Jasmine Portmann

European Surveillance for West Nile Virus in Mosquito Populations

A wide range of arthropod-borne viruses threaten both human and animal health either through their presence in Europe or through risk of introduction. Prominent among these is West Nile virus (WNV), primarily an avian virus, which has caused multiple outbreaks associated with human and equine mortality. Endemic outbreaks of West Nile fever have been reported in Italy, Greece, France, Romania, Hungary, Russia and Spain, with further spread expected. Most outbreaks in Western Europe have been due to infection with WNV Lineage 1. In Eastern Europe WNV Lineage 2 has been responsible for human and bird mortality, particularly in Greece, which has experienced extensive outbreaks over three consecutive years. Italy has experienced co-circulation with both virus lineages. The ability to manage this threat in a cost-effective way is dependent on early detection. Targeted surveillance for pathogens within mosquito populations offers the ability to detect viruses prior to their emergence in livestock, equine species or human populations. In addition, it can establish a baseline of mosquito-borne virus activity and allow monitoring of change to this over time. Early detection offers the opportunity to raise disease awareness, initiate vector control and preventative vaccination, now available for horses, and encourage personal protection against mosquito bites. This would have major benefits through financial savings and reduction in equid morbidity/mortality. However, effective surveillance that predicts virus outbreaks is challenged by a range of factors including limited resources, variation in mosquito capture rates (too few or too many), difficulties in mosquito identification, often reliant on specialist entomologists, and the sensitive, rapid detection of viruses in mosquito pools. Surveillance for WNV and other arboviruses within mosquito populations varies between European countries in the extent and focus of the surveillance. This study reviews the current status of WNV in mosquito populations across Europe and how this is informing our understanding of virus epidemiology. Key findings such as detection of virus, presence of vector species and invasive mosquito species are summarized, and some of the difficulties encountered when applying a cost-effective surveillance programme are highlighted.

Unique human immune signature of Ebola virus disease in Guinea

Despite the magnitude of the Ebola virus disease (EVD) outbreak in West Africa, there is still a fundamental lack of knowledge about the pathophysiology of EVD. In particular, very little is known about human immune responses to Ebola virus. Here we evaluate the physiology of the human T cell immune response in EVD patients at the time of admission to the Ebola Treatment Center in Guinea, and longitudinally until discharge or death. Through the use of multiparametric flow cytometry established by the European Mobile Laboratory in the field, we identify an immune signature that is unique in EVD fatalities. Fatal EVD was characterized by a high percentage of CD4+ and CD8+ T cells expressing the inhibitory molecules CTLA-4 and PD-1, which correlated with elevated inflammatory markers and high virus load. Conversely, surviving individuals showed significantly lower expression of CTLA-4 and PD-1 as well as lower inflammation, despite comparable overall T cell activation. Concomitant with virus clearance, survivors mounted a robust Ebola-virus-specific T cell response. Our findings suggest that dysregulation of the T cell response is a key component of EVD pathophysiology.

Analysis of Diagnostic Findings From the European Mobile Laboratory in Guéckédou, Guinea, March 2014 Through March 2015

Background. A unit of the European Mobile Laboratory (EMLab) consortium was deployed to the Ebola virus disease (EVD) treatment unit in Guéckédou, Guinea, from March 2014 through March 2015. Methods. The unit diagnosed EVD and malaria, using the RealStar Filovirus Screen reverse transcription–polymerase chain reaction (RT-PCR) kit and a malaria rapid diagnostic test, respectively. Results. The cleaned EMLab database comprised 4719 samples from 2741 cases of suspected EVD from Guinea. EVD was diagnosed in 1231 of 2178 hospitalized patients (57%) and in 281 of 563 who died in the community (50%). Children aged <15 years had the highest proportion of Ebola virus–malaria parasite coinfections. The case-fatality ratio was high in patients aged <5 years (80%) and those aged >74 years (90%) and low in patients aged 10–19 years (40%). On admission, RT-PCR analysis of blood specimens from patients who died in the hospital yielded a lower median cycle threshold (Ct) than analysis of blood specimens from survivors (18.1 vs 23.2). Individuals who died in the community had a median Ct of 21.5 for throat swabs. Multivariate logistic regression on 1047 data sets revealed that low Ct values, ages of <5 and ≥45 years, and, among children aged 5–14 years, malaria parasite coinfection were independent determinants of a poor EVD outcome. Conclusions. Virus load, age, and malaria parasite coinfection play a role in the outcome of EVD.

Different features of Vδ2 T and NK cells in fatal and non-fatal human Ebola infections

Background Human Ebola infection is characterized by a paralysis of the immune system. A signature of αβ T cells in fatal Ebola infection has been recently proposed, while the involvement of innate immune cells in the protection/pathogenesis of Ebola infection is unknown. Aim of this study was to analyze γδ T and NK cells in patients from the Ebola outbreak of 2014–2015 occurred in West Africa, and to assess their association with the clinical outcome. Methodology/Principal findings Nineteen Ebola-infected patients were enrolled at the time of admission to the Ebola Treatment Centre in Guinea. Patients were divided in two groups on the basis of the clinical outcome. The analysis was performed by using multiparametric flow cytometry established by the European Mobile Laboratory in the field. A low frequency of Vδ2 T-cells was observed during Ebola infection, independently from the clinical outcome. Moreover, Vδ2 T-cells from Ebola patients massively expressed CD95 apoptotic marker, suggesting the involvement of apoptotic mechanisms in Vδ2 T-cell loss. Interestingly, Vδ2 T-cells from survivors expressed an effector phenotype and presented a lower expression of the CTLA-4 exhaustion marker than fatalities, suggesting a role of effector Vδ2 T-cells in the protection. Furthermore, patients with fatal Ebola infection were characterized by a lower NK cell frequency than patients with non fatal infection. In particular, both CD56bright and CD56dim NK frequency were very low both in fatal and non fatal infections, while a higher frequency of CD56neg NK cells was associated to non-fatal infections. Finally, NK activation and expression of NKp46 and CD158a were independent from clinical outcome. Conclusions/Significances Altogether, the data suggest that both effector Vδ2 T-cells and NK cells may play a role in the complex network of protective response to EBOV infection. Further studies are required to characterize the protective effector functions of Vδ2 and NK cells.

Determinants of antibody persistence across doses and continents after single-dose rVSV-ZEBOV vaccination for Ebola virus disease: an observational cohort study

BACKGROUND The recombinant vesicular stomatitis virus (rVSV) vaccine expressing the Zaire Ebola virus (ZEBOV) glycoprotein is efficacious in the weeks following single-dose injection, but duration of immunity is unknown. We aimed to assess antibody persistence at 1 and 2 years in volunteers who received single-dose rVSV-ZEBOV in three previous trials. METHODS In this observational cohort study, we prospectively followed-up participants from the African and European phase 1 rVSV-ZEBOV trials, who were vaccinated once in 2014-15 with 300 000 (low dose) or 10-50 million (high dose) plaque-forming units (pfu) of rVSV-ZEBOV vaccine to assess ZEBOV glycoprotein (IgG) antibody persistence. The primary outcome was ZEBOV glycoprotein-specific IgG geometric mean concentrations (GMCs) measured yearly by ELISA compared with 1 month (ie, 28 days) after immunisation. We report GMCs up to 2 years (Geneva, Switzerland, including neutralising antibodies up to 6 months) and 1 year (Lambaréné, Gabon; Kilifi, Kenya) after vaccination and factors associated with higher antibody persistence beyond 6 months, according to multivariable analyses. Trials and the observational study were registered at ClinicalTrials.gov (Geneva: NCT02287480 and NCT02933931; Kilifi: NCT02296983) and the Pan-African Clinical Trials Registry (Lambaréné PACTR201411000919191). FINDINGS Of 217 vaccinees from the original studies (102 from the Geneva study, 75 from the Lambaréné study, and 40 from the Kilifi study), 197 returned and provided samples at 1 year (95 from the Geneva study, 63 from the Lambaréné, and 39 from the Kilifi study) and 90 at 2 years (all from the Geneva study). In the Geneva group, 44 (100%) of 44 participants who had been given a high dose (ie, 10-50 million pfu) of vaccine and who were seropositive at day 28 remained seropositive at 2 years, whereas 33 (89%) of 37 who had been given the low dose (ie, 300 000 pfu) remained seropositive for 2 years (p=0·042). In participants who had received a high dose, ZEBOV glycoprotein IgG GMCs decreased significantly between their peak (at 1-3 months) and month 6 after vaccination in Geneva (p<0·0001) and Lambaréné (p=0·0298) but not in Kilifi (p=0·5833) and subsequently remained stable at all sites apart from Geneva, where GMC in those given a high dose of vaccine increased significantly between 6 months and 1 year (p=0·0264). Antibody persistence was similar at 1 year and at 6 months in those who had received a low dose of vaccine, with lower titres among participants from the Geneva study at 2 years than at 1 year after vaccination (GMC ratio 0·61, 95% CI 0·49-0·77; p<0·0001). In multivariable analyses, predictors of increased IgG GMCs beyond 6 months included high-dose versus low-dose vaccination (Geneva p=0·0133; Lambaréné p=0·008) and vaccine-related arthritis (p=0·0176), but not sex, age, or baseline seropositivity (all p>0·05). Neutralising antibodies seem to be less durable, with seropositivity dropping from 64-71% at 28 days to 27-31% at 6 months in participants from the Geneva study. INTERPRETATION Antibody responses to single-dose rVSV-ZEBOV vaccination are sustained across dose ranges and settings, a key criterion in countries where booster vaccinations would be impractical. FUNDING The Wellcome Trust and Innovative Medicines Initiative 2 Joint Undertaking.

Of a Storm in a Teacup and a Gutter Heater—Practical Aspects of VHP Room Fumigation

Room fumigation has traditionally been performed using formaldehyde. However, recently new methods have been developed, including vaporized hydrogen peroxide (VHP). VHP offers a number of advantages, such as degradation into non-toxic compounds (environmentally friendly), being non-corrosive (if used properly, with most common construction materials), leaving no residues, and being a vapor. On the other hand, it also poses new challenges in that the equipment is expensive (compared to formaldehyde), it has to be properly distributed inside the room, and fumigation cycles need to be developed for each room and situation. These challenges can be overcome as many laboratories have shown. In this article the authors outline the development of a VHP fumigation cycle and the validation of said cycle using the example of one laboratory at a new high-containment facility. Problems encountered as well as solutions that were or could have been implemented are also discussed. Even though the process was time-consuming and at times challenging, VHP room fumigation proved to be a versatile and robust process that may be used to efficiently decontaminate even complex laboratory setups.

The IndicatorSafe—A Simple Tool to Confirm Successful Fumigation of a HEPA Filter Housing:

International standards as well as national regulations often require the incorporation of HEPA filters in the exhaust of BSL-3 (unless work with non-airborne organisms is performed—Swiss Containment Ordinance [Federal Council, 2012]) and certainly BSL-4 laboratories (single HEPA filter on supply and double HEPA filter on exhaust). These may need to be decontaminated from time to time, for example prior to filter integrity testing (protection of the testing equipment in case filters or seals are damaged and thus leaking) or when exchanging filters (bag-in/bag-out may offer an alternative or an additional safety measure, respectively). However, HEPA filter housings are not generally equipped with sample ports that allow for placement of indicators to confirm a successful fumigation process. Additionally, a site-specific risk assessment concluded that since the filter boxes are situated in an area deemed to remain non-contaminated at all times (HEPA filter floor above laboratory suites), indicators may not be placed inside the filter housings as opening them prior to fumigation would open containment and thus pose a threat to the people working on the filter boxes as well as to the environment. In this article the authors describe the use of the IndicatorSafe, a simple and cheap solution to the above-mentioned problem. Placing the IndicatorSafe in the return line following the filter housing was shown to be an efficient alternative when fumigating filter housings with vaporized hydrogen peroxide (VHP), as shown during the validation of filter housing fumigation cycles, and may likely be adapted to other fumigation methods on the market.

Determination of host cell proteins constituting the molecular microenvironment of coronavirus replicase complexes by proximity-labeling

Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention.Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention.

Assessment of the zoonotic potential of the ruminant-associated influenza D virus

Influenza viruses are notorious pathogens that frequently cross the species barrier with often severe consequences for both animal and human health. In 2011, a novel member of the Orthomyxoviridae family, Influenza D virus (IDV), was identified in the respiratory tract of pigs with influenza-like symptoms and subsequently also in cattle, a species that previously never was associated with influenza virus infection. Epidemiological surveys among livestock demonstrated that IDV is worldwide distributed among ruminants, but the most striking observation is the detection of IDV-directed antibodies among humans with occupational exposure to livestock. As a first step toward identifying the zoonotic potential of the newly emerging IDV we determined the replication kinetics and cell tropism at the primary site of replication using an in vitro respiratory epithelium model of humans. The inoculation of IDV on human airway epithelial cell (hAEC) cultures revealed efficient replication kinetics and apical progeny virus release of IDV at different body temperatures. Intriguingly, the replication characteristics of IDV revealed many similarities to the human-associated Influenza C virus, including the predominant cell tropism for ciliated cells. Moreover, analysis of the host response during IDV infection revealed only a pronounced upregulation of Type III interferon (IFN) transcripts. Nevertheless, viral progeny virus is replication competent and can be efficiently sub-passaged in hAEC cultures from different donors. Highlighting, that there is no intrinsic impairment of IDV replication within the human respiratory epithelium and might explain why IDV-directed antibodies can be detected among humans with occupational exposure to livestock.Influenza viruses are notorious pathogens that frequently cross the species barrier with often severe consequences for both animal and human health. In 2011, a novel member of the Orthomyxoviridae family, Influenza D virus (IDV), was identified in the respiratory tract of diseased swine. Epidemiological surveys revealed that IDV is distributed worldwide among livestock and that IDV-directed antibodies are detected in humans with occupational exposure to livestock. To identify the transmission capability of IDV to humans, we determined the viral replication kinetics and cell tropism using an in vitro respiratory epithelium model of humans. The inoculation of IDV revealed efficient replication kinetics and apical progeny virus release at different body temperatures. Intriguingly, the replication characteristics of IDV revealed many similarities to the human-associated Influenza C virus, including the cell tropism preference for ciliated cells. Collectively, these results might indicate why IDV-directed antibodies are detected among humans with occupational exposure to livestock. One-sentence summary of the conclusion We show that the ruminant-associated Influenza D virus has direct transmission capability to humans.