The aim of this study was to produce E^rns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified E^rns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-E^rns was constructed based on the gene sequence of BVDV-1 NADL strain. The E^rns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-E^rns into CHO cells. The expression and purification of the E^rns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the E^rns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified E^rns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the E^rns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the E^rns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log₁₀ was induced in rabbits. In this study, purified BVDV E^rns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.
Rabbits ; Animals ; Cricetinae ; Cricetulus ; CHO Cells ; Antibodies, Viral ; Diarrhea Viruses, Bovine Viral/genetics* ; Antibodies, Monoclonal/genetics* ; Diarrhea ; Viral Vaccines/genetics*

OBJECTIVE: To explore the clinical characteristics and genetic basis for three children with Congenital chlorine diarrhea (CCD). METHODS: Three children with CCD who attended the Affiliated Children's Hospital of Capital Pediatric Institute from June 2014 to August 2020 were selected as the research subjects. Peripheral blood samples of the three children and their parents were collected for genetic testing. And the results were verified by Sanger sequencing. RESULTS: The clinical manifestations of the three children have included recurrent diarrhea, with various degrees of hypochloremia, hypokalemia and refractory metabolic alkalosis. Genetic testing revealed that the three children have all carried variants of the SLC26A3 gene, including homozygous c.1631T>A (p.I544N) variants, c.2063_1G>T and c.1039G>A (p.A347T) compound heterozygous variants, and c.270_271insAA(p.G91kfs*3) and c.2063_1G>T compound heterozygous variants. Sanger sequencing confirmed that all of the variants were inherited from their parents. CONCLUSION The variants of the SLC26A3 gene probably underlay the CCD in these children. Above finding has enriched the spectrum of SLC26A3 gene variants.
Humans ; Child ; Chlorine ; Genetic Testing ; Hypokalemia/genetics* ; Homozygote ; Diarrhea/genetics* ; Mutation

OBJECTIVE: To explore the genetic basis of a child with Very early onset inflammatory bowel disease (VEOIBD). METHODS: A female child who had presented at the Children's Hospital of Fudan University on May 23, 2018 due to occurrence of diarrhea and fever 6 days after birth was selected as the study subject. Clinical data of the child was collected. Family-based whole-exome sequencing (WES) was carried out. Candidate variant was verified by Sanger sequencing and PCR of the patient and her parents. RESULTS: The child had developed the symptoms 6 days after birth, with main manifestations including diarrhea, fever, failure to thrive, rectovestibular fistula and hypothyroidism. An enterostomy was performed at the age of 3.5 months due to severe intestinal adhesion and obstruction. Based on her clinical manifestations, colonoscopic finding, and results of biopsies, she was diagnosed with VEOIBD in conjunct with congenital hypothyroidism. Replacement treatment of levothyroxine was given since one month of age. Family-based WES revealed that the child has harbored compound heterozygous variants of the DUOX2 gene, namely c.2654G>T (p.R885L) and c.505C>T (p.R169W), in addition with a heterozygous c.301C>T (p.R101W) variant of the IL10RA gene. Re-analysis of the WES data revealed that the patient also had a 333 bp deletion spanning exon 1 of the IL10RA gene (Chr11: 117857034_117857366). CONCLUSION For patients with VEOIBD, genetic testing is recommended. Presence of additional DUOX2 gene variants might have exacerbated the clinical symptoms in this patient. Above finding has facilitated genetic counseling and prenatal diagnosis for this family, and raised clinicians' awareness of this rare disease.
Female ; Humans ; Infant ; Pregnancy ; Diarrhea ; Dual Oxidases/genetics* ; Exons ; Failure to Thrive ; Inflammatory Bowel Diseases/genetics*

OBJECTIVETo develop a multiplex real-time PCR for the detection of Salmonella invasion protein A gene (invA), enterotoxigenic Escherichia coli (ETEC) heat-labile I enterotoxin gene (elt), and Shigella or enteroinvasive E. coli (EIEC) invasive plasmid antigen H gene (ipaH).

METHODSUnder the optimized reaction conditions of the multiplex real-time PCR, invA, elt, and ipaH were determined in 10-fold series of dilution of DNA extracted from Salmonella enterica serovar Typhimurium, ETEC 44815 strain and Shigella F301 strain. The three genes were examined in 90 fecal samples from diarrhea patients using the multiplex real-time PCR. When PCR-positive samples were found, the target strains were isolated and identified.

RESULTSThe detectable concentration for this multiplex real-time PCR was 10 CFU/microl for Shigella F301 strain, 10(2) CFU/microl for S. enterica serovar Typhimurium and ETEC 44815 strain, respectively. Out of 90 fecal samples from diarrhea patients, thirteen were found positive for elt gene (14.4%), and five were found positive for ipaH gene (5.6%). Three E. coli strains positive for elt gene and four E. coli strains positive for ipaH gene were isolated successfully from the PCR-positive samples mentioned above. The detection of invA, elt and ipaH genes was completed in 10 h, which included an enrichment period of 6 h.

CONCLUSIONThe multiplex real-time PCR assay can detect invA, elt, ipaH simultaneously in a single reaction, moreover, it can detect for virulence genes in strains of Salmonella, ETEC, and Shigella or EIEC and screen these pathogens in fecal specimens from patients with diarrhea with a high specificity.

DNA, Bacterial ; analysis ; Diarrhea ; microbiology ; Escherichia coli ; genetics ; Feces ; microbiology ; Humans ; Polymerase Chain Reaction ; methods ; Salmonella ; genetics ; Shigella ; genetics

OBJECTIVETo analyze the etiological characteristics, virulence genes and plasmids that carrying diarrhea-causing Proteus mirabilis and to assess their relationship with drug resistance and pathogenicity.

METHODSProteus mirabilis coming from six different sources (food poisoning, external environment and healthy people) were analyzed biochemically, on related susceptibility and pulsed-field gel electrophoresis (PFGE). Virulence genes were detected by PCR. Plasmids were extracted and sequenced after gel electrophoresis purification.

RESULTSThe biochemical characteristics of Proteus mirabilis from different sources seemed basically the same, and each of them showed having common virulence genes, as ureC, rsmA, hpmA and zapA. However, the PFGE patterns and susceptibility of these strains were different, so as the plasmids that they carried. Plasmid that presented in the sequenced strain showed that the 2 683 bp length plasmid encodes qnrD gene was associated with the quinolone resistance.

CONCLUSIONEtiological characteristics and molecular characteristics of Proteus mirabilis gathered from different sources, were analyzed. Results indicated that traditional biochemical analysis and common virulence gene identification might be able to distinguish the strains with different sources. However, PFGE and plasmids analysis could distinguish the sources of strains and to identify those plasmids that commonly carried by the drug-resistant strains. These findings also provided theoretical basis for further study on the nature of resistance and pathogenicity in Proteus mirabilis.

Diarrhea ; microbiology ; Drug Resistance, Bacterial ; Genes, Bacterial ; Humans ; Plasmids ; genetics ; Proteus mirabilis ; genetics ; pathogenicity ; Virulence Factors ; genetics

Objective: To explore the characteristics of viral infections in children with diarrhea in Beijing from 2018 to 2022. Methods: Real-time PCR and enzyme-linked immunosorbent assay were used to detect viral nucleic acid of Norovirus (NoV), Sappovirus (SaV), Astrovirus (AstV), Enteric Adenovirus (AdV) or antigen of Rotavirus (RV) in 748 stool samples collected from Beijing Capital Institute of Pediatrics from January 2018 to December 2021. Subsequently, the reverse transcription PCR or PCR method was used to amplify the target gene of the positive samples after the initial screening, followed by sequencing, genotyping and evolution analysis, so as to obtain the characteristics of these viruses. Phylogenetic analysis was performed using Mega 6.0. Results: From 2018 to 2021, the overall detection rate of the above five common viruses was 37.6%(281/748)in children under 5 years old in Beijing. NoV, Enteric AdV and RV were still the top three diarrhea-related viruses, followed by AstV and SaV, accounting for 41.6%, 29.2%, 27.8%, 8.9% and 7.5%, respectively. The detection rate of co-infections with two or three diarrhea-related viruses was 4.7% (35/748). From the perspective of annual distribution, the detection rate of Enteric AdV was the highest in 2021, while NoV was predominant in the other 4 years. From the perspective of genetic characteristics, NoV was predominant by GⅡ.4, and after the first detection of GⅡ.4[P16] in 2020, it occupied the first two gene groups together with GⅡ.4[P31]. Although the predominant RV was G9P[8], the rare epidemic strain G8P[8] was first detected in 2021. The predominant genotypes of Enteric AdV and AstV were Ad41 and HAstV-1. SaV was sporadic spread with a low detection rate. Conclusion: Among the diarrhea-related viruses infected children under 5 years of age in Beijing, the predominant strains of NoV and RV have changed and new sub-genotypes have been detected for the first time, while the predominant strains of AstV and Enteric AdV are relatively stable.
Child, Preschool ; Humans ; Infant ; Beijing/epidemiology* ; Diarrhea/epidemiology* ; Feces ; Norovirus/genetics* ; Phylogeny ; Rotavirus/genetics* ; Virus Diseases/epidemiology* ; Viruses/genetics*

Child ; Child, Preschool ; Diarrhea ; virology ; Female ; Genotype ; Humans ; Infant ; Male ; Rotavirus ; classification ; genetics ; isolation & purification ; Seasons

OBJECTIVETo investigate the etiologic value of diarrheagenic E. coil harboring genomic O island 28(OI-28) containing five putative virulence genes (Z0608, Z0609, Z0615, Z0634 and Z0635), which were related to RTX (Repeat in toxin) toxin family isolated from children with diarrheal disease in Taiyuan.

METHODSIn the study, 257 fecal samples from children with diarrheal disease collected in Shanxi Children's Hospital. Diarrheagenic E. coli and enteropathogenic bacteria were isolated and identified by conventional bacterial culture and typing specific diarrheagenic E. coli (EPEC, EIEC, ETEC and EHEC) diagnostic serum, while diarrheagenic E. coli harboring genomic 01-28 containing five putative virulence genes (Z0608, Z0609, Z0615, Z0634 and Z0635) were detected by PCR and DNA southern blot hybridization.

RESULTS206 strains (80.16%) of enteropathogenic bacteria were detected from 257 children with diarrhea disease, containing 149 strains (57.98%) of diarrheagenic E. coli and 57 strains(22.18%) of other entero-pathogenic bacteria. Among 3 strains (2.01%) of EPEC, 2 strains (1.34%) of ETEC, 2 strains (1.34%) EHEC were detected by typing specific serum, while all of the 142 strains (95.30%) isolated were suspected to be diarrheagenic E. coli. 21 strains (14.09%) of diarrheagenic E. coil harboring genomic O1-28 containing five putative virulence genes (Z0608, Z0609, Z0615, Z0634 and Z0635) were detected by polymerase chain reaction and DNA southen blot hybridization, 8 strains (5.37%) of diarrheagenic E. coli containing only one genomic OI-28 virulence gene, 2 strains (1.34%) of diarrheagenic E. coli containing two genomic OI-28 virulence gene. 21 children with diarrhea diseases caused OI-28-harboring E. coli containing five important putative virulence genes were among 0 to 3 years old (80.95%). These children correlating with OI-28-harboring E. coli did not present special clinical symptoms or signs.

CONCLUSIONThe diarrheagenic E. coil harboring genomic OI-28 was one of the important etiology for children with diarrheal disease in summer season.

Child ; China ; Diarrhea ; microbiology ; Escherichia coli ; genetics ; pathogenicity ; Escherichia coli Infections ; complications ; Genes, Bacterial ; Humans ; Virulence

OBJECTIVETo study the prevalence, clinic features and epidemiologic characteristics of human adenovirus diarrhea in Nanjing.

METHODS730 stool specimens were collected from children with diarrhea in Nanjing Children's Hospital of Nanjing Medical University from June 2009 to June 2011. Polymerase chain reaction (PCR) was employed to detect human adenovirus. The total positive PCR products were typed by nest-PCR or multiple PCR.

RESULTS21 samples (21/730) were positive for human adenovirus of all 730 samples from June 2009 to June 2011 and enteric HAdV-41 is the predominant stain.

CONCLUSIONEnteric HAdV-41 and non-enteric adenovirus were the major etiological agents of viral diarrhea among infants and children in Nanjing from 2009 to 2011. We should take the long-term systematic surveillance seriously.

Adenoviruses, Human ; classification ; genetics ; Child, Preschool ; China ; Diarrhea ; virology ; Female ; Humans ; Infant ; Male ; Phylogeny

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.
Diarrhea ; virology ; Genomics ; Human bocavirus ; genetics ; isolation & purification ; physiology ; Humans ; Meningitis ; virology ; Respiratory Tract Diseases ; virology