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An equine parvovirus-hepatitis (EqPV-H) has been recently identified in association with equine serum hepatitis, also known as Theiler’s disease. The disease was first described by Arnold Theiler in 1918 and is often observed with parenteral use of blood products in equines. However, natural ways of viral circulation and potential risk factors for transmission still remain unknown. In this study, we investigated the occurrence of EqPV-H infections in Thoroughbred horses in northern and western Germany and aimed to identify potential risk factors associated with viral infections. A total of 392 Thoroughbreds broodmares and stallions were evaluated cross-sectionally for the presence of anti-EqPV-H antibodies and EqPV-H DNA using a luciferase immunoprecipitation assay (LIPS) and a quantitative PCR, respectively. In addition, data regarding age, stud farm, breeding history, and international transportation history of each horse were collected and analysed. An occurrence of 7% EqPV-H DNA positive and 35% seropositive horses was observed in this study cohort. The systematic analysis of risk factors revealed that age, especially in the group of 11–15-year-old horses, and breeding history were potential risk factors that can influence the rate of EqPV-H infections. Subsequent phylogenetic analysis showed a high similarity on nucleotide level within the sequenced Thoroughbred samples. In conclusion, this study demonstrates circulating EqPV-H infections in Thoroughbred horses from central Europe and revealed age and breeding history as risk factors for EqPV-H infections.

An equine parvovirus-hepatitis (EqPV-H) has been recently identified in association with equine serum hepatitis, also known as Theiler’s disease. This disease was first described by Arnold Theiler in 1918 and is often observed after applications with blood products in equines. So far, the virus has only been described in the USA and China. In this study, we evaluated the presence of EqPV-H in several commercial serum samples to assess the potential risk of virus transmission by equine serum-based products for medical and research applications. In 11 out of 18 commercial serum samples, EqPV-H DNA was detectable with a viral load up to 105 copies/mL. The same serum batches as well as three additional samples were also positive for antibodies against the EqPV-H VP1 protein. The countries of origin with detectable viral genomes included the USA, Canada, New Zealand, Italy, and Germany, suggesting a worldwide distribution of EqPV-H. Phylogenetic analysis of the EqPV-H NS1 sequence in commercial serum samples revealed high similarities in viral sequences from different geographical areas. As horse sera are commonly used for the production of anti-sera, which are included in human and veterinary medical products, these results implicate the requirement for diagnostic tests to prevent EqPV-H transmission.

Since its first discovery by Arnold Theiler in 1918, serum hepatitis also known as Theiler’s disease has been reported worldwide, causing idiopathic acute hepatitis and liver failure in horses. Recent studies have suggested a novel parvovirus, named equine parvovirus hepatitis (EqPV-H), to be associated with Theiler’s disease. Despite the severity and potential fatality of EqPV-H infection, little is known about the possibility of developing chronic infections and putative cross-species infection of equine sister species. In the present longitudinal study, we employed qPCR analysis, serology, and biochemical testing as well as pathology examination of liver biopsies and sequence analysis to investigate potential chronic EqPV-H infection in an isolated study cohort of in total 124 horses from Germany over five years (2013–2018). Importantly, our data suggest that EqPV-H viremia can become chronic in infected horses that do not show biochemical and pathological signs of liver disease. Phylogenetic analysis by maximum likelihood model also confirms high sequence similarity and nucleotide conservation of the multidomain nuclear phosphoprotein NS1 sequences from equine serum samples collected between 2013–2018. Moreover, by examining human, zebra, and donkey sera for the presence of EqPV-H DNA and VP1 capsid protein antibodies, we found evidence for cross-species infection in donkey, but not to human and zebra. In conclusion, this study provides proof for the occurrence of persistent EqPV-H infection in asymptomatic horses and cross-species EqPV-H detection in donkeys.

The clinical relevance of infections with the novel human coronavirus NL63 (HCoV-NL63) has not been investigated systematically. We therefore determined its association with disease in young children with lower respiratory tract infection (LRTI). Nine hundred forty-nine samples of nasopharyngeal secretions from children under 3 y of age with LRTIs were analysed by a quantitative HCoV-NL63-specific real-time PCR. The samples had been collected from hospitalised patients and outpatients from December 1999 to October 2001 in four different regions in Germany as part of the prospective population-based PRI.DE study and analysed for RNA from respiratory viruses. Forty-nine samples (5.2%), mainly derived from the winter season, were positive for HCoV-NL63 RNA. The viral RNA was more prevalent in samples from outpatients (7.9%) than from hospitalised patients (3.2%, p = 0.003), and co-infection with either respiratory syncytial virus or parainfluenza virus 3 was observed frequently. Samples in which only HCoV-NL63 RNA could be detected had a significantly higher viral load than samples containing additional respiratory viruses (median 2.1 x 10(6) versus 2.7 x 10(2) copies/ml, p = 0.0006). A strong association with croup was apparent: 43% of the HCoV-NL63-positive patients with high HCoV-NL63 load and absence of co-infection suffered from croup, compared to 6% in the HCoV-NL63-negative group, p < 0.0001. A significantly higher fraction (17.4%) of samples from croup patients than from non-croup patients (4.2%) contained HCoV-NL63 RNA. HCoV-NL63 infections occur frequently in young children with LRTI and show a strong association with croup, suggesting a causal relationship.

Although human immunodeficiency type 1 (HIV-1) infection induces strong antibody responses to the viral envelope glycoprotein (Env) only a few of these antibodies possess the capacity to neutralize a broad range of strains. The induction of such antibodies represents an important goal in the development of a preventive vaccine against the infection. Among the broadly neutralizing monoclonal antibodies discovered so far, three (2F5, Z13 and 4E10) target the short and hidden membrane proximal external region (MPER) of the gp41 transmembrane protein. Antibody responses to MPER are rarely observed in HIV-infected individuals or after immunization with Env immunogens. To initiate antibody responses to MPER in its membrane-embedded native conformation, we generated expression plasmids encoding the membrane-anchored ectodomain of gp41 with N-terminal deletions of various sizes. Following transfection of these plasmids, the MPER domains are displayed on the cell surface and incorporated into HIV virus like particles (VLP). Transfected cells displaying MPER mutants bound as efficiently to both 2F5 and 4E10 as cells transfected with a plasmid encoding full-length Env. Mice immunized with VLPs containing the MPER mutants produced MPER-specific antibodies, the levels of which could be increased by the trimerization of the displayed proteins as well as by a DNA prime-VLP boost immunization strategy. Although 2F5 competed for binding to MPER with antibodies in sera of some of the immunized mice, neutralizing activity could not be detected. Whether this is due to inefficient binding of the induced antibodies to MPER in the context of wild type Env or whether the overall MPER-specific antibody response induced by the MPER display mutants is too low to reveal neutralizing activity, remains to be determined.

Background Contamination of vertebrate cell lines with animal retroviruses has been documented repeatedly before. Although such viral contaminants can be easily identified with high sensitivity by PCR, it is impossible to screen for all potential contaminants. Therefore, we explored two novel methods to identify viral contaminations in cell lines without prior knowledge of the kind of contaminant. Results The first hint for the presence of contaminating retroviruses in one of our cell lines was obtained by electron microscopy of exosome-like vesicles released from the supernatants of transfected 293T cells. Random amplification of particle associated RNAs (PAN-PCR) from supernatant of contaminated 293T cells and sequencing of the amplicons revealed several nucleotide sequences showing highest similarity to either murine leukemia virus (MuLV) or squirrel monkey retrovirus (SMRV). Subsequent mass spectrometry analysis confirmed our findings, since we could identify several peptide sequences originating from monkey and murine retroviral proteins. Quantitative PCRs were established for both viruses to test currently cultured cell lines as well as liquid nitrogen frozen cell stocks. Gene fragments for both viruses could be detected in a broad range of permissive cell lines from multiple species. Furthermore, experimental infections of cells negative for these viruses showed that both viruses replicate rapidly to high loads. We decided to further analyze the genomic sequence of the MuLV-like contaminant virus. Surprisingly it was neither identical to MuLV nor to the novel xenotropic MuLV related retrovirus (XMRV) but showed 99% identity to a synthetic retrovirus which was engineered in the 1980s. Conclusion The high degree of nucleotide identity suggests unintended spread of a biosafety level 2 recombinant virus, which could also affect the risk assessment of gene-modified organisms released from contaminated cell cultures. The study further indicates that both mass spectrometry and PAN-PCR are powerful methods to identify viral contaminations in cell lines without prior knowledge of the kind of contaminant. Both methods might be useful tools for testing cell lines before using them for critical purposes.

The expression of connexin43 mRNA was detected in adult rat brains by in situ hybridization methods. Specific digoxigenin riboprobes were generated by in vitro transcription of two PCR-amplified fragments of connexin43 cDNA which lack homology with any other published connexin. Following immunohistochemical detection, the digoxigenin cRNA was found to occur in various neuronal populations including Purkinje cells of the cerebellum, pyramidal cells of the neocortex and the hippocampal formation, as well as granule cells of the dentate gyrus and various neurons of diverse hindbrain nuclei. This ubiquituous expression of connexin43 mRNA in adult neurons, in particular in neocortical pyramidal cells, is surprising insofar as gap junction communication in adult bains has been considered to be confined to specific subpopulations of neurons revealing a high incidence of synchronized electrical activities in contrast to the postnatal brain where interneuronal coupling via gap junctions precedes the formation of chemical transmission. In addition, connexin43 is regarded as being preferentially expressed in astrocytes, although its presence in adult neurons has not definitely been excluded. We propose that adult neurons preserve their capability of expressing functional gap junctions more frequently than presently considered and that connexin43 is a most likely neuronal gap junction protein candidate.

Cutaneous melanoma causes 55 500 deaths annually. The incidence and mortality rates of the disease differ widely across the globe depending on access to early detection and primary care. Once melanoma has spread, this type of cancer rapidly becomes life-threatening. For more than 40 years, few treatment options were available, and clinical trials during that time were all unsuccessful. Over the past 10 years, increased biological understanding and access to innovative therapeutic substances have transformed advanced melanoma into a new oncological model for treating solid cancers. Treatments that target B-Raf proto-oncogene serine/threonine-kinase (BRAF)V600 (Val600) mutations using selected BRAF inhibitors combined with mitogen-activated protein kinase inhibitors have significantly improved response and overall survival. Furthermore, advanced cutaneous melanoma has developed into a prototype for testing checkpoint-modulating agents, which has increased hope for long-term tumour containment and a potential cure. These expectations have been sustained by clinical success with targeted agents and antibodies that block programmed cell-death protein 1 in locoregional disease, which induces prolongation of relapse-free, distant-metastasis-free, and overall survival times.

Abstract Background Healthcare professionals (HPs) are the key figures to keep up the healthcare system during the COVID-19 pandemic and thus are one of the most vulnerable groups in this. To this point, the extent of this psychological burden, especially in Europe and Germany, remains unclear. This is the first study investigating German HPs after the COVID-19 outbreak. Methods We performed an online-based cross-sectional study after the COVID-19 outbreak in Germany (10–31 March 2020). In total, 2224 HPs (physicians n = 492, nursing staff n = 1511, paramedics n = 221) and 10 639 non-healthcare professionals (nHPs) were assessed including generalized anxiety (Generalized Anxiety Disorder-7), depression (Patient Health Questionnaire-2), current health status (EQ-5D-3L), COVID-19-related fear, subjective level of information regarding COVID-19. Results HPs showed less generalized anxiety, depression and COVID-19-related fear and higher health status and subjective level of information regarding COVID-19 than the nHPs. Within the HP groups, nursing staff were the most psychologically burdened. Subjective levels of information regarding COVID-19 correlated negatively with generalized anxiety levels across all groups. Among HPs, nursing staff showed the highest and paramedics the lowest generalized anxiety levels. Conclusions In the context of COVID-19, German HPs seem to be less psychological burdened than nHPs, and also less burdened compared with existing international data.

A recently described nuclear grading system predicted survival in patients with epithelioid malignant pleural mesothelioma. The current study was undertaken to validate the grading system and to identify additional prognostic factors. We analyzed cases of epithelioid malignant pleural mesothelioma from 17 institutions across the globe from 1998 to 2014. Nuclear grade was computed combining nuclear atypia and mitotic count into a grade of I–III using the published system. Nuclear grade was assessed by one pathologist for three institutions, the remaining were scored independently. The presence or absence of necrosis and predominant growth pattern were also evaluated. Two additional scoring systems were evaluated, one combining nuclear grade and necrosis and the other mitotic count and necrosis. Median overall survival was the primary endpoint. A total of 776 cases were identified including 301 (39%) nuclear grade I tumors, 354 (45%) grade II tumors and 121 (16%) grade III tumors. The overall survival was 16 months, and correlated independently with age ( P =0.006), sex (0.015), necrosis (0.030), mitotic count (0.001), nuclear atypia (0.009), nuclear grade (<0.0001), and mitosis and necrosis score (<0.0001). The addition of necrosis to nuclear grade further stratified overall survival, allowing classification of epithelioid malignant pleural mesothelioma into four distinct prognostic groups: nuclear grade I tumors without necrosis (29 months), nuclear grade I tumors with necrosis and grade II tumors without necrosis (16 months), nuclear grade II tumors with necrosis (10 months) and nuclear grade III tumors (8 months). The mitosis–necrosis score stratified patients by survival, but not as well as the combination of necrosis and nuclear grade. This study confirms that nuclear grade predicts survival in epithelioid malignant pleural mesothelioma, identifies necrosis as factor that further stratifies overall survival, and validates the grading system across multiple institutions and among both biopsy and resection specimens. An alternative scoring system, the mitosis–necrosis score is also proposed.