Diversity of Group A Human Rotavirus Types Circulating over a 4-Year Period in Madrid, Spain

Rotavirus infections can be diagnosed in stool samples by serological and molecular methods. We developed a novel reverse transcriptase PCR (RT-PCR) method for the amplification of rotavirus RNA and a reverse hybridization assay on a strip to detect amplimers and identify the specific G and P genotypes present in human stool specimens. An additional aim was to permit specific identification of the rotavirus G1P[8] strain, used in the Rotarix vaccine. Novel broad-spectrum PCR primers were developed for both VP4 and VP7, permitting the amplification of a wide range of rotavirus genotypes. Primer sets comprise mixtures of defined primer sequences. For the identification of G and P genotypes, two reverse hybridization strip assays were developed. Both the VP4 and the VP7 strip contain universal probes for the detection of VP4 and VP7 sequences, irrespective of the G or P genotype. The VP4 strip contains type-specific probes for P[4], P[6], P[8], P[9], and P[10]. The VP7 strip contains type-specific probes for G1, G2, G3, G4, G5, G6, G8, and G9. In addition, probes to distinguish between wild-type G1 and G1 vaccine strain sequences were present. Testing by analysis of multiple reference strains confirmed that both RT-PCR methods allowed the detection of a broad spectrum of genotypes. RT-PCR for VP7 was more sensitive than RT-PCR for VP4, but all samples identified as positive for rotavirus antigen by an enzyme-linked immunosorbent assay (ELISA) were also positive for both VP4 and VP7. The high specificity of the reverse hybridization method was confirmed by sequence analysis as well as by type-specific PCR, and the vaccine strain could also be specifically identified. The reverse hybridization method permits accurate identification of mixed infections with different genotypes. Rotavirus genotypes for which no type-specific probes were present on the strip were adequately identified by the universal detection probes. The assay was formally validated by analyses of specificity, sensitivity, precision, accuracy, and robustness. In a panel of 149 ELISA-positive stool samples, comparison with conventional type-specific RT-PCR methods revealed the superiority of the novel method, mainly in cases of mixed rotavirus infections. This novel method permits highly accurate detection and identification of human rotavirus infections in stool samples. This validated assay could be useful for large-scale epidemiological and clinical trials.

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“…In general, all recent studies confirm that the diversity of rotavirus strains is much greater than previously recognized (8,14,15,21,42).…”

Section: Discussionsupporting

confidence: 63%

Detection and Genotyping of Human Rotavirus VP4 and VP7 Genes by Reverse Transcriptase PCR and Reverse Hybridization

et al. 2009

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“…This was the first study to investigate the role of currently recognized viruses, especially NoVs, sapoviruses, and Aichi viruses, in infections of children. As was the case in a previous study (36), this two-year surveillance also showed that the vast majority (75.8%) of the children with gastroenteritis were less than 2 years old. Almost half of the infections (43.7%) were due to one or several viral agents, mainly to RVs and NoVs.…”

Section: Discussionsupporting

confidence: 50%

Acute Infantile Gastroenteritis Associated with Human Enteric Viruses in Tunisia

et al. 2008

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, investigated the incidence and the clinical role of various enteric viruses responsible for infantile gastroenteritis in 632 Tunisian children presenting in dispensaries (380 children) or hospitalized (252 children) for acute diarrhea. At least one enteric virus was found in each of 276 samples (43.7%). A single pathogen was observed in 234 samples, and mixed infections were found in 42 samples. In terms of frequency, rotavirus and norovirus were detected in 22.5 and 17.4% of the samples, respectively, followed by astrovirus (4.1%), Aichi virus (3.5%), adenovirus types 40 and 41 (2.7%), and sapovirus (1.0%). The seasonal distribution of viral gastroenteritis showed a winter peak but also an unusual peak from May to September. The severity of the diarrhea was evaluated for hospitalized infants. No significant differences were observed between rotavirus and norovirus infections with regard to the incidence and the clinical severity of the disease, especially in dehydration.Diarrhea is one of the major causes of morbidity and mortality among infants throughout the world, especially in developing countries, where malnutrition and poor local health service are factors responsible for the increased severity of the diarrhea. In infants, group A rotavirus (RV) is the major etiologic agent of viral gastroenteritis and is responsible for 29 to 45% of hospitalizations, depending on the income level of the country (32, 33). Other enteric viruses such as human norovirus (NoV), sapovirus, astrovirus (HAstV), adenovirus (HAdV) types 40 and 41, and Aichi virus are also associated with acute gastroenteritis, and their relative importance in high-income countries has been reported previously (2, 4).Human NoVs, which are members of the Caliciviridae family, are found in all age groups (7, 10) and are a major cause of food-and water-related outbreaks (11,22). Recent work has showed that NoVs are the second most frequent etiologic agents of viral gastroenteritis (16). Sapovirus, another genus of the family Caliciviridae, HAstV (41), and enteric type 40 and 41 HAdVs have also been associated with diarrhea. Aichi virus (42) has recently been classified into the Kobuvirus genus in the Picornaviridae family and has been associated with oyster consumption.Although the role of these viruses in outbreaks of gastroenteritis in industrialized countries has been established (7, 29), little is known about their contribution to outbreaks in developing countries and few data are currently available (25). In Tunisia, a middle-income Mediterranean country, earlier studies showed the contribution of RVs (8, 38) and more recently that of HAstVs and HAdVs (12) in cases of childhood diarrhea. However, the nature of the contribution of NoVs and Aichi viruses to outbreaks of gastroenteritis in Tunisia remains unknown.The aim of this study was to determine the incidence of these enteric viruses and their contribution to diarrheal diseases in Tunisian children. We conducted a prospective 2-year study of children who were hospitalized in or presented to ...

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“…While between 1985 and 2004, G2P [4], G4P [8], and G9P [8] strains varied in their distribution or completely disappeared for short or long periods of time, G1 strains have been continuously circulating in the infant population of Palermo, a pattern that has been described in various other geographical settings as well (6,9,11,25,35,46,58,59). However, unlike the G2P [4] and G4P [8] Italian rotavirus strains, a highly heterogeneous population of G1 strains has been circulating in Italy over the whole study period.…”

Section: Discussionmentioning

confidence: 92%

Heterogeneity and Temporal Dynamics of Evolution of G1 Human Rotaviruses in a Settled Population

Arista

Giammanco

Grazia

et al. 2006

J Virol

117

106

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A rotavirus sample collection from 19 consecutive years was used to investigate the heterogeneity and the dynamics of evolution of G1 rotavirus strains in a geographically defined population. Phylogenetic analysis of the VP7 gene sequences of G1P[8] human rotavirus strains showed the circulation of a heterogeneous population comprising three lineages and seven sublineages. Increases in the circulation of G1 rotaviruses were apparently associated with the introduction of novel G1 strains that exhibited multiple amino acid changes in antigenic regions involved in rotavirus neutralization compared to the strains circulating in the previous years. The emergence and/or introduction of G1 antigenic variants might be responsible for the continuous circulation of G1 rotaviruses in the local population, with the various lineages and sublineages appearing, disappearing, or cocirculating in an alternate fashion under the influence of immune-pressure mechanisms. Sequence analysis of VP4-encoding genes of the G1 strains revealed that the older strains were associated with a unique VP4 lineage, while a novel VP4 lineage emerged after 1995. The introduction of human rotavirus vaccines might alter the forces and balances that drive rotavirus evolution and determine the spread of novel strains that are antigenically different from those included in the vaccine formulations. The continuous emergence of VP7-VP4 gene combinations in human rotavirus strains should be taken into consideration when devising vaccination strategies.

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Diversity of Group A Human Rotavirus Types Circulating over a 4-Year Period in Madrid, Spain

Cited by 50 publications

References 41 publications

Detection and Genotyping of Human Rotavirus VP4 and VP7 Genes by Reverse Transcriptase PCR and Reverse Hybridization

Detection and Genotyping of Human Rotavirus VP4 and VP7 Genes by Reverse Transcriptase PCR and Reverse Hybridization

Acute Infantile Gastroenteritis Associated with Human Enteric Viruses in Tunisia

Heterogeneity and Temporal Dynamics of Evolution of G1 Human Rotaviruses in a Settled Population

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