antibody and T‐cell response to HCV proteins in vivo. Replication of CE1E2p7 chimera was observed in primary hepatocyte cultures by immunofluorescent staining in vitro. Conclusion: Infectious HCV chimeras causing chronic hepatitis in marmosets might constitute a small primate model suitable for evaluation of virus‐cell interaction, vaccination, and antiviral therapy against HCV infection. (Hepatology 2014;59:789–802)

High prevalence of anti-hepatitis B core antigen in hepatitis B virus-vaccinated Chinese blood donors suggests insufficient protection but little threat to the blood supply

In East Asia, individuals systematically vaccinated at birth to hepatitis B virus (HBV) are an increasing part of the blood donor population. Their environment presents a high risk of contact with HBV. HBV vaccine efficacy and potential safety risk carried by vaccinated donors were examined.

Seroprevalence of west nile virus in ghana.

The epidemiology of West Nile virus (WNV) in Ghana, sub-Saharan Africa, and its relevance to transfusion were newly assessed. A total of 1324 plasma samples from five Ghanaian populations, including 529 children (<6 y old, pre-transfusion) and 795 adults (236 blood donors, 226 HIV-infected or non-infected pregnant women, 203 HIV symptomatic patients, and 130 AIDS patients) were screened for WNV RNA. No WNV RNA was detected, but 4.8% (13/271) and 27.9% (127/455) carried specific IgG in children and adults, respectively, and 2.4% (4/167) of the children had IgM. The prevalence of IgG antibody to WNV increased progressively and peaked around 30% between ages 1 and 30 y, then stabilized. The absence of viremia in four WNV IgM-positive children, and of reactivation in HIV-infected patients suggests that once host immunity is established, it appears to be robust. In addition, there were no clinical reports of WNV infection in the hospital in Kumasi, Ghana, suggesting that WNV epidemiology in Ghana differs from that seen in the U.S. Most infections occur early in life, and as the window for infection is quite short, the risk of transmission by transfusion appears to be low, and the risk of pathogenicity in immunocompetent recipients appears to be limited in an endemic area such as Ghana.