Marina Höhne

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

ABSTRACT 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.

Sustained virological response in hepatitis C virus type 1b infected patients is predicted by the number of mutations within the NS5A-ISDR : a meta-analysis focused on geographical differences

Background and aims: There is growing evidence that the response of hepatitis C virus (HCV) genotype 1b infected patients towards interferon (IFN) therapy is influenced by the number of mutations within the carboxy terminal region of the NS5A gene, the interferon sensitivity determining region (ISDR). Patients and methods: In order to attain better insight into this correlation, a file comprising published data on ISDR strains from 1230 HCV genotype 1b infected patients, mainly from Japan and Europe, was constructed and analysed by logistic regression. Sustained virological response (SVR) was defined as negative HCV RNA six months after treatment. Results: The distribution of wild-, intermediate-, and mutant-type ISDR sequences differed significantly between Japanese (n = 655) (44.1%, 37.6%, and 18.3%) and European patients (n = 525) (24.8%, 63.4%, and 11.8%; p<0.001). There was a significant positive correlation between the number of ISDR mutations and SVR rate, irrespective of geographical region. The likelihood of SVR with each additional mutation within the ISDR was considerably more pronounced in Japanese compared with European patients (odds ratios 1.82 v 1.39; p<0.001). Pretreatment viraemia of <6.6 log copies/ml and ISDR mutant-type infection was associated with an SVR rate of 97.1% in Japanese patients but only 52.5% in European patients. Pretreatment viraemia was a stronger predictor of SVR than ISDR mutation number in Japanese patients whereas in European patients both parameters had similar predictive power. Conclusion: These data support the concept that mutant-type ISDR strains may represent a subtype within genotype 1b with a more favourable response towards IFN therapy.

Chronic Norovirus Infection after Kidney Transplantation: Molecular Evidence for Immune-Driven Viral Evolution

BACKGROUND Norovirus infection is the most common cause of acute self-limiting gastroenteritis. Only 3 cases of chronic norovirus infection in adult solid organ transplant recipients have been reported thus far. METHODS This case series describes 9 consecutive kidney allograft recipients with chronic norovirus infection with persistent virus shedding and intermittent diarrhea for a duration of 97-898 days. The follow-up includes clinical course, type of immunosuppression, and polymerase chain reaction for norovirus. Detailed molecular analyses of virus isolates from stool specimens over time were performed. RESULTS The intensity of immunosuppression correlated with the diarrheal symptoms but not with viral shedding. Molecular analysis of virus strains from each patient revealed infection with different variants of GII.4 strains in 7 of 9 patients. Another 2 patients were infected with either the GII.7 or GII.17 strain. No molecular evidence for nosocomial transmission in our outpatient clinic was found. Capsid sequence alignments from follow-up specimens of 4 patients showed accumulation of mutations over time, resulting in amino acid changes predominantly in the P2 and P1-2 region. Up to 25 amino acids mutations were accumulated over a 683-day period in the patient with an 898-day shedding history. CONCLUSION Norovirus infection may persist in adult renal allograft recipients with or without clinical symptoms. No evidence for nosocomial transmission in adult renal allograft recipients was found in our study. Molecular analysis suggests continuous viral evolution in immunocompromised patients who are unable to clear this infection.

A Mouse Model for Human Norovirus

ABSTRACT Human noroviruses (HuNoVs) cause significant morbidity and mortality worldwide. However, despite substantial efforts, a small-animal model for HuNoV has not been described to date. Since “humanized” mice have been successfully used to study human-tropic pathogens in the past, we challenged BALB/c mice deficient in recombination activation gene (Rag) 1 or 2 and common gamma chain (γc) (Rag-γc) engrafted with human CD34+ hematopoietic stem cells, nonengrafted siblings, and immunocompetent wild-type controls with pooled stool isolates from patients positive for HuNoV. Surprisingly, both humanized and nonhumanized BALB/c Rag-γc-deficient mice supported replication of a GII.4 strain of HuNoV, as indicated by increased viral loads over input. In contrast, immunocompetent wild-type BALB/c mice were not infected. An intraperitoneal route of infection and the BALB/c genetic background were important for facilitating a subclinical HuNoV infection of Rag-γc-deficient mice. Expression of structural and nonstructural proteins was detected in cells with macrophage-like morphology in the spleens and livers of BALB/c Rag-γc-deficient mice, confirming the ability of HuNoV to replicate in a mouse model. In summary, HuNoV replication in BALB/c Rag-γc-deficient mice is dependent on the immune-deficient status of the host but not on the presence of human immune cells and provides the first genetically manipulable small-animal model for studying HuNoV infection. IMPORTANCE Human noroviruses are a significant cause of viral gastroenteritis worldwide, resulting in significant morbidity and mortality. Antivirals and vaccines are currently not available, in part due to the inability to study these viruses in a genetically manipulable, small-animal model. Herein, we report the first mouse model for human noroviruses. This model will accelerate our understanding of human norovirus biology and provide a useful resource for evaluating antiviral therapies. Human noroviruses are a significant cause of viral gastroenteritis worldwide, resulting in significant morbidity and mortality. Antivirals and vaccines are currently not available, in part due to the inability to study these viruses in a genetically manipulable, small-animal model. Herein, we report the first mouse model for human noroviruses. This model will accelerate our understanding of human norovirus biology and provide a useful resource for evaluating antiviral therapies.

European Multicenter Evaluation of Commercial Enzyme Immunoassays for Detecting Norovirus Antigen in Fecal Samples

ABSTRACT A total of 2,254 fecal samples were tested in a European multicenter evaluation of commercially available norovirus antigen detection assays. Two commercial enzyme immunoassays, IDEIA Norovirus (Oxoid; Thermo Fisher Scientific, Ely, United Kingdom) and RIDASCREEN Norovirus (R-Biopharm, Darmstadt, Germany), were included in the evaluation, and their performance was compared with the results of reverse transcription-PCR (RT-PCR). Included in the evaluation were samples collected in sporadic cases of gastroenteritis, samples from outbreaks in which two or more samples were collected, well-characterized samples representing genotypes currently cocirculating within Europe, and samples collected from patients with gastroenteritis caused by a pathogen other than norovirus. The sensitivities and specificities of the IDEIA Norovirus and RIDASCREEN Norovirus assays were 58.93 and 43.81% and 93.91 and 96.37%, respectively, compared with RT-PCR. The sensitivities of both assays for outbreak investigations improved when six or more samples from an outbreak were examined. The IDEIA Norovirus assay exhibited reactivity to a broader range of norovirus genotypes than the RIDASCREEN Norovirus assay, which showed genotype-dependent sensitivities. The results indicate that, if used, these assays should serve as screening assays and the results should be confirmed by RT-PCR.

Detection and Typing of Norovirus from Frozen Strawberries Involved in a Large-Scale Gastroenteritis Outbreak in Germany

During September/October 2012, a norovirus gastroenteritis outbreak affecting about 11,000 people occurred in Germany. Epidemiological studies suggested that frozen strawberries represented the vehicle of infection. We describe here the analysis of frozen strawberries for the presence of norovirus. Samples were taken by applying a stratified subsampling scheme. Two different methods for virus extraction from strawberries were compared. First, viruses were eluted from strawberries under alkaline conditions and concentrated using a polyethylene glycol precipitation. Second, ultrafiltration was applied for concentration of viruses rinsed off of the berries. In both cases, RNA was extracted and analyzed by real-time RT-PCR. Application of the ultrafiltration method generally resulted in a lower detection rate. Noroviruses were detected in 7/11 samples derived from the lot of strawberries implicated in the outbreak using the precipitation method. Typing of norovirus revealed three different genotypes including a combination of norovirus genotype II.16 (viral polymerase) and II.13 (viral capsid). This genotype combination was also found in some of the patients that were involved in the outbreak, but that had not been reported in Germany so far. In conclusion, heterogeneously distributed noroviruses in frozen strawberries can be detected by applying an optimized combination of sampling procedures, virus extraction methods, and real-time RT-PCR protocols. The detection of several different genotypes in the strawberries may suggest contamination from sewage rather than from a single infected food handler.

Hepatitis GBV-C sequences in patients infected with HCV contaminated anti-D immunoglobulin and among i.v. drug users in Germany

BACKGROUND/AIMS A novel virus, GB virus-C (GBV-C), with a genome organization similar to those of the Flaviviridae family was identified in sera of patients diagnosed with hepatitis. Up to now little has been known about the prevalence of GBV-C sequences in German hepatitis C virus infected patients. METHODS We investigated two groups of patients chronically infected with hepatitis C virus: (i) Women infected in 1978/79 with HCV-contaminated anti-D immunoglobulin batches in former Eastern Germany, and (ii) i.v. drug users infected with different HCV subtypes. Nested polymerase chain reaction products amplified with GBV-C specific primer pairs within the helicase region were sequenced directly and compared with the GBV-C sequences reported recently. RESULTS GBV-C sequences of the putative NS 3 gene region were shown to occur in two randomly selected anti-D immunoglobulin batches of 1978 (GI and GII) and two sera of HCV-infected women drawn in 1979. In patient 3, the HCV-infected erythrocyte donor in 1978, specific GBV-C sequences were also evident in serum drawn in 1990. In the high-risk group of i.v. drug users, 49% were GBV-C RNA positive. Among the 21 GBV-C positive samples, 11 were coinfected with HCV subtype 3a and 10 with subtype 1 b. All isolates showed an overall homology to the GBV-C sequence reported by Simons of 75-81% at the nucleotide level and 94-100% at the amino acid level. CONCLUSION GBV-C sequences are detectable in the anti-D immunoglobulin batches which caused a hepatitis C virus outbreak in 1979, and a first hint of its transmission to recipients was shown. The detection of GBV-C in patient serum drawn 12 years after the onset of chronic liver disease confirms the persistence of the novel virus described here. In the group of i.v. drug users a high frequency of GBV-C sequences (49%) was shown, and the considerable variability of the nucleotide sequences indicates the existence of different GBV-C genotypes/subtypes.

Epidemiology of norovirus gastroenteritis in Germany 2001-2009: eight seasons of routine surveillance.

We analysed data on laboratory or epidemiologically confirmed cases (n = 856,539) and on outbreaks (n = 31,644) notified during week 31 (2001) to week 30 (2009), and performed molecular typing of specimens from 665 outbreaks. We aimed at identifying demographic and molecular characteristics to inform on potential additional approaches to prevent disease spread in the population. The mean incidence by norovirus season (week 31 in one year to week 30 in the following year) was 130 (range 19-300) cases/100,000 population and was highest in persons aged <5 years (430/100,000) and ≥ 75 years (593/100,000). The proportion hospitalized in community-acquired cases was 8-19% per season. The mean norovirus-associated mortality was 0.05/100,000 per season and 0.5/100,000 in the ≥ 75 years age group. Most outbreaks with known setting (75%) occurred in hospitals (32%), nursing homes (28%), households (24%) and childcare facilities (10%). GII strains dominated in the outbreak specimens. GII.4 strains were found in 82% of nursing home outbreaks, 85% of hospital outbreaks, and 33% of childcare facility and school outbreaks. Cases in younger individuals were notified earlier during the season than adult cases, and outbreaks in childcare facilities and schools preceded those in nursing/residential homes, hospitals and private households. We suggest future studies to investigate more closely potential transmission patterns between children and adults.

Seroepidemiology of TT virus, GBC-C/HGV, and hepatitis viruses B, C, and E among women in a rural area of Tanzania.

The seroprevalence and determinants of hepatitis B, C, and E virus infection, and of GBV‐C/hepatitis G virus and TT virus infection were investigated among women from a rural area of northeastern Tanzania. High seroprevalence rates were found for TTV (74%), HBV (74%), and GBV‐C/HGV (35%), whereas 7% of the women had evidence of HCV and HEV infection. The majority of TTV DNA sequences in the study population belonged to the genotypes 1 or 2. One sequence seems to represent a new subtype of genotype 4. The GBV‐C/HGV sequences either belonged to the genomic Group 1b or to the recently described Group 4. In multivariate analysis, the detection of TTV DNA was associated significantly with a larger number of children in the household and with older age. A history of injections of contraceptive hormones was an independent risk factor for HCV infection. The findings on TTV are consistent with fecal‐oral transmission, and recurrent infections may occur in adults. J. Med. Virol. 62:524–530, 2000.

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.