The European Virus Archive goes global: A growing resource for research.

The European Virus Archive (EVA) was created in 2008 with funding from the FP7-EU Infrastructure Programme, in response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry. Within three years, it developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. In 2014, the H2020 Research and Innovation Framework Programme (INFRAS projects) provided support for the transformation of the EVA from a European to a global organization (EVAg). The EVAg now operates as a non-profit consortium, with 26 partners and 20 associated partners from 21 EU and non-EU countries. In this paper, we outline the structure, management and goals of the EVAg, to bring to the attention of researchers the wealth of products it can provide and to illustrate how end-users can gain access to these resources. Organisations or individuals who would like to be considered as contributors are invited to contact the EVAg coordinator, Jean-Louis Romette, at jean-louis.romette@univmed.fr.

EBOLA Ag K-SeT rapid test: field evaluation in Sierra Leone.

OBJECTIVES: Efficient interruption of Ebola virus disease (EVD) transmission chains critically depends on reliable and fast laboratory diagnosis. We evaluated the performance of the EBOLA Virus Antigen Detection K-SeT (EBOLA Ag K-SeT), a new rapid diagnostic antigen test in field settings. METHODS: The study was conducted in a field laboratory located in Freetown (Sierra Leone) by the Italian National Institute for Infectious Diseases 'L. Spallanzani' and the EMERGENCY Onlus NGO. The EBOLA Ag K-SeT was tested on 210 residual plasma samples (EVD prevalence 50%) from patients hospitalized at the EMERGENCY Ebola treatment center in Goderich (Freetown), comparing the results with quantitative real-time PCR. RESULTS: Overall, the sensitivity of EBOLA Ag K-SeT was 88.6% (95% confidence interval (CI), 82.5-94.7), and the corresponding specificity was 98.1% (95% CI, 95.5-100.7). The positive and negative predictive values were 97.9% (95% CI, 95.0-100.8) and 89.6% (95% CI, 84-95.2), respectively. The sensitivity strongly increased up to 98.7% (95% CI, 96.1-101.2) for those samples with high virus load (≥6.2 log RNA copies/mL). CONCLUSIONS: Our results suggest that EBOLA Ag K-SeT could represent a new effective diagnostic tool for EVD, meeting a need for resource-poor settings and rapid diagnosis for individuals with suspected EVD.

Evaluation of a rapid and sensitive RT-qPCR assay for the detection of Ebola Virus.

BACKGROUND: The 2013-2016 Ebola virus disease (EVD) outbreak showed a lack of diagnostic point-of-care methods. Currently, EBOV diagnosis relies on quantitative reverse-transcription-PCR (RT- qPCR), highly specific and sensitive, but requiring skilled personnel and well-equipped laboratories. In field settings, these factors and others, such as samples' time of collection and transportation, determine a prolonged turnaround-time to final results. In outbreak scenarios, a rapid and transportable method could eliminate issues of cohorting suspected and actual EVD patients for lack of diagnostic certainty. The aim of this study was the field evaluation of the new fast, easy-to-use and reliable RT-qPCR assay and platform for EBOV detection, developed in the framework of the EbolaMoDRAD project by CLONIT S.r.l. and STMicroelectronics S.r.l. STUDY DESIGN: We evaluated its performance during the outbreak and in further studies in the EVD laboratory at the Princess Christian Maternity Hospital (PCMH) in Freetown (Sierra Leone) run by Emergency NGO and the Italian National Institute for Infectious Diseases (INMI). The assay was tested on residual aliquots of clinical specimens from EBOV-positive or -negative patients (n=116, EVD prevalence 37%). RESULTS AND CONCLUSION: Overall, the test was very easy-to-use and the instrument was robust and reliable in field-settings. The sensitivity of the assay was 100% and the specificity was 98.63% (95%CI: 96.34-100.92%). The positive and negative predictive values were 97.73 (95%CI:94.77-100.68%) and 100%, respectively. The high sensitivity and specificity of this new assay indicate that it is promising for laboratory diagnosis, especially in resource-limited settings.

Ebola virus disease: societal challenges and new treatments.

Ebola virus disease (EVD) is a zoonotic disease that causes severe haemorrhagic fever, with high fatality rates of up to 90% in humans. Today, there is no effective treatment available. Person-to-person transmission occurs through exposure to blood or body fluids, which can threaten other household members and first-line healthcare workers. The first cases of EVD in Guinea were identified on 22 March 2014. It was initially believed that this like previous outbreaks would be self-limiting. However, lack of public health infrastructure, delays in virus detection and late implementation of control interventions contributed to widespread transmission of EVD in a region inexperienced in dealing with the disease. Socio-cultural and economic factors probably also played a key role in the spread of the disease, resulting in the current large-scale outbreak. Some promising candidate treatments for this disease are now being developed.

International network for capacity building for the control of emerging viral vector-borne zoonotic diseases: ARBO-ZOONET.

Arboviruses are arthropod-borne viruses, which include West Nile fever virus (WNFV), a mosquito-borne virus, Rift Valley fever virus (RVFV), a mosquito-borne virus, and Crimean-Congo haemorrhagic fever virus (CCHFV), a tick-borne virus. These arthropod-borne viruses can cause disease in different domestic and wild animals and in humans, posing a threat to public health because of their epidemic and zoonotic potential. In recent decades, the geographical distribution of these diseases has expanded. Outbreaks of WNF have already occurred in Europe, especially in the Mediterranean basin. Moreover, CCHF is endemic in many European countries and serious outbreaks have occurred, particularly in the Balkans, Turkey and Southern Federal Districts of Russia. In 2000, RVF was reported for the first time outside the African continent, with cases being confirmed in Saudi Arabia and Yemen. This spread was probably caused by ruminant trade and highlights that there is a threat of expansion of the virus into other parts of Asia and Europe. In the light of global warming and globalisation of trade and travel, public interest in emerging zoonotic diseases has increased. This is especially evident regarding the geographical spread of vector-borne diseases. A multi-disciplinary approach is now imperative, and groups need to collaborate in an integrated manner that includes vector control, vaccination programmes, improved therapy strategies, diagnostic tools and surveillance, public awareness, capacity building and improvement of infrastructure in endemic regions.

Exogenous nitric oxide inhibits Crimean Congo hemorrhagic fever virus.

Crimean Congo hemorrhagic fever virus (CCHFV) is a geographically widespread pathogen that causes severe hemorrhagic fever with high mortality. Even though one of the main objectives focuses on the progress of antiviral agents, the research on CCHFV is strongly hampered due to its BSL-4 classification. Nitric oxide (NO), a mediator with broad biological effects, has been shown to possess inhibitory properties against various pathogens. The molecule constitutes a component of the innate immunity and serves to assist in the early immunological events where it contributes to clearance of microorganisms. In this study, we investigated the inhibitory properties of exogenous NO on CCHFV. We found that NO had a significant antiviral activity against CCHFV replication. By using the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) we were able to show up to 99% reduction in virion progeny yield. In contrast, 3-morpholinosydnonimine hydrochloride (SIN-1), a peroxynitrite donor, had no significant antiviral activity against CCHFV. Furthermore the expression of viral proteins; the nucleocapsid protein and the glycoprotein, were clearly reduced with increasing concentrations of SNAP. We have also shown that the amount of total vRNA in SNAP-treated cells was reduced by about 50% compared to the controls.

ATP is required for correct folding and disulfide bond formation of rotavirus VP7.

Rotavirus is one of very few viruses that utilize the endoplasmic reticulum (ER) for assembly, and therefore it has been used as an attractive model to study ER-associated protein folding. In this study, we have examined the requirements for metabolic energy (ATP) for correct folding of the luminal and ER-associated VP7 of rotavirus. We found that VP7 rapidly misfolds in an energy-depleted milieu and is not degraded within 60 min. We also found that VP7 attained a stable minimum-energy state soon after translation in the ER. Most surprisingly, energy-misfolded VP7 could be recovered and establish correct disulfide bonds and antigenicity following a shift to an ATP-rich milieu. Using a Semliki Forest virus expression system, we observed that VP7 requires ATP and cellular, but not viral, factors for correct disulfide bond formation. Our results show for the first time that the disulfide bond formation of rotavirus VP7 is an ATP-dependent process. It has previously been shown that chaperones hydrolyze ATP during interaction with newly synthesized polypeptides and prevent nonproductive intra- and intermolecular interactions. The most reasonable explanation for the energy requirement of VP7 is thus a close interaction during folding with an ATP-dependent chaperone, such as BiP (Grp78), and possibly with protein disulfide isomerase. Taken together, our observations provide new information about folding of ER-associated proteins in general and rotavirus VP7 in particular.

Encapsulation of rotavirus into poly(lactide-co-glycolide) microspheres.

Two small-scale double emulsion techniques for incorporation of formaldehyde-inactivated rotavirus particles (FRRV) into poly(lactide-co-glycolide) (PLG) microspheres were developed and optimised. The effects of high-speed homogenisation versus vortex mixing on the double emulsion stability, microsphere size, entrapment efficiency and in vitro release of FRRV in the second emulsification step were studied. A stable double emulsion was verified only when using vortex mixing in this step. Slow removal of the organic phase allowed measurement of the size of the emulsion droplets and subsequent prediction of the size of the resulting microspheres. Microspheres in the size range of 1-10 microm were prepared using both techniques. The homogenisation technique was sensitive to changes in the operating time, the emulsification energy and the volume of the outer aqueous phase, while the vortex technique was more robust. Rotavirus was released in vitro in a triphasic manner with both techniques. The more robust vortex technique was selected for preparation of PLG microspheres containing rotavirus for in vivo studies. After immunisation of mice with a single intramuscular injection, the PLG-FRRV microspheres elicited an IgG antibody response in serum detected by ELISA equally high as that elicited with FRRV alone. These results indicate that the antigenicity of FFRV was retained after incorporation into PLG microspheres using the vortex technique.

The molecular chaperone calnexin interacts with the NSP4 enterotoxin of rotavirus in vivo and in vitro.

Calnexin is an endoplasmic reticulum (ER)-associated molecular chaperone proposed to promote folding and assembly of glycoproteins that traverse the secretory pathway in eukaryotic cells. In this study we examined if calnexin interacts with the ER-associated luminal (VP7) and transmembrane (NSP4) proteins of rotavirus. Only glycosylated NSP4 interacted with calnexin and did so in a time-dependent manner (half-life, 20 min). In vitro translation experiments programmed with gene 10 of rhesus rotavirus confirmed that calnexin recognizes only glycosylated NSP4. Castanospermine (a glucosidase I and II inhibitor) experiments established that calnexin associates only with partly deglucosylated (di- or monoglucosylated) NSP4. Furthermore, enzymatic removal of the remaining glucose residues on the N-linked glycan units was essential to disengage the NSP4-calnexin complex. Novel experiments with castanospermine revealed that glucose trimming and the calnexin-NSP4 interaction were not critical for the assembly of infectious virus.

Carbohydrates facilitate correct disulfide bond formation and folding of rotavirus VP7.

It is well established that glycosylation is essential for assembly of enveloped viruses, but no information is yet available as to the function of carbohydrates on the nonenveloped but glycosylated rotavirus. We show that tunicamycin and, more pronouncedly, a combination of tunicamycin and brefeldin A treatment caused misfolding of the luminal VP7 protein, leading to interdisulfide bond aggregation. While formation of VP7 aggregates could be prevented under reducing conditions, they reoccurred in less than 30 min after a shift to an oxidizing milieu. Furthermore, while glycosylated VP7 interacted during maturation with protein disulfide isomerase, nonglycosylated VP7 did not, suggesting that glycosylation is a prerequisite for protein disulfide isomerase interaction. While native NSP4, which does not possess S-S bonds, was not dependent on N-linked glycosylation or on protein disulfide isomerase assistance for maturation, nonglycosylated NSP4 was surprisingly found to interact with protein disulfide isomerase, further suggesting that protein disulfide isomerase can act both as an enzyme and as a chaperone. In conclusion, our data suggest that the major function of carbohydrates on VP7 is to facilitate correct disulfide bond formation and protein folding.

Effect of brefeldin A on rotavirus assembly and oligosaccharide processing.

Rotavirus is one of very few viruses that utilizes the endoplasmic reticulum (ER) for maturation. The maturation process is unique not only because it involves translocation of subviral particles across the ER membrane, but also because mature virus is thought to be retained in the ER until cell lysis. Brefeldin A (BFA) is a compound that blocks protein export from the ER to the Golgi complex and causes disruption of the Golgi complex with relocation of resident Golgi proteins to the ER. We found that BFA had a pronounced effect on rotavirus assembly and oligosaccharide processing. Single-step growth experiments demonstrated that BFA reduced infectious progeny rotavirus yield by 99.9%. Immunohistochemical staining with monoclonal antibodies showed that all examined VP4, VP6, VP7, and NS28 epitopes remained unaffected by BFA. A novel observation from pulse-chase experiments was that BFA-treatment rapidly increased the molecular weight of the ER-associated VP7 followed by endo-beta-N-acetylglucosaminidase H (endo H) resistance. A novel observation was also that the trans-ER NS28 protein remained endo H sensitive through the course of BFA-treatment, but that the molecular weight varied during chase. Electron microscopy analysis revealed that BFA interfered in the transition from the intermediate enveloped particle to the mature double-shelled virus.