Miren Iturriza-Gomara

Uniformity of Rotavirus Strain Nomenclature Proposed by the Rotavirus Classification Working Group (RCWG)

In April 2008, a nucleotide-sequence-based, complete genome classification system was developed for group A rotaviruses (RVs). This system assigns a specific genotype to each of the 11 genome segments of a particular RV strain according to established nucleotide percent cutoff values. Using this approach, the genome of individual RV strains are given the complete descriptor of Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx. The Rotavirus Classification Working Group (RCWG) was formed by scientists in the field to maintain, evaluate and develop the RV genotype classification system, in particular to aid in the designation of new genotypes. Since its conception, the group has ratified 51 new genotypes: as of April 2011, new genotypes for VP7 (G20-G27), VP4 (P[28]-P[35]), VP6 (I12-I16), VP1 (R5-R9), VP2 (C6-C9), VP3 (M7-M8), NSP1 (A15-A16), NSP2 (N6-N9), NSP3 (T8-T12), NSP4 (E12-E14) and NSP5/6 (H7-H11) have been defined for RV strains recovered from humans, cows, pigs, horses, mice, South American camelids (guanaco), chickens, turkeys, pheasants, bats and a sugar glider. With increasing numbers of complete RV genome sequences becoming available, a standardized RV strain nomenclature system is needed, and the RCWG proposes that individual RV strains are named as follows: RV group/species of origin/country of identification/common name/year of identification/G- and P-type. In collaboration with the National Center for Biotechnology Information (NCBI), the RCWG is also working on developing a RV-specific resource for the deposition of nucleotide sequences. This resource will provide useful information regarding RV strains, including, but not limited to, the individual gene genotypes and epidemiological and clinical information. Together, the proposed nomenclature system and the NCBI RV resource will offer highly useful tools for investigators to search for, retrieve, and analyze the ever-growing volume of RV genomic data.

Full Genome-Based Classification of Rotaviruses Reveals a Common Origin between Human Wa-Like and Porcine Rotavirus Strains and Human DS-1-Like and Bovine Rotavirus Strains

ABSTRACT Group A rotavirus classification is currently based on the molecular properties of the two outer layer proteins, VP7 and VP4, and the middle layer protein, VP6. As reassortment of all the 11 rotavirus gene segments plays a key role in generating rotavirus diversity in nature, a classification system that is based on all the rotavirus gene segments is desirable for determining which genes influence rotavirus host range restriction, replication, and virulence, as well as for studying rotavirus epidemiology and evolution. Toward establishing such a classification system, gene sequences encoding VP1 to VP3, VP6, and NSP1 to NSP5 were determined for human and animal rotavirus strains belonging to different G and P genotypes in addition to those available in databases, and they were used to define phylogenetic relationships among all rotavirus genes. Based on these phylogenetic analyses, appropriate identity cutoff values were determined for each gene. For the VP4 gene, a nucleotide identity cutoff value of 80% completely correlated with the 27 established P genotypes. For the VP7 gene, a nucleotide identity cutoff value of 80% largely coincided with the established G genotypes but identified four additional distinct genotypes comprised of murine or avian rotavirus strains. Phylogenetic analyses of the VP1 to VP3, VP6, and NSP1 to NSP5 genes showed the existence of 4, 5, 6, 11, 14, 5, 7, 11, and 6 genotypes, respectively, based on nucleotide identity cutoff values of 83%, 84%, 81%, 85%, 79%, 85%, 85%, 85%, and 91%, respectively. In accordance with these data, a revised nomenclature of rotavirus strains is proposed. The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed (a) a common origin between human Wa-like rotavirus strains and porcine rotavirus strains and (b) a common origin between human DS-1-like rotavirus strains and bovine rotaviruses. These close evolutionary links between human and animal rotaviruses emphasize the need for close simultaneous monitoring of rotaviruses in animals and humans.

Recommendations for the classification of group A rotaviruses using all 11 genomic RNA segments.

Recently, a classification system was proposed for rotaviruses in which all the 11 genomic RNA segments are used (Matthijnssens et al. in J Virol 82:3204–3219, 2008). Based on nucleotide identity cut-off percentages, different genotypes were defined for each genome segment. A nomenclature for the comparison of complete rotavirus genomes was considered in which the notations Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx are used for the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 encoding genes, respectively. This classification system is an extension of the previously applied genotype-based system which made use of the rotavirus gene segments encoding VP4, VP7, VP6, and NSP4. In order to assign rotavirus strains to one of the established genotypes or a new genotype, a standard procedure is proposed in this report. As more human and animal rotavirus genomes will be completely sequenced, new genotypes for each of the 11 gene segments may be identified. A Rotavirus Classification Working Group (RCWG) including specialists in molecular virology, infectious diseases, epidemiology, and public health was formed, which can assist in the appropriate delineation of new genotypes, thus avoiding duplications and helping minimize errors. Scientists discovering a potentially new rotavirus genotype for any of the 11 gene segments are invited to send the novel sequence to the RCWG, where the sequence will be analyzed, and a new nomenclature will be advised as appropriate. The RCWG will update the list of classified strains regularly and make this accessible on a website. Close collaboration with the Study Group Reoviridae of the International Committee on the Taxonomy of Viruses will be maintained.

Evolutionary history and global spread of the emerging g12 human rotaviruses.

ABSTRACT G12 rotaviruses were first detected in diarrheic children in the Philippines in 1987, but no further cases were reported until 1998. However, G12 rotaviruses have been detected all over the world in recent years. Here, we report the worldwide variations of G12 rotaviruses to investigate the evolutionary mechanisms by which they managed to spread globally in a short period of time. We sequenced the complete genomes (11 segments) of nine G12 rotaviruses isolated in Bangladesh, Belgium, Thailand, and the Philippines and compared them with the genomes of other rotavirus strains. Our genetic analyses revealed that after introduction of the VP7 gene of the rare G12 genotype into more common local strains through reassortment, a vast genetic diversity was generated and several new variants with distinct gene constellations emerged. These reassortment events most likely took place in Southeast Asian countries and spread to other parts of the world. The acquirement of gene segments from human-adapted rotaviruses might allow G12 to better propagate in humans and hence to develop into an important emerging human pathogen.

Detection by PCR of eight groups of enteric pathogens in 4,627 faecal samples: re-examination of the English case-control Infectious Intestinal Disease Study (1993–1996)

The English case-control Infectious Intestinal Disease Study (1993–1996) failed to detect an enteric pathogen or toxin in 49% of cases of gastroenteritis. In the present study, polymerase chain reaction (PCR) assays were applied to DNA and cDNA generated from 4,627 faecal samples from cases and controls archived during the original study for the detection of norovirus, rotavirus, sapovirus, Campylobacter spp., Salmonella spp., enteroaggregative Escherichia coli, Cryptosporidium spp., and Giardia spp. The percentage of archived samples from cases and from controls in which at least one agent (or toxin) was detected increased from 53% in the original study to 75% and from 19 to 42%, respectively, after the application of PCR assays. Among cases, the following percentages of enteric pathogens were detected: norovirus 36%, rotavirus A 31%, sapovirus 4%, Salmonella spp. 6%, Campylobacter jejuni 13%, Campylobacter coli 2%, other Campylobacter spp. 8%, enteroaggregative E. coli 6%, Giardia spp. 2%, and Cryptosporidium spp. 2%. The present study provides additional insight into the aetiology of infectious intestinal disease in England and highlights the occurrence of viral infections in cases as well as in asymptomatic individuals. Other notable findings include the frequent presence of Campylobacter spp. other than C. jejuni or C. coli, the high frequency of multiple agents in 41% of cases and in 13% of controls, and the variation in the aetiology and rate of infection found for different age groups. The results demonstrate the greater sensitivity of PCR-based methods compared to current conventional methods.

Reassortment In Vivo: Driving Force for Diversity of Human Rotavirus Strains Isolated in the United Kingdom between 1995 and 1999

ABSTRACT The G and P genotypes of 3,601 rotavirus strains collected in the United Kingdom between 1995 and 1999 were determined (M. Iturriza-Gómara et al., J. Clin. Microbiol. 38:4394–4401, 2000). In 95.4% of the strains the most common G and P combinations, G1P[8], G2P[4], G3P[8], and G4P[8], were found. A small but significant number (2%) of isolates from the remaining strains were reassortants of the most common cocirculating strains, e.g., G1P[4] and G2P[8]. Rotavirus G9P[6] and G9P[8] strains, which constituted 2.7% of all viruses, were genetically closely related in their G components, but the P components of the G9P[8] strains were very closely related to those of cocirculating strains of the more common G types (G1, G3, and G4). In conclusion, genetic interaction by reassortment among cocirculating rotaviruses is not a rare event and contributes significantly to their overall diversity.

Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network

EuroRotaNet, a laboratory network, was established in order to determine the diversity of co-circulating rotavirus strains in Europe over three or more rotavirus seasons from 2006/2007 and currently includes 16 countries. This report highlights the tremendous diversity of rotavirus strains co-circulating in the European population during three years of surveillance since 2006/2007 and points to the possible origins of these strains including genetic reassortment and interspecies transmission. Furthermore, the ability of the network to identify strains circulating with an incidence of ≥1% allowed the identification of possible emerging strains such as G8 and G12 since the beginning of the study; analysis of recent data indicates their increased incidence. The introduction of universal rotavirus vaccination in at least two of the participating countries, and partial vaccine coverage in some others may provide data on diversity driven by vaccine introduction and possible strain replacement in Europe.

Diagnosing norovirus-associated infectious intestinal disease using viral load

BackgroundReverse transcription-polymerase chain reaction (RT-PCR) is the main method for laboratory diagnosis of norovirus-associated infectious intestinal disease (IID). However, up to 16% of healthy individuals in the community, with no recent history of IID, may be RT-PCR positive; so it is unclear whether norovirus is actually the cause of illness in an IID case when they are RT-PCR positive. It is important to identify the pathogen causing illness in sporadic IID cases, for clinical management and for community based incidence studies. The aim of this study was to investigate how faecal viral load can be used to determine when norovirus is the most likely cause of illness in an IID case.MethodsReal-time RT-PCR was used to determine the viral load in faecal specimens collected from 589 IID cases and 159 healthy controls, who were infected with genogroup II noroviruses. Cycle threshold (Ct) values from the real-time RT-PCR were used as a proxy measure of viral load. Receiver-operating characteristic (ROC) analysis was used to identify a cut-off in viral load for attributing illness to norovirus in IID cases.ResultsOne hundred and sixty-nine IID cases and 159 controls met the inclusion criteria for the ROC analysis. The optimal Ct value cut-off for attributing IID to norovirus was 31. The same cut-off was selected when using healthy controls, or IID cases who were positive by culture for bacterial pathogens, as the reference negative group. This alternative reference negative group can be identified amongst specimens routinely received in clinical virology laboratories.ConclusionWe demonstrated that ROC analysis can be used to select a cut-off for a norovirus real time RT-PCR assay, to aid clinical interpretation and diagnose when norovirus is the cause of IID. Specimens routinely received for diagnosis in clinical virology laboratories can be used to select an appropriate cut-off. Individual laboratories can use this method to define in-house cut-offs for their assays, to provide the best possible diagnostic service to clinicians and public health workers. Other clinical and epidemiological information should also be considered for patients with Ct values close to the cut-off, for the most accurate diagnosis of IID aetiology.

New oligonucleotide primers for P-typing of rotavirus strains: Strategies for typing previously untypeable strains

BACKGROUND The use of molecular methods for rotavirus characterisation provides increased sensitivity for typing, and allows the identification of putative reassortant strains. However, due to the constant accumulation of point mutations through genetic drift; and to the emergence of novel genotypes; and possibly zoonotic transmission and subsequent reassortment, the reagents and methods used for genotyping require close monitoring and updating. OBJECTIVES To design and evaluate a new VP4 consensus oligonucleotide primer pair that provides increased sensitivity and allows typing of strains that were untypeable using available methods. STUDY DESIGN A total of 489 rotavirus-positive faecal specimens from studies conducted between 1996 and 2006 were used for the evaluation of the new VP4 primers which was performed in the WHO Rotavirus Collaborating and Reference centres in the US, Australia, South Africa and the UK. RESULTS The new primer pair allowed P-typing of rotavirus strains and provided increased sensitivity, allowing typing of a significant number of strains that previously could not be P-typed. CONCLUSIONS This study highlights the importance of a constant reconsideration of primer sequences employed for the molecular typing of rotaviruses.

Analysis of Amino Acid Variation in the P2 Domain of the GII-4 Norovirus VP1 Protein Reveals Putative Variant-Specific Epitopes

Background Human noroviruses are a highly diverse group of viruses classified into three of the five currently recognised Norovirus genogroups, and contain numerous genotypes or genetic clusters. Noroviruses are the major aetiological agent of endemic gastroenteritis in all age groups, as well as the cause of periodic epidemic gastroenteritis. The noroviruses most commonly associated with outbreaks of gastroenteritis are genogroup II genotype 4 (GII-4) strains. The relationship between genotypes of noroviruses with their phenotypes and antigenic profile remains poorly understood through an inability to culture these viruses and the lack of a suitable animal model. Methodology/Principal Findings Here we describe a study of the diversity of amino acid sequences of the highly variable P2 region in the major capsid protein, VP1, of the GII-4 human noroviruses strains using sequence analysis and homology modelling techniques. Conclusions/Significance Our data identifies two sites in this region, which show significant amino acid substitutions associated with the appearance of variant strains responsible for epidemics with major public health impact. Homology modelling studies revealed the exposed nature of these sites on the capsid surface, providing supportive structural data that these two sites are likely to be associated with putative variant-specific epitopes. Furthermore, the patterns in the evolution of these viruses at these sites suggests that noroviruses follow a neutral network pattern of evolution.