Molecular characterization of human astroviruses isolated in Brazil, including the complete sequences of astrovirus genotypes 4 and 5

Summary. Human astroviruses (HAstV) are recognized as an important cause of gastroenteritis in young children worldwide. This study describes the molecular characteristics of astroviruses isolated in Brazil, using RT-PCR and molecular sequencing of segments of all three viral ORFs. Genetic analysis of a 348-nucleotide segment from ORF 2 demonstrated that the Brazilian isolates belong to HAstV genotypes 1 to 5 and 8. ORF 1b sequences displayed a high degree of nucleotide identity even between different genotypes, which disfavours HAstV genotyping in this region. ORF 1a sequence analysis classified all Brazilian samples as genogroup A. The complete sequences of HAstV genotype 4 (putative serotype 4) and genotype 5 (putative serotype 5) were determined for the first time.     

Comparisons of envelope through 5B sequences of infectious bronchitis coronaviruses indicates recombination occurs in the envelope and membrane genes

A 1.78 kb sequence, including the E, M, 5a and 5b genes, and the intergenic region between the M and 5a genes, of six US strains of infectious bronchitis (corona)virus (IBV) were sequenced and compared to the published sequences for two additional strains. The overall identities as determined through pairwise analyses of nucleotide sequences of the entire 1.78 kb region ranged from 90 to 99%, with the 5b open reading frame (ORF) having the greatest identity (94–99%) while the identities of the E, 5a and M ORFs ranged from 87 to 100%. Nucleotide sequencing of recent field isolates from Alabama (Ala1) and California (Cal3) revealed distinct shifts in homology in the M gene, indicating two apparent recombination events between the Holland 52/Mass41-like strain and an Ark-like strain, both origins of commonly used vaccine strains. Putative sites of recombination could also be identified in both the E and M ORFs of laboratory strains of IBV.     

Molecular epidemiology and genetic diversity of human astrovirus in South Korea from 2002 to 2007

The present study was conducted to survey the prevalence and genotypic distribution of human astrovirus (HAstV) circulating in South Korea. Of 160,027 patients with acute gastroenteritis, 2,057 (1.3%) were positive for HAstV antigen. We determined the genotypes of 187 HAstV strains collected from laboratories across the country. Genetic analysis revealed genotype 1 to be the most prevalent, accounting for 72.19% of the strains, followed by genotypes 8 (9.63%), 6 (6.95%), 4 (6.42%), 2 (3.21%) and 3 (1.60%). Our findings indicate that HAstV is less common but, even so, a potentially important viral agent of gastroenteritis in South Korea, with significant genetic diversity among circulating HAstV strains.     

Astrovirus infection in hospitalized children: Molecular,clinical and epidemiological features

BackgroundAstrovirus (HAstV) is a common viral pathogen that causes gastroenteritis worldwide. It is classified into eight classical human types (HAstV-1/8) and seven other less prevalent types, described as HAstV VA1, VA2, VA3, VA4, MLB-1, MLB-2 and MLB-3. During outbreaks, the elderly and children are the most affected, and the spread of the virus is associated with person-to-person contact, food ingestion and contaminated water.ObjectivesThe aim of the present study was to investigate the prevalence of infection and genetic diversity of HAstV strains. Samples were collected from children with acute gastroenteritis admitted to a large pediatric hospital during a surveillance period of three years (2008–2011) in Belém city, Pará State, Amazon Region, Northern Brazil.Study designScreening and genotyping tests were conducted using RT-PCR to detect the classical and non-classical HAstV types using specific primers. A semi-nested RT-PCR protocol was developed to improve viral detection in samples with a low viral load.ResultsThe overall positivity observed in this study was 3.9% (19/483). The age distribution showed a high prevalence of positive cases in children under one year old (5.3%). We found vomiting associated with 75% of the positive cases, fever with 82.3%, and dehydration with 76.9%. Most patients with positive cases demonstrated two to five days of diarrhea, two to three episodes of vomiting during hospitalization, and three bowel movements per day. Co-infection with HAstV and norovirus was observed in three cases (15.8%), and no pattern of seasonality or any relationship between the HAstV positivity rate and climate variables was observed. Eighteen positive samples (94.7%–18/19) were genotyped based on the ORF 2 region, and the greatest prevalence was of HAstV-1a (66.6%–12/18), followed by HAstV-2 (22.2%–4/18, comprising two type-2b and two type-2c genotypes), HAstV-3c (5.6%–1/18) and HAstV-4c (5.6%–1/18). No non-classical types were detected in the clinical samples analyzed.ConclusionsThe present study showed that although HAstV infections occur at low frequency, they are involved in severe pediatric cases of acute gastroenteritis presenting with a high diversity of strains, including the lineages 3c and 4c, which were never before detected in Brazil.     

Analysis of the ORF2 of human astroviruses reveals lineage diversification,recombination and rearrangement and provides the basis for a novel sub-classification system

Canonical human astroviruses (HAstVs) are important enteric pathogens that can be classified genetically and antigenically into eight types. Sequence analysis of small diagnostic regions at either the 5′ or 3′ end of ORF2 (capsid precursor) is a good proxy for prediction of HAstV types and for distinction of intratypic genetic lineages (subtypes), although lineage diversification/classification has not been investigated systematically. Upon sequence and phylogenetic analysis of the full-length ORF2 of 86 HAstV strains selected from the databases, a detailed classification of HAstVs into lineages was established. Three main lineages could be defined in HAstV-1, four in HAstV-2, two in HAstV-3, three in HAstV-4, three in HAstV-5 and two in HAstV-6. Intratypic (inter-lineages) ORF2 recombinant strains were identified in type 1 (1b/1d) and type 2 (2c/2b) with distinct crossover points. Other potential intratypic recombinant strains were identified in type 3, type 5 and type 6. In addition, a type-1b strain with a large insertion (~600 bp) of heterologous RNA in the N-terminal region and a type-6 strain with a large RNA rearrangement in the hypervariable region were identified. The classification scheme was integrated in a novel nomenclature system suitable for designation of HAstV strains.     

Development of real-time and nested RT-PCR to detect astrovirus and one-year survey of astrovirus in Jiangmen City, China

Human astroviruses (HAstVs) are one of the major viral agents of acute gastroenteritis (AGE) in all age groups, especially in young children. In this study, new one-step real-time RT-PCR and nested RT-PCR assays were developed to detect HAstVs. HAstVs were identified in 46 (8.75%) of 526 stool samples in Jiangmen City over 1 year, including 43 (9.15%) of 470 children and 3 (5.4%) of 56 adults, and HAstV-1 was the most predominant strain. This finding suggests that HAstVs infections are common in Jiangmen City, China. Further detailed molecular epidemiological studies are required for understanding the prevalence of HAstVs infection and gaining knowledge about the circulating genotypes.     

Identification of new subtype of astrovirus type 3 from an infant with diarrhea in Wuhan, China

Human astrovirus is one of the important causes for viral gastroenteritis in young children. In previous study where we examined the molecular epidemiology of human astrovirus (HAstV) infection in infants in Wuhan City, we isolated and identified a new subtype (WH1859) of HAstV genotype 3 from an infant with diarrhea. The sequence analysis of this strain showed that the complete region of ORF2 of WH1859 contains 2385-bp of nucleotides that encode 795 amino acids. Because WH1859 strain has the identity of less than 95% with the distance of more than 0.05 to the reference strains of HAstV-3, WH1859 represents a distinct subtype within HAstV-3 strains. Further studies are needed to determine the role of this new subtype strain of HAstV in viral gastroenteritis among young children.     

Characterisation of a South African human astrovirus as type 8 by antigenic and genetic analyses.

Human astroviruses (HAstV) can, on the basis of immunoassays using type-specific rabbit antisera, be classified into eight serotypes that correlate with genotypes. Very few isolates of HAstV type 8 have been described and there is a paucity of data available with regard to the antigenic and genetic relationships between HAstV type 8 (HAstV-8) and HAstV types 1 (HAstV-1) to 7 (HAstV-7). A wild-type HAstV from a South African paediatric patient with diarrhoea was analysed antigenically, by immune electron microscopy and enzyme immunoassay, and genetically in selected regions of the ORF1a, ORF1b and ORF2 and characterised as a HAstV-8. This HAstV-8 strain exhibited greatest homology with HAstV-4 in the 5' end of the capsid gene and ORF1a and 1b, and greatest homology with HAstV-5 in the 3' end of the capsid region. This study confirms, by both antigenic and genetic analyses, that HAstV-8 represents a distinct antigenic and genotype and is the first report of a HAstV-8 from a hospitalised paediatric patient with diarrhoea in southern Africa.     

Genetic Heterogeneity and Recombination in Human Type 2 Astroviruses

Novel lineages of human astrovirus (HAstV) types 2, 2c, and 2d have been identified. Upon sequencing of the 3′ end of the genome, the type 2c and 2d HAstVs were found to be open reading frame 1b (ORF1b)-ORF2 recombinant, with ORF1b being derived from type 3 and type 1 HAstVs, respectively. An ORF2 interlineage recombinant strain, 2c/2b, was also identified.     

Detection of GII‐4/2006b variant and recombinant noroviruses in children with acute gastroenteritis,South Korea

Norovirus (NoV), a single‐stranded, positive RNA virus, is an important etiologic agent of acute gastroenteritis in children worldwide. In this study, a total of 434 fecal samples collected from 434 children with acute gastroenteritis in Seoul, between September 2007 and July 2008 were tested to determine the molecular epidemiology of NoVs and characterize recombinant strains by using RT‐PCR followed by sequencing. Of the 434 specimens, NoV, rotavirus, and adenovirus were detected in 155 (35.8%), 72 (16.6%), and 19 specimens (4.3%), respectively. NoV GI was detected in 7 specimens (1.6%) and GII in 148 (34.1%) specimens. Phylogenetic analysis of capsid sequences in the GII‐positive specimens revealed the presence of the following strains: GII‐4, 111 (75.0%); GII‐3, 35 cases (23.6%); GII‐6b, 1 case; and GII‐16, 1 case. Most of the GII‐4 strains were grouped with the GII‐4/2006b variant with 98–100% nucleotide identity. Eleven strains were identified as recombinant (GII‐4/GII‐3 in 10 cases and GII‐b polymerase/GII‐16 capsid in 1 case) by sequencing based on the RdRP and capsid genes. The putative recombination point in the recombinant strains was the ORF‐1/ORF2 overlap, located at nucleotide 5,046 with reference to Lordsdale. In conclusion, GII‐4/2006b variants were detected predominantly and a new recombinant strain (GII‐4/GII‐3) was found in the Korean children with gastroenteritis. Continuous monitoring of the genetic diversity of NoVs is important to determine the trend of the predominant genotype and new recombinant strain. J. Med. Virol. 82:146–152, 2010. © 2009 Wiley‐Liss, Inc.     

Genetic analysis of the hypervariable region of the human astrovirus nsP1a coding region: design of a new RFLP typing method

Human astroviruses (HAstV) are causative agents of viral gastroenteritis worldwide. A hypervariable region (HVR) is located close to the C-terminus of the nsP1a, and recent data support the involvement of the HVR-containing nonstructural protein in viral RNA replication processes, suggesting a correlation between variability in this region and pathogenic properties. The HVR of the C-terminal nsP1a coding region of 104 wild-type and reference isolates of HAstV was sequenced. A phylogenetic analysis was performed to identify different genotypes, and a restriction fragment length polymorphism (RFLP) method was designed. An extensive nucleotide and deduced amino acid sequence variability was observed, as well as many insertions and deletions that retained the reading frame. The resultant phylogenetic tree supported the subdivision of HAstV into the two previously described major genetic groups, genogroup A and B, and the identification of 12 genotypes (9 within genogroup A, and 3 within genogroup B), which could be identified by RFLP. A correlation analysis was performed between genotype information and viral load using information from 35 clinical samples. Significant differences were observed between the viral load in clinical samples and certain HAstV genotypes that belonged to the same serotype, confirming the influence of C-terminal nsP1a variability on the viral replication phenotype. The use of the new RFLP typing method based on the HVR of the C-terminal nsP1a coding region by diagnosticians would help to understand the relationship between different genotypes and the severity of the gastroenteritis.