Detection and genetic characterization of Mamastrovirus 5 from Brazilian dogs

Abstract Mamastrovirus 5 (MAstV5), belonging to the Astroviridae (AstV) family, previously known as canine astrovirus or astrovirus-like particles, has been reported in several countries to be associated with viral enteric disease in dogs since the 1980s. Astroviruses have been detected in fecal samples from a wide variety of mammals and birds that are associated with gastroenteritis and extra enteric manifestations. In the present study, RT-PCR was used to investigate the presence of MAstV5 in 269 dog fecal samples. MAstV5 was detected in 26% (71/269) of the samples. Interestingly, all MAstV5-positive samples derived from dogs displaying clinical signs suggestive of gastroenteritis, other enteric viruses were simultaneously detected (canine parvovirus, canine distemper virus, canine coronavirus, canine adenovirus and canine rotavirus). Based on genomic sequence analysis of MAstV5 a novel classification of the species into four genotypes, MAstV5a-MAstV5d, is proposed. Phylogenetic analyses based on the ORF2 amino acid sequences, samples described herein grouped into the putative genotype 'a' closed related with Chinese samples. Other studies are required to attempt the clinical and antigenic implications of these astrovirus genotypes in dogs.

Characteristics of molecular epidemiology of human astrovirus in hospitalized and outpatient children with acute diarrhea / 中华传染病杂志

Objective To compare the molecular epidemic characteristics of human astrovirus (HAstV) between outpatient and hospitalized children with acute diarrhea,and to investigate the relationship between HAstY infection and diarrhea in children.Methods A total of 298 cases were randomly collected from hospitalized children from January 2008 to December 2010 in Children's Hospital of Fudan University,and 360 specimens were collected from outpatients with acute diarrhea from August 2010 to July 2011.Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect rotavirus (RV),human calicivirus (HuCV),HAstV and human adenovirus (HAdV).H AstV genotype was determined by gene sequencing and phylogenetic analysis.Results Epidemiology of HAstV in hospitalized children was as follows:among the included 298 samples,HAstV was detected in 27.2％ (81/298) of the patients,compared with 33.9％ (42/124),33.8％ (25/74) and 14.0％ (14/100),respectively from 2008 to 2010.HAstV diarrhea occurred throughout the year and peaked in January,March,and April.95.1％ (77/81) of the infected children were 0-35 months old.All the episodes of HAstV were mixed with other diarrhea virus infection.Molecular epidemiology of HAstV in outpatient children with diarrhea was as follows:the overall incidence of HAstV was 1.9 ％ among the 360 cases (7/360).The seasonal distribution of HAstV's gastroenteritis showed a peak in November.All the outpatient children were 0-35 months old.Three cases were single infection with HAstV and the others were coinfection with RV (3 cases) or HAdV (1 case).All of the detected HAstV,either in inpatients or outpatients,belonged to HAstV-1.Conclusions The detection rate of HAstV in hospitalized children is significantly higher than that in outpatients.Most HAstV infections in hospitalized children are ascribed to nosocomial infections.Most episodes of HAstV infection were accompanied with other diarrhea viruses infection.HAstY single infection is seen in outpatient children while the detection rate is very low,implying that HAstV co-infection with other viruses plays a main role in diarrhea in most instances.

Comparing the multiplex RT-PCR method and liquichip technology in the detection of diarrhea-related virus / 中华检验医学杂志

Objective To compare the detection efficiency between multiplex RT-PCR method and liquichip technology for screening the viral etiological agents of diarrhea.Methods The development of the multiplex RT-PCR method.A total of 107 feces samples from patients who suffered from diarrhea and attended to Zhujiang Hospital of Southern University from September 2013 to February 2014 were collected and tested in parallel by both multiplex RT-PCR and xTAG Gastrointestinal Pathogen Panel ( xTAG GPP) for Adenovirus, Norovirus genogroupⅠandⅡ, as well as by both multiplex RT-PCR and monoplex RT-PCR for Astrovirus and Sapovirus.To evaluate the sensitivity and specificity of multiplex RT-PCR, xTAG GPP and monoplex RT-PCR were used as reference.Kappa coefficient test was used to evaluate the consistency among the methods.The detection limit and accuracy of multiplex RT-PCR were evaluated by detection of serial dilution of positive plasmids and products sequencing for the five viral agents.Results The multiplex RT-PCR showed high consistency with xTAG GPP and monoplex RT-PCR, in which Kappa value was 0.885 and 1.000 respectively( P=0.000 ).Compared to xTAG GPP, the sensitivity and specificity of the multiplex RT-PCR were at average of 80.8%( 21/26 ) and 100%( 295/295 ) respectively.The detection limit and accuracy of multiplex RT-PCR were 104 copies /μl-106 copies/μl.Conclusion The high consistency indicated that both the multiplex RT-PCR and xTAG GPP are useful as a special,sensitive, high throughput and rapid diagnostic tools for the detection of the major viral pathogens related to diarrhea in clinical laboratory.

Distribution and clinical features of gastrointestinal virus infection in infants with acute diarrhea / 中华临床感染病杂志

Objective To analyze the distribution and clinical characteristics of gastrointestinal virus infection in infants with acute diarrhea.Methods Stool samples and clinical data were collected from 900 infants (≤5 years old) with acute diarrhea in outpatient department of Beilun District People' s Hospital during July 2012 and July 2013.Specimens were tested for 5 gastrointestinal virus including group A/B/C rotavirus (RV),adenovirus (AdV),astrovirus (AstV),sapovirus (SV) and norovirus (NV) by the multiplex PCR assay.Chi-square test was performed to compare the positive rates of virus infection among children with different genders and ages.Results Among 900 stool samples,369 were positive of gastrointestinal virus,of which 291 were positive for single virus and 78 for mixed virus.In single virus infection,NV was detected with the highest positive rate of 19.4％ (4.9％ for G Ⅰ and 14.6％ for G Ⅱ),followed by RV-A (8.2％),SV (2.9％),AstV (1.0％) and AdV (0.8％).RV-B and C type were not found.In 78 cases with mixed infections,RV-A plus NV infection was the most common one with a prevalent rate of 5.8％.The positive rate in age group ≤2 years old was 51.0％,which was significantly higher than that of age group ＞ 2-5 years old (22.1％,x2 =70.404,P ＜ 0.01).In 369 children with positive gastrointestinal virus,fever was present in 24.1％,and vomit in 35.2％ of children.Fever,vomit and fever plus vomit was more common symptoms in children with mixed infections (x2 =17.878,21.869 and 14.155,P ＜ 0.01).Conclusion NV and RV-A are the most common pathogens in infants with acute diarrhea in Beilun district,especially in children younger than 2 years old.

Identification of Astrovirus type 1 as the pathogen that caused an outbreak of neonatal diarrhea / 中华传染病杂志

Objective To identify the pathogen of an acute epidemic gastroenteritis outbreak in newborn room. Methods Forty five samples were collected from 38 newborn patients at the peak of a diarrhea outbreak, which happened in a newborn room in Inner Mongolia Autonomous Region during December 2008 to February 2009. The presence of rotavirus antigen, Adeno-like virus antigen and Astrovirus antigen were detected by enzyme-linked immunosorbent assay (ELISA). The presence of Astrovirus RNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR) with universal primer of Astrovirus. Thirteen samples positive for Astrovirus nucleic acid were analyzed by sequencing and phylogenetic tree. Four samples positive for both Astrovirus antigen and Astrovirus nucleic acid were observed by immune electron microscopy. Results Both rotavirus antigen and Adeno-like virus antigen were negative in 45 faecal samples. Thirty samples were positive for Astrovirus antigen when checked by ELISA, which resulted in a positive rate of 66.7%. Thirty-one samples were positive for Astrovirus RNA when check by RT-PCR, which resulted in a positive rate of 68.9%. The genotype results confirmed all patients were infected with genotype 1 Astrovirus. The gene sequences of thirteen samples were compared with reference strains of Astrovirus type 1 in GeneBank and the homology rate of nucleotide sequence was 90.9 %- 96.3 %. The homology rate of intra these thirteen sample was 94.7%-100.0%. Four positive samples were randomly selected and observed by immune electron microscopy and a large amount of Astrovirus particles were found in two of these samples. Conclusion Genotype 1 Astrovirus is the pathogen of this diarrhea outbreak in newborn.

Characteristics of astrovirus infection and typing characteristics analysis among children hospitalized with diarrhea in Tianjin / 中华检验医学杂志

Objective To study the pathogenic prevalence and genotypes of astrovirus among children under 5 years old hospitalized with diarrhea in Tianjin. Methods A total 837 stool specimens were collected from children with diarrhea hospitalized in Tianjin children's hospital from May 2008 to April 2009. Astrovirus antigens were detected using ELISA and the postive specimens were inoculated in CaCo-2cells. After the CPE caused by virus were observed, the total RNA of virus was extracted, then the genomc fragments of the strains were amplified by using RT-PCR and confirmed by sequencing of the RT-PCR products. Detection of rotavirus was employed by Colloidal Gold Device. Results Astrovirus antigen was found positive in 3.0% of the patients. The coinfection rate of astrovirus and rotavirus was 0. 7% (6/837).Ninety-six persent of children with astrovirus diarrhea were younger than 2 years of age, Forty-eight persent of children with astrovirus diarrhea were younger than 6 months. The astrovirus infections occurred mainly between August 2008 and April 2009. Of the 21 astrovirus positive specimens, 11 cases were successfully identified by RT-PCR and they were all serotype 1. Conclusion Astrovirus is a major cause of nonbacterical diarrhea between 2008 and 2009 in Tianjin, and the predominant serotype is type 1.