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Additional file 1.Table S1: ORF2-Primer sequences for genotyping HAstV classic and MLB (region ORF1-ORF2).

Nigeria having approximately 50 000 Rotavirus A (RVA) deaths annually is yet to introduce RVA vaccine into routine national immunization; therefore surveillance of RVA strains circulating before vaccine introduction is essential in evaluating impact of the intervention. Stool samples and sociodemographic data of diarrhoeic children

Two rotavirus (RV) vaccines were introduced to the European market in 2006. To support the decision-making process of the German Standing Committee on Vaccination ("Standige Impfkommission", STIKO) regarding adoption of routine RV vaccination into the national vaccination schedule in Germany relevant scientific background was reviewed. According to STIKO’s Standard Operating Procedures for the development of evidence-based vaccination recommendations, a set of key questions was addressed and systematic reviews were performed with a focus on the efficacy, effectiveness, impact and safety of RV vaccines. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology was applied to assess the quality of available evidence. Data from 5 randomized controlled trials demonstrated a high efficacy of RV vaccines in preventing severe RV-associated gastroenteritis (91%) and hospitalization (92%) in settings comparable to Germany. Post-marketing observational studies confirmed these findings. In several countries, impact studies suggest that age groups not eligible for vaccination might also benefit from herd effects and demonstrated a decrease in the number of nosocomial RV infections after RV vaccine introduction. The vaccines were considered safe, except for a slightly increased risk of intussusception shortly after the first dose, corresponding to 1-2 additional cases per 100,000 infants vaccinated (relative risk =1.21, 95% confidence interval [CI] 0.68-2.14). RV case-fatality is extremely low in Germany. However, RV incidence among children aged

Norovirus infection is the main cause of epidemic non-bacterial gastroenteritis in humans. Although human norovirus (HuNoV) infection is self-limiting, it can persist for extended periods of time in immune deficient patients. Due to the lack of robust cell culture and small animal systems, little is known about HuNoV pathogenicity. However, murine norovirus (MNV) can be propagated in cell culture and is used as a model to study norovirus infection. Several MNV are known to persist in mice. In this study, we show that the MNV strain MNV-S99 persists in wild type inbred (C57BL/6J) mice over a period of at least 5 weeks post infection. Viral RNA was detectable in the jejunum, ileum, cecum, and colon, with the highest titers in the colon and cecum. To characterize the effect of MNV-S99 on the innate immune response, Stat1 phosphorylation and IFN-β production were analyzed and compared to the non-persistent strain MNV-1.CW3. While MNV-S99 and MNV-1.CW3 showed comparable growth characteristics in vitro, Stat1 phosphorylation and IFN-β release is strongly decreased after infection with MNV-S99 compared to MNV-1.CW3. In conclusion, our results show that unlike MNV-1.CW3, MNV-S99 establishes a persistent infection in mice, possibly due to interfering with the innate immune response.

Low-density lipoprotein receptor (LDLR) is involved in the entry of hepatitis C virus (HCV) in host cells. We investigated whether three single-nucleotide alterations within LDLR might be associated with the course of hepatitis C infection and response to antiviral therapy. We enrolled 651 individuals with chronic HCV infection who had received interferon-based combination therapy, 174 individuals with self-limited HCV infection, and 516 healthy controls. LDLR c.1171G>A, c.1413G>A, and c.*52G>A genotyping was performed by real-time PCR-based assays. HCV genotype 1-infected individuals who were homozygous for 3'UTR c.*52G were at increased risk for virologic non-response to antiviral therapy compared to virologic responders (66.3% vs. 51.0%, p=0.001). Furthermore, compared to healthy controls, self-limited HCV genotype 1 infection was significantly associated with c.1171A (15.1% vs. 6.6%, p=0.006) and negatively associated with c.1413G>A heterozygosity (33.0% vs. 46.1%, p=0.023). The data indicate that LDLR alterations are correlated with response to interferon-based combination therapy and with self-limitation of HCV 1 infection.

Summary 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.

Infectious hepatitis C virus (HCV) particles bind to host lipoproteins such as low-density lipoproteins (LDLs). Low-density lipoprotein receptors (LDLR) have been termed candidate receptors for HCV-LDL complexes. Functional host genetic single nucleotide polymorphisms (SNPs) in the apolipoprotein E (APOE) gene encoding apolipoprotein E (apoE) - a major structural LDL component and natural ligand of LDLR - likely influence the course of HCV infection. We investigated the prevalence of APOE SNPs in two large and independent cohorts of patients with chronic HCV infection compared to respective controls.We genotyped 996 chronically HCV-infected patients; 179 patients with spontaneous HCV clearance; 283 individuals with non-HCV-associated liver disease; and 2 234 healthy controls.APOE genotype proportions in patients with persistent HCV infection significantly differed from healthy controls (P = 0.007) primarily because of a substantial under-representation of APOE4 alleles in chronically HCV-infected patients (10.2%) compared to 13.0% in healthy controls (P = 0.001). The distribution of APOE4 allele positive genotypes (?2?4, ?3?4, ?4?4) also significantly differed between chronically HCV-infected patients and healthy controls (1.4%, 17%, 1% vs. 2.4%, 20.5%, 1.7%; P = 0.001), suggesting a protective effect of the APOE4 allele in HCV infection. This was confirmed by a significant over-representation of the APOE4 allele in patients with spontaneous HCV clearance (17.6%; P = 0.00008). The APOE4 allele distribution in patients with non-HCV-associated liver disease (14.0%) was very similar to healthy controls and also differed from chronically HCV-infected patients (P = 0.012), suggesting HCV specificity.Our findings suggest that the APOE4 allele may confer a protective effect in the course of HCV infection.

Human norovirus is the main cause of non-bacterial gastroenteritis worldwide. It is transmitted from person to person, by fecally contaminated food or water or through virus containing aerosols originating during vomiting of infected persons. In September and October 2012, the largest foodborne norovirus outbreak in Germany so far spread over 5 Federal States (Berlin, Brandenburg, Saxony, Saxony-Anhalt, and Thuringia) affecting nearly 11,000 people mainly in schools and child care facilities. Epidemiological and trace-back investigations supported the assumption that a batch of frozen strawberries imported from China was the likely source of the outbreak. Sequence analysis of the capsid region encoding the P2 domain was used successfully for identification of transmission routes and epidemiologic relationship but was hampered by a lack of universal primers for all known genotypes so far. In the present study, a molecular approach was designed to track outbreak-related samples from the affected states of the large foodborne outbreak in Germany. Therefore, sequence analysis within the highly variable P2 domain of the capsid gene using newly developed universal P2 primers for genogroup I and genogroup II strains in combination with sequencing of the polymerase gene (region A) and the orf1/orf2 junction (region c) was used. The sequence analysis of 138 norovirus positive stool samples suspected to be outbreak-related revealed a considerable genomic diversity. At least 3 strains of genogroup I (I.3, I.4, and I.9) and 5 strains of genogroup II (II.6, II.7, II. 8, and recombinants II.P7_II.6, and II.P16_II.13) as well as 19 samples containing mixtures of these strains were detected. Six samples were considered as not linked to the outbreak. The most prevalent genotype was GI.4 (48/132; 36%). Genotype I.9 and the recombinant strain II.P16_II.13 were detected for the first time in Germany. Notably, the genotype II.P16_II.13 could also be determined in one of the samples of the frozen strawberry lot suspected as infection source. Especially, due to the good concordance of the P2 sequences from infected patients of 5 Federal States the outbreak-relation of the strains could be demonstrated. The high diversity of virus strains and the occurrence of sub-clusters within genotypes I.3, II.8, II.P16_II.13, and II.7 revealed the complex mixture of the outbreak source suggesting a possible waterborne fecal contamination of the strawberries. The typing system described here is in general useful for analysis of outbreaks caused by mixed infection sources. Extensive sequence analysis of different gene regions including the highly variable P2 domain in a sufficient number of cases is required to confirm the epidemiological relation of samples from outbreaks with high diversity of strains spreading over several geographic locations.

To investigate the effect of pulse cyclophosphamide treatment on hepatitis C virus (HCV) kinetics and quasispecies in interferon alpha (IFNalpha) resistant HCV related cryoglobulinaemic vasculitis.Reports on two patients with severe manifestations of HCV related cryoglobulinaemia who failed to respond to interferon alpha are given. Both patients were treated with pulse cyclophosphamide (750-1000 mg/month for six and 11 months, respectively). HCV RNA was quantified and HCV quasispecies determined in cryoprecipitates and supernatants before and during treatment.Cryocrit and complement activation decreased in both patients with rebound of cryocrit in one case during continuing pulse cyclophosphamide treatment. Vasculitic symptoms improved. Alanine aminotransferase (ALT) levels and HCV viral load (0.2-0.4 log) increased slightly and reached pretreatment levels after cyclophosphamide was stopped. A highly heterogeneous quasispecies was found in the cryoprecipitate and supernatant of one patient, whereas the viral population was homogeneous in the other patient. After six cycles of cyclophosphamide, viral distances decreased non-significantly. However, phylogenetic analysis showed the evolution of distinct viral strains in one patient and replacement of the main viral population by another population in the second patient.Immunosuppressive treatment with pulse cyclophosphamide has a temporary limited effect on HCV associated cryoglobulinaemia and leads to a reversible increase of ALT levels and HCV viral load. Short term immunosuppression does not affect the viral heterogeneity as measured by amino acid and nucleotide distances in the hypervariable region 1 of HCV. A change of quasispecies was observed, but further studies are needed to evaluate if this does affect the outcome of IFNalpha treatment in such patients.

Both a double-stranded RNA-dependent protein kinase (PKR)-phosphorylation homology domain (PePHD) within the E2 protein and a PKR-binding domain within the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) genotype 1 isolates inhibit the function of the interferon alfa (IFN-alpha)-induced antiviral effector protein PKR in vitro. We investigated whether the mutational pattern of the E2 region (codons 618-681, including PePHD) of 81 HCV genotype 1-infected patients (HCV-1b [n = 54], HCV-1a [n = 27]) influences the response to IFN-alpha. Initial viral decline (DeltaHCV RNA) was determined at week 1 hereby covering the effector reactions of IFN-alpha-mediated first phase and the immune-mediated second phase. DeltaHCV RNA less than 50% (group 1); DeltaHCV RNA greater than 50% but less than 90% (group 2); and DeltaHCV RNA > or =90% (group 3) were differentiated. The PePHD region was highly conserved; the few mutations (5 patients) did not correlate with DeltaHCV RNA or sustained virologic response to IFN-alpha. Within the flanking regions before and after PePHD (codons 618-681) 72 of 81 patients (89%) had 2.6+/-0.17 mutations (median, 3; range, 1-8) that did not correlate with treatment response. Sequence analysis of the NS5A protein (codons 2,209-2,274, including interferon sensitivity determining region [ISDR]) in 39 of 81 patients showed a higher mean number of mutations in the ISDR (codons 2,209-2,248) in groups 2 (1.28+/- 0.43 [n = 18]) and 3 (1.89+/-0.54 [n = 9]) than in group 1 (0.67+/- 0.19 [n = 12]; P =.049 group 1 vs. 3) and a mutant type ISDR (e.g., > or =4 mutations) was significantly more frequent in sustained virologic responders than in nonresponders or relapsers (2 of 4 [50%] vs. 2 of 35 [6%]; P =.045). Thus, NS5A appears to be functionally relevant in IFN-alpha-induced effector reactions.

Background The principle of a capture ELISA is binding of specific capture antibodies (polyclonal or monoclonal) to the surface of a suitable 96 well plate. These immobilized antibodies are capable of specifically binding a virus present in a clinical sample. Subsequently, the captured virus is detected using a specific detection antibody. The drawback of this method is that a capture ELISA can only function for a single virus captured by the primary antibody. Human Apolipoprotein H (ApoH) or β2-glycoprotein 1 is able to poly-specifically bind viral pathogens. Replacing specific capture antibodies by ApoH should allow poly-specific capture of different viruses that subsequently could be revealed using specific detection antibodies. Thus, using a single capture ELISA format different viruses could be analysed depending on the detection antibody that is applied. In order to demonstrate that this is a valid approach we show detection of group A rotaviruses from stool samples as a proof of principle for a new method of capture ELISA that should also be applicable to other viruses. Results Stool samples of different circulating common human and potentially zoonotic group A rotavirus strains, which were pretested in commercial EIAs and genotyped by PCR, were tested in parallel in an ApoH-ELISA set-up and by quantitative real-time PCR (qPCR). Several control samples were included in the analysis. The ApoH-ELISA was suitable for the capture of rotavirus-particles and the detection down to 1,000 infectious units (TCID50/ml). Subsets of diagnostic samples of different G- and P-types were tested positive in the ApoH-ELISA in different dilutions. Compared to the qPCR results, the analysis showed high sensitivity, specificity and low cross-reactivity for the ApoH-ELISA, which was confirmed in receiver operating characteristics (ROC) analysis. Conclusions In this study the development of a highly sensitive and specific capture ELISA was demonstrated by combining a poly-specific ApoH capture step with specific detection antibodies using group A rotaviruses as an example.