Tao Hung

Inhibition of Severe Acute Respiratory Syndrome Virus Replication by Small Interfering RNAs in Mammalian Cells

ABSTRACT Severe acute respiratory syndrome (SARS) is an acute respiratory infectious disease that spread worldwide in early 2003. The cause was determined as a novel coronavirus (CoV), SARS-associated CoV (SARS-CoV), with a single-stranded, plus-sense RNA. To date, no effective specific treatment has been identified. To exploit the possibility of using RNA interference as a therapeutic approach to fight the disease, plasmid-mediated small interfering RNAs (siRNAs) were generated to target the SARS-CoV genome. The expression of siRNAs from two plasmids, which specifically target the viral RNA polymerase, effectively blocked the cytopathic effects of SARS-CoV on Vero cells. These two plasmids also inhibited viral replication as shown by titer assays and by an examination of viral RNA and protein levels. Thus, our results demonstrated the feasibility of developing siRNAs as effective anti-SARS drugs.

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.

Systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector‐based vaccines carrying the spike protein of Middle East respiratory syndrome coronavirus

An ideal vaccine against mucosal pathogens such as Middle East respiratory syndrome coronavirus (MERS‐CoV) should confer sustained, protective immunity at both systemic and mucosal levels. Here, we evaluated the in vivo systemic and mucosal antigen‐specific immune responses induced by a single intramuscular or intragastric administration of recombinant adenoviral type 5 (Ad5) or type 41 (Ad41) ‐based vaccines expressing the MERS‐CoV spike (S) protein. Intragastric administration of either Ad5‐S or Ad41‐S induced antigen‐specific IgG and neutralizing antibody in serum; however, antigen‐specific T‐cell responses were not detected. In contrast, after a single intramuscular dose of Ad5‐S or Ad41‐S, functional antigen‐specific T‐cell responses were elicited in the spleen and pulmonary lymphocytes of the mice, which persisted for several months. Both rAd‐based vaccines administered intramuscularly induced systemic humoral immune responses (neutralizing IgG antibodies). Our results show that a single dose of Ad5‐S‐ or Ad41‐S‐based vaccines represents an appealing strategy for the control of MERS‐CoV infection and transmission.

TRIM38 Negatively Regulates TLR3-Mediated IFN-β Signaling by Targeting TRIF for Degradation

Toll-like receptors (TLRs) mediated immune response is crucial for combating pathogens and must be tightly controlled. Tripartite motif (TRIM) proteins are a family of proteins that is involved in a variety of biological and physiological processes. Some members of the TRIM family are important in the regulation of innate immunity. Although it has been shown that TRIM38 negatively regulates innate immunity, the mechanisms by which it does so have not been fully addressed. In this study, we demonstrated that TRIM38 negatively regulates Toll-like receptor 3 (TLR3)-mediated type I interferon signaling by targeting TIR domain-containing adaptor inducing IFN-β (TRIF). We found that overexpression of TRIM38 inhibits TLR3-mediated type I interferon signaling, whereas knockdown of TRIM38 has the reverse effects. We further showed that TRIM38 targets TRIF, a critical adaptor protein downstream of TLR3. TRIF is co-immunoprecipitated with TRIM38, and domain mapping experiments show that PRYSPRY of TRIM38 interacts with the N-terminus of TRIF. Overexpression of TRIM38 decreased expression of overexpressed and endogenous TRIF. This effect could be inhibited by MG132 treatment. Furthermore, the RING/B-box domain of TRIM38 is critical for K48-linked polyubiquitination and proteasomal degradation of TRIF. Collectively, our results suggest that TRIM38 may act as a novel negative regulator for TLR3-mediated type I interferon signaling by targeting TRIF for degradation.

Rotavirus nonstructural protein 1 antagonizes innate immune response by interacting with retinoic acid inducible gene I

BackgroundThe nonstructural protein 1 (NSP1) of rotavirus has been reported to block interferon (IFN) signaling by mediating proteasome-dependent degradation of IFN-regulatory factors (IRFs) and (or) the β-transducin repeat containing protein (β-TrCP). However, in addition to these targets, NSP1 may subvert innate immune responses via other mechanisms.ResultsThe NSP1 of rotavirus OSU strain as well as the IRF3 binding domain truncated NSP1 of rotavirus SA11 strain are unable to degrade IRFs, but can still inhibit host IFN response, indicating that NSP1 may target alternative host factor(s) other than IRFs. Overexpression of NSP1 can block IFN-β promoter activation induced by the retinoic acid inducible gene I (RIG-I), but does not inhibit IFN-β activation induced by the mitochondrial antiviral-signaling protein (MAVS), indicating that NSP1 may target RIG-I. Immunoprecipitation experiments show that NSP1 interacts with RIG-I independent of IRF3 binding domain. In addition, NSP1 induces down-regulation of RIG-I in a proteasome-independent way.ConclusionsOur findings demonstrate that inhibition of RIG-I mediated type I IFN responses by NSP1 may contribute to the immune evasion of rotavirus.

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.

Identification of a Highly Conserved H1 Subtype-Specific Epitope with Diagnostic Potential in the Hemagglutinin Protein of Influenza A Virus

Subtype specificity of influenza A virus (IAV) is determined by its two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). For HA, 16 distinct subtypes (H1–H16) exist, while nine exist for NA. The epidemic strains of H1N1 IAV change frequently and cause annual seasonal epidemics as well as occasional pandemics, such as the notorious 1918 influenza pandemic. The recent introduction of pandemic A/H1N1 IAV (H1N1pdm virus) into humans re-emphasizes the public health concern about H1N1 IAV. Several studies have identified conserved epitopes within specific HA subtypes that can be used for diagnostics. However, immune specific epitopes in H1N1 IAV have not been completely assessed. In this study, linear epitopes on the H1N1pdm viral HA protein were identified by peptide scanning using libraries of overlapping peptides against convalescent sera from H1N1pdm patients. One epitope, P5 (aa 58–72) was found to be immunodominant in patients and to evoke high titer antibodies in mice. Multiple sequence alignments and in silico coverage analysis showed that this epitope is highly conserved in influenza H1 HA [with a coverage of 91.6% (9,860/10,767)] and almost completely absent in other subtypes [with a coverage of 3.3% (792/23,895)]. This previously unidentified linear epitope is located outside the five well-recognized antigenic sites in HA. A peptide ELISA method based on this epitope was developed and showed high correlation (χ2 = 51.81, P<0.01, Pearson correlation coefficient R = 0.741) with a hemagglutination inhibition test. The highly conserved H1 subtype-specific immunodominant epitope may form the basis for developing novel assays for sero-diagnosis and active surveillance against H1N1 IAVs.

Large-scale seroprevalence analysis of human metapneumovirus and human respiratory syncytial virus infections in Beijing, China

BackgroundHuman metapneumovirus (hMPV), a recently identified virus, causes acute respiratory tract infections (ARTIs) in infants and children. However, studies on the seroepidemeology of hMPV are very limited in China. To assess the seroprevalence of hMPV infection in China, we tested a total of 1,156 serum specimens for the presence of anti-hMPV IgG antibody in children and adults free of acute respiratory illness in Beijing, China by using hMPV nucleocapsid (N) protein as an antigen. As a control, we used the human serum antibody against the N protein of human respiratory syncytial virus (hRSV), the most important viral agent responsible for ARIs in children.ResultsThe seropositive rate for hMPV increased steadily with age from 67% at 1-6 mo to 100% at age 20. However, the rate dropped slightly between 6 mo and 1 yr of age. The seropositive rate for hRSV also increased steadily with age from 71% at 1-6 mo to 100% at age 20. In children aged six months to six years, the seropositive rates for the anti-hRSV IgG antibody were significantly higher than those for hMPV. Additionally, IgG antibody titers to hMPV and hRSV were significantly higher in adults than in young children. Consistent with the seropositive rates, the geometric mean titer of anti-hMPV IgG antibody was lower than that of anti-hRSV IgG antibody in children aged six months to six years.ConclusionsOur results indicate that similar to hRSV, exposure to hMPV is ubiquitous in the Beijing population. However, the seroprevalence of anti-hMPV IgG antibody is lower than that of hRSV in children between six months and six years old, which suggests a different number of repeat infections or a different response to infections.

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.

Lysosome-associated membrane glycoprotein 3 is involved in influenza A virus replication in human lung epithelial (A549) cells

BackgroundInfluenza A virus mutates rapidly, rendering antiviral therapies and vaccines directed against virus-encoded targets ineffective. Knowledge of the host factors and molecular pathways exploited by influenza virus will provide further targets for novel antiviral strategies. However, the critical host factors involved in influenza virus infection have not been fully defined.ResultsWe demonstrated that LAMP3, a member of lysosome-associated membrane glycoprotein (LAMP) family, was significantly induced in human lung epithelial (A549) cells upon influenza A virus infection. Knockdown of LAMP3 expression by RNA interference attenuated production of viral nucleoprotein (NP) as well as virus titers. Confocal microscopy results demonstrated that viral NP is colocalized within LAMP3 positive vesicles at early stages of virus infection. Furthermore, knockdown of LAMP3 expression led to a reduction in nuclear accumulation of viral NP and impeded virus replication.ConclusionsLAMP3 is an influenza A virus inducible gene, and plays an important role in viral post-entry steps. Our observations may provide insights into the mechanism of influenza virus replication and potential targets for novel anti-influenza therapeutics.