Adolescent ; Child ; China ; epidemiology ; Disease Outbreaks ; Female ; Humans ; Male ; Mamastrovirus ; drug effects ; genetics ; pathogenicity ; Phylogeny ; Viral Proteins ; genetics

Astroviruses are widely detected in pigs but their detection in wild boars is rather sporadic. In this study, astroviruses were detected in organ homogenates of wild boars by applying nested reverse transcriptase polymerase chain reaction, and the typing was carried out by phylogenetic analysis. Overall, 30/200 (15.0%) homogenates were positive for astroviruses. Genetic typing revealed that of 13 amplicons analyzed, 8 were typed as porcine astrovirus lineage 2 (PAstV-2), 2 as lineage 4 (PAstV-4), 2 identical sequences were grouped with chicken astrovirus, and 1 sequence belonged to a bat astrovirus lineage. This first identification of chicken and bat astroviruses in wild boars indicates interspecies transmission.
Avastrovirus ; Chickens ; Reverse Transcriptase Polymerase Chain Reaction ; Sus scrofa ; Swine

Human astrovirus (HastV) is recognized as one of the leading causes of acute viral diarrhea in infants. The HastV non-structural protein, nsPla, and C-terminal protein, nsPla/4, contain various conserved functional domains,and may play an important role in virus replication, transcription and the virus-host interactions of HastV. This study used an E. coli system to investigate the expression of nsPla and nsPla/4 proteins. Firstly,the nsPla and nsPla/4 genes of HAstV-1 were cloned into the prokaryotic expression vector,PGEX-4T-1, to build the PGEX-4T-1a and PGEX-4T-la/4 fusion protein plasmids. Then, the recombinant plasmids were transformed into Escherichia coli BL21 (DE3) and induced with isopropyl-β-D-thiogalactopyranoside (IPTG). The optimal expression conditions of the two fusion proteins were identified and then analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting, respectively. The results showed that the pGEX-4T-la fusion protein was maximally expressed at 30 °C after 12 hours of induction with 1.0 mM IPTG. The pGEX-4T-la/4 fusion protein was maximally expressed at 20 °C after 8 hours of induction with 0.5 mM IPTG. Western blot analysis showed that the two fusion proteins specificity reacted with the anti-nsPla and anti-GST monoclonal antibodies, respectively. This study successfully obtained the HAstV non-structural protein, nsP1a, and its C-terminal protein nsP1a/4 protein using an E. coli system. This novel study lays the foundation for future research into the pathogenic mechanisms of human astrovirus and the functions of its non-structural protein.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Mamastrovirus ; genetics ; metabolism ; Viral Nonstructural Proteins ; genetics ; metabolism

OBJECTIVETo investigate the etiological characteristics of human rotavirus (HRV), human calicivirus (HuCV), human astrovirus (HAstV) and human enteral adenovirus (HAdV) in Ningxia province during 2011.

METHODSStool specimen was collected from acute diarrhea case of Ningxia during 2011. HRV was detected by ELISA and serotype/genotype identified on those RT-PCR positive specimens. HuCV, HAstV and HAdV were detected by RT-PCR.

RESULTSIn this study, a total of 690 specimens were detected, with the infection rates of HRV, HuCV, HAstV and HAdV as 2.17%, 21.74%, 3.19% and 6.52%, respectively. Co-infections were found in 4.20% of all the samples being tested. Among 15 HRV positive cases, serotypes G1, G3 and P[4] were the most predominant strains.

CONCLUSIONChildren who were under 2 years of age were the majority among patients infected by diarrhea viruses while HuCV was recognized as the main pathogen responsible for the viral diarrhea cases in Ningxia, 2011.

Adenoviruses, Human ; isolation & purification ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Caliciviridae ; isolation & purification ; Child ; Child, Preschool ; China ; epidemiology ; Diarrhea ; virology ; Humans ; Infant ; Infant, Newborn ; Mamastrovirus ; isolation & purification ; Middle Aged ; Rotavirus ; isolation & purification ; Young Adult

The prevalence of human astroviruses was tested in patients with acute gastroenteritis by using conventional duplex reverse transcription (RT)-PCR and electrophoresis. Diarrheal fecal samples were collected from 9,597 patients at local hospitals in Seoul. The prevalence of astroviruses was 1.0% (94/9,597 patients; mostly infants), and that of sapoviruses was 0.1% (14/9,597 patients). Age- and gender-wise analyses were carried out on 29 astrovirus-positive patients having complete information on file regarding their age, gender, and other particulars. The results were higher in patients of ages 0 to 14 yr, and 69.0% of the astrovirus-positive patients were females, of which 69.2% were infants (0 to 12 months), and 61.5% were 1-4 yr old. Notably, in the case of 5 to 78-yr-old acute gastroenteritis patients, 100% were females.
Acute Disease ; Adolescent ; Adult ; Age Factors ; Aged ; Astroviridae Infections/complications/*epidemiology/virology ; Child ; Child, Preschool ; DNA, Viral/analysis ; Feces/virology ; Female ; Gastroenteritis/complications/*diagnosis ; Humans ; Infant ; Male ; Mamastrovirus/genetics/*isolation & purification ; Middle Aged ; Prevalence ; Reverse Transcriptase Polymerase Chain Reaction ; Sapovirus/genetics/isolation & purification ; Sex Factors ; Young Adult

OBJECTIVETo establish a one-step four multiplex reverse transcription polymerase chain reaction (RT-PCR) method based on Homo-Tag Assisted Non-Dimer System (HAND) system for simultaneous detection of 4 diarrhea viruses of rotavirus, astrovirus, norovirus and sapovirus.

METHODSPrimers were designed according to the conserved genome sequence of the 4 viruses and the homologous tail sequences were added to the 5' end. The multiplex RT-PCR system was constructed by optimizing the PCR parameters such as the concentration of universal tag primer and genome-specific Homo-Tailed primers. The specificity, stability and sensitivity of the method were evaluated systematically.

RESULTSThe 4 multiplex RT-PCR methods based on HAND system was established successfully. Specificity analysis showed no cross reaction between the 4 diarrhea viruses. The sensitivity analysis showed detection limits for rotavirus, astrovirus, norovirus and sapovirus of 48, 1.92, 9.6 and 48 pg per reaction, respectively.

CONCLUSIONThe established HAND system-based multiplex RT-PCR assay allows simple, rapid, specific, sensitive, and stable for detection of the 4 common diarrhea viruses at low costs and is suitable for application in general medical laboratories.

Astroviridae ; genetics ; isolation & purification ; Diarrhea ; virology ; Feces ; virology ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Norovirus ; genetics ; isolation & purification ; RNA, Viral ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Rotavirus ; genetics ; isolation & purification ; Sapovirus ; genetics ; isolation & purification

Human astrovirus (HAstV) is one of the leading causes of actue virual diarrhea in infants. HAstV-induced epithdlial cell apoptosis plays an important role in the pathogenesis of HAstV infection. Our previous study indicated that HAstV non-structural protein nsPla C-terminal protein nsPla/4 was the major apoptosis functional protein and probably contained the main apoptosis domains. In order to screen for astrovirus encoded apoptotic protien, nsPla/4 and six turncated proteins, which possessed nsPla/4 protein different function domain ,were cloned into green fluorescent protein (GFP) vector pEG-FP-N3. After 24-72 h transfection, the fusion protein expression in BHK21 cells, was analysis by fluorescence microscope and Western blot. The results indicated seven fusion proteins were observed successfully in BHK21 cell after transfected for 24 h. Western blot analysis showed that the level of fusion protein expressed in BHK21 cells was increased significantly at 72h compared to 48h in transfected cells. The successful expression of deletion mutants of nsPla/4 protein was an important foundation to gain further insights into the function of apoptosis domains of nsPla/4 protein and it would also provide research platform to further confirm the molecule pathogenic mechanism of human astrovirus.
Amino Acid Motifs ; Astroviridae Infections ; virology ; Humans ; Mamastrovirus ; genetics ; metabolism ; Mutation ; Sequence Deletion ; Transfection ; Viral Nonstructural Proteins ; chemistry ; genetics ; metabolism

A molecular study of intestinal samples from 21 broiler flocks with a history of enteritis revealed that 23.8% and 14.3% were positive for chicken astrovirus (CAstV) and avian rotavirus (ARV), respectively. CAstV and group A ARV were simultaneously detected in only one broiler flock. Birds in this group developed the significant intestinal lesions characterized by frothy contents, paleness, and thin intestinal walls. In this report we present an unusual case of runting stunting syndrome (RSS) with a history of high mortality and growth retardation in broiler chickens. We also make the first identification of CAstV and group A ARV in broiler chickens in Korea.
Animals ; Astroviridae Infections/diagnosis/epidemiology/*veterinary/virology ; Avastrovirus/classification/*genetics/isolation & purification/metabolism ; *Chickens/growth & development ; Enteritis/diagnosis/pathology/veterinary/virology ; Intestines/pathology/virology ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases/*diagnosis/epidemiology/virology ; Republic of Korea/epidemiology ; Rotavirus/classification/*genetics/isolation & purification/metabolism ; Rotavirus Infections/diagnosis/epidemiology/*veterinary/virology

Human astrovirus (HAstV) is a major cause of acute diarrhea among children, resulting in outbreaks of diarrhea and occasionally hospitalization. Improved surveillance and application of sensitive molecular diagnostics have further defined the impact of HAstV infections in children. These studies have shown that HAstV infections are clinically milder (diarrhea, vomiting, fever) than infections with other enteric agents. Among the 8 serotypes of HAstV identified, serotype 1 is the predominant strain worldwide. In addition to serotype 1, the detection rate of HAstV types 2 to 8 has increased by using newly developed assays. HAstV is less common compared with other major gastroenteritis viruses, including norovirus and rotavirus; however, it is a potentially important viral etiological agent with a significant role in acute gastroenteritis. A better understanding of the molecular epidemiology and characteristics of HAstV strains may be valuable to develop specific prevention strategies.
Child ; Diarrhea ; Disease Outbreaks ; Gastroenteritis ; Hospitalization ; Humans ; Mamastrovirus ; Molecular Epidemiology ; Norovirus ; Pathology, Molecular ; Sprains and Strains ; Vomiting

Human astrovirus (HAstV) is a major cause of acute diarrhea among children, resulting in outbreaks of diarrhea and occasionally hospitalization. Improved surveillance and application of sensitive molecular diagnostics have further defined the impact of HAstV infections in children. These studies have shown that HAstV infections are clinically milder (diarrhea, vomiting, fever) than infections with other enteric agents. Among the 8 serotypes of HAstV identified, serotype 1 is the predominant strain worldwide. In addition to serotype 1, the detection rate of HAstV types 2 to 8 has increased by using newly developed assays. HAstV is less common compared with other major gastroenteritis viruses, including norovirus and rotavirus; however, it is a potentially important viral etiological agent with a significant role in acute gastroenteritis. A better understanding of the molecular epidemiology and characteristics of HAstV strains may be valuable to develop specific prevention strategies.
Child ; Diarrhea ; Disease Outbreaks ; Gastroenteritis ; Hospitalization ; Humans ; Mamastrovirus ; Molecular Epidemiology ; Norovirus ; Pathology, Molecular ; Sprains and Strains ; Vomiting