Max Ciarlet

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.

Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in Asia: a randomised, double-blind, placebo-controlled trial

BACKGROUND Rotavirus vaccine has proved effective for prevention of severe rotavirus gastroenteritis in infants in developed countries, but no efficacy studies have been done in developing countries in Asia. We assessed the clinical efficacy of live oral pentavalent rotavirus vaccine for prevention of severe rotavirus gastroenteritis in infants in Bangladesh and Vietnam. METHODS In this multicentre, double-blind, placebo-controlled trial, undertaken in rural Matlab, Bangladesh, and urban and periurban Nha Trang, Vietnam, infants aged 4-12 weeks without symptoms of gastrointestinal disorders were randomly assigned (1:1) to receive three oral doses of pentavalent rotavirus vaccine 2 mL or placebo at around 6 weeks, 10 weeks, and 14 weeks of age, in conjunction with routine infant vaccines including oral poliovirus vaccine. Randomisation was done by computer-generated randomisation sequence in blocks of six. Episodes of gastroenteritis in infants who presented to study medical facilities were reported by clinical staff and from parent recollection. The primary endpoint was severe rotavirus gastroenteritis (Vesikari score >or=11) arising 14 days or more after the third dose of placebo or vaccine to end of study (March 31, 2009; around 21 months of age). Analysis was per protocol; infants who received scheduled doses of vaccine or placebo without intervening laboratory-confirmed naturally occurring rotavirus disease earlier than 14 days after the third dose and had complete clinical and laboratory results were included in the analysis. This study is registered with ClinicalTrials.gov, number NCT00362648. FINDINGS 2036 infants were randomly assigned to receive pentavalent rotavirus vaccine (n=1018) or placebo (n=1018). 991 infants assigned to pentavalent rotavirus vaccine and 978 assigned to placebo were included in the per-protocol analysis. Median follow up from 14 days after the third dose of placebo or vaccine until final disposition was 498 days (IQR 480-575). 38 cases of severe rotavirus gastroenteritis (Vesikari score >or=11) were reported during more than 1197 person-years of follow up in the vaccine group, compared with 71 cases in more than 1156 person years in the placebo group, resulting in a vaccine efficacy of 48.3% (95% CI 22.3-66.1) against severe disease (p=0.0005 for efficacy >0%) during nearly 2 years of follow-up. 25 (2.5%) of 1017 infants assigned to receive vaccine and 20 (2.0%) of 1018 assigned to receive placebo had a serious adverse event within 14 days of any dose. The most frequent serious adverse event was pneumonia (vaccine 12 [1.2%]; placebo 15 [1.5%]). INTERPRETATION In infants in developing countries in Asia, pentavalent rotavirus vaccine is safe and efficacious against severe rotavirus gastroenteritis, and our results support expanded WHO recommendations to promote its global use. FUNDING PATH (GAVI Alliance grant) and Merck.

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.

Zoonotic aspects of rotaviruses.

Rotaviruses are important enteric pathogens of humans and animals. Group A rotaviruses (GARVs) account for up to 1 million children deaths each year, chiefly in developing countries and human vaccines are now available in many countries. Rotavirus-associated enteritis is a major problem in livestock animals, notably in young calves and piglets. Early in the epidemiological GARV studies in humans, either sporadic cases or epidemics by atypical, animal-like GARV strains were described. Complete genome sequencing of human and animal GARV strains has revealed a striking genetic heterogeneity in the 11 double stranded RNA segments across different rotavirus strains and has provided evidence for frequent intersections between the evolution of human and animal rotaviruses, as a result of multiple, repeated events of interspecies transmission and subsequent adaptation.

VP6-sequence-based cutoff values as a criterion for rotavirus species demarcation

Indirect immunofluorescence techniques targeting the rotavirus (RV) protein VP6 are used to differentiate RV species. The ICTV recognizes RV species A to E and two tentative species, F and G. A potential new RV species, ADRV-N, has been described. Phylogenetic trees and pairwise identity frequency graphs were constructed with more than 400 available VP6 sequences and seven newly determined VP6 sequences of RVD strains. All RV species were separated into distinct phylogenetic clusters. An amino acid sequence cutoff value of 53% firmly permitted differentiation of RV species, and ADRV-N was tentatively assigned to a novel RV species H (RVH).

Rotavirus disease and vaccination: impact on genotype diversity

Temporal and spatial fluctuations in the genotype distribution of human rotaviruses are continuously observed in surveillance studies. New genotypes, such as G9 and G12, have emerged and spread worldwide in a very short time span. In addition, reassortment events have the potential to contribute substantially to genetic diversity among human and animal rotaviruses. With the recent introduction of the two rotavirus vaccines, RotaTeq and Rotarix, in many countries, it appears that the total number of hospitalizations due to rotavirus infections is being reduced, at least in developed countries that implemented a universal immunization program. However, continued surveillance is warranted, especially regarding the long-term effects of the vaccines. No data analyses are available to clarify whether rotavirus vaccine introduction would allow other rotavirus P and G genotypes, which are not covered by the current vaccines, to emerge into the human population and fill the apparent gap. This kind of data analysis is essential, but its interpretation is hampered by natural and cyclical genotype fluctuations.

Rotavirus antigenaemia and viraemia: a common event?

BACKGROUND Rotavirus infection is thought to be confined to the intestine. Reports of rotavirus RNA in the cerebral spinal fluid and serum of children infected with rotavirus suggest the possibility that rotavirus escapes the intestine into the circulatory system. We assessed whether rotavirus antigen, RNA, or both, were present in serum samples from immunocompetent rotavirus-infected children and animals. METHODS We obtained sera from immunocompetent mice, rats, rabbits, and calves 1-10 days after inoculation with rotavirus or matched vehicle. We obtained sera retrospectively from immunocompetent children diagnosed with rotavirus diarrhoea (n=33), healthy children (n=6) and adults (n=12), children convalescing from rotavirus (n=6), and children with non-rotavirus diarrhoea (n=11). Samples were analysed for the presence of rotavirus antigen or RNA by EIA or RT-PCR, respectively. FINDINGS Rotavirus antigen was present in sera from rotavirus-infected animals, but not in sera from control animals. Infectious rotavirus or rotavirus RNA was detected in sera of mice and calves, respectively. Antigen was present in 22 of 33 serum samples from children with confirmed rotavirus infection but in none of 35 samples from controls. Detection of serum antigen was inversely related to the number of days between symptom onset and sample collection, and directly related to stool antigen concentration. Rotavirus RNA was detected by RT-PCR in three of six rotavirus-positive sera. INTERPRETATION Rotavirus can escape the gastrointestinal tract in children, resulting in antigenaemia and possible viraemia. This finding is important for the understanding of the pathogenesis, immunology, and clinical manifestations of rotavirus infection.

Full Genomic Analysis of Human Rotavirus Strain B4106 and Lapine Rotavirus Strain 30/96 Provides Evidence for Interspecies Transmission

ABSTRACT The Belgian rotavirus strain B4106, isolated from a child with gastroenteritis, was previously found to have VP7 (G3), VP4 (P[14]), and NSP4 (A genotype) genes closely related to those of lapine rotaviruses, suggesting a possible lapine origin or natural reassortment of strain B4106. To investigate the origin of this unusual strain, the gene sequences encoding VP1, VP2, VP3, VP6, NSP1, NSP2, NSP3, and NSP5/6 were also determined. To allow comparison to a lapine strain, the 11 double-stranded RNA segments of a European G3P[14] rabbit rotavirus strain 30/96 were also determined. The complete genome similarity between strains B4106 and 30/96 was 93.4% at the nucleotide level and 96.9% at the amino acid level. All 11 genome segments of strain B4106 were closely related to those of lapine rotaviruses and clustered with the lapine strains in phylogenetic analyses. In addition, sequence analyses of the NSP5 gene of strain B4106 revealed that the altered electrophoretic mobility of NSP5, resulting in a super-short pattern, was due to a gene rearrangement (head-to-tail partial duplication, combined with two short insertions and a deletion). Altogether, these findings confirm that a rotavirus strain with an entirely lapine genome complement was able to infect and cause severe disease in a human child.

Are Human P[14] Rotavirus Strains the Result of Interspecies Transmissions from Sheep or Other Ungulates That Belong to the Mammalian Order Artiodactyla?

ABSTRACT A limited number of human G6P[14] rotavirus strains that cause gastroenteritis in humans have been isolated in Europe and Australia. The complete genome sequences were determined for five of these human strains—B10925-97 (isolated in Belgium in 1997), 111/05-27 (Italy, 2005), PA169 (Italy, 1987), MG6 (Australia, 1993), and Hun5 (Hungary, 1997)—and their genetic relatedness to animal rotavirus strains was evaluated by sequencing the complete genome of the sheep rotavirus OVR762 (G8P[14]; Spain, 2002), the guanaco (Lama guanicoe) rotavirus strains Arg/Chubut/99 and Arg/Río Negro/98 (G8P[14] and G8P[1], respectively; Argentina, 1999 and 1998), the sable antelope strain RC-18/08 (G6P[14]; South Africa, 2008), and the bovine rotavirus strain Arg/B383/98 (G15P[11]; Argentina, 1998). These analyses revealed an overall consensus genomic constellation (G6/G8)-P[14]-I2-(R2/R5)-C2-M2-(A3/A11)-N2-T6-(E2/E12)-H3, together with a few gene reassortments, and the phylogenetic analyses confirmed that the P[14] human strains evaluated in this study were closely related to rotavirus strains isolated from sheep, cattle, goats, guanacos, and antelopes and to rabbits (albeit to a lesser extent), suggesting that one (or more) of these animal species might be the source of the human G6P[14] strains. The main feature of the genotype and phylogenetic analyses was the close overall genomic relatedness between the five human G6P[14] rotavirus strains and the ovine and antelope rotavirus strains. Taken together, these data strongly suggest a common origin for the human P[14] strains and those of the even-toed ungulates belonging to the mammalian order Artiodactyla, with sheep probably playing a key role in the interspecies transmission responsible for the introduction of P[14] rotavirus strains into the human population.