Single-cell PCR and sequencing of full-length Ig heavy (Igh) and Igk and Igl light chain genes is a powerful tool to measure the diversity of antibody repertoires and allows the functional assessment of B-cell responses through direct Ig gene cloning and the generation of recombinant mAbs. However, the current methodology is not high-throughput compatible. Here we developed a two-dimensional bar-coded primer matrix to combineKeywords: Antibody repertoire r B cell r Index cell sorting Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionAntibodies are B-cell antigen receptors encoded by two independent Ig heavy (Igh) and light (Igk or Igl) chain genes. Therefore, the accurate assessment of antibody repertoires and clonal relationships between individual B cells requires the combined analysis Correspondence: Hedda Wardemann e-mail: wardemann@mpiib-berlin.mpg.de of Igh and Igk/Igl genes from the same cell. Conventional singlecell (sc) based antibody repertoire studies use Sanger sequencing of Igh and corresponding Igk/Igl PCR products to assess V(D)J gene usage, levels of somatic hypermutations, and B-cell clonality [1][2][3][4][5]. This process is highly efficient, unbiased, and has been widely used to assess antibody repertoires in health and disease in mice and humans. Available single-cell PCR (scPCR) protocols provide full-length gene information and are compatible with the direct cloning of Igh and corresponding Igk/Igl genes [3,4]. This facilitates the production of recombinant mAbs, which have beenwww.eji-journal.eu 598Christian E. Busse et al. Eur. J. Immunol. 2014. 44: 597-603 used to study B-cell repertoires based on Ig reactivity and function. Here we combined the scPCR-based amplification of Igh and Igk/Igl transcripts and NGS into a unified pipeline that allows the high-throughput antibody repertoire analysis of thousands of single isolated B cells. The platform is scalable and provides highquality sequence information by consensus building of multiple reads originating from the same cell. It integrates sequencing and flow cytometry data obtained by indexed cell sorting into a single data set and thereby allows for the retrospective phenotypic characterization of B cells according to their antibody genotype. The strategy provides full-length Ig gene sequences of corresponding Igh and Igk/Igl genes and is therefore ideally suited for phylogenetic B-cell repertoire analyses and fully compatible with established procedures for antibody cloning and expression [3]. Importantly, the tag matrix can be used universally to barcode primer sets for the single-cell-based amplification of Ig genes from any species including humans [4]. Results Primer matrix designThe aim of our experimental strategy was to combine the advantages of scPCR and NGS to facilitate the massive parallel analysis of B-cell antibody repertoires (Fig. 1). To perform high-throughput parallel NGS of pooled PCR products without loss of Igh and Igk/Igl linkage, we desig...

BackgroundIn Germany, 17 % of the general human population have antibodies to hepatitis E virus (HEV) (recomLine HEV-IgG/IgM immunoassay [Mikrogen GmbH]). Wild boars represent an animal reservoir for HEV genotype 3, which is the common genotype in Germany. We estimated the seroprevalence among hunters with contact to wild boars to identify factors that may be associated with past or present HEV infection.MethodsIn 2013, the local veterinarian authority in a district in Central Germany attended meetings of hunters who provided blood specimens and completed a questionnaire collecting information on age, sex, hunting-related activities and consumption of wild boar meat. Specimens of wild boars were taken during drive hunts in this district during the season 2012/2013. All specimens were tested for HEV RNA and anti-HEV IgM and IgG antibodies. Log-binomial regression was used to estimate prevalence ratios (PR) for the hunters.ResultsOf 126 hunters (median age 55; 94 % male) 21 % tested positive for anti-HEV IgG antibodies (95 % confidence interval [CI] 13–28 %) (recomWell HEV IgG assay [Mikrogen GmbH]). Anti-HEV prevalence was highest in the age group of the 70–79-year-olds (67 %; 95 % CI 39–95 %). Wild boars showed an average anti-HEV prevalence of 41 %. HEV RNA was detected in 4/22 (18 %) liver specimens and in 1/22 (4.5 %) muscle specimens. Most wild boars were tested positive for HEV RNA (3/10; 30 %) and HEV-specific antibodies (7/15; 47 %) in the southwestern part of the district. Hunters preferring this hunting ground had a lower anti-HEV prevalence when gloves were frequently used during disembowelling of wild boars compared to hunters using gloves never or infrequently (age-adjusted PR 0.12; 95 % CI 0.02–0.86).ConclusionsHunters may benefit from wearing gloves when in contact with blood or body fluids of HEV animal reservoirs. Anti-HEV prevalence among the hunters of this study did not significantly differ from that of the general population suggesting that other factors play a major role in the epidemiology of HEV in Germany.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-015-1199-y) contains supplementary material, which is available to authorized users.

BackgroundHerpes simplex infections (HSV1/2) are characterized by recurrent symptoms, a risk of neonatal herpes, and the facilitation of HIV transmission. In Germany, HSV1/2 infections are not notifiable and data are scarce. A previous study found higher HSV1/2 seroprevalences in women in East Germany than in women in West Germany. We assessed changes in the HSV1/2 seroprevalences over time and investigated determinants associated with HSV1/2 seropositivity to guide prevention and control.MethodsThe study was based on the German Health Interview and Examination Survey for Adults (DEGS; 2008–2011) and the German National Health Interview and Examination Survey (GNHIES; 1997–1999). We tested serum samples from DEGS participants for HSV1 and HSV2 immunoglobulin G. We used Pearson’s χ2 test to compare the HSV1/HSV2 seroprevalences in terms of sex, age, and region of residence (East/West Germany) and investigated potential determinants by calculating prevalence ratios (PR) with log-binomial regression. All statistical analyses included survey weights.ResultsIn total, 6627 DEGS participants were tested for HSV1, and 5013 were also tested for HSV2. Overall, HSV1 seroprevalence decreased significantly from 1997–1999 (82.1%; 95%CI 80.6–83.6) to 2008–2011 (78.4%; 95%CI 77.8–79.7). In the same period, overall HSV2 seroprevalence decreased significantly from 13.3% (95%CI 11.9–14.9) to 9.6% (95%CI 8.6–10.8), notably in 18–24-year-old men (10.4 to 0%) in East Germany. Women were more likely than men to be seropositive for HSV1 (PR 1.1) or HSV2 (PR 1.6). A lower level of education, smoking, and not speaking German were associated with HSV1 in both sexes. Women of older age, who smoked, or had a history of abortion and men of older age or who had not attended a nursery school during childhood were more often seropositive for HSV2.ConclusionThe reduced seroprevalences of HSV1 and HSV2 leave more people susceptible to genital HSV1/2 infections. Practitioners should be aware of HSV infection as a differential diagnosis for genital ulcers. We recommend educational interventions to raise awareness of the sexual transmission route of HSV1/2, possible consequences, and prevention. Interventions should especially target pregnant women, their partners, and people at risk of HIV.

Shape matching has many applications in computer vision, such as shape classification, object recognition, object detection, and localization. In 2D cases, shape instances