Study of the association between histo-blood group antigens and norovirus infection in Chinese children.

Human caliciviruses (HuCVs) have been recognized as a major cause of sporadic viral diarrhea in children, among which norovirus genotype GII.4 is the most prevalent genotype. Stool and saliva samples were collected from 295 children with acute diarrhea and 150 asymptomatic children at a hospital in China. The HuCV detection rate was 10.85% (32/295) among the children with acute diarrhea, and all of these 32 children were either HBGA secretors (12/32) or partial secretors (20/32). HuCV was detected in two (1.33%) of the 150 samples obtained from the asymptomatic children. Of the norovirus-GII.3-positive children, 60% had blood type O, but only 17.29% of the symptomatic patients had blood type O, indicating that type O individuals could be at higher risk of GII.3 infection. However, due to the limited number of individuals in this study, further studies with a larger number of subjects should be conducted to verify this hypothesis.

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.

[Genome amplification and sequence analysis of human bocavirus 2].

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.

[Molecular and epidemiological study on among children under 5 years old in Nanjing].

OBJECTIVE: To study the infected information, clinical symptom and molecular epidemiological characteristics of HuCV infection among children under 5 years old in Nanjing. METHODS: In Nanjing Children's Hospital of Nanjing Medical University from July 2010 to June 2011, we collected 428 stool specimens from children with diarrhea and 428 asymptomatic controls. Human Calicivirus were tested by using RT-PCR. Then we sequenced the nucleic acid of PCR amplifications and identified the genotype and gene group of prevalent strains. RESULTS: 63 (14.72%) out of 428 stool samples were detected as HuCV. 58 were norovirus and 5 were sapovirus, while GII-4 2006b was the predominant strain of NoV. In the 428 control samples, 19 samples were positive for calicivirus, there were 8 NoV and 13 SaV (Including 3 co-infection cases). CONCLUSION: Human caliciviruses with different genotypes circulated among children in Nanjing,and GII. 2006b is the dominant genotype.

Genome characterization of a novel porcine bocavirus.

Using a high-throughput DNA sequencing method, one DNA sequence (contig01006), suspected to belong to a novel porcine bocavirus (PBoV), was found with a high rate of detection (19.6 %) in fecal samples from healthy piglets. Moreover, a novel PBoV (tentatively named PBoV3C) with a nearly complete genome sequence (5235 bp) was identified. PBoV3C exhibits typical genome characteristics of bocaviruses and shows the highest genomic sequence identity (78 % to 81 %) to PBoV3A/B (PBoV3/4-UK) and PBoV3D/E (PBoV3/4-HK), respectively. Phylogenetic and recombination analysis indicated high diversity, prevalence and complexity among the PBoVs. The phospholipase A2 (PLA2) site of VP1 and the secondary structure of VP2 of PBoV3C were also analyzed. Additionally, we propose a uniform method of PBoV nomenclature based on the VP1 gene.

Simultaneous detection of seven enteric viruses associated with acute gastroenteritis by a multiplexed Luminex-based assay.

Rapid and broad diagnostic methods are needed for the identification of viral agents of gastroenteritis. In this study, we used Luminex xMAP technology to develop a multiplexed assay for the simultaneous identification of major enteric viral pathogens, including rotavirus A (RVA), noroviruses (NoVs) (including genogroups GI and GII), sapoviruses (SaV), human astrovirus (HAstV), enteric adenoviruses (EAds), and human bocavirus 2 (HBoV2). The analytical sensitivity allowed detection of 10(3) (EAds, HBoV2, and RVA) and 10(4) (NoV GI and GII, SaV, and HAstV) copies per reaction mixture. Compared to conventional PCR, the Luminex-based assay yielded greater than 75% sensitivity and 97% specificity for each virus, and the kappa correlation for detection of all viruses ranged from 0.75 to 1.00. In conclusion, this multiplexed Luminex-based assay provides a potentially rapid, high-throughput, and maneuverable diagnostic tool for major viral pathogens associated with gastroenteritis.

High prevalence of human bocavirus 2 and its role in childhood acute gastroenteritis in China.

BACKGROUND: Acute gastroenteritis (AGE) is one of the leading causes of death in children worldwide. Human bocavirus 2 (HBoV2) was recently identified in stool samples and is involved in the pathogenesis of AGE, but the current data were too limited to clarify this issue. OBJECTIVE: We conducted a case-control study on 632 children with diarrhea and 162 healthy controls in Lanzhou, China, to assess the role of HBoV2 in gastroenteritis. STUDY DESIGN: Viruses known or suspected to be agents of AGE, including RV, HucV, AdV, AstV, and HBoVs, were detected. Viral loads of HBoV2 were quantified by Real-time PCR. RESULTS: HBoV2 was detected in 129 (20.4%) and 20 (12.3%) of the gastroenteritis and control samples, respectively. The association between HBoV2 and gastroenteritis was weaker (OR = 1.269, CI= 0.704-2.288) than that between gastroenteritis and RV, HucV, AdV, or AstV, as determined by multivariate logistic regression analysis. The data also suggested that infection with HBoV2 did not exacerbate the clinical symptoms of gastroenteritis. Mean HBoV2 viral load in the case and control groups was fewer than 55 copies/ml extract. CONCLUSIONS: HBoV2 exhibit different epidemiological features from HBoV1 and HBoV3. The data presented herein do not support a causative role for HBoV2 in AGE, despite its high prevalence in stool samples.

Genetic diversity of noroviruses in Chinese adults: potential recombination hotspots and GII-4/Den Haag-specific mutations at a putative epitope.

Little is known about the role of noroviruses (NVs) in sporadic cases of acute gastroenteritis in adults. The GII-4 NVs are currently the globally dominant genotype with diverse genetic makeups. The mechanism(s) underlying the persistence and rapid evolution of the viruses are not yet clear. In this study we collected 547 specimens from adult of >14 years of age with acute gastroenteritis in Beijing, China from September 2007 to Febraury 2008. NVs were screened and sequenced to determine their genotypes. Bioinformatics methods were used to detect NV recombination and their breakpoints. The residue variations of the capsid proteins between GII-4/Den Haag and previous predominant variants of GII-4 were compared to identify mutations that are likely important for current epidemic wave. Putative epitopes were predicted based upon the crystal structure. 106 (19.4%) NVs were identified among 547 specimens. While GII-4 remains predominant, at least six other genotypes were observed. Two recombinant types were identified with both predicted breakpoints locating within the 24-27 bp region upstream the start codon of ORF2. We found the emergent mutations H414P/Q of the capsid protein are specific for GII-4/Den Haag and this site lies within a predicted antibody-binding epitope. Our data demonstrated that NVs were an important cause of acute gastroenteritis in Chinese adults. The shared breakpoints identified in the GI and GII recombinants imply the presence of recombination hotspots in NVs. The mutations at residue 414 and its location within a putative antigenic epitope suggested a possible mechanism that may allow GII-4 NVs to escape from herd immunity.

[A novel method for multiplex detection of gastroenteritis-associated viruses].

To develop and optimize a simultaneous detection method of RotavirusA, Norovirus GI, GII, Sapovirus, human astrovirus, enteric adenoviruses and HBoV2 with GenomeLab GeXP analysis system. The sensitivity was verified to be 10(4) copies/microL with plasmids containing the viral targets in triplicate on different days, and no cross-reaction with enterovirus71, human Parechovirus and PicobirnavirusII was observed. Finally, we successfully developed a high throughout, rapid and maneuverable multiplex RT-PCR assay for simultaneous detection of seven viruses related with viral gastroenteritis, which provide a novel method for the molecular diagnosis of diarrhea-associated virus.

[Molecular and epidemiological study on viral diarrhea among infants in Lanzhou].

OBJECTIVE: To study the epidemiologic characteristics of viral diarrhea in children under 5 years old in Lanzhou, understand the four major virus in children of distribution. METHODS: In the first hospital of Lanzhou university from Jul 2009 to Jun 2010,we collected 290 stool specimens from children with diarrhea and 114 asymptomatic controls. Rotavirus was detected by ELISA,further strain characterization was carried out by nested PCR. The human calicivirus, astrovirus, adenovirus were detected by RT-multiplex PCR and PCR. RESULTS: At least one of the four viral agents was found in 60% of the specimens. Rotavirus, human calicivirus, adenovirus, and astrovirus were identified in 39.31%, 11.38%, 10.69%, and 4.83% in 290 specimens respectively. Rotavirus G3 was the most prevailing serotype, P [8] was the most common genotype. In the 114 control samples, 7 sample was positived for calicivirus, 5 samples were positived for human adenovirus and 1 sample was positived for astrovirus. CONCLUSION: The results indicated clearly the impact of viral agents causing diarrhea and the importance of long-term systematic surveillance.

[Detection and molecular characterization of human parechovirus (HPeV) in children with acute gastroenteritis].

OBJECTIVE: To study HPeV from stool samples of children with acute gastroenteritis under 5 years old. METHODS: We conducted a real-time PCR to detect HPeV from stool samples and to amply VP1 sequence by nested RT-PCR to identify HPeV type. RESULTS: The results showed that 27 of 306 (8.82%) children with acute gastroenteritis were infected HPeV. 11 strains were typed. 9 strains HPeV1, both HPeV2 and HPeV4 was 1 strain. HPeV was mostly identified in autumn season with a peak in July. HPeV seemed relevant in children >2 years old. The range of nucleotide identity between all isolated strains with reference strains was 79%-92%. CONCLUSION: Epidemiology characteristic of HPeV in Jilin was concordance with that of reports. HPeV3 wasnt detected. It's significant to conduct the large scale and long-term surveillance of HPeV.

Development of a real-time PCR assay for detecting and quantifying human bocavirus 2.

Human bocavirus 2 (HBoV2) is a parvovirus that has been recently identified in stool samples from children. Any association between the virus and clinical disease is unclear. A rapid, reliable diagnostic method is necessary to address this issue. In this study, we developed a sensitive and specific HBoV2 quantitative real-time PCR assay that targets the HBoV2 NP-1 gene, based on the TaqMan method. The assay could reproducibly detect 10 copies of a recombinant DNA plasmid containing a partial region of the HBoV2 genome, with a dynamic range of 8 log units (10(1) to 10(8) copies). A clinical evaluation detected HBoV2 in 85 (24.6%) of 345 children with gastroenteritis, with viral loads ranging from 1.67 × 10(2) to 4.27 × 10(9) copies per ml of stool specimen.

Identification and nearly full-length genome characterization of novel porcine bocaviruses.

The genus bocavirus includes bovine parvovirus (BPV), minute virus of canines (MVC), and a group of human bocaviruses (HBoV1-4). Using sequence-independent single primer amplification (SISPA), a novel bocavirus group was discovered with high prevalence (12.59%) in piglet stool samples. Two nearly full-length genome sequences were obtained, which were approximately 5,100 nucleotides in length. Multiple alignments revealed that they share 28.7-56.8% DNA sequence identity with other members of Parvovirinae. Phylogenetic analyses indicated their closest neighbors were members of the genus bocavirus. The new viruses had a putative non-structural NP1 protein, which was unique to bocaviruses. They were provisionally named porcine bocavirus 1 and 2 (PBoV1, PBoV2). PBoV1 and PBoV2 shared 94.2% nucleotide identity in NS1 gene sequence, suggesting that they represented two different bocavirus species. Two additional samples (6V, 7V) were amplified for 2,407 bp and 2,434 bp products, respectively, including a partial NP1 gene and the complete VP1 gene; Phylogenetic analysis indicated that 6Vand 7V grouped with PBoV1 and PBoV2 in the genus of bocavirus, but were in the separate clusters. Like other parvoviruses, PBoV1, PBoV2, 6Vand 7V also contained a putative secretory phospholipase A(2) (sPLA(2)) motif in the VP1 unique region, with a conserved HDXXY motif in the catalytic center. The conserved motif YXGXF of the Ca(2+)-binding loop of sPLA2 identified in human bocavirus was also found in porcine bocavirus, which differs from the YXGXG motif carried by most other parvoviruses. The observation of PBoV and potentially other new bocavirus genus members may aid in molecular and functional characterization of the genus bocavirus.

[Analysis on epidemiologic characteristics of viral diarrhea among infants in Taiyuan, Shanxi province, 2007-2008].

OBJECTIVE: To study the epidemiologic characteristics of virus-induced acute diarrhea in children under 5 years old in Taiyuan, Shanxi province. METHODS: Stool specimens and clinical data were collected from 346 inpatients with acute diarrhea from children less than 5 years old. Rotavirus-positive specimens were identified by ELASA kit. Calicivirus and astrovirus were detected by reverse transcription-polymerase chain reaction (RT-PCR). Adenovirus was done by polymerase chain reaction (PCR). RESULTS: Of the 346 specimens, the percentage of samples with Rotavirus, Calicivirus, Astrovirus, and Adenovirus was 40.8%, 7.5%, 6.4% and 3.2%. Among 141 rotavirus positive samples, serotype G1 (42.6%) was the predominant strain. More than 95% of viral diarrhea patients under hospitalization occurred among children younger than 2 years. CONCLUSION: Rotavirus is the major pathogen contributing to the acute diarrhea. The disease generally peaks at autumn/winter. The predominant rotavirus strain circulated was G1P[8].

[Detection of Aichi virus in stool samples from children in Lanzhou].

OBJECTIVE: To identification and analysis Aichi virus from diarrhea and normal children in Lanzhou, and discuss the relationship between Aichi virus and Infant Diarrhea. METHODS: According to the literature published data, Using RT-PCR method to amplified Aichi virus 3CD fragment and the positive products were sequenced and determined, and made the alignment analysis between the nucleotide sequences of the amplified fragment with the known sequence of this virus. RESULTS: There was 1 case detection of Aichi virus in the 46 hospitalized children with diarrhea and 299 children with diarrhea out-patients specifically, Overall detection rate was 0.06%, and there was no Aichi virus was detected in normal control children. 2 viral 3CD gene and the known reference strains of nucleotide sequences were 97%, while phylogenetic analysis showed that genotype of 2 viral belongs to the B. CONCLUSIONS: There existed B Genotype of Aichi virus in China, and more research is needed to clarified the etiology and epidemiology of Aichi virus characteristics.

[Sequence the complete sequence of bocavirus I with SISPA-PCR].

OBJECTIVE: To sequence the complete sequence of bocavirus I with sequence independent single primer amplification (SISPA-PCR). METHODS: To exclude the co-effection samples, all clinical samples of diarrhea cases were screened with special primers of rotavirus, astrovirus, adenovirus, calicivirus and bocavirus I. The virus were enriched through ultracentrifugation. Other nucleic acids, such as human and bacteria genomes, were degradated by DNase I and RNase. DNA of bocavirus was Amplificated with SISPA-PCR, then purificated, cloned and sequenced. The sequences were alighmented in nr with blastn and assembled with DNAstar. RESULTS: A 4834bp sequence of bocavirus I were assembled. CONCLUSION: SISPA-PCR is an economical and efficient technique for sequence a virus complete genome.

[Pathogens of high incidence of other infectious diarrhea in Guangdong Province, from November 2008 to January 2009].

From November 2008 to January 2009, a sharp increase of diarrhea in children in Guangdong province appeared, we randomly collected 53 stool specimens from out-patient children with dirrhea in 3 major hospitals (Guangzhou City Children's Hospital, Shenzhen Baoan District Maternal and Child Health Hospital, First Affiliated Hospital of Shantou University). Rotavirus and calicivirus were screened by ELISA and RT-PCR. We found 29 cases of rotavirus infection with diverse serotypes. Only four cases were identified as calicivirus infection. The result indicated that rotavirus was a major pathogen of this high incidence of diarrhea from November 2008 to January 2009 in Guangdong Province.

Epidemiological study of human calicivirus infection in children with gastroenteritis in Lanzhou from 2001 to 2007.

Stool specimens were collected from 1,195 young children with acute diarrhea in Lanzhou, China, from 2001 to 2007. RT-PCR was used to detect human calicivirus (HucV). One hundred seventeen specimens were found positive for HucV. The infection rate was noticeably higher during 2006-2007 compared to the other years studied. Ninety-six specimens were sequenced to determine the genotypes of HucV. Eighty-six were norovirus and 10 were sapovirus, while GII/4 was the predominant strain of NV, followed by GII/3. The subtype of NV GII/4 changed from the Farmington Hills strain to the Bristol strain, and then to the Hunter strain and variant 2006b strain, over time. Variant 2006b has become the major epidemic strain in Lanzhou and should be closely monitored in the coming years.

Viral agents associated with acute gastroenteritis in children hospitalized with diarrhea in Lanzhou, China.

BACKGROUND: Gastroenteritis is a major cause of childhood morbidity and mortality worldwide. Rotavirus, human caliciviruses (HucV), adenovirus, and astrovirus are recognized as common etiologies of acute gastroenteritis. OBJECTIVES: To use antigen detection and molecular methods to determine the viral etiology of childhood diarrhea in Lanzhou, China, 2005-2007. STUDY DESIGN: 544 stool specimens were collected from children hospitalized with diarrhea. ELISA, RT-PCR, or PCR were used to detect viruses commonly causing diarrhea. RESULTS: Group A rotavirus, norovirus, sapovirus, astrovirus, and adenovirus, were detected in 54.0%, 9.2%, 1.1%, 3.3%, and 4.4%, respectively. No group B or group C rotaviruses were detected. The relative contribution of these viruses changed greatly over 2 years. The percentage of rotavirus and adenovirus dropped from 61.2% and 5.4% to 47.6% and 1.4%, whereas HucV increased from 5.0% to 15.0%. G1 and P[8] were the predominant rotavirus strains, and P[6] was detected for the first time in this area. The predominant norovirus strain changed from GII3 to GII4, and the subtypes of GII4 changed from the Hunter strain to the variant 2006b strain. CONCLUSIONS: The distribution of viruses and genotypes of individual viruses causing gastroenteritis in Lanzhou, China changed greatly during 2005-2007.