Carl D. Kirkwood

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.

Serotype Diversity and Reassortment between Human and Animal Rotavirus Strains: Implications for Rotavirus Vaccine Programs

The development of rotavirus vaccines that are based on heterotypic or serotype-specific immunity has prompted many countries to establish programs to assess the disease burden associated with rotavirus infection and the distribution of rotavirus strains. Strain surveillance helps to determine whether the most prevalent local strains are likely to be covered by the serotype antigens found in current vaccines. After introduction of a vaccine, this surveillance could detect which strains might not be covered by the vaccine. Almost 2 decades ago, studies demonstrated that 4 globally common rotavirus serotypes (G1-G4) represent >90% of the rotavirus strains in circulation. Subsequently, these 4 serotypes were used in the development of reassortant vaccines predicated on serotype-specific immunity. More recently, the application of reverse-transcription polymerase chain reaction genotyping, nucleotide sequencing, and antigenic characterization methods has confirmed the importance of the 4 globally common types, but a much greater strain diversity has also been identified (we now recognize strains with at least 42 P-G combinations). These studies also identified globally (G9) or regionally (G5, G8, and P2A[6]) common serotype antigens not covered by the reassortant vaccines that have undergone efficacy trials. The enormous diversity and capacity of human rotaviruses for change suggest that rotavirus vaccines must provide good heterotypic protection to be optimally effective.

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.

Metagenomic analysis of human diarrhea: Viral detection and discovery

Worldwide, approximately 1.8 million children die from diarrhea annually, and millions more suffer multiple episodes of nonfatal diarrhea. On average, in up to 40% of cases, no etiologic agent can be identified. The advent of metagenomic sequencing has enabled systematic and unbiased characterization of microbial populations; thus, metagenomic approaches have the potential to define the spectrum of viruses, including novel viruses, present in stool during episodes of acute diarrhea. The detection of novel or unexpected viruses would then enable investigations to assess whether these agents play a causal role in human diarrhea. In this study, we characterized the eukaryotic viral communities present in diarrhea specimens from 12 children by employing a strategy of “micro-mass sequencing” that entails minimal starting sample quantity (<100 mg stool), minimal sample purification, and limited sequencing (384 reads per sample). Using this methodology we detected known enteric viruses as well as multiple sequences from putatively novel viruses with only limited sequence similarity to viruses in GenBank.

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.

Reduction in Rotavirus-associated Acute Gastroenteritis Following Introduction of Rotavirus Vaccine Into Australia's National Childhood Vaccine Schedule

Introduction: Rotavirus vaccines were introduced into the funded Australian National Immunization Program (NIP) in July 2007. Due to purchasing arrangements, individual states and territories chose either a 2-dose RV1 (Rotarix, GSK) regimen or 3-dose RV5 (Rotateq, Merck/CSL) regimen. This allowed comparison of both vaccines in similar populations with high infant vaccination coverage. Methods: Admission and rotavirus identification data from the major pediatric hospitals in 3 states (2 using RV5, 1 RV1), together with state-based hospitalization and vaccination data from Queensland (RV5) were analyzed for the years before, and up to 30 months following rotavirus vaccine introduction. Emergency encounters and short-stay unit admissions for gastroenteritis are also described. Results: Rotavirus vaccine coverage in Australia is high, with 87% of infants receiving at least 1 dose. Hospital admissions for both rotavirus gastroenteritis and nonrotavirus-coded gastroenteritis were reduced following vaccine introduction in all states, not only for the age group eligible for NIP rotavirus vaccination, but also for children born prior. RV5 vaccine efficacy in Queensland has been estimated at 89.3%. Marked reductions in acute gastroenteritis emergency presentations and short-stay unit admissions have also been observed. Conclusions: Early evidence from the NIP in Australia has demonstrated high rotavirus coverage with both RV1 and RV5. The introduction of both vaccines has been associated with a marked reduction in gastroenteritis admissions, supportive of both direct vaccine protection, as well as with indirect herd protection.

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.

Distribution of rotavirus genotypes after introduction of rotavirus vaccines, Rotarix® and RotaTeq®, into the National Immunization Program of Australia.

Background: Rotavirus vaccines, RotaTeq and Rotarix, were introduced into the Australian National Immunization Program on July 1, 2007. The simultaneous introduction in different Australian states and territories provides a unique opportunity to compare the affect of each vaccine on the types of circulating rotavirus strains. This report describes the rotavirus genotypes responsible for the hospitalization of children during the first 2-year period after vaccine introduction. Methods: A total of 764 rotavirus-associated diarrheal cases were collected from children presenting to hospital in 10 Australian centers. Rotavirus genotype was determined using reverse transcription polymerase chain reaction assays. Results: G1P[8] was the dominant genotype nationally (52%), followed by G2P[4] (19.8%), G9P[8] (12.2%), and G3P[8] (11%). Differences in the prevalence rates of G2P[4] and G3P[8] were seen in the various states. G2P[4] strains were more prevalent in states using Rotarix, whereas G3P[8] strains were more prevalent in states using RotaTeq. Conclusions: Differences in rotavirus genotypes were observed across Australia, which suggest that different immune pressures are exerted by the different vaccines, but do not necessarily imply lack of protection by either vaccine. These differences may simply be related to the variation that can occur because of natural annual fluctuation in rotavirus strain prevalence.

Comparative Evolution of GII.3 and GII.4 Norovirus over a 31-Year Period

ABSTRACT Noroviruses are the most common cause of epidemic gastroenteritis. Genotype II.3 is one of the most frequently detected noroviruses associated with sporadic infections. We studied the evolution of the major capsid gene from seven archival GII.3 noroviruses collected during a cross-sectional study at the Childrens Hospital in Washington, DC, from 1975 through 1991, together with capsid sequence from 56 strains available in GenBank. Evolutionary analysis concluded that GII.3 viruses evolved at a rate of 4.16 × 10−3 nucleotide substitutions/site/year (strict clock), which is similar to that described for the more prevalent GII.4 noroviruses. The analysis of the amino acid changes over the 31-year period found that GII.3 viruses evolve at a relatively steady state, maintaining 4% distance, and have a tendency to revert back to previously used residues while preserving the same carbohydrate binding profile. In contrast, GII.4 viruses demonstrate increasing rates of distance over time because of the continued integration of new amino acids and changing HBGA binding patterns. In GII.3 strains, seven sites acting under positive selection were predicted to be surface-exposed residues in the P2 domain, in contrast to GII.4 positively selected sites located primarily in the shell domain. Our study suggests that GII.3 noroviruses caused disease as early as 1975 and that they evolve via a specific pattern, responding to selective pressures induced by the host rather than presenting a nucleotide evolution rate lower than that of GII.4 noroviruses, as previously proposed. Understanding the evolutionary dynamics of prevalent noroviruses is relevant to the development of effective prevention and control strategies.

Mycobacterium avium subspecies paratuberculosis in children with early-onset Crohn's disease.

Background: Mycobacterium avium subspecies paratuberculosis (MAP) is the most enduring infectious candidate that may be associated with inflammatory bowel disease (IBD). It is possible that the inconsistencies in the prevalence studies of MAP in adults reflect clinical differences in adult patients studied, including duration of disease and treatment regimens, and also in lack of specificity of some of the assays used. The aim was to determine the presence of MAP in children with symptoms of Crohns disease (CD) and ulcerative colitis (UC), using gut biopsy tissue and peripheral blood mononuclear cells (PBMC) collected at initial endoscopic examination prior to clinical treatment. Methods: Mucosal biopsies and/or PBMC specimens were collected from a total of 142 children, comprising 62 with CD, 26 with UC, and 54 with non‐IBD. MAP‐specific IS900 polymerase chain reaction (PCR) analysis was performed on all biopsies and PBMC specimens. Conventional MAP culture technique was performed on a subset of 10 CD, 2 UC, and 4 non‐IBD patients to isolate MAP. Results: MAP was identified by IS900 PCR significantly more often in mucosal biopsies from CD 39% (22/56) than from non‐IBD 15% (6/39) patients (P < 0.05), and in PBMC from CD 16% (8/50) than from non‐IBD 0% (0/31) patients (P < 0.05). Viable MAP were cultured from mucosal biopsies from 4/10 CD, 0/2 UC, and 0/4 non‐IBD patients, but were not cultured from PBMC specimens. Conclusions: This unique study on the occurrence of MAP in gut tissue and blood from pediatric IBD patients suggests the possible involvement of MAP in the early stages of development of CD in children. Inflamm Bowel Dis 2009