Boping Zhou

Serology of Severe Acute Respiratory Syndrome: Implications for Surveillance and Outcome

Abstract Background. Severe acute respiratory syndrome (SARS) is a novel infectious disease. No information is currently available on host-specific immunity against the SARS coronavirus (CoV), and detailed characteristics of the epidemiology of SARS CoV infection have not been identified. Methods. ELISA was used to detect antibody to SARS CoV. Reverse-transcriptase polymerase chain reaction was used to detect SARS CoV RNA. T cells in peripheral blood of patients were quantified by flow cytometry. Results. Of 36 patients with probable SARS CoV infection, 30 (83.3%) were positive for IgG antibody to SARS CoV; in contrast, only 3 of 48 patients with suspected SARS CoV infection, 0 of 112 patients with fever but without SARS, and 0 of 96 healthy control individuals were positive for it. IgG antibody to SARS CoV was first detected between day 5 and day 47 after onset of illness (mean ± SD, 18.7±10.4). Conclusion. Detection of antibody to SARS CoV is useful in the diagnosis of SARS; however, at the incubation and initial phases of the illness, serological assay is of little value, because of late seroconversion in most patients.

Elevated IL-6 Receptor Expression on CD4+ T Cells contributes to the increased Th17 Responses in patients with Chronic Hepatitis B

BackgroundIncreased numbers of Interleukin-17-producing CD4+ T cells (Th17) have been found in association with hepatitis B virus (HBV)-induced liver injury. However, the mechanism underlying the increase of Th17 responses in patients with HBV infection remains unclear. In this study, we investigate the possible regulatory mechanisms of increased Th17 responses in patients with chronic hepatitis B(CHB).MethodsTh17 response and IL-6R expression on CD4+ T cells in peripheral blood samples were determined by flow cytometry. Cytokines TGF-β, IL-1β, IL-6 and IL-17 in plasma and/or supernatant samples were determined by ELISA and the IL-17 and IL-6R mRNA levels were quantified by quantitative real-time reverse polymerase chain reaction.ResultsAll these data indicated that the frequency of periphery Th17 cells is significantly correlated with the percentage of CD4+ T cells expressing IL-6R in CHB patients. CD4+ T cells from patients with CHB, but not those from healthy donors, produced higher levels of IL-17 and had more IL-6R expression upon stimulation with the HBV core antigen (HBcAg) in vitro. The PMA/ionomycin and HBcAg -stimulated up-regulation of IL-17 production by CD4+ T cells could be reversed by a neutralizing antibody against IL-6R.Conclusionwe showed that enhancement of IL-6R expression on CD4+ T cells upon HBV infection contributes to increased Th17 response in patients with CHB.

The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely.

HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing tool in vitro. In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus.

Roles of circulating soluble interleukin (IL)-6 receptor and IL-6 receptor expression on CD4+ T cells in patients with chronic hepatitis B.

OBJECTIVES The objective of this study was to investigate the potential clinical roles of circulating soluble interleukin (IL)-6 receptor (sIL-6R) and IL-6R expression on CD4+ T cells (CD4+ IL-6R+ T cells) in chronic hepatitis B (CHB) patients. METHODS One hundred and thirty-three subjects, including 72 CHB patients, 27 asymptomatic carriers, eight acute hepatitis B (AHB) patients, and 26 healthy donors were included in this study. Plasma IL-6 and sIL-6R levels were measured by enzyme-linked immunosorbent assay (ELISA); the frequency of CD4+ IL-6R+ T cells was detected by flow cytometry analysis. RESULTS Our data showed a significant increase in plasma sIL-6R levels and the frequency of CD4+ IL-6R+ T cells in peripheral blood in CHB patients compared to asymptomatic carriers and healthy controls (both p<0.05). The elevated prevalence of CD4+ IL-6R+ T cells was positively associated with increased serum alanine aminotransferase levels in CHB patients (r = 0.316, p = 0.007), but was not correlated with serum hepatitis B virus (HBV) DNA load. Moreover, CHB patients with an HBV DNA load >1.0 × 10(6) copies/ml had a lower level of plasma sIL-6R than those with an HBV DNA load <1.0 × 10(6) copies/ml. CONCLUSIONS Circulating sIL-6R and CD4+ IL-6R+ T cells were increased in CHB patients. Elevated plasma sIL-6R is probably associated with HBV elimination, and CD4+ IL-6R+ T cells in peripheral blood might contribute to the pathogenesis of liver injury in CHB patients.

Genetic variation in interleukin 28B and response to antiviral therapy in patients with dual chronic infection with hepatitis B and C viruses.

Concurrent infection with hepatitis C virus (HCV) and hepatitis B virus (HBV) was not uncommon in China. To date, information on predictors of response to treatment of dually-infected HCV/HBV is limited. The aim of this study was to evaluated whether determination of the interleukin 28B (IL-28B) polymorphism statuses sufficient to predict treatment response of interferon (IFN)-based therapy in patients chronically infected with both hepatitis B and C viruses. We investigated the role of IL28B variations (rs8099917 and rs12979860) in response to IFN-based treatment and evaluated its association with the risk of the null virological response (NVR) in HCV /HBV dually-infected patients. We found that the overall distributions of the genotypes among the sustained virological response (SVR), NVR groups were significantly different (P<0.001): patients with the rs8099917 TG genotype had an increased risk of NVR (odds ratio [OR] =2.37 95% confidence interval [CI] =1.16–4.83, P =0.017), and those with the GG genotype had a further increased risk of NVR (OR=4.23, 95% CI =1.17-15.3, P=0.027). The rs12979860 allele was also highly associated with treatment failure (CT/TT vs. CC; OR =2.04, 95%CI =1.05-3.97, P =0.037). Moreover, we found that IL28B rs8099917 G variants (TG+GG) interact with HCV genotype 1(G1) to result in higher risk of NVR (P=0.009), and that they are also associated with HBV DNA reactivation (TG+GG vs. TT, P=0.005). Furthermore, multivariate regression analysis show that the rs8099917 G allele was the most important factor significantly associated with a NVR in HCV G1 patients. This study suggest that IL28B genotyping may be a valid pretreatment predictor of which patients are likely to respond to treatment in this group of difficult-to-treat HCV/HBV dually-infected patients.

Combined acoustic radiation force impulse, aminotransferase to platelet ratio index and Forns index assessment for hepatic fibrosis grading in hepatitis B

AIM To investigate the combined diagnostic accuracy of acoustic radiation force impulse (ARFI), aspartate aminotransferase to platelet ratio index (APRI) and Forns index for a non-invasive assessment of liver fibrosis in patients with chronic hepatitis B (CHB). METHODS In this prospective study, 206 patients had CHB with liver fibrosis stages F0-F4 classified by METAVIR and 40 were healthy volunteers were measured by ARFI, APRI and Forns index separately or combined as indicated. RESULTS ARFI, APRI or Forns index demonstrated a significant correlation with the histological stage (all P < 0.001). According to the AUROC of ARFI and APRI for evaluating fibrotic stages more than F2, ARFI showed an enhanced diagnostic accuracy than APRI (P < 0.05). The combined measurement of ARFI and APRI exhibited better accuracy than ARFI alone when evaluating ≥ F2 fibrotic stage (Z = 2.77, P = 0.006). Combination of ARFI, APRI and Forns index did not obviously improve the diagnostic accuracy compared to the combination of ARFI and APRI (Z = 0.958, P = 0.338). CONCLUSION ARFI + APRI showed enhanced diagnostic accuracy than ARFI or APRI alone for significant liver fibrosis and ARFI + APRI + Forns index shows the same effect with ARFI + APRI.

Clinical Characteristics and Treatment Experiences in 2 Serious Cases ofInfluenza A (H5N6) Virus Infection

Since April 2014, a fatal human infection with a novel reassortant H5N6 avian influenza virus was identified in China. Eight of 10 patients died from this virus. Two H5N6 patients were hospitalized in Shenzhen Third People’s Hospital in China, with one patient recovered. Here, we describe their clinical characteristics and treatment strategies. We suggest these strategies in the early stage may be important for severe avian influenza patients, including early combined antiviral therapy, oxygen therapy and ventilator support treatment, short-term usage of low-dose corticosteroid and appropriate immune globulin and keeping negative fluid balance in the progression period.

Investigation on Factors Associated with Severe Hand, Foot and Mouth Disease

Abstract Objective To analyze the clinical and laboratory features of patients with mild and severe HFMD to identify early predictive or diagnostic markers for severe cases. Methods Samples of feces, nasopharyngeal-swab specimens, peripheral blood, serum and cerebral spinal fluid were collected. Postmortem pathological examination was conducted on 2 dead patients with complication due to neurogenic pulmonary edema. Reverse transcription-polymerase chain-reaction (RT-PCR), culture and isolation of enterovirus 71 (EV71) were performed to detect EV71 infection. Both univariate and multivariate logistic analysis were used to identify factors associated with severe cases. Results EV71 was mainly responsible for HFMD. In this study, 5 isolated EV71 strains belonged to C4 gene subtype. Compared with mild patients, EV71-RNA detection rate was higher and CoxA16 detection rate was lower among severe patients (P < 0.01). Inflammatory cell infiltration in the lung, cardiac and liver tissues were mild by postmortem pathological examination. It was found that body temperature, vomitting, limb tremor, neutrophil, blood glucose and EV71 infection were significantly related to the severe cases by univariate logistic analysis. However, after multivariate logistic regression analysis, only vomiting (OR 16.1, CI 2.3-110.5, P < 0.01) and limb tremor (OR 117.6, CI 13.8-1004.5, P < 0.01) were significantly and independently correlated with the severe cases. Conclusions EV71 was mainly responsible for HFMD, particularly for severe cases. Vomiting and limb tremor were predictive markers for severe cases.

Original Article. Dynamic Changes of Multiple Immune Cells in Chronic Hepatitis B Patients Undergoing Antiviral Treatment

Abstract Objective Various immune cells in patients with CHB have been demonstrated to play critical roles in HBV infection. The goal of this study is to observe changes in Th17, Treg, Th1 and B lymphocytes from peripheral blood and to evaluate immune status of CHB patients undergoing antiviral treatment. Methods Total of 49 CHB patients, 19 asymptomatic carriers and 29 healthy donors were included in our present study. The frequencies of peripheral Th17 cells (CD3+CD4+IL-17+Tcells), Treg cells (CD3+CD4+CD25+CD127- T cells), Th1 cells (CD3+CD4+IFN-γ T cells) and B lymphocytes in chronic hepatitis B (CHB) were analyzed by flow cytometry. Results The frequency of Th17 cells increased after treatment for 6 months, but there was no statistically significant difference of IL-17 expression between baseline and 6 months after treatment. The frequencies of Treg cells, momory B cells and total CD19+ B cells decreased after antiviral treatment. The frequencies of Th1 cells and plasma cells increased after antiviral treatment. Conclusions This study highlights that the reestablishment of immune function during antiviral treatment in CHB patients, which caused by the antiviral drugs or the patients themselves. CHB patients may exhibit varied responses to these antiviral drugs. It is essential to supplement immune therapy during the antiviral treatment, but Th17 may play a limited role in inflammation during antiviral treatment, targeting Th17 therapy may not be useful for CHB treatment. More time and more experiments are critical to explain it.

Reply to Tso et al

1. Chen X, Zhou B, Li M, et al. Serology of severe acute respiratory syndrome: implications for surveillance and outcome. J Infect Dis 2004; 189:1158–63. 2. Chen W, Xu Z, Mu J, et al. Antibody response and viraemia during the course of severe acute respiratory syndrome (SARS)–associated coronavirus infection. J Med Microbiol 2004; 53: 435–8. 3. Tso EYK, Tsang OTY, Choi KW, et al. Persistence of physical symptoms in and abnormal laboratory findings for survivors of severe acute respiratory syndrome [letter]. Clin Infect Dis 2004; 38:1338. 4. Lee HK, Tso EY, Chau TN, Tsang OT, Choi KW, Lai TS. Asymptomatic severe acute respiratory syndrome–associated coronavirus infection. Emerg Infect Dis 2003; 9:1491–2. 5. Wesley R. Neutralizing antibody decay and lack of contact transmission after inoculation of 3and 4-day-old piglets with porcine respiratory coronavirus. J Vet Diagn Invest 2002; 14:525–7. 6. Sui J, Li W, Murakami A, et al. Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association. Proc Natl Acad Sci USA 2004; 101: 2536–41. 7. Wong VW, Dai D, Wu AK, Sung JJ. Treatment of severe acute respiratory syndrome with convalescent plasma. Hong Kong Med J 2003; 9: 199–201.