GII.13/21 Noroviruses Recognize Glycans with a Terminal ß-Galactose via an Unconventional Glycan Binding Site.

Human noroviruses (huNoVs) recognize histo-blood group antigens (HBGAs) as host susceptibility factors. GII.13 and GII.21 huNoVs form a unique genetic lineage that emerged from mainstream GII NoVs via development of a new, nonconventional glycan binding site (GBS) that binds Lea antigen. This previous finding raised the question of whether the new GII.13/21 GBS really has such a narrow glycan binding spectrum. In this study, we provide solid phenotypic and structural evidence indicating that this new GBS recognizes a group of glycans with a common terminal ß-galactose (ß-Gal). First, we found that P domain proteins of GII.13/21 huNoVs circulating at different times bound three glycans sharing a common terminal ß-Gal, including Lec, lactose, and mucin core 2. Second, we solved the crystal structures of the GII.13 P dimers in complex with Lec and mucin core 2, which showed that ß-Gal is the major binding saccharide. Third, nonfat milk and lactose blocked the GII.13/21 P domain-glycan binding, which may explain the low prevalence of GII.13/21 viruses. Our data provide new insight into the host interactions and epidemiology of huNoVs, which would help in the control and prevention of NoV-associated diseases.IMPORTANCE Evidence from both phenotypic binding assay and structural study support the observed interactions of human noroviruses (huNoVs) with histo-blood group antigens (HBGAs) as receptors or attachment factors, affecting their host susceptibility. GII.13 and GII.21 genotypes form a unique genetic lineage that differs from the mainstream GII huNoVs in their unconventional glycan binding site. Unlike the previous findings that GII.13/21 genotypes recognize only Lea antigen, we found in this study that they can interact with a group of glycans with a common terminal ß-Gal, including Lec, lactose, and mucin core 2. However, this wide glycan binding spectrum in a unique binding mode of the GII.13/21 huNoVs appears not to increase their prevalence, probably due to the existence of decoy glycan receptors in human gastrointestinal tract limiting their infection. Our findings shed light on the host interaction and epidemiology of huNoVs, which would impact the strategy of huNoV control and prevention.

Glycan binding patterns of human rotavirus P[10] VP8* protein.

BACKGROUND: Rotaviruses (RVs) are a major cause of acute children gastroenteritis. The rotavirus P [10] belongs to P[I] genogroup of group A rotaviruses that mainly infect animals, while the rotavirus P [10] was mainly identified from human infection. The rotavirus P [10] is an unusual genotype and the recognition pattern of cellular receptors remains unclear. METHODS: We expressed and purified the RV P [10] VP8* protein and investigated the saliva and oligosaccharide binding profiles of the protein. A homology model of the P [10] VP8* core protein was built and the superimposition structural analysis of P [10] VP8* protein on P [19] VP8* in complex with mucin core 2 was performed to explore the possible docking structural basis of P [10] VP8* and mucin cores. RESULTS: Our data showed that rotavirus P [10] VP8* protein bound to all ABO secretor and non-secretor saliva. The rotavirus P [10] could bind strongly to mucin core 2 and weakly to mucin core 4. The homology modeling indicated that RV P [10] VP8* binds to mucin core 2 using a potential glycan binding site that is the same to P [19] VP8* belonging to P[II] genogroup. CONCLUSION: Our results suggested an interaction of rotavirus P [10] VP8* protein with mucin core 2 and mucin core 4. These findings offer potential for elucidating the mechanism of RV A host specificity, evolution and epidemiology.

The suppression of innate immune response by human rhinovirus C.

Rhinovirus C (RV-C), a newly identified group of human rhinoviruses (RVs), is associated with exacerbation of severe asthma. The type I interferon (IFN) response induced by this virus and the mechanisms of evasion of IFN-mediated innate immunity for RV-C remain unclear. In this study, we constructed a full-length cDNA clone of RV-C (LZ651) from a clinical sample. IFN-ß mRNA and protein levels were not elevated in differentiated Human bronchial epithelial (HBE) cells at the air-liquid interface infected with RV-C, except in the early stage of infection. The ability to attenuate IFN-ß activation was ascribed to 3Cpro of RV-C, and the 40-His site of 3Cpro played an important role. Furthermore, RIG-I was degraded by 3Cpro in a caspase-dependent manner and 3Cpro cleaved MAVS at 148 Q/A, which inhibited IFN signaling. Taken together, our results demonstrate the mechanism by which RV-C circumvents the production of type I IFN in infected cells.

Quantitative detection of human Malawi polyomavirus in nasopharyngeal aspirates, sera, and feces in Beijing, China, using real-time TaqMan-based PCR.

BACKGROUND: Human Malawi polyomavirus (MWPyV) was discovered in 2012, but its prevalence and clinical characteristics are largely unknown. METHODS: We used real-time TaqMan-based PCR to detect MWPyV in the feces (n = 174) of children with diarrhea, nasopharyngeal aspirates (n = 887) from children with respiratory infections, and sera (n = 200) from healthy adults, and analyzed its clinical characteristics statistically. All the MWPyV-positive specimens were also screened for other common respiratory viruses. RESULTS: Sixteen specimens were positive for MWPyV, including 13 (1.47%) respiratory samples and three (1.7%) fecal samples. The samples were all co-infected with other respiratory viruses, most commonly with influenza viruses (69.2%) and human coronaviruses (30.7%). The MWPyV-positive children were diagnosed with bronchopneumonia or viral diarrhea. They ranged in age from 12 days to 9 years, and the most frequent symptoms were cough and fever. CONCLUSIONS: Real-time PCR is an effective tool for the detection of MWPyV in different types of samples. MWPyV infection mainly occurs in young children, and fecal-oral transmission is a possible route of its transmission.

Gene Knockdown in Human Rhinovirus 1B Using 2'-OMe-modified siRNAs Results in the Reactivation of the Interferon Response.

The aim of this study was to investigate the knockdown efficiency of 2'-O-methylated (2'-OMe)-modified small interfering RNAs (siRNAs) on human rhinovirus 1B (HRV1B) replication and the interferon response. Thus, 24 2'-OMe-modified siRNAs were designed to target HRV1B. The RNA levels of HRV1B, Toll-like receptor 3, melanoma differentiation-associated gene 5, retinoic acid inducible gene-I, and interferons were determined in HRV1B-infected HeLa and BEAS-2B epithelial cells transfected with 2'-OMe-modified siRNAs. The results revealed that all 2'-OMe-modified siRNAs interfered with the replication of HRV1B in a cell-specific and transfection efficiency-dependent manner. Viral activation of Toll-like receptor 3, melanoma differentiation-associated gene 5, retinoic acid inducible gene-I, and the interferon response was detected. In conclusion, the 2'-OMe-modified siRNAs used in this study could interfere with HRV1B replication, possibly leading to the reactivation of the interferon response.

Norovirus Infection and Histo-blood Group Antigens in Children Hospitalized with Diarrhea in Lulong and Chenzhou in China.

Norovirus (NoV) is a pathogen that commonly causes viral diarrhea in children. Studies indicate that NoV recognizes human histo-blood group antigens (HBGAs) as cell attachment factors. In order to explore the correlation between of NoV infection and HBGAs, a cross-sectional study was conducted in children less than five years old who were hospitalized with diarrhea in two areas of China between November 2014 and February 2015. Of the paired stool and saliva samples taken from 424 children, NoV was detected in 24 (6%) children, with viral genotypes GII.3 (n=5), GII.4 (n=14), GII.12 (n=1), and GII.17 (n=4). All of the individuals having NoV infection were either secretors (Lea-b+/Lex-y+) or partial secretors (Lea+b+/Lex+y+) except one GII.3 infection of a non-secretor (Lea+b-/Lex+y-). These results suggest that secretor positive is associated with NoV infection, although non-secretors are not absolutely protected from NoV infection.

Outbreak of febrile illness caused by coxsackievirus A4 in a nursery school in Beijing, China.

BACKGROUND: Coxsackievirus A4 (CV-A4) is classified as human enterovirus A according to its serotype. CV-A4, an etiological agent of hand, foot, and mouth disease, affects children worldwide and can circulate in closed environments such as schools and hospitals for long periods. FINDINGS: An outbreak of febrile illness at a nursery school in Beijing, China, was confirmed to be caused by CV-A4. Phylogenetic analysis of the complete genome of the isolated strain showed that the virus belongs to the same cluster as the predominant CV-A4 strain in China. This outbreak was controlled by effective measures. CONCLUSIONS: The early identification of the pathogen and timely intervention may be the most critical factors in controlling an outbreak caused by CV-A4 in a preschool.

Identification and genetic characterization of two porcine astroviruses from domestic piglets in China.

Porcine astrovirus are divided into five genotypes. In this study, we identified two porcine astroviruses (AstV-LL-1 and AstV-LL-2) by using sequence-independent single-primer amplification (SISPA) on faecal specimens of healthy domestic piglets younger than 15 days. The detection rate for both was 2.82% (14/497). AstV-LLs were then sequenced and characterised. Phylogenetic analysis revealed that they have the characteristics of porcine astrovirus (PastV) 2 and 5 and have some unique genetic features. Our findings show that the two astroviruses are novel lineages of PAstV2 and 5. The findings may be helpful in evaluating the ecology and evolution of astroviruses in pigs.

Cytokine responses and correlations thereof with clinical profiles in children with enterovirus 71 infections.

BACKGROUND: Severe complications associated with EV71 infections caused many infants death. However, the pathogenesis of EV71 infection in the severe cases remained poorly understood. METHODS: In this study we collected plasma and cerebrospinal fluid (CSF) specimens drawn in the acute and/or recovery phases from EV71-infected individuals, and plasma specimens from healthy children served as normal controls. We compared the levels of cytokines and chemokines determined by a Luminex-based cytokine bead array. RESULTS: The plasma levels of IL-1ß and IL-6 were significantly higher in severe and critical cases than in mild patients and normal controls. Higher plasma levels of IL-6, IL-10, and IL-8 were evident in critical than severe cases. The CSF levels of IL-6, IL-8, and IP-10 were higher, and that of RANTES lower (compared to plasma), in severe and critical patients. Significantly lower CSF levels of cytokines and chemokines were recorded in the recovery than the acute phase in severe and critical cases treated with intravenous immunoglobulin (IVIG) and glucocorticoids. Only the CSF levels of IL-6, IP-10, and IL-8 were significantly correlated with white blood cell counts, and absolute neutrophil and monocyte counts, in severe cases. Furthermore, the CSF levels of IL-6 were correlated with temperature in both cases. CONCLUSIONS: These data indicate that a major cytokine response and inflammation, in both plasma and the CNS, are features of disease caused by EV71 infection. Systemic inflammation caused by EV71 infection exacerbated the deterioration of the disease, and resulted in the disease progression to the critical illness stage.

The induction and suppression of type I and type III interferons by human group H rotavirus.

Group H Rotavirus (RVH) is associated with human diarrhea gastroenteritis. The interferon (IFN) response induced by RVH remains unclear. In this study, we first studied the characteristic feature of RVH and found J19 strain of RVH grew less efficiently compared with the G6P1 strain of RVA. Next, we found that infection with the J19 virus resulted in the secretion of IFN-λ1, but not IFN-ß, while both IFN-ß and IFN-λ1 could inhibit J19 replication significantly in Caco-2 cells. NSP1 played an important role in the suppression of type I and type III IFN response, and NSP5 protein significantly inhibited activation of IFN-λ1. J19 NSP1 suppressed the induction of IFN-ß obviously than G6P1 NSP1, while G6P1 NSP1 reduced IFN-λ1 induction to the greatest extent compared with G9P8, Wa, and J19 NSP1s. Our studies reveal the propagation feature of RVH and interferon induction and suppression by group H rotavirus.

Molecular characterization of unusual human G5P[6] rotaviruses identified in China.

BACKGROUND: We found an unusual human rotavirus, LL36755 of G5P[6] genotype, in a stool sample collected in Lulong County, Hebei Province, China. This is the first detection of rotavirus serotype G5 in Asia. OBJECTIVES: To identify and characterize G5 rotaviruses in 988 stool samples collected from children under 5 years old with acute gastroenteritis. STUDY DESIGN: We analyzed 459 rotavirus-positive samples with RT-PCR using G5 genotype-specific primers. The G5 strains were sequenced. RESULTS: Two additional G5-positive samples (LL3354 and LL4260) were identified. VP7, VP4, VP6 and NSP4 genes of LL3354, LL4260 and LL36755 strains were sequenced. The VP4 sequences formed a group with porcine P[6] strains. The VP6 sequences of strains LL3354 and LL36755 were phylogenetically close to the major clusters of SGI and SGII rotaviruses, respectively. The deduced VP6 protein of strain LL4260 had characteristics of both SGI and SGII strains, but best fit with a cluster of atypical SGI viruses. In addition, based on NSP4 sequences, the three G5 strains belonged to genogroup B and were closest to human strain Wa. CONCLUSION: These results indicate a dynamic interaction of human and porcine rotaviruses and suggest that reassortment could result in the stable introduction and successful spread of porcine gene alleles into human rotaviruses.

Human bocavirus infection in children hospitalized with acute gastroenteritis in China.

BACKGROUND: Human bocavirus (HBoV) was first identified in children with acute respiratory-tract infections, but recent studies have revealed that HBoV is also frequently detected in fecal specimens from children with gastroenteritis. OBJECTIVES: To investigate the prevalence of HBoV in children hospitalized with gastroenteritis in different areas of China. STUDY DESIGN: Employing ELISA, RT-PCR or PCR, we evaluated 1216 fecal samples for common diarrheal agents from children aged less than 5-year-old hospitalized with acute gastroenteritis. MEGA software was used to construct phylogenetic trees of the VP1/VP2 partial sequences of the HBoV genome. RESULTS: There were 67 HBoV-positive specimens, 52 (77.6%) were co-infected with rotavirus, norovirus, astrovirus, or enteric adenovirus. Statistical analysis of the clinical data indicated that children infected with both rotavirus and bocavirus did not have more severe clinical symptoms than children infected with rotavirus. The phylogenetic analysis of the VP1/VP2 partial sequences of the HBoV genome revealed a single genetic lineage. CONCLUSIONS: Despite its high infection rate, there was no statistically significant a causual relationship between HBoV and gastroenteritis in children.

Faecal shedding of rotavirus vaccine in Chinese children after vaccination with Lanzhou lamb rotavirus vaccine.

Lanzhou lamb rotavirus vaccine (LLR) is an oral live attenuated vaccine first licensed in China in 2000. To date, > 60 million doses of LLR have been distributed to children. However, very little is known about faecal shedding of LLR in children. Therefore, faecal samples (n = 1,184) were collected from 114 children for 15 days post-vaccination in September-November 2011/2012. Faecal shedding and viral loads were determined by an enzyme immunoassay kit (EIA) and real-time RT-PCR. The complete genome was sequenced and the vaccine strain was isolated by culture in MA104 cells. Approximately 14.0% (16/114) of children had rotavirus-positive samples by EIA for at least 1 day post-vaccination. Viral loads in EIA-positive samples ranged from < 1.0 × 103 to 1.9 × 108 copies/g. Faecal shedding occurred as early as post-vaccination day 2 and as late as post-vaccination day 13 and peaked on post-vaccination day 5-10. One LLR strain was isolated by culture in MA104 cells. Sequence analysis showed 99% identity with LLR prototype strain. Faecal shedding of LLR in stool is common within 15 days of LLR vaccination, indicating vaccine strains can replicate in human enteric tissues.

Susceptibility of human tonsillar epithelial cells to enterovirus 71 with normal cytokine response.

A recent histopathologic study implicated human tonsillar crypt epithelium as an important site for EV71 replication in EV71-caused fatal cases. This study aimed to confirm the susceptibility of human tonsillar epithelium to EV71. Two human tonsillar epithelial cell lines (UT-SCC-60A and UT-SCC-60B) were susceptive to EV71, and PI3K/AKT, p38, ERK1/2, and JNK1/2 signal pathways were activated. Interferon-α, IL-8, IL-1ß, IL-6 and IL-12p40 were induced and regulated by PI3K/AKT, p38, ERK1/2, and JNK1/2 signal pathways. PI3K/AKT pathway activation appeared to suppress the induction of TNF-α, which induced cell survival by inhibiting GSK-3ß. The activation of NF-κB was observed but inhibited by these pathways in EV71 infection. Furthermore, ERK1/2 and JNK1/2 were essential for efficient EV71 replication. Human tonsillar epithelial cells support EV71 replication and display innate antiviral immunity in vitro, indicating that human tonsillar epithelial cells may be novel targets for EV71 infection and replication in vivo.

Binding Patterns of Rotavirus Genotypes P[4], P[6], and P[8] in China with Histo-Blood Group Antigens.

Rotaviruses (RVs) are an important cause of severe gastroenteritis in children. It has been found that RV may recognize the histo-blood group antigens (HBGAs) as ligands or receptors and bind HBGAs in a type-dependent manner. In this study, we investigated the binding specificity of VP8* proteins from human rotaviruses (RV) that are prevalent in China including genotypes P[4], P[6], and P[8]. Through the saliva- and oligosaccharide-based binding assays, we found that the VP8* proteins of P[4] and P[8] RV showed similar reactivity with the Leb and H type 1 antigens, while P[6] RV weakly bound the Leb antigen. These findings may facilitate further research into RV host specificity and vaccine development.

Prevalence of Coxsackievirus A6 and Enterovirus 71 in Hand, Foot and Mouth Disease in Nanjing, China in 2013.

BACKGROUND: Although hand, foot and mouth disease (HFMD) has been strongly associated with enterovirus 71 (EV71), coxsackievirus A16 (CVA16) and other enteroviruses, studies regarding coxsackievirus A6 (CVA6) infection in HFMD are limited. The aim of this study was to identify the major etiological agents causing HFMD in Nanjing in 2013 and explore the clinical and genetic characteristics of the prevalent enterovirus (EV) types in HFMD. METHODS: A total of 394 throat swabs were collected from hospitalized children diagnosed with HFMD from April to July 2013. EVs were detected by reverse transcription polymerase chain reaction of 5' UTR sequences. Genotyping and phylogenetic analysis were based on VP4 sequences. Demographic and clinical data were obtained. RESULTS: Of the specimens, 68.5% (270/394) were positive for EVs. The genotypes and detection rates were CVA6, 30.00% (81/270); EV71, 17.41% (47/270); HRV, 11.11% (30/270); CVA10, 3.33% (9/270); CVA2, 1.11% (3/270); CVA16, 0.74% (2/270); EV68, 0.37% (1/270); echovirus 6, 0.37% (1/270); echovirus 9, 0.37% (1/270), respectively. Patients infected with CVA6 displayed symptoms atypical of HFMD, including larger vesicles on their limbs and buttocks. Phylogenetic analysis revealed 2 genetically distinct CVA6 strains that circulated independently within the region. Patients infected with CVA6 were more likely to have abnormal periphery blood white blood cell and C-reactive protein levels, while EV71 was more likely to infect the central nervous system, as indicated by clinical manifestations and white blood cell analysis of cerebrospinal fluid. CONCLUSIONS: Multiple EV genotypes contributed to HFMD in Nanjing in 2013, and CVA6 was the dominant genotype. The clinical presentation of CVA6 infection differs from that of EV71 infection in HFMD.

[A review of detection methods for human bocaviruses].

Human bocavirus (HBoV) 1-4 have been detected both in respiratory and stool samples since the first HBoV was discovered in 2005. HBoV-1 is mostly associated with respiratory infection, while HBoV 2-4 are usually associated with intestinal tract infection. A variety of signs and symptoms have been described in patients with HBoV infection, including cough, wheezing, pneumonia, and diarrhea, but the research on pathogenic mechanism of HBoV is limited because HBoV cannot be cultured in vitro due to the lack of appropriate host cells. Three-dimensional epithelial cell culture, reverse genetics, and viral metagenomics are identified as novel tools that may promote the research on pathogenic mechanism of HBoV and the discovery of new viruses. This review summaries currently available diagnostic approaches such as electron microscopy, cell culture, PCR, and immunoassay in order to provide a method reference for indepth research on HBoV.

[Research progress in receptors involved in rotavirus infection].

Rotaviruses, which are recognized as one of the major etiological agents among infants and young children with diarrhea, consist of three concentric layers of protein capsid with the enclosed double-stranded RNA genome. Rotaviruses infect host cells mainly by identifying the specific receptors on cell surfaces and binding to them. Therefore, receptors are important factors for viruses infecting cells. So far, there have been many receptors found to be involved in rotavirus infection, including sialic acid, integrin, Toll-like receptor, and blood group antigen. This article provides an overview of receptors involved in rotavirus infection.