Franco Maria Ruggeri

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.

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.

Detection of Multiple Noroviruses Associated with an International Gastroenteritis Outbreak Linked to Oyster Consumption

ABSTRACT An international outbreak linked to oyster consumption involving a group of over 200 people in Italy and 127 total subjects in 13 smaller clusters in France was analyzed using epidemiological and clinical data and shellfish samples. Environmental information from the oyster-producing area, located in a lagoon in southern France, was collected to investigate the possible events leading to the contamination. Virologic analyses were conducted by reverse transcription-PCR (RT-PCR) using the same primer sets for both clinical and environmental samples. After sequencing, the data were analyzed through the database operated by the scientific network FoodBorne Viruses in Europe. The existence of an international collaboration between laboratories was critical to rapidly connect the data and to fully interpret the results, since it was not obvious that one food could be the link because of the diversity of the several norovirus strains involved in the different cases. It was also demonstrated that heavy rain was responsible for the accidental contamination of seafood, leading to a concentration of up to hundreds of genomic copies per oyster as detected by real-time RT-PCR.

Analysis of Integrated Virological and Epidemiological Reports of Norovirus Outbreaks Collected within the Foodborne Viruses in Europe Network from 1 July 2001 to 30 June 2006

ABSTRACT The Foodborne Viruses in Europe network has developed integrated epidemiological and virological outbreak reporting with aggregation and sharing of data through a joint database. We analyzed data from reported outbreaks of norovirus (NoV)-caused gastroenteritis from 13 European countries (July 2001 to July 2006) for trends in time and indications of different epidemiology of genotypes and variants. Of the 13 countries participating in this surveillance network, 11 were capable of collecting integrated epidemiological and virological surveillance data and 10 countries reported outbreaks throughout the entire period. Large differences in the numbers and rates of reported outbreaks per country were observed, reflecting the differences in the focus and coverage of national surveillance systems. GII.4 strains predominated throughout the 5-year surveillance period, but the proportion of outbreaks associated with GII.4 rose remarkably during years in which NoV activity was particularly high. Spring and summer peaks indicated the emergence of genetically distinct variants within GII.4 across Europe and were followed by increased NoV activity during the 2002-2003 and 2004-2005 winter seasons. GII.4 viruses predominated in health care settings and in person-to-person transmission. The consecutive emergence of new GII.4 variants is highly indicative of immune-driven selection. Their predominance in health care settings suggests properties that facilitate transmission in settings with a high concentration of people such as higher virus loads in excreta or a higher incidence of vomiting. Understanding the mechanisms driving the changes in epidemiology and clinical impact of these rapidly evolving RNA viruses is essential to design effective intervention and prevention measures.

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.

Virus hazards from food, water and other contaminated environments

Abstract Numerous viruses of human or animal origin can spread in the environment and infect people via water and food, mostly through ingestion and occasionally through skin contact. These viruses are released into the environment by various routes including water run‐offs and aerosols. Furthermore, zoonotic viruses may infect humans exposed to contaminated surface waters. Foodstuffs of animal origin can be contaminated, and their consumption may cause human infection if the viruses are not inactivated during food processing. Molecular epidemiology and surveillance of environmental samples are necessary to elucidate the public health hazards associated with exposure to environmental viruses. Whereas monitoring of viral nucleic acids by PCR methods is relatively straightforward and well documented, detection of infectious virus particles is technically more demanding and not always possible (e.g. human norovirus or hepatitis E virus). The human pathogenic viruses that are most relevant in this context are nonenveloped and belong to the families of the C aliciviridae, A denoviridae, H epeviridae, P icornaviridae and R eoviridae. Sampling methods and strategies, first‐choice detection methods and evaluation criteria are reviewed.

Lactobacilli Expressing Variable Domain of Llama Heavy-Chain Antibody Fragments (Lactobodies) Confer Protection against Rotavirus-Induced Diarrhea

BACKGROUND Rotavirus-induced diarrhea poses a worldwide medical problem in causing substantial morbidity and mortality among children in developing countries. We therefore developed a system for passive immunotherapy in which recombinant lactobacilli constitutively express neutralizing variable domain of llama heavy-chain (VHH) antibody fragments against rotavirus. METHODS VHH were expressed in Lactobacillus paracasei, in both secreted and cell surface-anchored forms. Electron microscopy was used to investigate the binding efficacy of VHH-expressing lactobacilli. To investigate the in vivo function of VHH-expressing lactobacilli, a mouse pup model of rotavirus infection was used. RESULTS Efficient binding of the VHH antibody fragments to rotavirus was shown by enzyme-linked immunosorbent assay and scanning electron microscopy. VHH fragments expressed by lactobacilli conferred a significant reduction in infection in cell cultures. When administered orally, lactobacilli-producing surface-expressed VHH markedly shortened disease duration, severity, and viral load in a mouse model of rotavirus-induced diarrhea when administered both fresh and in a freeze-dried form. CONCLUSIONS Transformed lactobacilli may form the basis of a novel form of prophylactic treatment against rotavirus infections and other diarrheal diseases.

Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy

In July 2000, an outbreak of gastroenteritis occurred at a tourist resort in the Gulf of Taranto in southern Italy. Illness in 344 people, 69 of whom were staff members, met the case definition. Norwalk-like virus (NLV) was found in 22 of 28 stool specimens tested. The source of illness was likely contaminated drinking water, as environmental inspection identified a breakdown in the resort water system and tap water samples were contaminated with fecal bacteria. Attack rates were increased (51.4%) in staff members involved in water sports. Relative risks were significant only for exposure to beach showers and consuming drinks with ice. Although Italy has no surveillance system for nonbacterial gastroenteritis, no outbreak caused by NLV has been described previously in the country.

Rotavirus Surveillance in Europe, 2005–2008: Web-Enabled Reporting and Real-Time Analysis of Genotyping and Epidemiological Data

BACKGROUND The first European rotavirus surveillance network, EuroRotaNet, comprising 16 laboratories in 15 European countries, has been established. METHODS Fecal samples from gastroenteritis cases positive for group A rotavirus antigen were collected from multiple European countries from 2005 to mid-2008 and were subjected to G and P genotyping. Epidemiological data collected included age, sex, geographical location, setting, dates of onset and sample collection, and clinical symptoms. RESULTS A total of 8879 rotavirus-positive samples were characterized: 2129 cases were from the 2005-2006 season, 4030 from the 2006-2007 season, and 2720 from the ongoing 2007-2008 season. A total of 30 different G and P type combinations of strains circulated in the region from 2005 through 2008. Of these strains, 90% had genotypes commonly associated with human infections-G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]-and 1.37% represented potential zoonotic introductions. G1P[8] remained the most prevalent genotype in Europe as a whole, but the incidence of infection with G1P[8] rotavirus strains was <50% overall, and all 3 seasons were characterized by a significant diversity of cocirculating strains. The peak incidence of rotavirus infection occurred from January through May, and 81% of case patients were aged <2.5 years. Conclusions. Data gathered through EuroRotaNet will provide valuable background information on the rotavirus strain diversity in Europe before the introduction of rotavirus vaccines, and the network will provide a robust method for surveillance during vaccine implementation.

European Multicenter Evaluation of Commercial Enzyme Immunoassays for Detecting Norovirus Antigen in Fecal Samples

ABSTRACT A total of 2,254 fecal samples were tested in a European multicenter evaluation of commercially available norovirus antigen detection assays. Two commercial enzyme immunoassays, IDEIA Norovirus (Oxoid; Thermo Fisher Scientific, Ely, United Kingdom) and RIDASCREEN Norovirus (R-Biopharm, Darmstadt, Germany), were included in the evaluation, and their performance was compared with the results of reverse transcription-PCR (RT-PCR). Included in the evaluation were samples collected in sporadic cases of gastroenteritis, samples from outbreaks in which two or more samples were collected, well-characterized samples representing genotypes currently cocirculating within Europe, and samples collected from patients with gastroenteritis caused by a pathogen other than norovirus. The sensitivities and specificities of the IDEIA Norovirus and RIDASCREEN Norovirus assays were 58.93 and 43.81% and 93.91 and 96.37%, respectively, compared with RT-PCR. The sensitivities of both assays for outbreak investigations improved when six or more samples from an outbreak were examined. The IDEIA Norovirus assay exhibited reactivity to a broader range of norovirus genotypes than the RIDASCREEN Norovirus assay, which showed genotype-dependent sensitivities. The results indicate that, if used, these assays should serve as screening assays and the results should be confirmed by RT-PCR.