[Analysis of CYP17A1 gene mutation in a child patient with 17 alpha-hydroxylase/17, 20-lyase deficiency].

OBJECTIVE: To analyze CYP17A1 gene mutations in a child patient with 17 alpha-hydroxylase/17, 20-lyase deficiency (17OHD), and to review characteristics of CYP17A1 gene mutations in Chinese patients with 17OHD. METHODS: Clinical data were collected. PCR and DNA sequencing were performed to detect mutations in the patient. RESULTS: The patient has presented classical features of 17OHD including hypertension, hypokalemia, decreased sex hormones and plasma cortisol, and elevated blood adrenocorticotrophic hormone. A compound heterozygous mutation c.987C>A and c.985del was detected in the CYP17A1 gene, which resulted in two premature stop codons at positions 328 and 417. CONCLUSION: A compound mutation, c.987C>A and c.985del, has been identified in a patient with 17OHD. Among CYP17A1 gene mutations identified in Chinese patients, missence mutations have been most common, and exons 5 and 8 have been the mutation hotspots.

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.

Analysis and characterization of the complete genome of a member of a new species of kobuvirus associated with swine.

A virus belonging to a new species in the genus Kobuvirus, family Picornaviridae, was first isolated in 2008 from apparently healthy pigs in Hungary and China. We report the complete genome sequence and the genetic organization of the novel porcine kobuvirus strain Y-1-CHI, which was identified in China. The RNA genome of strain Y-1-CHI contains 8210 nucleotides (nt) and has an organization similar to that of other picornaviruses. The full-length nucleotide sequence of Y-1-CHI was 88.62%, 58.66%, and 48.86% identical to those of S-1-HUN, U-1, and Aichi virus, respectively. No positive results were found in 454 stool samples from children with acute gastroenteritis. Dendrograms indicated that Y-1-CHI and S-1-HUN are most closely related to each other and belong to the same species. Our results suggest that members of this novel species have the typical genome characteristics of members of the genus Kobuvirus and may be distributed globally in swine.

Human bocavirus in children hospitalized for acute gastroenteritis: a case-control study.

BACKGROUND: Human bocavirus (HBoV) was recently discovered in children with respiratory tract disease and gastroenteritis. The causative role of HBoV in human gastroenteritis remains uncertain, and, to our knowledge, no previous case-control study has studied the relationship between HBoV and gastroenteritis. METHODS: We conducted a case-control study that examined stool samples from 397 children with diarrhea and from 115 asymptomatic control subjects. HBoV was detected using polymerase chain reaction. Real-time polymerase chain reaction was used to quantify the HBoV loads in case and control groups. Common enteric viruses were examined using enzyme-linked immunosorbent assays, polymerase chain reaction, and reverse-transcription polymerase chain reaction. RESULTS: At least 1 viral agent was discovered in 60.2% of cases. HBoV was detected in 14 samples, and 9 were coinfected with either rotavirus (7 of 14 samples) or human calicivirus (2 of 14). Many (8 [57.1%] of 14) of the HBoV infections occurred during September-December 2006. Most (12 [85.7%]) of the HBoV-infected children were 7-18 months of age. The percentage of children with HBoV infection did not differ significantly between case patients and control subjects (3.5% vs. 3.5%), and the statistical analysis did not support a correlation between HBoV infection and more-severe clinical symptoms. The viral load differences between the 2 groups were not statistically significant (P = .09, by log-normal Student's t test). In addition, the VP1/VP2 partial gene of HBoV from case patients and control subjects showed minimal sequence variation. CONCLUSIONS: A single genetic lineage of HBoV was revealed in persons in China. Despite its high prevalence in stool samples, our study does not support a causative role of HBoV in gastroenteritis.

Emergence of the GII4/2006b variant and recombinant noroviruses in China.

Noroviruses are an important cause of acute gastroenteritis. Increasing data showed that the GII-4 strains are predominant worldwide and new GII-4 variants emerge every 1-2 years causing major epidemics. Surveillance of gastroenteritis in hospitalized children under 5 years of age in China is described. Among 1,110 specimens, 114 (10.3%) were positive for noroviruses, which was higher than adenoviruses (7.6%), astroviruses (3.5%), and sapoviruses (0.9%) and only lower than group A rotaviruses (40.6%). Thirty-eight of the 114 positive norovirus cases were co-infected with other enteric viruses. Five norovirus genotypes (GI-2, GI-4, GII-3, GII-4, and GII-14) were detected, with GII-4/2006b the most predominant type (64.9%). The reported recombinant of GII-3 capsid and GII-4 polymerase described previously was also detected frequently and a recombinant of GII-14 capsid and GII-6 polymerase was found for the first time. This study suggests that continual surveillance focusing on strain variation and dynamic change is important for understanding the epidemiology and development of a strategy for disease control and prevention.

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.

[Detection and typing assay of norovirus in acute hospitalizations among children less than 5 years old from 2008 to 2009 in Lulong, Hebei province].

OBJECTIVE: To investigate the molecular epidemiologic characteristics and genotypes of norovirus in children less than 5 years of age in Lulong area from 2008 to 2009. METHODS: 325 stool specimens and epidemiological data from hospitalized children with diarrhea less than 5 years of age were collected. Rotavirus was detected by using the ELISA kit. Norovirus, adenovirus and astrovirus were detected by multiple reverse transcription-polymerase chain reaction (RT-PCR). Partial norovirus strains were sequenced and the tree was conducted by using the phylogenetic analyses. RESULTS: Norovirus was detected in 37 out of 325 (11.3%) specimens,ranked only second to rotavirus (48.6%), and higher than adenovirus (6.5%) and astrovirus (4.3%). Norovirus predominantly infected children less than 2 years of age and the season peak of norovirus occurred in November. Phylogenetic analysis showed that the predominant strain was the GII. 4/2006b variant. Interestingly, a novel unreported GII-4 variant was found in this study. CONCLUSION: Norovirus was one of the most important pathogens causing acute gastroenteritis from 2008 to 2009 in Lulong area. The GII. 4/2006b vairant was still the predominant strain. It is important to keep on monitoring the novel GII. 4 variant.

[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.

[Genetic diversity of porcine sapoviruses from Lulong county in China].

Porcine sapoviruses (SaVs), which belong to the family Caliciviridae, have been considered potential zoonotic agents for human infection, and several cases have been reported in Asian countries. In this study, a total of 200 porcine fecal samples collected from Lulong county of China were tested. Among 200 samples, porcine sapoviruses were detected by RT-PCR in 17 samples (8.5%) showing their circulation in China. 14 out of 17 positive sapovirus strains were genetically related to the genogroup III (GIII) and were further divided into three different clusters or genotypes according to the phylogenetic analysis. In addition, the remaining three sapovirus strains belonged to GVII (one strain) and a potential novel genogroup (two strains) according to the phylogenetic analysis and the nucleotide identity and amino acid identity. These data suggested the genetic diversity of porcine sapoviruses in China.

[Study on the epidemiological of rotavirus diarrhea in Lulong in 2008-2009].

OBJECTIVE: To analyze the feature of epidemiological of rotavirus diarrhea in Lulong county, Hebei province. METHODS: 426 stool specimens were collected from inpatant with acute diarrhea from children less than 5 years old. Rotavirus-positive specimens were identified by ELISA kit. G/P typing assays were confirmed with multiplex seminested RT-PCR. RESULTS: Rotavirus was detected in 202 of 426 (47.42%) specimens. Genotyping of rotavirus showed that G3 was predominant (57.9%), followed by Gmix (16.3%), G9 (14.9% ), G1 (7.9%), G4 (1%), G2 (0.5%), P-genotyping showed that P [8], Pmix, P [4], P [9], type were found in 58.4%, 28.7%, 6.9% and 1% respectively. The most common G/P combination identified was G3P [8]. CONCLUSION: Group A rotaviruses was a major pathogen of diarrhea in Children in Lulong. G3P [8] was the predominant type in 2009, Gmix and Pmix abound, and G9 serotypes has become the second predominant after G3 strain in the region.

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.

[Outbreaks of noroviral gastroenteritis and their molecular characteristics in China, 2006 - 2007].

OBJECTIVE: To acknowledge the epidemiology of gastroenteritis outbreaks caused by noroviruses and their genotypes. METHODS: Epidemiologic data and specimens were collected from 19 gastroenteritis outbreaks. 201 specimens were detected for norovirus, rotavirus, astrovirus, adenovirus and sapovirus by RT-PCR methods and PCR products were sequenced. Sequence alignment and phylogenetic analysis were performed by Clustal X 1.83 and MEGA 4.0 programs. RESULTS: Noroviruses were one of the most predominant pathogens causing viral gastroenteritis outbreaks (12 of 19 outbreaks, accounting for 63.2%). Variant GII-4/2006b was the predominant strain responsible for 11 of the 12 NV-associated outbreaks. Other genotypes would include GII-17, GII-6 and GII-3. The NV-associated gastroenteritis outbreaks occurred mainly in winter and spring between December 2006 and April 2007. These gastroenteritis outbreaks caused by noroviruses would involve all age groups in various locations. Meantime, 2 out of 12 outbreaks were caused by norovirus or other viruses. In addition, multiple viruses and multiple genotypes of noroviruses were found in the same outbreak. CONCLUSION: Noroviruses were one of the most major pathogens causing gastroenteritis outbreaks while GII-4/2006b variant was identified as the predominant strain in China.

[Study on effect of enzyme linked immunosorbent assay kits of norovirus].

OBJECTIVE: Effects of RIDASCREEN Norovirus (C 1401) 3rd Generation kit (R-biopham AG, darmstadt, Germany) and IDEIA NLV kit (DAKOCytomation., Ely, UK) were compared for detecting human norovirus (HuNV) in fecal sample. METHODS: The performance of the ELISA was compared with that of the reverse transcription polymerase chain reaction (RT-PCR) by testing a panel of 308 fecal samples collected from patients involved in outbreaks of gastroenteritis in Chang Chun and Guang Zhou. Gene sequencing was performed to positive samples tested by RT-PCR to determine genotype compared with standard sequences. RESULTS: RT-PCR is gold standard, RIDASCREEN Norovirus (C 1401) 3rd Generation kit had a high sensitivity of 96.10% but a specificity of 93.51%, and Dako kit had a low sensitivity of 95.83% but a high specificity of 95.76%. CONCLUSION: RIDASCREEN Norovirus (C 1401) 3rd Generation kit is more Satisfactory for a preliminary screening.

[Analysis of epidemiologic feature and genetic sequence of Sapovirus in China].

To investigate epidemiologic feature and genetic variance of Sapovirus among children in China, fecal specimens were collected from children under 5 years old with acute diarrhea from Feb 2006 to Jan 2007 in nine provinces including Anhui, Fujian et al. A total of 1,110 fecal samples were detected for Sapovirus by reverse transcriptase-PCR (RT-PCR). Ten samples (0.9%) were positive for Sapovirus. The PCR products were then sequenced and analysed by phylogenetic tree. The results indicated that the detected Sapovirus strains were classified into two genogroups and three genotypes, including G I/1, G I/3, G II/3.

[Molecular epidemiology of rotavirus among children under 5 years old hospitalized for diarrhea in China].

OBJECTIVE: To study molecular epidemiology of Rotavirus among children under 5 years of age in china. METHODS: Stool specimens were collected from 4047 inpatients under 5 years of age with diarrhea in our 9 hospital-based surveillance sites from January 2006 to December 2007 following the WHO Rotavirus surveillance protocol. Rotavirus were detected by ELISA, Further strain characterization of rotavirus was carried out with RT-PCR. RESULTS: A total of 4047 stool samples were collected and 3862 of total stools were tested among which 1700 was positive. The Rotavirus positive rate is 44.0%. A peak admission of rotavirus diarrhea was observed from November to next January. More than 95.4% of viral diarrhea patients occurred in their first 2 years. The incidence rates of rotavirus diarrhea were highest in 12-17 months of age. The most common rotavirus strain was P[8]G3(58.3%); followed by P[8] G1(22.1%), P[4]G1 (3.0%), P[8]G9 (2.4%). G4 was not detected in this study. The four common strains were 80.8% in the world. CONCLUSION: Rotavirus diarrhea was an important infectious disease among children under 5 years of age in China. Safe and effective rotavirus vaccines for the prevention of rotavirus diarrhea and reduction of treatment costs are of significant importance to China.

[A method of detect the virus which caused diarrhea].

OBJECTIVE: Building a method which can examines virus pathogenic in gastroenteritis excrement specimen. METHODS: Choosing six positive specimens which tested in our laboratory, include adenovirus, calicivirus, rotavirus, bocavirus, astrovirus and enterovirus. Through sequence-independent single primer amplification(SISPA) constructs a gene bank. Looks up the viral gene fragment in gene bank. RESULTS: Obtaining corresponding viral acid sequence in six specimens. CONCLUSION: This research can examine enterovirus and the virus which cause diarrhea, It make a foundation for further studies the viral cause of disease which the examination not yet discovered at present.

[Detection of human parechovirus in children hospitalized for acute gastroenteritis].

OBJECTIVE: To detect human parechovirus (HPeV) from stool samples of hospitalized children for acute gastroenteritis of undetectable etiology. METHODS: We conducted a real-time PCR to detect HPeV. RESULT: The results showed that 24 of 99 (24%) children with gastroenteritis of undetectable etiology were detected with HPeV. Four known HPeV types (HPeV1, 3, 4, 6) were detected in the present study. HPeV1 (50%) was frequently identified as the predominant strain and follow by HPeV3 (25%), HPeV4 (8.3%) and HPeV6 (4.2%). We were unable to type 3 samples. CONCLUSION: HPeV was prevalent in hospitalized children for acute gastroenteritis of undetectable etiology in China. Further study is needed for clarifying the role of HPeV in gastroenteritis.