Jingdong Song

Genetic analysis of norovirus in children affected with acute gastroenteritis in Beijing, 2004–2007

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. OBJECTIVES To determine the dominant circulating NoV genotypes and strains associated with pediatric cases of acute gastroenteritis in Beijing, China. STUDY DESIGN 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. RESULTS 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. CONCLUSIONS 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.

Intranasal immunization with a replication-deficient adenoviral vector expressing the fusion glycoprotein of respiratory syncytial virus elicits protective immunity in BALB/c mice

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.

Differential Seroprevalence of Human Bocavirus Species 1-4 in Beijing, China

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

Molecular characterization of astrovirus infection in children with diarrhea in Beijing, 2005-2007.

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.

Electron microscopy: essentials for viral structure, morphogenesis and rapid diagnosis

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.

Novel recombinant adenovirus type 41 vector and its biological properties

Human adenovirus serotype 41 (Ad41) is a natural pathogen of the digestive tract and can cause gastroenteritis. There has been interest in reconstructing Ad41 as a gene delivery vector targeting the gastrointestinal tract, which is hampered by its fastidiousness.

A recombinant adenovirus prime-virus-like particle boost regimen elicits effective and specific immunities against norovirus in mice.

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.

A prime-boost vaccination strategy using attenuated Salmonella typhimurium and a replication-deficient recombinant adenovirus vector elicits protective immunity against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV), for which no clinically approved vaccine is available yet, is globally a serious pediatric pathogen of the lower respiratory tract. Several approaches have been used to develop vaccines against RSV, but none of these have been approved for use in humans. An efficient vaccine-enhancing strategy for RSV is still urgently needed. We found previously that oral SL7207/pcDNA3.1/F and intranasal FGAd/F were able to induce an effective protective immune response against RSV. The heterologous prime-boost immunization regime has been reported recently to be an efficient vaccine-enhancing strategy. Therefore, we investigated the ability of an oral SL7207/pcDNA3.1/F prime and intranasal (i.n.) FGAd/F boost regimen to generate immune responses to RSV. The SL7207/pcDNA3.1/F prime-FGAd/F boost regimen generated stronger RSV-specific humoral and mucosal immune responses in BALB/c mice than the oral SL7207/pcDNA3.1/F regimen alone, and stronger specific cellular immune responses than the i.n. FGAd/F regimen alone. Histopathological analysis showed an increased efficacy against RSV challenge by the heterologous prime-boost regimen. These results suggest that such a heterologous prime-boost strategy can enhance the efficacy of either the SL7207 or the FGAd vector regimen in generating immune responses in BALB/c mice.

Isolation and identification of a natural reassortant mammalian orthoreovirus from least horseshoe bat in China.

Background Mammalian orthoreoviruses (MRVs) have a wide geographic distribution and can infect virtually all mammals. Infections in humans may be either symptomatic or asymptomatic. This study describes the isolation and identification of a natural reassortant MRV from least horseshoe bats (Rhinolophus pusillu) in China, referred to as RpMRV-YN2012. Methods and Results The RpMRV-YN2012 was obtained from urine samples of Rhinolophus pusillus by cell culture. Negative-staining electron microscopy revealed that RpMRV-YN2012 was a non-enveloped icosahedral virus with ∼75 nm in diameter. Polyacrylamide gel electrophoresis (PAGE) migration patterns of the genome segments showed that RpMRV-YN2012 contained 10 segments in a 3:3:4 arrangement. The whole genome sequence of RpMRV2012 was determined. The consensus terminal sequences of all segments of 5’-GCUAh…yUCAUC-3’ (h = A, U or C; y = C or U) were similar to the MRV species within the genus Orthoreovirus. Its evolution and evidence of genetic reassortment were analyzed by sequence comparison and phylogenetic analysis. The results showed that RpMRV-YN2012 is a novel serotype 2 MRV that may have originated from reassortment among bat, human, and/or pig MRV strains which associated with diarrhea, acute gastroenteritis and necrotizing encephalopathy in animals and humans. Conclusions RpMRV-YN2012 is a novel bat reassortant MRV, which may have resulted from a reassortment involving MRVs known to infect humans and animals. It is necessary to identify whether RpMRV-YN2012 is associated with diarrhea, acute gastroenteritis and necrotizing encephalopathy in clinical patients. In addition, we should carefully monitor its evolution and virulence in real time.

Human adenovirus type 41 possesses different amount of short and long fibers in the virion

To determine the ratio of short fiber (sfiber) to long fiber (lfiber) in human adenovirus type 41 (HAdV-41) virions, sfiber and lfiber were expressed in E. coli, quantified, and used as loading standards in Western blot. Densitometric analyses of the standard and target bands indicated that the ratio of sfiber to lfiber in virions was 5.7±0.7. Sfiber-deleted HAdV-41, HAdV-41-DSF-GFP, was constructed, and Western blot analysis showed that the amount of lfiber in HAdV-41-DSF-GFP was about 7.3±1.9 times of that in HAdV-41 virions, confirming a ratio of approximate 6 for sfiber to lfiber in HAdV-41. In HAdV-41-infected cells, mRNAs of the sfiber and lfiber were comparable in quantity, while the expression at protein level was significantly different. Our results suggested an unequal number of short and long fibers, which might result from their differential protein expression during HAdV-41 packaging. The method used here could be extended to quantify other trace proteins.