[show abstract][hide abstract] ABSTRACT: Electron microscopy (EM) should be used in the front line for detection of agents in emergencies and bioterrorism, on accounts of its speed and accuracy. However, the number of EM diagnostic laboratories has decreased considerably and an increasing number of people encounter difficulties with EM results. Therefore, the research on viral structure and morphologyant in EM diagnostic practice. EM has several technological advantages, and should be a fundamental tool in clinical diagnosis of viruses, particularly when agents are unknown or unsuspected. In this article, we review the historical contribution of EM to virology, and its use in virus differentiation, localization of specific virus antigens, virus-cell interaction, and viral morphogenesis. It is essential that EM investigations are based on clinical and comprehensive pathogenesis data from light or confocal microscopy. Furthermore, avoidance of artifacts or false results is necessary to exploit fully the advantages while minimizing its limitations.
Science China. Life sciences 05/2013; 56(5):421-30. · 2.02 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 01/2012; 7(6):e39644. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human astroviruses (HAstVs) have been recognized as one of the major causes of acute gastroenteritis in children. To provide more insight into the prevalence of HAstV gastroenteritis in China, 664 fecal samples were collected from children affected with acute gastroenteritis in Beijing from March 2005 to November 2007. The samples were analyzed genetically. All eight serotypes (genotypes) of HAstVs were screened using RT-PCR assays targeting the ORF2 region in the study. The assays detected HAstVs in 52 (7.8%) of the patients, with HAstV-1 (50/52) being the dominant genotype during the study period. Two minor genotypes, HAstV-6 and HAstV-3, were also detected. Partial sequencing of the 50 HAstV-1 strains showed that the homology of the nucleotide sequence of the ORF1a region between these strains was 88.4-100%, whereas the homology of the amino acid sequences was 95.6-100%. In the ORF2 partial region, the nucleotide identities ranged from 91.5% to 100%, and amino acid identities ranged from 97.3% to 100%. The identity of the whole genome sequence between four randomly examined HAstV-1 strains was 91-99%. No recombination events were observed in HAstVs in this study. The findings of this study will provide baseline data for HAstVs surveillance and control. J. Med. Virol. 82:415-423, 2010. (c) 2010 Wiley-Liss, Inc.
Journal of Medical Virology 03/2010; 82(3):415-23. · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: To study the precise role of the neuraminidase (NA), and its stalk region in particular, in the assembly, release, and entry of influenza virus, we deleted the 20-aa stalk segment from 2009 pandemic H1N1 NA (09N1) and inserted this segment, now designated 09s60, into the stalk region of a highly pathogenic avian influenza (HPAI) virus H5N1 NA (AH N1). The biological characterization of these wild-type and mutant NAs was analyzed by pseudotyped particles (pseudoparticles) system. Compared with the wild-type AH N1, the wild-type 09N1 exhibited higher NA activity and released more pseudoparticles. Deletion/insertion of the 09s60 segment did not alter this relationship. The infectivity of pseudoparticles harboring NA in combination with the hemagglutinin from HPAI H5N1 (AH H5) was decreased by insertion of 09s60 into AH N1 and was increased by deletion of 09s60 from 09N1. When isolated from the wild-type 2009H1N1 virus, 09N1 existed in the forms (in order of abundance) dimer>tetramer>monomer, but when isolated from pseudoparticles, 09N1 existed in the forms dimer>monomer>tetramer. After deletion of 09s60, 09N1 existed in the forms monomer>dimer. AH N1 from pseudoparticles existed in the forms monomer>dimer, but after insertion of 09s60, it existed in the forms dimer>monomer. Deletion/insertion of 09s60 did not alter the NA glycosylation pattern of 09N1 or AH N1. The 09N1 was more sensitive than the AH N1 to the NA inhibitor oseltamivir, suggesting that the infectivity-enhancing effect of oseltamivir correlates with robust NA activity.
PLoS ONE 01/2010; 5(12):e15825. · 3.73 Impact Factor
[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: The fastidious property of human adenovirus type 41 (Ad41) may be resulted from inadequate expression of protein V (pV), the minor core protein of adenovirus, in packaging cells. In this report, we prepared antiserum to pV of Ad41 and for study the mechanism of Ad41 fastidiousness.
Coding sequence of pV was amplified by PCR with the genome DNA of wild Ad41 (NIVD103) as template, and cloned into pET30a(+) vector to generate a recombinant plasmid called pET-pV. His-tag-fused pV was expressed in pET-pV-transformed E. Coli strain BL21(DE3) by adding the inducer of Isopropy beta-D-1-Thiogalactopyranoside (IPTG) and purified with the method of immobilized metal ion affinity chromatography (IMAC). Antiserums to pV were collected from pV inclusion bodies-immunized mice and evaluated by Western blot.
The sequencing assay showed that the cloned pV gene was highly homologous with that of Ad41 Tak strain, and there were only three residues changed in the corresponding amino-acid sequence. pV was expressed as inclusion bodies or in soluble form in BL21(DE3) cells under inducing condition of 1 mmol/L IPTG, 37 degrees C, 4 h or 0.5 mmol/L IPTG, 25 degrees C, 8 h, respectively. Antiserums to pV from most immunized mice were highly effective for Western blot assay. After infected with equivalent Ad41, 293E12, an Ad41 E1B55K-transfected 293 cell line, expressed more pV than 293 cells.
We successfully prepared antiserums to Ad41 pV and it could be used in Western blot assay to study the fastidious property of Ad41.
[show abstract][hide abstract] ABSTRACT: The baculovirus expression system was employed to prepare the virus-like particles (VLPs) of human parvovirus B19. The synthesized VP2 gene of B19 was inserted into the multi-cloning site (MCS) of pFastBac1 vector; the resulting plasmid was transferred to the Escherichia coli DH10Bac competent cells, which contain a baculovirus shuttle vector (Bacmid), to generate Bacmid-VP2 by site-specific transposition. Recombinant baculovirus carrying VP2 gene (rBac-VP2) was then rescued from Bacmid-VP2-transfected Sf9 cells. Indirect immunofluorescence and Western blotting were used to identify the VP2 protein in rBac-VP2-infected Sf9 cells, and the VLPs were observed under transmission electron microscope after being enriched by ultracentrifugation. The B19 VLPs were successfully produced in insect cells with baculovirus expression system, which will facilitate the development of diagnostic reagents to detect the antibody against B19 virus in human serum.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 05/2009; 25(4):575-9.
[show abstract][hide abstract] ABSTRACT: Human respiratory syncytial virus (RSV) is a serious pediatric pathogen of the lower respiratory tract worldwide. There is currently no clinically approved vaccine against RSV infection. Recently, it has been shown that a replication-deficient first generation adenoviral vector (FGAd), which encodes modified RSV attachment glycoprotein (G), elicits long-term protective immunity against RSV infection in mice. The major problem in developing such a vaccine is that G protein lacks MHC-I-restricted epitopes. However, RSV fusion glycoprotein (F) is a major cytotoxic T-lymphocyte epitope in humans and mice, therefore, an FGAd-encoding F (FGAd-F) was constructed and evaluated for its potential as an RSV vaccine in a murine model. Intranasal (i.n.) immunization with FGAd-F generated serum IgG, bronchoalveolar lavage secretory IgA, and RSV-specific CD8+ T-cell responses in BALB/c mice, with characteristic balanced or mixed Th1/Th2 CD4+ T-cell responses. Serum IgG was significantly elevated after boosting with i.n. FGAd-F. Upon challenge, i.n. immunization with FGAd-F displayed an effective protective role against RSV infection. These results demonstrate FGAd-F is able to induce effective protective immunity and is a promising vaccine regimen against RSV infection.
Biochemical and Biophysical Research Communications 03/2009; 381(4):528-32. · 2.41 Impact Factor
[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. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Background
Noroviruses (NoVs) are a major cause of acute gastroenteritis in children, but prevalence and circulation of NoVs in China have not been well characterized.
Journal of Clinical Virology - J CLIN VIROL. 01/2009; 44(1):94-98.
[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.