[Show abstract][Hide abstract] ABSTRACT: The recently identified human infections with a novel avian influenza H7N9 virus in China raise important questions regarding possible risk to humans. However, the entry properties and tropism of this H7N9 virus were poorly understood. Moreover, neuraminidase inhibitor resistant H7N9 isolates were recently observed in two patients and correlated with poor clinical outcomes. In this study, we aimed to elucidate the entry properties of H7N9 virus, design and evaluate inhibitors for H7N9 virus entry. We optimized and developed an H7N9-pseudotyped particle system (H7N9pp) that could be neutralized by anti-H7 antibodies and closely mimicked the entry process of the H7N9 virus. Avian, human and mouse-derived cultured cells showed high, moderate and low permissiveness to H7N9pp, respectively. Based on influenza virus membrane fusion mechanisms, a potent anti-H7N9 peptide (P155-185-chol) corresponding to the C-terminal ectodomain of the H7N9 hemagglutinin protein was successfully identified. P155-185-chol demonstrated H7N9pp-specific inhibition of infection with IC50 of 0.19 µM. Importantly, P155-185-chol showed significant suppression of A/Anhui/1/2013 H7N9 live virus propagation in MDCK cells and additive effects with NA inhibitors Oseltamivir and Zanamivir. These findings expand our knowledge of the entry properties of the novel H7N9 viruses, and they highlight the potential for developing a new class of inhibitors targeting viral entry for use in the next pandemic.
PLoS ONE 09/2014; 9(9):e107235. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human enterovirus (EV) 68 is a member of EV-D species which belongs to the EV genus of the Picornaviridae family. Over the past several years, there have been increasingly documented outbreaks in respiratory disease associated with EV68. As a globally emerging pathogen, EV68 infects both adults and children. However, the molecular basis of EV68 pathogenesis is unknown. Here, we report that EV68 inhibits Toll-like receptor 3 (TLR3)-mediated innate immune response by targeting TIR domain-containing adaptor inducing beta interferon (TRIF). In infected HeLa cells, EV68 inhibits poly(I:C)-induced interferon regulatory factor 3 (IRF3) activation and the interferon-β (IFN-β) expression. Further investigations reveal that TRIF, a critical adaptor downstream of TLR3, is targeted by EV68. When expressed alone, 3C(pro), an EV68-encoded protease, cleaves TRIF. 3C(pro) mediates TRIF cleavage at Q312 and Q653, two sites in the amino- and carboxyl-terminal domains, respectively. This cleavage relies on its cysteine protease activity. Cleavage of TRIF abolishes the capacity of TRIF to activate NF-κB and IFN-β signaling. These results suggest that control of TRIF by 3C(pro) may be a mechanism by which EV68 subverts host innate immune response.
EV 68 is a globally emerging pathogen, but the molecular basis of EV68 pathogenesis is unclear. Here, we report that EV68 inhibits TLR3-mediated innate immune response by targeting TRIF. Further investigations reveal that TRIF is cleaved by 3C(pro). These results suggest that control of TRIF by 3C(pro) may be a mechanism by which EV68 impairs type I IFN production in response to TLR3 activation.
[Show abstract][Hide abstract] ABSTRACT: Understanding host antibody response is crucial for predicting disease severity and for vaccine development. We investigated antibody responses against influenza A(H7N9) virus in 48 serum samples from 21 patients, including paired samples from 15 patients. IgG against subtype H7 and neutralizing antibodies (NAbs) were not detected in acute-phase samples, but ELISA geometric mean titers increased in convalescent-phase samples; NAb titers were 20-80 (geometric mean titer 40). Avidity to IgG against subtype H7 was significantly lower than that against H1 and H3. IgG against H3 was boosted after infection with influenza A(H7N9) virus, and its level in acute-phase samples correlated with that against H7 in convalescent-phase samples. A correlation was also found between hemagglutinin inhibition and NAb titers and between hemagglutinin inhibition and IgG titers against H7. Because of the relatively weak protective antibody response to influenza A(H7N9), multiple vaccinations might be needed to achieve protective immunity.
[Show abstract][Hide abstract] ABSTRACT: Human bocavirus species 1-4 (HBoV1-4) have been associated with respiratory and enteric infections in children. However, the immunological mechanisms in response to HBoV infections are not fully understood. Though previous studies have shown cross-reactivities between HBoV species, the epitopes responsible for this phenomenon remain unknown. In this study, we used genomic and immunologic approaches to identify the reactive epitopes conserved across multiple HBoV species and explored their potential as the basis of a novel diagnostic test for HBoVs.
We generated HBoV1-3 VP2 gene fragment phage display libraries (GFPDLs) and used these libraries to analyze mouse antisera against VP2 protein of HBoV1, 2, and 3, and human sera positive for HBoVs. Using this approach, we mapped four epitope clusters of HBoVs and identified two immunodominant peptides-P1 ((1)MSDTDIQDQQPDTVDAPQNT(20)), and P2 ((162)EHAYPNASHPWDEDVMPDL(180))-that are conserved among HBoV1-4. To confirm epitope immunogenicity, we immunized mice with the immunodominant P1 and P2 peptides identified in our screen and found that they elicited high titer antibodies in mice. These two antibodies could only recognize the VP2 of HBoV 1-4 in Western blot assays, rather than those of the two other parvoviruses human parvovirus B19 and human parvovirus 4 (PARV4). Based on our findings, we evaluated epitope-based peptide-IgM ELISAs as potential diagnostic tools for HBoVs IgM antibodies. We found that the P1+P2-IgM ELISA showed a higher sensitivity and specificity in HBoVs IgM detection than the assays using a single peptide.
The identification of the conserved B-cell epitopes among human bocavirus species contributes to our understanding of immunological cross-reactivities of HBoVs, and provides important insights for the development of HBoV diagnostic tools.
PLoS ONE 01/2014; 9(1):e86960. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Human adenoviruses (HAdVs) have been implicated as important agents in a wide range of human illnesses. To date, 58 distinct HAdV serotypes have been identified and can be grouped into six species. For the immunological diagnosis of adenoviruses, the hexon protein, a structural protein, has been used. The potential of other HAdV proteins has not been fully addressed. METHODOLOGYPRINCIPAL FINDINGS: In this study, a nonstructural antigenic protein, the DNA binding protein (DBP) of human adenovirus 5 and 35 (Ad5, Ad35) - was identified using immunoproteomic technology. The expression of Ad5 and Ad35 DBP in insect cells could be detected by rhesus monkey serum antibodies and healthy adult human serum positive for Ad5 and Ad35. Recombinant DBPs elicited high titer antibodies in mice. Their conserved domain displayed immunological cross-reactions with heterologous DBP antibodies in Western blot assays. DBP-IgM ELISA showed higher sensitivity adenovirus IgM detection than the commercial Adenovirus IgM Human ELISA Kit. A Western blot method developed based on Ad5 DBP was highly consistent with (χ(2) = 44.9, P<0.01) the Western blot assay for the hexon protein in the detection of IgG, but proved even more sensitive. CONCLUSIONSSIGNIFICANCE: The HAdV nonstructural protein DBP is an antigenic protein that could serve as an alternative common antigen for adenovirus diagnosis.
PLoS ONE 03/2013; 8(3):e56708. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Four species of human bocaviruses (HBoV1-4) have been identified based on phylogenetic analysis since its first report in 2005. HBoV1 has been associated with respiratory disease, whereas HBoV2-4 are mainly detected in enteric infections. Although the prevalence of HBoVs in humans has been studied in some regions, it has not been well addressed globally.
Cross-reactivity of anti-VP2 antibodies was detected between HBoV1, 2, 3, and 4 in mouse and human serum. The prevalence of specific anti-VP2 IgG antibodies against HBoV1-4 was determined in different age groups of healthy individuals aged 0-70 years old in Beijing, China, using a competition ELISA assay based on virus-like particles of HBoV1-4. The seroprevalence of HBoV1-4 was 50%, 36.9%, 28.7%, and 0.8%, respectively, in children aged 0-14 years (n = 244); whereas the seroprevalence of HBoV1-4 was 66.9%, 49.3%, 38.7% and 1.4%, respectively, in healthy adults (≥ 15 years old; n = 142). The seropositive rate of HBoV1 was higher than that of HBoV2, HBoV3, and HBoV4 in individuals older than 0.5 years. Furthermore, IgG seroconversion of HBoV1 (10/31, 32.3%), HBoV2 (8/31, 25.8%), and HBoV3 (2/31, 6.5%) was found in paired sera collected from children with respiratory tract infections who were positive for HBoV1 according to PCR analysis.
Our data indicate that HBoV1 is more prevalent than HBoV2, HBoV3, and HBoV4 in the population we sampled in Beijing, China, suggesting that HBoV species may play differential roles in disease.
PLoS ONE 06/2012; 7(6):e39644. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the prevalence and clinical manifestations of human metapneumovirus (hMPV) in immunocompetent Chinese adults with acute respiratory tract infections (ARTIs).
A reverse transcription PCR (RT-PCR) assay targeting the P gene was developed in this study and used to detect hMPV in nasal and throat swabs collected from 2936 immunocompetent adult patients with ARTIs in Beijing, China between July 2008 and June 2010.
Among the 2936 patients studied, 49 (1.7%) were positive for hMPV, of whom 14 (28.6%) were positive for hMPV_A2b, 19 (38.8%) for hMPV_B1, and 16 (32.6%) for hMPV_B2. hMPV_A1 was not detected. An average detection rate of 6.6% was observed in the peak months of the two epidemic seasons studied. The hMPV prevalence was higher in the sampled elderly (>65 years, 3.2%) than in middle aged adults (25-65 years; 2.0%) and teenagers (14-25 years; 0.9%). During the study period, hMPV infections showed a biennial rhythm of seasonality, peaking from November to March in 2008/09 and from March to June in 2010.
hMPV infection plays an important role in immunocompetent adults in its epidemic season. The demographic and clinical data presented in this study improves our understanding of the pathogenesis and clinical burden of hMPV infection in adults.
The Journal of infection 11/2011; 64(1):96-103. · 4.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To the Editor: Four species of human bocavirus (HBoV1-4) have been identified since 2005 (1-4). Several reports have documented that HBoV1 are prevalent in respiratory tract samples. Although there may be many asymptomatic carriers, HBoV1 has been shown to cause respiratory tract diseases (1,5,6). HBoV2 has mainly been detected in fecal samples and has been linked to gastroenteritis (3,7,8). HBoV3 and HBoV4 have recently also been detected in fecal samples (3,4), although no link to disease has been established for these 2 species.
[Show abstract][Hide abstract] ABSTRACT: Influenza viruses are known for their ability to change their antigenic structure and create new viral strains. Hemagglutinin (HA), for which 16 subtypes have been identified, is a major antigenic determinant essential for the pathogenesis of influenza A viruses. To predict and monitor future epidemics, it is critical to produce various HA subtype antigens conveniently and rapidly. A simple, effective, and economic method to generate subunit HA1 of all 16 HA subtypes as recombinant proteins (rHA1) is reported. rHA1 proteins are expressed in insect cells as secretory proteins after integration into a baculovirus expression vector containing a 6×His tag element and the signal peptide of the GP67 protein, a membrane glycoprotein identified in Autographa californica nuclear polyhedrosis virus. The proteins can be purified to ≥90% purity using a single Ni(2+)-chelating affinity chromatography step, yielding a recovery rate of about 50%. The rHA1 proteins elicit high titer antibodies in mice and show high specificity in Western blots. This study paves the way for subtype specific detection methods and for future studies of the immune relationships among the subtypes of influenza A virus HA proteins.
Journal of virological methods 07/2011; 177(2):160-7. · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human parainfluenza viruses (HPIVs) are a leading cause of acute respiratory tract infections (ARTIs). Although HPIV-4 has been associated with mild ARTIs for years, recent investigations have also associated HPIV-4 infection with severe respiratory syndromes and with outbreaks of ARTIs in children.
To characterize the role of HPIV-4 and its clinical features in children with acute lower respiratory tract infections (ALRTIs) in Beijing, China.
Nasopharyngeal aspirates were collected from 2009 hospitalized children with ALRTIs between March 2007 and April 2010. RT-PCR and PCR analyses were used to identify HPIV types and other known respiratory viruses.
HPIVs were detected in 246 (12.2%) patients, of whom 25 (10.2%) were positive for HPIV-4, 11 (4.5%) for HPIV-2, 51 (20.7%) for HPIV-1, 151 (61.4%) for HPIV-3, and 8 (3.3%) were co-detected with different types of HPIVs. Like HPIV-3, HPIV-4 was detected in spring, summer, and late fall over the study period. Seasonal incidence varied for HPIV-1 and -2. The median patient age was 20 months for HPIV-4 infections and 7-11 months for HPIV-1, -2, and -3 infections, but the clinical manifestations did not differ significantly between HPIV-1, -2, -3, and -4 infections. Moreover, co-detection of HPIV-4 (44%) with other respiratory viruses was lower than that of HPIV-1 (62.7%), HPIV-2 (63.6%), and HPIV-3 (72.7%).
HPIV-4 plays an important role in Chinese paediatric ALRTIs. The epidemiological and clinical characteristics reported here improve our understanding of the pathogenesis associated with HPIV-4.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 07/2011; 51(3):209-12. · 3.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human coronaviruses (HCoVs) are a common etiological agent of acute respiratory tract infections. HCoV infections, especially those caused by the two HCoVs identified most recently, NL63 and HKU-1, have not been characterized fully. To evaluate the prevalence and clinical presentations of HKU1 and NL63 in adults with acute respiratory tract infections, an investigation of HCoV infections in Beijing, China from 2005 to 2009 was performed by using reverse transcriptase PCR assays and sequencing analysis. Among 8,396 respiratory specimens studied, 87 (1%) clinical samples were positive for HCoVs, of which 50 samples (0.6% of the total) were positive for HCoV-OC43, 15 (0.2%) for HCoV-229E, 14 (0.2%) for HCoV-HKU1, and 8 (0.1%) for HCoV-NL63. The prevalence of HCoV infection in adults exhibited distinct seasonal fluctuations during the study period. In addition, patients positive for HCoV-229E infections were more likely to be co-infected with other respiratory viruses. Enterovirus, rhinovirus, and parainfluenza virus type 3 were the most common viruses found in patients with HCoV infections. The demographic and clinical data present in this study of HCoV infections in adults with acute respiratory tract infections should improve our understanding of the pathogenesis of HCoVs.
Journal of Medical Virology 02/2011; 83(2):291-7. · 2.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rotavirus (RV) is the main cause of severe gastroenteritis in children. An effective vaccination regime against RV can substantially reduce morbidity and mortality. Previous studies have demonstrated the efficacy of virus-like particles formed by RV VP2 and VP6 (VLP2/6), as well as that of recombinant adenovirus expressing RV VP6 (rAd), in eliciting protective immunities against RV. However, the efficacy of such prime-boost strategy, which incorporates VLP and rAd in inducing protective immunities against RV, has not been addressed. We assessed the immune effects of different regimens in mice, including rAd prime-VLP2/6 boost (rAd+VLP), VLP2/6 prime-rAd boost (VLP+rAd), rAd alone, and VLP alone.
Mice immunized with the VLP+rAd regimen elicit stronger humoral, mucosal, and cellular immune responses than those immunized with other regimens. RV challenging experiments showed that the highest reduction (92.9%) in viral shedding was achieved in the VLP+rAd group when compared with rAd+VLP (25%), VLP alone (75%), or rAd alone (40%) treatment groups. The reduction in RV shedding in mice correlated with fecal IgG (r = 0.95773, P = 0.04227) and IgA (r = 0.96137, P = 0.038663).
A VLP2/6 prime-rAd boost regimen is effective in conferring immunoprotection against RV challenge in mice. This finding may lay the groundwork for an alternative strategy in novel RV vaccine development.
[Show abstract][Hide abstract] ABSTRACT: Prime-boost vaccination using recombinant viral vectors and proteins has emerged as a highly effective strategy for protecting against viral pathogens. However, the ability of such regimens to provide immunity against norovirus (NV), an important cause of acute epidemic gastroenteritis worldwide, has never been assessed. In this study, we analyzed NV-specific humoral, mucosal, and cellular immune responses following intranasal immunization with the recombinant adenovirus expressing the NV GGII4 capsid protein (rAd) prime-NV virus-like particle (VLP) boost, VLP prime-rAd boost, or repeated NV VLP regimens. Our results show that mice primed with rAd and boosted with VLP had stronger humoral, mucosal, and interferon-gamma responses than those immunized with VLP prime-rAd boost or VLP alone. These results demonstrate that adenovirus prime-VLP boost vaccination is an effective strategy for induction of immune responses against NV and is a promising strategy to improve current VLP-based NV vaccine development.
[Show abstract][Hide abstract] ABSTRACT: Noroviruses (NoVs) are a major cause of acute gastroenteritis in children, but prevalence and circulation of NoVs in China have not been well characterized.
To determine the dominant circulating NoV genotypes and strains associated with pediatric cases of acute gastroenteritis in Beijing, China.
Fecal samples were obtained from 1126 children affected with acute gastroenteritis in Beijing from March 2004 to November 2007. NoV RNA was amplified, sequenced, and phylogenetically analyzed to determine the dominant circulating genotypes and strains.
NoVs were detected in 8.88% of patients, GII.4 being the dominant genotype. Ehime/05-30 was the dominant strain during 2004-2005, whereas 2006b dominated during 2006-2007. The homology of nucleotide and amino acid sequences among full-length VP1 of 15 randomly selected NoV strains was 91.6-99.6% and 94.5-99.6%, respectively. Recombination between NoV genotypes was frequent among the isolates.
The predominant circulating genotype of NoV infections in Beijing is GII.4, but the dominant strains of this virus responsible for gastroenteritis epidemics are evolving rapidly. A global surveillance network may be needed to identify trends in molecular evolution of NoVs for prevention of future epidemics.
Journal of Clinical Virology 01/2009; 44(1):94-8. · 3.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Norovirus (NV) is a major cause of acute, epidemic nonbacterial gastroenteritis in individuals of all ages. The immunological mechanism of NV infection and the approaches used to prevent infection remain to be elucidated. In this study, the specific immune responses of BALB/c mice were assessed following intranasal immunization with a recombinant adenovirus vector expressing the genogroup II4 (GGII/4) norovirus capsid protein. Analysis of IgM, IgG, and IgA antibodies specific for the recombinant virus-like particles (VLPs) of NV demonstrated that a high level of humoral immunity developed following immunization. Mucosal immune responses were also detectable in stool, intestinal homogenates, lung homogenates, and lung lavage samples. Specific cellular immune responses were observed in NV VLPs-restimulated splenocytes by ELISPOT and Th1/Th2 cytokine cytometric array (CBA). Serum IgG subclass analysis showed that a balanced Th1- and Th2-like cellular immune response was induced in BALB/c mice following immunization with recombinant adenovirus. These findings demonstrate that the intranasal immunization of a recombinant adenovirus expressing the NV capsid protein is an efficient strategy to stimulate systemic, mucosal, and cellular Th1/Th2 immune responses in mice, and could serve as a novel approach for designing NV vaccines.