Mark Zeller

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.

Phylodynamic Analyses of Rotavirus Genotypes G9 and G12 Underscore Their Potential for Swift Global Spread

Rotaviruses (RVs) are responsible for more than 600,000 child deaths each year. The worldwide introduction of two life oral vaccines RotaTeq and Rotarix is believed to reduce this number significantly. Before the licensing of both vaccines, two new genotypes, G9 and G12, emerged in the human population and were able to spread across the entire globe in a very short time span. To quantify the VP7 mutation rates of these G9 and G12 genotypes and to estimate their most recent common ancestors, we used a Bayesian Markov chain Monte Carlo framework. Based on 356 sequences for G9 and 140 sequences for G12, we estimated mutation rates (nt substitutions/site/year) of 1.87 × 10(-3) (1.45-2.27 × 10(-3)) for G9 and 1.66 × 10(-3) (1.13-2.32 × 10(-3)) for G12. For both the G9 and G12 strains, one particular (sub) lineage was able to disseminate and cause disease across the world. The most recent common ancestors of these particular lineages were dated back to 1989 (1986-1992) and 1995 (1992-1998) for the G9 and G12 genotypes, respectively. These estimates suggest that a single novel RV (e.g., a vaccine escape mutant) can spread worldwide in little more than a decade. These results re-emphasize the need for thorough and continued RV surveillance in order to detect such potential spreading events at an early stage.

Rotavirus incidence and genotype distribution before and after national rotavirus vaccine introduction in Belgium

Rotarix™ was introduced into the Belgian market in 2006 and RotaTeq™ in 2007, quickly reaching more than 85% vaccine coverage of all newborns in Belgium. The incidence of rotavirus gastroenteritis has been monitored in the Gasthuisberg University Hospital (GUH), Belgium since 1986, and since 1999 the genotypes of circulating rotavirus strains have been determined. The average percentage of rotavirus positive cases out of all hospitalized gastro-enteritis cases tested (>95% of these cases are younger than 5 years old) at the GUH between 1986 and 2006 was 19.0%. This percentage dropped to 12.4%, 9.6% and 6.4% in the three seasons post vaccine introduction (2006-2009), which is a decline of 34.7%, 49.4% and 66.3% respectively. In addition the rotavirus season was found to be shortened and delayed. The prevalence of the G2 genotype sharply increased in the 2006-2007 rotavirus season compared to the previous seasons and remained high (30-40%) in the 2007-2008 and 2008-2009 seasons. Rotavirus vaccines have strongly reduced the number of children hospitalized due to a rotavirus infection at the GUH; it is however unclear if the predominance of G2 genotypes is related to the vaccine introduction, or if this is attributable to normal genotype fluctuations. Continued surveillance will be pivotal to answer this question in the future.

Evolutionary dynamics of human rotaviruses: balancing reassortment with preferred genome constellations.

Group A human rotaviruses (RVs) are a major cause of severe gastroenteritis in infants and young children. Yet, aside from the genes encoding serotype antigens (VP7; G-type and VP4; P-type), little is known about the genetic make-up of emerging and endemic human RV strains. To gain insight into the diversity and evolution of RVs circulating at a single location over a period of time, we sequenced the eleven-segmented, double-stranded RNA genomes of fifty-one G3P[8] strains collected from 1974 to 1991 at Childrens Hospital National Medical Center, Washington, D. C. During this period, G1P[8] strains typically dominated, comprising on average 56% of RV infections each year in hospitalized children. A notable exception was in the 1976 and 1991 winter seasons when the incidence of G1P[8] infections decreased dramatically, a trend that correlated with a significant increase in G3P[8] infections. Our sequence analysis indicates that the 1976 season was characterized by the presence of several genetically distinct, co-circulating clades of G3P[8] viruses, which contained minor but significant differences in their encoded proteins. These 1976 lineages did not readily exchange gene segments with each other, but instead remained stable over the course of the season. In contrast, the 1991 season contained a single major clade, whose genome constellation was similar to one of the 1976 clades. The 1991 clade may have gained a fitness advantage after reassorting with as of yet unidentified RV strain(s). This study reveals for the first time that genetically distinct RV clades of the same G/P-type can co-circulate and cause disease. The findings from this study also suggest that, although gene segment exchange occurs, most reassortant strains are replaced over time by lineages with preferred genome constellations. Elucidation of the selective pressures that favor maintenance of RVs with certain sets of genes may be necessary to anticipate future vaccine needs.

Genetic Analyses Reveal Differences in the VP7 and VP4 Antigenic Epitopes between Human Rotaviruses Circulating in Belgium and Rotaviruses in Rotarix and RotaTeq

ABSTRACT Two live-attenuated rotavirus group A (RVA) vaccines, Rotarix (G1P[8]) and RotaTeq (G1-G4, P[8]), have been successfully introduced in many countries worldwide, including Belgium. The parental RVA strains used to generate the vaccines were isolated more than 20 years ago in France (G4 parental strain in RotaTeq) and the United States (all other parental strains). At present, little is known about the relationship between currently circulating human RVAs and the vaccine strains. In this study, we determined sequences for the VP7 and VP4 outer capsid proteins of representative G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8] RVAs circulating in Belgium during 2007 to 2009. The analyses showed that multiple amino acid differences existed between the VP7 and VP4 antigenic epitopes of the vaccine viruses and the Belgian isolates, regardless of their G and P genotypes. However, the highest variability was observed among the circulating G1P[8] RVA strains and the G1 and P[8] components of both RVA vaccines. In particular, RVA strains of the P[8] lineage 4 (OP354-like) showed a significant number of amino acid differences with the P[8] VP4 of both vaccines. In addition, the circulating Belgian G3 RVA strains were found to possibly possess an extra N-linked glycosylation site compared to the G3 RVA vaccine strain of RotaTeq. These results indicate that the antigenic epitopes of RVA strains contained in the vaccines differ substantially from those of the currently circulating RVA strains in Belgium. Over time, these differences might result in selection for strains that escape the RVA neutralizing-antibody pressure induced by vaccines.

Multiple reassortment and interspecies transmission events contribute to the diversity of feline, canine and feline/canine-like human group A rotavirus strains

RNA-RNA hybridization assays and complete genome sequence analyses have shown that feline rotavirus (FRV) and canine rotavirus (CRV) strains display at least two distinct genotype constellations (genogroups), represented by the FRV strain RVA/Cat-tc/AUS/Cat97/1984/G3P[3] and the human rotavirus (HRV) strain RVA/Human-tc/JPN/AU-1/1982/G3P3[9], respectively. G3P[3] and G3P[9] strains have been detected sporadically in humans. The complete genomes of two CRV strains (RVA/Dog-tc/ITA/RV198-95/1995/G3P[3] and RVA/Dog-tc/ITA/RV52-96/1996/G3P[3]) and an unusual HRV strain (RVA/Human-tc/ITA/PA260-97/1997/G3P[3]) were determined to further elucidate the complex relationships among FRV, CRV and HRV strains. The CRV strains RV198-95 and RV52-96 were shown to possess a Cat97-like genotype constellation. However, 3 and 5 genes of RV198-95 and RV52-96, respectively, were found in distinct subclusters of the same genotypes, suggesting the occurrence of reassortment events among strains belonging to this FRV/CRV/HRV genogroup. Detailed phylogenetic analyses of the HRV strain PA260-97 showed that (i) 8 genome segments (VP3, VP4, VP6, VP7 and NSP2-5) clustered closely with RV198-95 and/or RV52-96; (ii) 2 genome segments (VP1 and VP2) were more closely related to HRV AU-1; and (iii) 1 genome segment (NSP1) was distantly related to any other established NSP1 genotypes and was ratified as a new NSP1 genotype, A15. These findings suggest that the human strain PA260-97 has a history of zoonotic transmission and is likely a reassortant among FRV/CRV strains from the Cat97 and AU-1-like genogroups. In addition, a potential third BA222-05-like genogroup of FRV and HRV strains should be recognized, consisting of rotavirus strains with a stable genetic genotype constellation of genes also partially related to bovine rotavirus (BRV) and bovine-like rotaviruses. The detailed phylogenetic analysis indicated that three major genotype constellations exist among FRV, CRV and feline/canine-like HRV strains, and that reassortment and interspecies transmission events contribute significantly to their wide genetic diversity.

Reassortment of human rotavirus gene segments into G11 rotavirus strains.

These viruses may become human pathogens.

Modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis.

A major limitation for better understanding the role of the human gut virome in health and disease is the lack of validated methods that allow high throughput virome analysis. To overcome this, we evaluated the quantitative effect of homogenisation, centrifugation, filtration, chloroform treatment and random amplification on a mock-virome (containing nine highly diverse viruses) and a bacterial mock-community (containing four faecal bacterial species) using quantitative PCR and next-generation sequencing. This resulted in an optimised protocol that was able to recover all viruses present in the mock-virome and strongly alters the ratio of viral versus bacterial and 16S rRNA genetic material in favour of viruses (from 43.2% to 96.7% viral reads and from 47.6% to 0.19% bacterial reads). Furthermore, our study indicated that most of the currently used virome protocols, using small filter pores and/or stringent centrifugation conditions may have largely overlooked large viruses present in viromes. We propose NetoVIR (Novel enrichment technique of VIRomes), which allows for a fast, reproducible and high throughput sample preparation for viral metagenomics studies, introducing minimal bias. This procedure is optimised mainly for faecal samples, but with appropriate concentration steps can also be used for other sample types with lower initial viral loads.

Complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium, january 2015.

ABSTRACT Porcine epidemic diarrhea virus (PEDV) is a member of the family Coronaviridae and can cause severe outbreaks of diarrhea in piglets from different age groups. Here, we report the complete genome sequence (28,028 nt) of a PEDV strain isolated during a novel outbreak in Belgium.

Complete molecular genome analyses of equine rotavirus A strains from different continents reveal several novel genotypes and a largely conserved genotype constellation

In this study, the complete genome sequences of seven equine group A rotavirus (RVA) strains (RVA/Horse-tc/GBR/L338/1991/G13P[18], RVA/Horse-wt/IRL/03V04954/2003/G3P[12] and RVA/Horse-wt/IRL/04V2024/2004/G14P[12] from Europe; RVA/Horse-wt/ARG/E30/1993/G3P[12], RVA/Horse-wt/ARG/E403/2006/G14P[12] and RVA/Horse-wt/ARG/E4040/2008/G14P[12] from Argentina; and RVA/Horse-wt/ZAF/EqRV-SA1/2006/G14P[12] from South Africa) were determined. Multiple novel genotypes were identified and genotype numbers were assigned by the Rotavirus Classification Working Group: R9 (VP1), C9 (VP2), N9 (NSP2), T12 (NSP3), E14 (NSP4), and H7 and H11 (NSP5). The genotype constellation of L338 was unique: G13-P[18]-I6-R9-C9-M6-A6-N9-T12-E14-H11. The six remaining equine RVA strains showed a largely conserved genotype constellation: G3/G14-P[12]-I2/I6-R2-C2-M3-A10-N2-T3-E2/E12-H7, which is highly divergent from other known non-equine RVA genotype constellations. Phylogenetic analyses revealed that the sequences of these equine RVA strains are related distantly to non-equine RVA strains, and that at least three lineages exist within equine RVA strains. A small number of reassortment events were observed. Interestingly, the three RVA strains from Argentina possessed the E12 genotype, whereas the three RVA strains from Ireland and South Africa possessed the E2 genotype. The unusual E12 genotype has until now only been described in Argentina among RVA strains collected from guanaco, cattle and horses, suggesting geographical isolation of this NSP4 genotype. This conserved genetic configuration of equine RVA strains could be useful for future vaccine development or improvement of currently used equine RVA vaccines.