Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections / 生物医学与环境科学（英文）

OBJECTIVE@#Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.@*METHODS@#A platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.@*RESULTS@#Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.@*CONCLUSION@#Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

Gene Knockdown in Human Rhinovirus 1B Using 2'-OMe-modified siRNAs Results in the Reactivation of the Interferon Response / 生物医学与环境科学（英文）

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 / 生物医学与环境科学（英文）

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.

Progress on development and research of human bocavirus 1 / 病毒学报

Human bocavirus 1 (HBoV1) is a novel virus that mainly causes respiratory tract infection, and it has the characteristic of genome of Parvovirus, containing three open reading frames that encode non-structural proteins NS1 and NP1 and structural proteins VP1 and VP2. Circular episome is present during the rolling circle replication of HBoV1, which provides the possibility of full genome amplification and infectious clone construction to save HBoV1. The recombination between HBoV1 and HBoV2-4 occurs frequently. With the three-dimensional culture, in vitro culture of HBoV1 provides a powerful tool for research on the pathogenesis of HBoV1. This review focuses on the molecular characteristics, association with diseases, in vitro culture, diagnosis and treatment of HBoV1.

Whole genome analysis of human group A rotavirus G9p8 strains in Hebei lulong region, 2009-2011 / 病毒学报

Abstract:This study aims to investigate the genetic characteristics of group A rotavirus (GARV) G9P[8] strains from infantile diarrhea samples in Hebei Lulong region from 2009 to 2011. We randomly selected five GARV G9P[8] strains in Hebei Lulong region from 2009 to 2011, amplified the 11 gene fragments of GARVs by RT-PCR, and analyz their full-genome sequences by homology and phylogenetic analysis with DNAStar and MEGA. The nucleotide homology between strains LL11131077 and LL11131083 in 2011 was significantly higher than hat etween them and the other three strains in 2009 and 2010. The G9P[8] GARVs circulating in Hebei Lulong region from 2009 to 2011 elenged to the same genotype as the prevalent G9P[8] GARVs in other parts of the world. However,the two strains in 2011, compared with those in 2009 and 2010, were located in a different sub-branch of the phylogenetic tree and had amino acid mutations at many sites.

Establishment and primary application of a novel resequencing pathogen microarray-based assay for detecting pathogens in patients with unexplained diarrhea / 病毒学报

In this study, a novel resequencing pathogen microarray (RPM)-based multi-pathogen detection assay was developed to simultaneously detect 14 rotaviruses, 7 caliciviruses, 8 astroviruses, 28 enteroviruses, and 16 rare diarrhea viruses in patients with diarrhea syndrome. The specificity of the assay was examined using confirmed virus-positive specimens, and the sensitivity was evaluated by serial ten-fold dilutions of in vitro transcribed RNA. RPM assay could detect and differentiate virus types/subtypes at 20-2000 copies/microL. The detection threshold of RPM was determined by adjusting the reference concentration, and the detection steps were optimized to type Enterovirus. The nucleic acids of 10 stool samples from patients with unexplained diarrhea were screened, and 6 of them showed positive results. The RPM results were further verified by singleplex PCR followed by sequencing, and no difference was found between the two assays. In conclusion, we have established a high-throughput RPM assay with high specificity and sensitivity, which demonstrates a great potential for the identification of pathogens in patients with unexplained diarrhea and the management of emerging epidemic.

Research progress in human adenovirus / 病毒学报

Human adenovirus (HAdV) is one of the most important pathogens in infants and young children with acute respiratory infections and other diseases. This article reviews the literature on HAdV, including its molecular biological characteristics, detection and typing, and pathogenic mechanism, the clinical features and epidemiological characteristics of HAdV-related diseases, and the prevention and control of HAdV infections. So far, 67 types of HAdV have been identified, including recombinant variants discovered in recent years. The major epidemic strains that cause acute respiratory infections are HAdV-3 and HAdV-7, both of which belong to the subgroup B. HAdV often leads to acute respiratory infections, but it also causes diseases of other systems. HAdV-related diseases have similar clinical manifestations as those caused by other respiratory viruses, but often accompanied by gastrointestinal symptoms. The pathogenic mechanism of HAdV remains unclear, especially for the new recombinant variants, due to few studies on their association with diseases. Because there are no prospective, large randomized controlled trials of HAdV infections, the treatment of HAdV infections is controversial. Vaccine is the most effective measure to reduce respiratory HAdV infections, but it is still not commercially available.

Genome amplification and sequence analysis of human bocavirus 2 / 病毒学报

To obtain the genome sequence of human bocavirus 2 (HBoV2), different regions of HBoV2 genome were amplified through PCR in fecal specimens which had been identified as single-positive for HBoV2 in 2010. A genome sequence of HBoV2 (HBoV2-NC, 5444 bp) was obtained after sequence assembly. The phylogenetic analysis showed that HBoV2-NC had the closest evolutionary relationship with HBoV2 Lanzhou strain. The predication of inverted terminal repeats of HBoV2-NC by DINAMelt showed that inverted terminal repeats were contained in HBoV2-NC 5' terminal, which had the typical stem-loop structure in other parvoviruses. Finally, some flanking sequences of HBoV2-NC were amplified by linker-PCR.

A review of detection methods for human bocaviruses / 病毒学报

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 / 病毒学报

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.

Etiological study of human bocavirus 1-4 in children with acute diarrhea in Lanzhou, China / 病毒学报

This study aimed to study the epidemiological and clinical characteristics of human bocavirus 1-4 (HBoV1-4) in children with acute diarrhea in Lanzhou and to investigate the association between HBoV and acute gastroenteritis. A total of 331 stool samples were collected from children aged under 5 years with acute diarrhea at the Department of Pediatrics, the First Hospital, Lanzhou University, between July 2012 and June 2013. Nested PCR was used to screen for HBoV and a general PCR was employed to screen other common diarrhea viruses. We found human bocavirus 1, 2, 3 and 4 in 26, 15, 7 and 1 cases, respectively. There was no specific seasonal distribution of HBoV, with infections occurring throughout the year. HBoV was mostly found in children aged between 7 and 12 months, with a mean age of 11.04 months (+/- 6.92 months), and 93.88% of affected children were aged under 2 years. Overall, 71.3% of mixed infections were mixed and the majority of other infections were caused by rotavirus. There was no statistical difference in the incidence of fever and vomiting associated with HBoV infection. A rare virus strain, HBoV4 (LZFB086), was identified, which showed highest levels of nucleotide sequence identity (99.0%) with a single Thai HBoV strain (JQ267789). No case of HBoV2B was found. In conclusion, HBoV1 was a major etiological pathogen of HBoV in pediatric cases in Lanzhou. HBoV4 was detected in feces for the first time in China. The rate of mixed infections was high and rotavirus was dominant. The data presented suggests that HBoV is not a major causative agent of gastroenteritis.

Evolutionary relationships of G3 GARV isolated from pigs and humans in Lulong County, Hebei Province, China / 病毒学报

This study aimed to amplify major genome segments (VP7, VP4, VP6, VP2 and NSP2-5) of porcine G3 group A rotavirus (GARV) LLZ212 isolated in our laboratory, determine their genotypes, and explore the evolutionary relationships between G3 GARV strains isolated from humans and pigs in Lulong County, Hebei Province, China. Major genome segments of seven GARV strains were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and the segments were sequenced. The genome segments of seven GARV strains were determined by the online RotaC genotyping tool (RotaC v2.0). The reference sequences of each GARV genome segment were downloaded from GenBank. Homology and phylogenetic evolutionary analyses were conducted using the MEGA 5.0 and DNAStar software packages. LLZ212 isolated from pigs in Lulong had the following genotype: G3-P[8]-I5-C1-N1-T1-E1-H1. All human GARV strains had the following genotype: G3-P[8]-I1-C1-N1-T1-E1-H1. The VP7, VP4, NSP4 and NSP5 genes of the LLZ212 strain had the highest nucleotide identities with the human GARV E885, CMH014/07, Wa and RMC321 strains, respectively, and these clustered together in a sublineage. The VP6, NSP4 and NSP5 genes of the LLZ212 strain shared the highest nucleotide identities with the porcine GARV PRG921 strain, while VP2 associated most closely with porcine GARV OSU strain, and these also clustered in a sublineage. A rare porcine G3-P[8]-I5-C1-N1-T1-E1-H1 GARV strain was identified, which may represent a reassortment between porcine and human viruses. In conclusion, the VP7, VP4, NSP4 and NSP5 genes of LLZ212 share high levels of sequence identity with human GARV, while VP2, VP6, NSP2 and NSP3 cluster with porcine GARV.

Current research on picornavirus 3C protease / 病毒学报

The picornavirus family comprises many small viruses, several of which are important pathogens of humans and livestock. The 3C protease (3Cpro) of different species and genera of picornavirus contains the classic G-X-C-G motif and Cys-His-Asp/Glu catalytic triad. 3Cpro conducts maturation cleavage in the regions of VP2-VP3 and VP3-VP1 in P1, 2A-2B and 2B-2C in P2 and the whole P3. Picornavirus 3Cpro has been shown to have significant substrate preference in Q-G/S/A/V/H/R and E-S/G/R/M as well as species and genera specificity through analyses of the maturation cleavage of picornavirus polyproteins. Innate immune adaptors such as TRIF, MAVS, IRF3, IRF7 and NEMO have various potential cleavage sites in picornavirus 3Cpro (TRIF and NEMO show considerable diversity in their cleavage sites). Useful information will be provided for the development of broad-spectrum antiviral agents as well as evasion mechanisms of the innate immune system against picornavirus 3Cpro through continued research of picornavirus 3Cpro.

Detection of human bocavirus in children with acute respiratory tract infections in Lanzhou and Nanjing, China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>The aim of this study was to explore the prevalent characteristics of HBoV1 and its co-infection.</p><p><b>METHODS</b>PCR was used to detect HBoV1-DNA (HBoV1) and other viruses. A multivariate logistic regression model was used to explore possibility of co-detected for related viruses.</p><p><b>RESULTS</b>The positivity rates in Nanjing and Lanzhou were 9.38% (74/789) and 11.62% (161/1386), respectively (P>0.05). The HBoV1 positive group was younger than negative group (P<0.05). Seasonal differences were noted, with a higher frequency of infection in December and July. HBoV1-positive children [72.34% (169/235)] were co-infected with other respiratory viruses. Multifactorial analysis showed no correlations between HBoV1 and the clinical classification, region, gender, age, or treatment as an outpatient or in a hospital. Correlations were identified between HBoV1 infections with ADV (OR=1.53, 95% CI 1.03-2.28), RSV (OR=0.71, 95% CI 0.52-0.98), and IFVA (OR=1.77, 95% CI 1.00-3.13).</p><p><b>CONCLUSION</b>Presence of HBoV1 in nasopharyngeal aspirates did not correlate with region or gender, although the prevalence of HBoV1 was higher in younger children. There were no correlations between HBoV1 and other variables, except for the season and ADV, RSV, or IFVA infections.</p>

Viral etiology in children with acute lower respiratory tract infections plus platelet disorders in Changsha, China: an analysis of 255 cases / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the viral etiology in hospitalized children with acute lower respiratory tract infections (ALRTI) plus platelet disorders.</p><p><b>METHODS</b>A total of 255 children with ALRTI plus platelet disorders and 442 children with ALRTI and normal platelets, all of whom were hospitalized between March 2010 and February 2011, were included in the study. Their nasopharyngeal aspirate samples were collected, and RT-PCR or PCR was performed to detect 14 viruses.</p><p><b>RESULTS</b>Of 255 ALRTI patients with platelet disorders, thrombocytosis was found in 253 cases (99.2%) and thrombocytopenia in 2 cases (0.8%). Among ALRTI patients with platelet disorders, 173 (67.8%) were infected with at least one virus, with human rhinovirus as the most common one, followed by parainfluenza virus type 3 (PIV3) and respiratory syncytial virus (RSV). The detection rate of PIV3 in the abnormal platelet group was significantly higher than in the normal platelet group (P<0.05). In contrast, the detection rate of influenza virus B (IFVB) in the abonormal platelet group was significantly lower than in the normal platelet group (P<0.05). The age distribution showed significant difference between the abnormal and normal platelet groups (P<0.01). Platelet disorders were mainly found in children under one year of age (P<0.01).</p><p><b>CONCLUSIONS</b>Thrombocytosis is often found in children with ALRTI caused by viruses, especially PIV3, but infection with IFVB seldom causes platelet disorders. Hospitalized children with ALRTI under one year tend to develop platelet disorders.</p>

Establish A duplex fluorescent quantitative one-step RT-PCR system for the detection of norovirus genogroup I and II / 病毒学报

The object of this study is to develop a duplex fluorescent quantitative one-step RT-PCR assay for detection and quantitation of GI and GII norovirus. The specific primers, Taqman probes, optimized reaction solution and condition were used to develop the duplex fluorescent quantitative one-step RT-PCR assay. The sensitivity, specificity and reproducibility of the assay were evaluated. The assay was evaluated by testing the 100 specimen samples and compared with the reference assay conventional RT-PCR. The assay possessed high specificity for norovirus detection without any evident cross-reaction with enteric adenovirus, rotavirus or astrovirus. The detection limit of the real-time RT-PCR assay, for GI and GII norovirus was up to 10(3) copy/microL respectively. Compared with the conventional RT-PCR assay, the assay in this study had higher sensitivity with higher detection rate of norovirus in stool specimens. The duplex fluorescent quantitative one-step RT-PCR assay provides rapid, sensitive and reliable detection of GI and GII norovirus, and could be used as a laboratory diagnosis of norovirus in acute gastroenteritis patients.

Research progress of real-time quantitative PCR method for group A rotavirus detection / 病毒学报

Group A rotavirus is one of the most significant etiological agents which causes acute gastroenteritis among infants and young children worldwide. So far, several method which includes electron microscopy (EM), enzyme immunoassay (EIA), reverse transcription-polymerase chain reaction (RT-PCR)and Real-time Quantitative PCR has been established for the detection of rotavirus. Compared with other methods, Real-time quantitative PCR have advantages in specificity, sensitivity, genotyping and quantitative accuracy. This article shows a overview of the application of real-time quantitative PCR technique to detecte group A rotavirus.

Status of enterovirus infection in children with acute lower respiratory tract infection / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the status of enterovirus (EV) infection in children with acute lower respiratory tract infection (ALRTI).</p><p><b>METHODS</b>A total of 404 samples (with odd numbers) of nasopharyngeal aspirates were collected from the children who were hospitalized in the Children's Medical Center, Hunan Provincial People's Hospital due to ALRTI between September 2007 and April 2008. The conserved sequence in the 5'-noncoding region of EV was used to design the primer, and nested RT-PCR was performed to detect EV in the samples.</p><p><b>RESULTS</b>Of the 404 samples, 19 (4.7%) were EV-positive, and mostly taken from children under 3 years of age (95%); there was no significant difference in the detection rate between male and female children. Of the EV-positive children, 13 (68%) were clinically diagnosed with bronchial pneumonia, and 6 (32%) with bronchiolitis; 90% of them showed symptoms of fever, 84% had a cough, 63% had asthma, and 63% had complications mainly including diarrhea (6 cases), granulocytopenia (4 cases), and acute respiratory distress syndrome (2 cases). In addition, 26% of the EV-positive children had leukocyte disorder, more than half had liver dysfunction, and a few had myocardial involvement.</p><p><b>CONCLUSIONS</b>EV is a pathogen that should not be neglected in children with ALRTI. For these children, close attention should be paid to the epidemiological status and clinical features of EV infection, and blood routine examination, liver function test and myocardial enzyme assay should be carried out periodically to improve prognosis.</p>

Virus detection in bronchoalveolar lavage fluid of 122 children with severe pneumonia / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the prevalence of viral pathogen in children with severe pneumonia in Hunan.</p><p><b>METHOD</b>Bronchoalveolar lavage fluid [BALF] were collected from 122 hospitalized children with severe pneumonia in People's Hospital of Hunan province from January 2011 to December 2011. Nested- or reverse transcription Polymerase chain reaction (PCR or RT-PCR) was used to screen Adenovirus (ADV), Human Bocavirus (HBoV), Parainfluenzaviruses1-4 (PIV1-4), Human Respiratory Syneytial virus (RSV), Influenza virus A (IFVA), Influenza virus B (IFVB), Human Rhinovirus(HRV), Human Metapneumovirus (HMPV), human coronaviruses NL63 and HKU1 (HCoV-NL63, HCoV- HKU1).</p><p><b>RESULTS</b>Among the 122 bronchoalveolar lavage fluid, viral agents were detected in 60 samples(49.1%), among which ADV (40.98%) was the most common virus, followed by RSV (7.37%) and HBoV (7.37%). Two viruses were detected in 21 individual (35%) samples, of which 20 were dual positive for ADV (40%).</p><p><b>CONCLUSION</b>ADV is the most frequently detected viral etiology of severe pneumonia in children in Hunan during this year. And its Coinfection with other respiratory viruses was common.</p>

Enzyme-linked immunospot test detected specific cellular immune response induced by human bocavirus VP2 virus-like particles / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To discuss the enzyme linked immune spot test (ELISPOT) detected the cellular immune response induced by human Bocavirus (HBoV) VP2 virus-like particles (VLPs).</p><p><b>METHODS</b>After immunized by HBoV VP2 VLPs, the specific cellular immune response in mice were detected by ELISPOT assay, observe the ELISPOT results at the conditions of different polypeptide stimulate, different cell culture time, different cell concentration and different specific stimulus peptide concentration, then screening the right ELISPOT experimental conditions and establish the ELISPOT method.</p><p><b>RESULTS</b>The spots induced by HBoV1 VLPs immunized mice spleen lymphocytes stimulate with polypeptide P3 (GYIPIENEL) and P5 (LYQMPFFLL)were 233 spots/10(6) cells and 157 spots/10(6) cells,spots induced by HBoV2 VLPs immunized mice spleen lymphocytes stimulate with polypeptide P8 (GYIPVIHEL) were 113 spots/10(6) cells; 24 hours is the best time for culture, at this time HBoV1 and HBoV2 groups specificity secretion IFN-gamma ratio were 232 spots/10(6) cells and 119/10(6) cells; Best concentration of mice spleen lymphocyte is 5 x 10(5), right now HBoV1 and HBoV2 group specificity secretion IFN-gamma ratio were 232 spots/10(6) cells and 108/10(6) cells; Best concentration of polypeptides is 10 microg/ml, HBoV1 and HBoV2 group specificity secretion IFN -gamma ratio were 233 spots/10(6) cells and 96/10(6) cells.</p><p><b>CONCLUSIONS</b>HBoV1 and HBoV2 specificT-cell epitope in BABL/c mice were P3, P5 (HBoV1) and P8 (HBoV2). The best experiment condition were: cell cultivated for 24 h, cells concentration for 5 x 10(5) cells/well, stimulating polyperides concentration for 10 microg/ml, it can use to study the cellular immune induced by HBoV in mice.</p>