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.

Structural and functional changes of the duodenum in human norovirus infection.

BACKGROUND: Norovirus infection is the most frequent cause of infectious diarrhoea in the western world. This study aimed to characterise functionally and histomorphologically the diseased duodenum in human biopsies. METHODS: Norovirus infection was diagnosed by the Kaplan criteria and confirmed by PCR of stool samples. Duodenal biopsies were obtained endoscopically. In miniaturised Ussing chambers, short circuit current, flux measurements and impedance spectroscopy were performed. Histological analysis including apoptosis staining and characterisation of intraepithelial lymphocytes was performed. Tight junction proteins were quantified by immunoblotting. RESULTS: In norovirus infection, epithelial resistance decreased from (mean (SEM)) 24 (2) Omega cm(2) in controls to 10 (1) Omega cm(2). Mannitol flux increased from 113 (24) nmol h(-1) cm(-2) in controls to 242 (29) nmol h(-1) cm(-2). Microdissection revealed a villus surface area reduced by 47% (6.6%). Intraepithelial lymphocytes were increased to 63 (7) per 100 enterocytes, with an increased rate of perforin-positive cytotoxic T cells. Expression of tight junctional proteins occludin, claudin-4 and claudin-5 was reduced. The epithelial apoptotic ratio was doubled in norovirus infection. Furthermore, the basal short circuit current was increased in norovirus infection and could be reduced by bumetanide and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). CONCLUSIONS: Norovirus infection leads to epithelial barrier dysfunction paralleled by a reduction of sealing tight junctional proteins and an increase in epithelial apoptosis, which may partly be mediated by increased cytotoxic intraepithelial lymphocytes. Furthermore, active anion secretion is markedly stimulated. Thus, the diarrhoea in norovirus infection is driven by both a leak flux and a secretory component.

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.

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.

A functional toll-like receptor 8 variant is associated with HIV disease restriction.

BACKGROUND: Toll-like receptors (TLRs) play an important role in the innate immune response to pathogens. TLR8 has been found to recognize RNA derived from various viruses, including human immunodeficiency virus (HIV). Presently, very little is known about the influence of TLR8 genetic variation on susceptibility to and progression of HIV disease. METHODS AND RESULTS: We genotyped a population of 782 HIV-positive adults and 550 healthy control subjects for 3 nonsynonymous TLR8 single-nucleotide polymorphisms. We found that the presence of the most frequent TLR8 polymorphism, TLR8 A1G (rs3764880), confers a significantly protective effect regarding progression of the disease. In overexpression assays, we demonstrated that this receptor variant displays impaired NF-kappaB activation in vitro. Furthermore, we analyzed different cell types obtained from individuals differing in their TLR8 genotype and assessed their response to TLR8 ligands in vitro. The presence of the mutated receptor variant was associated with modulation of cytokine secretion profiles and lipid mediator synthesis patterns in monocytes and neutrophils. CONCLUSIONS: This first report of a functional TLR8 variant associated with a different clinical course of an RNA viral disease may have implications for the individual risk assessment of patients infected with HIV and other RNA viruses as well as for future HIV vaccine development.

Data quality of 5 years of central norovirus outbreak reporting in the European Network for food-borne viruses.

BACKGROUND: The food-borne viruses in Europe (FBVE) network database was established in 1999 to monitor trends in outbreaks of gastroenteritis due to noroviruses (NoVs), to identify major transmission routes of NoV infections within and between participating countries and to detect diffuse international food-borne outbreaks. METHODS: We reviewed the total of 9430 NoV outbreak reports from 13 countries with date of onset between 1 January 2002 and 1 January 2007 for representativeness, completeness and timeliness against these objectives. RESULTS: Rates of reporting ranged from a yearly average of 1.8 in 2003 to 11.6 in 2006. Completeness of reporting of an agreed minimum dataset improved over the years, both for epidemiological and virological data. For the 10 countries that provided integrated (epidemiological AND virological) reporting over the 5-year period, the completeness of the minimum dataset rose from 15% in 2003 to 48% in 2006. Two countries have not been able to combine both data types due to the structure of the national surveillance system (England and Wales and Germany). Timeliness of reporting (median days between the onset of an outbreak and the date of reporting to the FBVE database) differed greatly between countries, but gradually improved to 47 days in 2006. CONCLUSION: The outbreaks reported to the FBVE reflect the lack of standardization of surveillance systems across Europe, making direct comparison of data between countries difficult. However, trends in reported outbreaks per country, distribution of NoV genotypes, and detection of diffuse international outbreaks were used as background data in acute questions about NoV illness and the changing genotype distribution during the 5-year period, shown to be of added value. Integrated reporting is essential for these objectives, but could be limited to sentinel countries with surveillance systems that allow this integration. For successful intervention in case of diffuse international outbreaks, completeness and timeliness of reporting would need to be improved and expanded to countries that presently do not participate.

A foodborne norovirus outbreak due to manually prepared salad, Austria 2006.

BACKGROUND: Norovirus is increasingly being recognized as a leading cause of foodborne disease. Nevertheless, well documented foodborne outbreaks due to norovirus are rarely found in the literature. MATERIAL AND METHODS: A retrospective cohort study was conducted for identifying the source of a gastroenteritis outbreak. A total of 325 persons were identified as the at risk group. RESULTS: The overall attack rate was 56% (182/325). Of the four working days of possible foodborne exposure to norovirus (Monday till Thursday), Wednesday (risk ratio [RR]: 18.82; 95%CI 11.82-29.96) and Thursday (RR 2.14; 95%CI 1.65-2.79) turned out to be the most likely days on which infections with norovirus occurred. The day-by-day food specific cohort analyses yielded consumption of salad on Wednesday (adjusted RR 2.82; 95%CI 1.0-7.94) to be associated with highest risk of illness. The most likely source of food contamination is a kitchen assistant having prepared salad manually. She fell ill with symptoms of gastroenteritis on Wednesday during the early working hours and continued working. Human stool samples obtained from five out of six outbreak cases, including the sick kitchen assistant, were RT-PCR positive for norovirus genotype GGII.7 (Leeds-like). CONCLUSION: This foodborne norovirus outbreak underlines the drastic consequences of neglecting the rules of basic kitchen hygiene. Food handlers working despite manifest diarrhea or vomiting--often in fear of job loss--are a common cause of foodborne norovirus outbreaks.

Group A rotavirus genotypes in Germany during 2005/2006.

During the 2005/2006 winter season a total of 802 group A positive rotavirus specimens of patients from different regions throughout Germany were genotyped. Amplicons from a one-tube RT-PCR were typed by analysis of their (type-specific) size using type-specific primers, fluorescent consensus primers and a capillary sequencer for detection. While G1P[8] was predominant (45.8%), G9P[8] has emerged as the second most frequent genotype combination (37.7%). The distribution of genotypes was heterogeneous, regional frequencies regarding G1 and G9 were ranging from 15.0 to 89.3% and from 7.1 to 67.7%, respectively. Furthermore, a few human rotavirus G10P[6] and G10P[8] infections were observed.

Genetic diversity and recombination of murine noroviruses in immunocompromised mice.

Murine noroviruses (MNV) are newly identified pathogens which infect laboratory mice. In this study, we found a high prevalence (64.3%) of MNV in various breeding colonies of immunocompromised, transgenic and wild-type mouse lines. All mice survived infection with no signs of clinical disease. Faeces samples were collected from animals housed in two separate laboratory mouse colonies in Berlin, Germany, and screened using quantitative reverse transcription (RT)-PCR. We have determined the complete nucleotide sequences of 3 novel MNV strains. Furthermore, we sequenced two subgenomic regions within open reading frames (ORFs) 1 and 2 that are suitable for genotyping. Sequence analysis of the full-length and partial genomes obtained from naturally infected mice yielded valuable data on genetic diversity of murine noroviruses. The discordance of genotype affiliation of some MNVs shown in ORF1 and ORF2 suggests intertypic recombination events in vivo.

[The German Health Interview and Examination Survey for Children and Adolescents (KiGGS): state of immunity against poliomyelitis in German children]. / Kinder- und Jugendgesundheitssurvey (KiGGS): Immunitätslage gegen Poliomyelitis.

One of the goals of the WHO is the worldwide eradication of poliomyelitis in the coming years. Europe was declared poliofree in 2002, but increasing migration may lead to a come-back of circulating polioviruses. A high level of population immunity protects against imported wild viruses from endemic areas. The first seroprevalence data since the switch from live to inactivated vaccine in Germany (OPV to IPV) are provided. A serum panel was tested (n=2,046) in order to study the serological status against poliomyelitis. The microneutralization test on RD cells was used. Overall, neutralizing antibodies against poliovirus types 1, 2 and 3 were detected in 97.4%, 97.6%, and 93.6% of samples, respectively. Of the test persons, 91.7% had antibodies against all three virus types. Only 26 children simultaneously lacked neutralizing antibodies for all three serotypes (1.3%). No significant correlation between gender, region (East/West)), migration status (with/without migration background) and antibody prevalence to polioviruses was found. The seroprevalence of antibodies against all three types of polioviruses indicates a very high level of population immunity in German children. It must be maintained through consequently performed vaccination programmes.

Outbreak of hepatitis A in two federal states of Germany: bakery products as vehicle of infection.

In April 2004, increased numbers of hepatitis A were noted in six neighbouring districts in Germany. Exploratory interviews showed that patients had consumed bakery products from company X where two employees had been diagnosed with hepatitis A in February. A case-control study of consumption of products of company X was carried out through telephone interviews. Altogether, 64 cases were identified. Fifty-two cases and 112 controls aged >or=16 years were included in the case-control study. In total, 46/52 cases and 37/112 controls had consumed company X products [odds ratio (OR) 15.5, 95% confidence interval (CI) 6.1-39.7]. Of these, 36/46 cases and 16/37 controls had consumed pastries (OR 4.7, 95% CI 1.8-12.3), 25/46 cases and 12/37 controls had consumed filled doughnuts (OR 2.5, 95% CI 1.0-6.1). Sequence analysis of the VP1-2A junction region indicated 100% strain homology between cases and an infected employee of company X. We recommended reinforcement of hygiene precautions, and consideration of a prolongation of compulsory work absence after post-exposure vaccination.

[Norovirus infections in Germany]. / Norovirusinfektionen in Deutschland.

Noroviruses are responsible for the majority of acute viral gastroenteritis infections worldwide. Transmission may be faecal-oral or through contaminated food and water or airborne by virus-containing aerosols. Characteristics of noroviruses that facilitate their spread are their high concentration in stool and vomitus, their extreme environmental stability, their low infectious dose as well as the lack of long-lasting immunity. The majority of norovirus infections occur in large outbreaks among persons living in institutional settings, such as hospitals and nursing homes, although sporadic cases also occur. Children and elderly persons are most often affected. Illness is characterized by acute onset of projectile vomiting. For prevention and control of norovirus outbreaks strict control management is necessary. Based on the high genomic variability new variant noroviruses with different pathogenic factors can arise. Depending on the circulating variant the extent of the usual winter peak can vary enormously. Available diagnostic methods include RT-PCR assays for detection of viral RNA, electron microscopy and enzyme immunoassays (EIA) for detection of viral antigens. The implicated virus can be subtyped through nucleotide sequencing and linked to a specific outbreak. With the enactment of the Protection against Infection Act in January 2001 a mandatory reporting system of norovirus infections was established. Analysis of surveillance data from this system permits a detailed overview of the nationwide epidemiology of this disease in Germany.

Molecular characterization of the first Aichi viruses isolated in Europe and in South America.

The occurrence of Aichi virus, a picornavirus associated with acute gastroenteritis, has so far only been described in Asian countries. This study reports the first finding of Aichi virus in clinical specimens from Germany and Brazil. The nucleotide sequences of both a German and a Brazilian isolate were determined, analyzed, and compared to known Aichi sequences. The German strain turned out to be a member of genogroup A, while the Brazilian belonged to genogroup B. For a primary assessment of the epidemiological importance of Aichi virus in Germany, a panel of 485 German serum samples was screened for antibody to Aichi virus, and a seroprevalence of 76% was found.

Molecular characterization of human astroviruses isolated in Brazil, including the complete sequences of astrovirus genotypes 4 and 5.

Human astroviruses (HAstV) are recognized as an important cause of gastroenteritis in young children worldwide. This study describes the molecular characteristics of astroviruses isolated in Brazil, using RT-PCR and molecular sequencing of segments of all three viral ORFs. Genetic analysis of a 348-nucleotide segment from ORF 2 demonstrated that the Brazilian isolates belong to HAstV genotypes 1 to 5 and 8. ORF 1b sequences displayed a high degree of nucleotide identity even between different genotypes, which disfavours HAstV genotyping in this region. ORF 1a sequence analysis classified all Brazilian samples as genogroup A. The complete sequences of HAstV genotype 4 (putative serotype 4) and genotype 5 (putative serotype 5) were determined for the first time.

Nosocomial transmission of hepatitis C virus from an anesthesiologist to three patients--epidemiologic and molecular evidence.

Acute hepatitis C occurred in three patients who had undergone gynecologic surgery in an outpatient clinic on a single day. Epidemiologic and virologic investigations revealed virus transmission from an anesthesiologist with chronic hepatitis C. HCV sequencing demonstrated that all four persons were infected by the same strain. The precise mode of transmission could not be elucidated but hygienic standards had been severely neglected.

An outbreak of viral meningitis associated with a public swimming pond.

From July to October 2001, 215 cases of aseptic meningitis occurred among the inhabitants of the German city of Kassel and neighbouring counties. A matched case-control study identified bathing in a public, nature-like pond during the beginning of the outbreak as a risk factor for disease [matched odds ratio (mOR) 44.8, 95% confidence interval (CI) 3.9-515.6]. Among bathers, patients with meningitis spent more time in the water (mOR 18.8, 95% CI 2.0-174.1) and swallowed water more frequently (mOR = 7.3, 95% CI 0.7-81.8). Of 30 cerebrospinal fluid samples tested, echovirus 30 was cultured from 16, and echovirus 13 from seven. An echovirus 30 sequence obtained from one pond water sample showed a 99% nucleotide and 100% amino-acid homology with patient isolates. This outbreak demonstrates the potential of nature-like swimming ponds to cause widespread community infection with substantial public health impact.

Generation of recombinant norovirus-like particles (VLP) in the human endothelial kidney cell line 293T.

Noroviruses (NVs) are the major cause of non-bacterial outbreaks of gastroenteritis. Here we report a new alternative system to generate recombinant NV virus-like particles (rNV-VLP) in a human endothelial kidney cell line (HEK). Transfecting HEK-293T cells with an expression vector coding for the ORF-2 gene lead to the expression of the viral structural protein VP1 which spontaneously assembled into virus-like particles (VLP), as shown by electron microscopy. The transfected cells did not show a cytopathic effect and released rNV-VLP into the culture medium. The HEK-293T cell derived particles were morphologically indistinguishable to the rNV-VLP produced from baculovirus and the Venezuelan equine encephalitis virus (VEE)-replicon. The produced particles were stable for at least 2.5 months at 4 degrees C.

Detection of enteric viruses and bacterial indicators in German environmental waters.

A German mining lake and the supplying surface waters, which are located downstream of a sewage plant, were examined regarding their microbiological and virological quality. Between October 2002 and September 2003, specific PCR methods were used to determine the occurrence of enteric viruses in 123 water specimens drawn at different sites downstream of the waste water treatment plant and in 9 samples from the sewage plant influent. Detection rates in sewage plant effluents and surface water samples depended on sampling sites and were: 29-76% for enterovirus (EntV), 24-42% (astrovirus, AstV), 15-53% (norovirus, NV), 3-24% (rotavirus, RoV), 5-20% (hepatitis A virus, HAV) and 20% (adenovirus, AdV). AstV genome load of selected samples was between 3.7 x 10(3) to 1.2 x 10(8) genome equivalents per liter (gen.equ./l), depending on sampling location; NV average genome load ranged from 1.8 x 10(4) to 9.7 x 10(5) gen.equ./l. Cell culture methods showed that three out of 18 PCR positive samples contained infectious EntV. Even though microbiological parameters such as Escherichia coli, enterococci and coliphages indicated acceptable microbiological water quality, the virological data of this study suggest the possibility that surface waters may be a source for enteric viral infections.