Comparative analysis of the Rotarix™ vaccine strain and G1P[8] rotaviruses detected before and after vaccine introduction in Belgium.

G1P[8] rotaviruses are responsible for the majority of human rotavirus infections worldwide. The effect of universal mass vaccination with rotavirus vaccines on circulating G1P[8] rotaviruses is still poorly understood. Therefore we analyzed the complete genomes of the Rotarix™ vaccine strain, and 70 G1P[8] rotaviruses, detected between 1999 and 2010 in Belgium (36 before and 34 after vaccine introduction) to investigate the impact of rotavirus vaccine introduction on circulating G1P[8] strains. All rotaviruses possessed a complete Wa-like genotype constellation, but frequent intra-genogroup reassortments were observed as well as multiple different cluster constellations circulating in a single season. In addition, identical cluster constellations were found to circulate persistently over multiple seasons. The Rotarix™ vaccine strain possessed a unique cluster constellation that was not present in currently circulating G1P[8] strains. At the nucleotide level, the VP6, VP2 and NSP2 gene segments of Rotarix™ were relatively distantly related to any Belgian G1P[8] strain, but other gene segments of Rotarix™ were found in clusters also containing circulating Belgian strains. At the amino acid level, the genetic distance between Rotarix™ and circulating Belgian strains was considerably lower, except for NSP1. When we compared the Belgian G1P[8] strains collected before and after vaccine introduction a reduction in the proportion of strains that were found in the same cluster as the Rotarix™ vaccine strain was observed for most gene segments. The reduction in the proportion of strains belonging to the same cluster may be the result of the vaccine introduction, although natural fluctuations cannot be ruled out.

Emergence of human G2P[4] rotaviruses containing animal derived gene segments in the post-vaccine era.

The introduction of Rotarix into the Belgian immunization program in June 2006 coincided with an increase of the relative prevalence of G2P[4] strains. However, the genetic composition of these persistent G2P[4] strains has not been investigated. Therefore, we have investigated the NSP4 gene of 89 Belgian G2P[4] strains detected between 1999 and 2013, covering both pre- and post-vaccination periods. The NSP4 genes were divided over seven separate clusters of which six were more closely related to animal than to human strains. The NSP4 genes that clustered more closely to animal DS-1-like strains were isolated after 2004-2005 and were found throughout multiple seasons. Complete genome sequencing of 28 strains identified several other gene segments that clustered more closely to animal than to human DS-1-like strains. These findings suggest that frequent interspecies reassortments may have played a role in the spread of G2P[4] rotaviruses in the post-vaccination period in Belgium.

Spatio-temporal analysis of Nova virus, a divergent hantavirus circulating in the European mole in Belgium.

Over the last decade, the recognized host range of hantaviruses has expanded considerably with the discovery of distinct hantaviruses in shrews, moles and bats. Unfortunately, in-depth studies of these viruses have been limited. Here we describe a comprehensive analysis of the spatial distribution, genetic diversity and evolution of Nova virus, a hantavirus that has the European mole as its natural host. Our analysis demonstrated that Nova virus has a high prevalence and widespread distribution in Belgium. While Nova virus displayed relatively high nucleotide diversity in Belgium, amino acid changes were limited. The nucleocapsid protein was subjected to strong purifying selection, reflecting the strict evolutionary constraints placed upon Nova virus by its host. Spatio-temporal analysis using Bayesian evolutionary inference techniques demonstrated that Nova virus had efficiently spread in the European mole population in Belgium, forming two distinct clades, representing east and west of Belgium. The influence of landscape barriers, in the form of the main waterways, on the dispersal velocity of Nova virus was assessed using an analytical framework for comparing Bayesian viral phylogenies with environmental landscape data. We demonstrated that waterways did not act as an environmental resistance factor slowing down Nova virus diffusion in the mole population. With this study, we provide information about the spatial diffusion of Nova virus and contribute sequence information that can be applied in further functional studies.

Molecular characterization of equine rotaviruses isolated in Europe in 2013: implications for vaccination.

Equine group A rotavirus (RVAs) mainly cause disease in foals under the age of 3 months. Only sporadic data are available on the circulation of RVAs in equine populations in Europe. In this study, 65 diarrheic samples from foals under 4 months of age were collected in Belgium (n=32), Germany (n=17), Slovenia (n=5), Sweden (n=4), Hungary (n=3), Italy (n=2), France (n=1) and The Netherlands (n=1). Forty percent of these samples (n=26) were found to be RVA positive by a quantitative RT-PCR assay. The viral load in 11 of these samples was sufficiently high to be (partially) genotyped. G3, G14 and P[12] were the main genotypes detected, and phylogenetic analyses revealed that they were closely related to contemporary equine RVA strains detected in Europe as well as in Brazil and South Africa. Regional variation was observed with only G14 and P[12] being detected in Germany, whereas mainly G3P[12] was encountered in Belgium. Surprisingly the only G14P[12] RVA strain detected in Belgium was also found to possess the very rare P[18] genotype, which has been described only once from equine RVA strain L338 detected in the UK in 1991. Despite the identification of this uncommon P[18] genotype, G3P[12] and G14P[12] RVA strains remained the most important genotypes in Europe during the study period. Based on this finding and the knowledge that G3P[12] and G14P[12] serotypes are partially cross-reactive it can be assumed that a vaccine based on an inactivated virus of the G3P[12] genotype is still relevant in the current European epidemiological situation, although the addition of a G14 strain would most likely be beneficial.

A decade of norovirus genetic diversity in Belgium.

Outbreaks of norovirus-associated gastroenteritis occur during all seasons and in various locations, and are recognized as one of the most common causes of nonbacterial food-borne infections. The molecular epidemiology of norovirus infections has not been well characterized in Belgium. To study the incidence of norovirus infections and the nature of the circulating genotypes, 3080 specimens were collected from patients with acute gastroenteritis between 2004 and 2014. Norovirus was detected with RT-PCR in 554 samples (18%). The circulating strains were genotyped based on the variability in the 5' end of the capsid gene (region C). The GII.4 genotype, which is detected predominantly worldwide, was also the most prevalent genotype in our study (87%). This study shows a high frequency and genetic diversity of norovirus in patients with acute gastroenteritis in health care facilities in Flanders, Belgium.

Rotavirus surveillance in Kisangani, the Democratic Republic of the Congo, reveals a high number of unusual genotypes and gene segments of animal origin in non-vaccinated symptomatic children.

Group A rotavirus (RVA) infections form a major public health problem, especially in low-income countries like the Democratic Republic of the Congo (COD). However, limited data on RVA diversity is available from sub-Saharan Africa in general and the COD in particular. Therefore, the first aim of this study was to determine the genetic diversity of 99 RVAs detected during 2007-2010 in Kisangani, COD. The predominant G-type was G1 (39%) and the most predominant P-type was P[6] (53%). A total of eight different G/P-combinations were found: G1P[8] (28%), G8P[6] (26%), G2P[4] (14%), G12P[6] (13%), G1P[6] (11%), G9P[8] (4%), G4P[6] (2%) and G8P[4] (1%). The second aim of this study was to gain insight into the diversity of P[6] RVA strains in the COD. Therefore, we selected five P[6] RVA strains in combination with the G1, G4, G8 (2x) or G12 genotype for complete genome analysis. Complete genome analysis showed that the genetic background of the G1P[6] and G12P[6] strains was entirely composed of genotype 1 (Wa-like), while the segments of the two G8P[6] strains were identified as genotype 2 (DS-1-like). Interestingly, all four strains possessed a NSP4 gene of animal origin. The analyzed G4P[6] RVA strain was found to possess the unusual G4-P[6]-I1-R1-C1-M1-A1-N1-T7-E1-H1 constellation. Although the majority of its genes (if not all), were presumably of porcine origin, this strain was able to cause gastro-enteritis in humans. The high prevalence of unusual RVA strains in the COD highlights the need for continued surveillance of RVA diversity in the COD. These results also underline the importance of complete genetic characterization of RVA strains and indicate that reassortments and interspecies transmission among human and animal RVAs strains occur regularly. Based on these data, RVA vaccines will be challenged with a wide variety of different RVA strain types in the COD.

Complete genetic characterization of human G2P[6] and G3P[6] rotavirus strains.

During the 2008-2009 rotavirus season, 10 G3P[6] rotavirus strains were isolated for the first time in Belgium, while an outbreak of G2P[6] strains occurred in the USA in 2005-2006. Partial sequencing of the 11 genome segments of the 10 Belgian G3P[6] strains revealed a clonal origin. Two of these strains, and a G2P[6] strain representative of the American outbreak, were selected and sequenced completely to analyze their evolutionary relationships. Genetic analysis revealed that all strains possessed a DS-1-like genotype constellation. The 2 Belgian G3P[6] strains showed >99% sequence identity at the nucleotide level and the American G2P[6] strain was phylogenetically closely related to the Belgian P[6] strains. These data suggest that reassortment(s) involving VP7 occurred recently, and that the prevalence of DS-1-like P[6] rotavirus strains need to be closely monitored because the currently licensed RVA vaccines contain neither the P[6] genotype nor strains with a complete human DS-1 genotype constellation.

Full genome characterization of a porcine-like human G9P[6] rotavirus strain isolated from an infant in Belgium.

Interspecies transmissions of group A rotavirus (RVA) strains among animals and humans are thought to take place frequently. During a RVA surveillance study in Belgium we isolated an unusual G9P[6] RVA strain, RVA/human-wt/BEL/BE2001/2009/G9P[6], from a 1month old boy, which did not cluster with other G9 or P[6] strains isolated in Belgium. In this study we sequenced and characterized the complete genome of this unusual G9P[6] strain BE2001. Phylogenetic analyses of all 11 genes revealed a unique genotype constellation: G9-P[6]-I5-R1-C1-M1-A8-N1-T7-E1-H1. The VP6 and NSP1 genotypes I5 and A8 are genotypes commonly found in porcine RVA strains, while the VP7 and VP4 genes clustered only distantly to human lineages of G9 and P[6], respectively. The VP1, VP2, VP3, NSP2, NSP4 and NSP5 genes all belonged to Wa-like genotypes, but clustered more closely to porcine strains than to human strains. NSP3 belonged to the rare T7 genotype. Thus far, T7 genotypes have only been detected in one porcine-like human strain (RVA/human-tc/CHN/R479/2004/G4P[6]), one bovine-like human strain (RVA/human-xx/IND/mani-265/2007/G10P[6]) and one bovine RVA strain (RVA/cow-tc/GBR/UK/1973/G6P7[5]). Sequence analysis of the BE2001 NSP5 gene segment revealed a 300 nucleotide duplication in the 3' end non-coding region. BE2001 is most likely a direct interspecies transmission between a pig and a human. Inquiry with the patient's physician revealed that the father of the patient had been working on a pig farm in the week the patient became ill, providing a plausible route of transmission.

Genetic analyses reveal differences in the VP7 and VP4 antigenic epitopes between human rotaviruses circulating in Belgium and rotaviruses in Rotarix and RotaTeq.

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.

Exhaled breath condensate sampling is not a new method for detection of respiratory viruses.

BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections.

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.