Asymptomatic arbovirus and campylobacter infections in German travelers to Asia.

The true risk for many travel diseases is unknown because most studies do not detect asymptomatic infections. In this study, we performed ELISA for dengue virus (DENV), chikungunya virus (CHIKV), Zika virus (ZIKV), hepatitis E virus (HEV), and Campylobacter jejuni on samples from 81 healthy Germans before and after they traveled to Asia. ELISA found five seroconversions for C. jejuni, two for DENV, one for ZIKV, and zero for HEV. For CHIKV, three subjects were positive before travel and negative afterwards. None had symptoms. These infections would have gone unnoticed by retrospective studies. Therefore, the risk for these infections may be higher than previously estimated.

Real-time, portable genome sequencing for Ebola surveillance.

The Ebola virus disease epidemic in West Africa is the largest on record, responsible for over 28,599 cases and more than 11,299 deaths. Genome sequencing in viral outbreaks is desirable to characterize the infectious agent and determine its evolutionary rate. Genome sequencing also allows the identification of signatures of host adaptation, identification and monitoring of diagnostic targets, and characterization of responses to vaccines and treatments. The Ebola virus (EBOV) genome substitution rate in the Makona strain has been estimated at between 0.87 × 10(-3) and 1.42 × 10(-3) mutations per site per year. This is equivalent to 16-27 mutations in each genome, meaning that sequences diverge rapidly enough to identify distinct sub-lineages during a prolonged epidemic. Genome sequencing provides a high-resolution view of pathogen evolution and is increasingly sought after for outbreak surveillance. Sequence data may be used to guide control measures, but only if the results are generated quickly enough to inform interventions. Genomic surveillance during the epidemic has been sporadic owing to a lack of local sequencing capacity coupled with practical difficulties transporting samples to remote sequencing facilities. To address this problem, here we devise a genomic surveillance system that utilizes a novel nanopore DNA sequencing instrument. In April 2015 this system was transported in standard airline luggage to Guinea and used for real-time genomic surveillance of the ongoing epidemic. We present sequence data and analysis of 142 EBOV samples collected during the period March to October 2015. We were able to generate results less than 24 h after receiving an Ebola-positive sample, with the sequencing process taking as little as 15-60 min. We show that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks.

Increased Seroprevalence of Campylobacter jejuni, but not HEV, in healthcare workers in gastroenterological endoscopy.

BACKGROUND: The exposure of healthcare workers (HCW) to fecal-orally transmitted pathogens like hepatitis E Virus (HEV), Campylobacter jejuni or Helicobacter pylori is still not known. The potential risk for employees or patients to acquire these infections through asymptomatic infected healthcare personnel has not yet been studied. Physicians and nurses in gastroenterology working in endoscopic workspaces were recruited. Employees from cardiology, presumed to possess a lower exposure, served as controls. The cytomegalovirus (CMV) seroprevalence was analyzed as a control pathogen without fecal-oral route of transmission. This study provides an objective view onto the potential exposure risk for HCW and patients in endoscopic workspaces. We hypothesize that HCW in gastroenterological endoscopy show a higher seroprevalence for fecal-oral pathogens like HEV, C. jejuni and H. pylori compared to HCW in cardiology. OBJECTIVE: Primary objective was the assessment of antibody titers against HEV, C. jejuni and H. pylori in serum of HCW from gastroenterological endoscopy as well as cardiology. As a secondary objective we analyzed the seroprevalence against CMV. METHODS: 65 HCW were from gastroenterological endoscopy (n=42) and cardiology (n=23) in three medical centers in the German federal states of Brandenburg, Hamburg and Schleswig-Holstein and were prospectively studied. Antibody titers were determined via ELISA in serum. RESULTS: HCW in gastroenterological endoscopy showed a significantly higher C. jejuni seroprevalence for IgG (19.1 %) compared to HCW from the field of cardiology (8.7 %; p=0.04). IgA titers against C. jejuni were negligible. HEV seroprevalence for IgG did not differ significantly between HCW in gastroenterological endoscopy (7.1 %) and cardiology (8.7 %), respectively. IgA and IgM titers against HEV were also negligible. All other antibody titers against CMV and H. pylori showed no significant difference. CONCLUSIONS: Only the C. jejuni seroprevalence was significantly increased in HCW from the field of gastroenterological endoscopy. HEV seroprevalence showed no differences. The results for CMV and H. pylori were without pathological findings. However, there is no elevated risk for HEV exposure in medical staff working at an endoscopy unit, but for C. jejuni the protective measures might need to be improved.

Laboratory Findings, Compassionate Use of Favipiravir, and Outcome in Patients With Ebola Virus Disease, Guinea, 2015-A Retrospective Observational Study.

BACKGROUND: In 2015, the laboratory at the Ebola treatment center in Coyah, Guinea, confirmed Ebola virus disease (EVD) in 286 patients. The cycle threshold (Ct) of an Ebola virus-specific reverse transcription-polymerase chain reaction assay and 13 blood chemistry parameters were measured on admission and during hospitalization. Favipiravir treatment was offered to patients with EVD on a compassionate-use basis. METHODS: To reduce biases in the raw field data, we carefully selected 163 of 286 patients with EVD for a retrospective study to assess associations between potential risk factors, alterations in blood chemistry findings, favipiravir treatment, and outcome. RESULTS: The case-fatality rate in favipiravir-treated patients was lower than in untreated patients (42.5% [31 of 73] vs 57.8% [52 of 90]; P = .053 by univariate analysis). In multivariate regression analysis, a higher Ct and a younger age were associated with survival (P < .001), while favipiravir treatment showed no statistically significant effect (P = .11). However, Kaplan-Meier analysis indicated a longer survival time in the favipiravir-treated group (P = .015). The study also showed characteristic changes in blood chemistry findings in patients who died, compared with survivors. CONCLUSIONS: Consistent with the JIKI trial, this retrospective study revealed a trend toward improved survival in favipiravir- treated patients; however, the effect of treatment was not statistically significant, except for its influence on survival time.

Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII.

Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections.IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and MKRN1 proteins may prime MKRN1 for proteasomal degradation, because the MKRN1 protein is efficiently degraded during the late phase of HAdV-C5 infection. Since MKRN1 protein accumulation is also reduced in measles virus- and vesicular stomatitis virus-infected cells, our results signify the general strategy of viruses to target MKRN1.

Ebola Virus Persistence in Breast Milk After No Reported Illness: A Likely Source of Virus Transmission From Mother to Child.

A 9-month-old infant died from Ebola virus (EBOV) disease with unknown epidemiological link. While her parents did not report previous illness, laboratory investigations revealed persisting EBOV RNA in the mother's breast milk and the father's seminal fluid. Genomic analysis strongly suggests EBOV transmission to the child through breastfeeding.

The Plant-Derived Naphthoquinone Droserone Inhibits In Vitro Measles Virus Infection.

The naphthoquinone droserone (1) is a natural product occurring in dicotyledonous plants. We have now observed that the addition of 1 during infection of tissue culture cells with measles virus considerably reduced the infection. Interestingly, the infection was inhibited only when droserone (1) was added during virus entry, but not when added to the cells prior to virus uptake or after virus uptake. These findings suggest that 1 interacts with viral particles to reduce infectivity. The formation of progeny measles virus particles was inhibited to 50 % by droserone (1) at a concentration (IC50) of approximately 2 µM with a half-maximal cytotoxicity (CC50) of about 60 µM for Vero cells. Other tested naphthoquinone derivatives, among them the likewise natural plumbagin (2), but also synthetic analogs, were either more cytotoxic or not as effective as 1. Thus, our data do not support the development of naphthoquinone derivatives into antiviral compounds, but suggest that they may be interesting research tools to study measles virus entry into cells.

Monoclonal antibodies to various epitopes of hepatitis B surface antigen inhibit hepatitis B virus infection.

BACKGROUND AND AIM: Antibodies against the "a" determinant of hepatitis B surface antigen (HBsAg) are able to neutralize circulating hepatitis B virus (HBV) particles and prevent HBV infection. It has been proposed that a single amino acid exchange may allow the virus to escape the immune response. We used a set of monoclonal antibodies (MAbs) to investigate whether a single mutation may account for virus escape from humoral immunity. METHODS: Nine murine HBsAg-specific MAbs were raised. Reactivity of all antibodies with 14 recombinant mutants of HBsAg was assessed by ELISA. HBV infection of HepaRG cells was used to evaluate viral neutralization capacity of MAbs in vitro. RESULTS: All MAbs were able to inhibit the establishment of HBV infection in a dose-dependent fashion, but recognition of HBsAg variants varied. The MAbs were classified into three subgroups based on their pattern of reactivity to the HBsAg variants. Accordingly, three MAbs showed weak reactivity (< 40%) to variants with mutations within the first loop of "a" determinant, five MAbs displayed negligible binding to variants with mutations within the second loop, and one MAb lost its binding to variants having mutations in both loops of the "a" determinant. CONCLUSIONS: Our results indicate that antibodies against different epitopes of the "a" determinant of HBsAg are able to neutralize HBV. It seems that mutations within a single or a limited number of amino acids within this determinant can hardly result in viral escape. These results have important implications for the development of antibody-based therapies against HBV.

Efficient stabilization of therapeutic hepatitis B vaccine components by amino-acid formulation maintains its potential to break immune tolerance.

Background & Aims: Induction of potent, HBV-specific immune responses is crucial to control and finally cure HBV. The therapeutic hepatitis B vaccine TherVacB combines protein priming with a Modified Vaccinia virus Ankara (MVA)-vector boost to break immune tolerance in chronic HBV infection. Particulate protein and vector vaccine components, however, require a constant cooling chain for storage and transport, posing logistic and financial challenges to vaccine applications. We aimed to identify an optimal formulation to maintain stability and immunogenicity of the protein and vector components of the vaccine using a systematic approach. Methods: We used stabilizing amino acid (SAA)-based formulations to stabilize HBsAg and HBV core particles (HBcAg), and the MVA-vector. We then investigated the effect of lyophilization and short- and long-term high-temperature storage on their integrity. Immunogenicity and safety of the formulated vaccine was validated in HBV-naïve and adeno-associated virus (AAV)-HBV-infected mice. Results: In vitro analysis proved the vaccine's stability against thermal stress during lyophilization and the long-term stability of SAA-formulated HBsAg, HBcAg and MVA during thermal stress at 40 °C for 3 months and at 25 °C for 12 months. Vaccination of HBV-naïve and AAV-HBV-infected mice demonstrated that the stabilized vaccine was well tolerated and able to brake immune tolerance established in AAV-HBV mice as efficiently as vaccine components constantly stored at 4 °C/-80 °C. Even after long-term exposure to elevated temperatures, stabilized TherVacB induced high titre HBV-specific antibodies and strong CD8+ T-cell responses, resulting in anti-HBs seroconversion and strong suppression of the virus in HBV-replicating mice. Conclusion: SAA-formulation resulted in highly functional and thermostable HBsAg, HBcAg and MVA vaccine components. This will facilitate global vaccine application without the need for cooling chains and is important for the development of prophylactic as well as therapeutic vaccines supporting vaccination campaigns worldwide. Impact and implications: Therapeutic vaccination is a promising therapeutic option for chronic hepatitis B that may enable its cure. However, its application requires functional cooling chains during transport and storage that can hardly be guaranteed in many countries with high demand. In this study, the authors developed thermostable vaccine components that are well tolerated and that induce immune responses and control the virus in preclinical mouse models, even after long-term exposure to high surrounding temperatures. This will lower costs and ease application of a therapeutic vaccine and thus be beneficial for the many people affected by hepatitis B around the world.

CD9 clustering and formation of microvilli zippers between contacting cells regulates virus-induced cell fusion.

Members of the tetraspanin family including CD9 contribute to the structural organization and plasticity of the plasma membrane. K41, a CD9-specific monoclonal antibody, inhibits the release of HIV-1 and canine distemper virus (CDV)- but not measles virus (MV)-induced cell-cell fusion. We now report that K41, which recognizes a conformational epitope on the large extracellular loop of CD9, induces rapid relocation and clustering of CD9 in net-like structures at cell-cell contact areas. High-resolution analyses revealed that CD9 clustering is accompanied by the formation of microvilli that protrude from either side of adjacent cell surfaces, thus forming structures like microvilli zippers. While the cellular CD9-associated proteins beta(1)-integrin and EWI-F were co-clustered with CD9 at cell-cell interfaces, viral proteins in infected cells were differentially affected. MV envelope proteins were detected within CD9 clusters, whereas CDV proteins were excluded from CD9 clusters. Thus, the tetraspanin CD9 can regulate cell-cell fusion by controlling the access of the fusion machinery to cell contact areas.

Clearance of measles virus from persistently infected cells by short hairpin RNA.

Subacute sclerosing panencephalitis (SSPE) is a demyelinating central nervous system disease caused by a persistent measles virus (MV) infection of neurons and glial cells. There is still no specific therapy available, and in spite of an intact innate and adaptive immune response, SSPE leads inevitably to death. In order to select effective antiviral short interfering RNAs (siRNAs), we established a plasmid-based test system expressing the mRNA of DsRed2 fused with mRNA sequences of single viral genes, to which certain siRNAs were directed. siRNA sequences were expressed as short hairpin RNA (shRNA) from a lentiviral vector additionally expressing enhanced green fluorescent protein (EGFP) as an indicator. Evaluation by flow cytometry of the dual-color system (DsRed and EGFP) allowed us to find optimal shRNA sequences. Using the most active shRNA constructs, we transduced persistently infected human NT2 cells expressing virus-encoded HcRed (piNT2-HcRed) as an indicator of infection. shRNA against N, P, and L mRNAs of MV led to a reduction of the infection below detectable levels in a high percentage of transduced piNT2-HcRed cells within 1 week. The fraction of virus-negative cells in these cultures was constant over at least 3 weeks posttransduction in the presence of a fusion-inhibiting peptide (Z-Phe-Phe-Gly), preventing the cell fusion of potentially cured cells with persistently infected cells. Transduced piNT2 cells that lost HcRed did not fuse with underlying Vero/hSLAM cells, indicating that these cells do not express viral proteins any more and are "cured." This demonstrates in tissue culture that NT2 cells persistently infected with MV can be cured by the transduction of lentiviral vectors mediating the long-lasting expression of anti-MV shRNA.

A Broad-Spectrum Antiviral Peptide Blocks Infection of Viruses by Binding to Phosphatidylserine in the Viral Envelope.

The ongoing threat of viral infections and the emergence of antiviral drug resistance warrants a ceaseless search for new antiviral compounds. Broadly-inhibiting compounds that act on elements shared by many viruses are promising antiviral candidates. Here, we identify a peptide derived from the cowpox virus protein CPXV012 as a broad-spectrum antiviral peptide. We found that CPXV012 peptide hampers infection by a multitude of clinically and economically important enveloped viruses, including poxviruses, herpes simplex virus-1, hepatitis B virus, HIV-1, and Rift Valley fever virus. Infections with non-enveloped viruses such as Coxsackie B3 virus and adenovirus are not affected. The results furthermore suggest that viral particles are neutralized by direct interactions with CPXV012 peptide and that this cationic peptide may specifically bind to and disrupt membranes composed of the anionic phospholipid phosphatidylserine, an important component of many viral membranes. The combined results strongly suggest that CPXV012 peptide inhibits virus infections by direct interactions with phosphatidylserine in the viral envelope. These results reiterate the potential of cationic peptides as broadly-acting virus inhibitors.

Lipase inhibitor orlistat prevents hepatitis B virus infection by targeting an early step in the virus life cycle.

Hepatitis B Virus (HBV) is a strictly hepatotropic pathogen which is very efficiently targeted to the liver and into its host cell, the hepatocyte. The sodium taurocholate co-transporting polypeptide (NTCP) has been identified as a key virus entry receptor, but the early steps in the virus life cycle are still only barely understood. Here, we investigated the effect of lipase inhibition and lipoprotein uptake on HBV infection using differentiated HepaRG cells and primary human hepatocytes. We found that an excess of triglyceride rich lipoprotein particles in vitro diminished HBV infection and a reduced hepatic virus uptake in vivo if apolipoprotein E is lacking indicating virus transport along with lipoproteins to target hepatocytes. Moreover, we showed that HBV infection of hepatocytes was inhibited by the broadly active lipase inhibitor orlistat, approved as a therapeutic agent which blocks neutral lipid hydrolysis activity. Orlistat treatment targets HBV infection at a post-entry step and inhibited HBV infection during virus inoculation strongly in a dose-dependent manner. In contrast, orlistat had no effect on HBV gene expression or replication or when added after HBV infection. Taken together, our data indicate that HBV connects to the hepatotropic lipoprotein metabolism and that inhibition of cellular hepatic lipase(s) may allow to target early steps of HBV infection.

Persistence and clearance of Ebola virus RNA from seminal fluid of Ebola virus disease survivors: a longitudinal analysis and modelling study.

BACKGROUND: By January, 2016, all known transmission chains of the Ebola virus disease (EVD) outbreak in west Africa had been stopped. However, there is concern about persistence of Ebola virus in the reproductive tract of men who have survived EVD. We aimed to use biostatistical modelling to describe the dynamics of Ebola virus RNA load in seminal fluid, including clearance parameters. METHODS: In this longitudinal study, we recruited men who had been discharged from three Ebola treatment units in Guinea between January and July, 2015. Participants provided samples of seminal fluid at follow-up every 3-6 weeks, which we tested for Ebola virus RNA using quantitative real-time RT-PCR. Representative specimens from eight participants were then inoculated into immunodeficient mice to test for infectivity. We used a linear mixed-effect model to analyse the dynamics of virus persistence in seminal fluid over time. FINDINGS: We enrolled 26 participants and tested 130 seminal fluid specimens; median follow up was 197 days (IQR 187-209 days) after enrolment, which corresponded to 255 days (228-287) after disease onset. Ebola virus RNA was detected in 86 semen specimens from 19 (73%) participants. Median duration of Ebola virus RNA detection was 158 days after onset (73-181; maximum 407 days at end of follow-up). Mathematical modelling of the quantitative time-series data showed a mean clearance rate of Ebola virus RNA from seminal fluid of -0·58 log units per month, although the clearance kinetic varied greatly between participants. Using our biostatistical model, we predict that 50% and 90% of male survivors clear Ebola virus RNA from seminal fluid at 115 days (90% prediction interval 72-160) and 294 days (212-399) after disease onset, respectively. We also predicted that the number of men positive for Ebola virus RNA in affected countries would decrease from about 50 in January 2016, to fewer than 1 person by July, 2016. Infectious virus was detected in 15 of 26 (58%) specimens tested in mice. INTERPRETATION: Time to clearance of Ebola virus RNA from seminal fluid varies greatly between individuals and could be more than 13 months. Our predictions will assist in decision-making about surveillance and preventive measures in EVD outbreaks. FUNDING: This study was funded by European Union's Horizon 2020 research and innovation programme, Directorate-General for International Cooperation and Development of the European Commission, Institut national de la santé et de la recherche médicale (INSERM), German Research Foundation (DFG), and Innovative Medicines Initiative 2 Joint Undertaking.

Matrix conditions and KLF2-dependent induction of heme oxygenase-1 modulate inhibition of HCV replication by fluvastatin.

BACKGROUND & AIMS: HMG-CoA-reductase-inhibitors (statins) have been shown to interfere with HCV replication in vitro. We investigated the mechanism, requirements and contribution of heme oxygenase-1(HO-1)-induction by statins to interference with HCV replication. METHODS: HO-1-induction by fluva-, simva-, rosuva-, atorva- or pravastatin was correlated to HCV replication, using non-infectious replicon systems as well as the infectious cell culture system. The mechanism of HO-1-induction by statins as well as its relevance for interference with HCV replication was investigated using transient or permanent knockdown cell lines. Polyacrylamide(PAA) gels of different density degrees or the Rho-kinase-inhibitor Hydroxyfasudil were used in order to mimic matrix conditions corresponding to normal versus fibrotic liver tissue. RESULTS: All statins used, except pravastatin, decreased HCV replication and induced HO-1 expression, as well as interferon response in vitro. HO-1-induction was mediated by reduction of Bach1 expression and induction of the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) cofactor Krueppel-like factor 2 (KLF2). Knockdown of KLF2 or HO-1 abrogated effects of statins on HCV replication. HO-1-induction and anti-viral effects of statins were more pronounced under cell culture conditions mimicking advanced stages of liver disease. CONCLUSIONS: Statin-mediated effects on HCV replication seem to require HO-1-induction, which is more pronounced in a microenvironment resembling fibrotic liver tissue. This implicates that certain statins might be especially useful to support HCV therapy of patients at advanced stages of liver disease.

A small-molecule inhibitor of Nipah virus envelope protein-mediated membrane fusion.

Nipah virus (NiV), a highly pathogenic paramyxovirus, causes respiratory disease in pigs and severe febrile encephalitis in humans with high mortality rates. On the basis of the structural similarity of viral fusion (F) proteins within the family Paramyxoviridae, we designed and tested 18 quinolone derivatives in a NiV and measles virus (MV) envelope protein-based fusion assay beside evaluation of cytotoxicity. We found five compounds successfully inhibiting NiV envelope protein-induced cell fusion. The most active molecules (19 and 20), which also inhibit the syncytium formation induced by infectious NiV and show a low cytotoxicity in Vero cells, represent a promising lead quinolone-type compound structure. Molecular modeling indicated that compound 19 fits well into a particular protein cavity present on the NiV F protein that is important for the fusion process.

Tetraspanins: Small transmembrane proteins with big impact on membrane microdomain structures.

Members of the tetraspanin family of transmembrane proteins including CD9, CD37, CD53, CD63, CD81, CD82, CD151, etc., contribute to the structural organization of the plasma membrane by forming microdomain structures, influencing cell fusion and regulating cell motility. Interestingly, K41, a CD9-specific monoclonal antibody (mAb), inhibits the release of human immunodeficiency virus (HIV-1), and the canine distemper virus (CDV)-, but not measles virus (MV)-induced cell-cell fusion. This mAb, which recognizes a conformational epitope on the large extracellular loop (LEL) of CD9, induced rapid relocation and clustering of CD9 in net-like structures at cell-cell contact areas.1 High-resolution analyses revealed that CD9 clustering is accompanied by the formation of microvilli that protrude from either side of adjacent cell surfaces, thus forming structures like microvilli zippers. While the cellular CD9-associated proteins beta1-integrin and EWI-F were co-clustered with CD9 at cell-cell interfaces, viral proteins in infected cells were differentially affected. MV envelope proteins were detected within, whereas CDV proteins were excluded from CD9 clusters, and thus, the tetraspanin CD9 can regulate cell-cell fusion by controlling the access of the viral fusion machinery to cell contact areas.