A putative diacidic motif in the SARS-CoV ORF6 protein influences its subcellular localization and suppression of expression of co-transfected expression constructs

<p>Abstract</p> <p>Background</p> <p>The ORF6 protein is one of the eight accessory proteins of the severe acute respiratory syndrome coronavirus (SARS-CoV). Numerous properties of ORF6 have been documented and this study focuses on two of these, namely, its ability to suppress the expression of co-transfected expression constructs and its subcellular localization to vesicular structures.</p> <p>Results</p> <p>Using a transient transfection system, ORF6's ability to suppress the expression of co-transfected expression constructs was measured in a quantitative manner. While ORF6 does not have a global effect on protein synthesis, quantitative real-time PCR revealed that it down-regulated the mRNA level of the co-transfected myc-nsp8 gene. Furthermore, alanine substitution of a diacidic cluster motif (aa53-56) in the ORF6 gene caused a reduction in the suppression of expression of co-transfected myc-nsp8 gene. Our previous study revealed that ORF6 localized to vesicular structures in SARS-CoV infected Vero E6 cells. Here, ORF6 was observed to be localized to similar vesicular structures in Vero E6 cells which have been transiently transfected with a mammalian expression plasmid encoding for untagged ORF6. ORF6 showed partial colocalization with cellular proteins CD63 and Lamp1, suggesting that the vesicular structures may be a subpopulation of endosomal/lysosomal vesicles. The alanine substitution of the diacidic cluster motif also altered the subcellular localization of the ORF6 protein, indicating a potential relationship between the subcellular localization of the ORF6 protein and its ability to suppress the expression of co-transfected expression constructs.</p> <p>Conclusions</p> <p>By combining quantitative real-time PCR and transient transfection system, a simple and safe method is established to measure ORF6's ability to suppress the expression of co-transfected myc-nsp8. In addition, immunofluorescence analysis revealed that the subcellular localization of ORF6 when expressed on its own is similar to that observed in SARS-CoV infected cells. Through the use of these two assays, a putative diacidic motif in the ORF6 protein was found to influence its subcellular localization and ability to suppress the expression of co-transfected expression constructs.</p

Identification and validation of internal reference genes for real-time quantitative polymerase chain reaction-based studies in Hyalomma anatolicum ticks

Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne viral disease caused by the orthonairovirus CCHF virus (CCHFV). Ticks of the genus Hyalomma are the viral reservoir and they represent the main vector transmitting the virus to their hosts during blood feeding. However, how CCHFV replicates in its natural arthropod host cells and the nature of virus/host interactions are still largely unknown. With the aim of developing tools for use in this field, we identified and validated expression of four candidate endogenous control tick genes in a Hyalomma anatolicum-derived cell line. These genes will be useful for normalization of viral/cellular transcripts in infection/expression studies or as internal controls in molecular epidemiology surveys of pathogens transmitted by Hyalomma ticks

Rapid bedside inactivation of Ebola virus for safe nucleic acid tests

Rapid bedside inactivation of Ebola virus would be a solution for the safety of medical and technical staff, risk containment, sample transport and high-throughput or rapid diagnostic testing during an outbreak. We show that the commercially available MagNA Pure lysis/binding buffer used for nucleic acid extraction inactivates Ebola virus. A rapid bedside inactivation method for nucleic acid tests is obtained by simply adding MagNA Pure lysis/binding buffer directly into vacuum blood collection EDTA-tubes using a thin needle and syringe prior to sampling. The ready-to-use inactivation vacuum tubes are stable for more than 4 months and Ebola virus RNA is preserved in the MagNA Pure lysis/binding buffer for at least 5 weeks independent of the storage temperature. We also show that Ebola virus RNA can be manually extracted from MagNA Pure lysis/binding buffer-inactivated samples using the QIAamp Viral RNA mini kit. We present an easy and convenient method for bedside inactivation using available blood collection vacuum tubes and reagents. We propose to use this simple method for fast, safe and easy bedside inactivation of Ebola virus for safe transport and routine nucleic acid detection

Fiber-optic immunosensor for detection of Crimean-Congo Hemorrhagic fever IgG antibodies in patients

Crimean-Congo hemorrhagic fever (CCHF) is a severe viral disease with high fatality rate. CCHF virus is endemic in parts of Africa, Asia, Middle East and southeastern Europe. Rapid diagnostics of CCHF is vital for appropriate clinical management and prevention of secondary spread from human-to-human. Currently, diagnostics relies on Real-Time RT-PCR and antibody or antigen detection using ELISA. These methods require trained personnel and expensive equipment and are not appropriate for point-of-care (POC) diagnostics. Furthermore there are no POC assays available for CCHF. We developed a fiber-optic biosensor for the detection of CCHF IgG antibodies. In order to improve sensitivity, we optimized both the bioreceptor immobilization protocol and the chemiluminescence substrate formulation. The resulting protocol showed a 100-fold greater sensitivity for detection of CCHF antibodies. Finally, we evaluated the fiber-optic biosensor with two CCHF patient sera. We showed that the fiber-optic biosensor is 10-times more sensitive that colorimetric ELISA and is able to detect both patients with high and low levels of IgG antibodies. We believe that the fiber-optic biosensor is a suitable alternative to ELISA as it is much more sensitive and makes it possible to detect small amount of antibodies at an early stage of infection, and can be integrated as a point-of-care diagnostic system of CCHF

Comparison of Zaire ebolavirus realtime RT-PCRs targeting the nucleoprotein gene

In last five years, the Africa has faced two outbreaks of Zaire ebolavirus. These outbreaks have been the largest so far, and latest outbreak is still ongoing and affecting the Democratic Republic of the Congo. We tested in parallel three different Zaire ebolavirus (EBOV) realtime RT-PCRs targeting the nucleoprotein gene (EBOV NP-RT-qPCRs) described by Trombley et al. (2010); Huang et al. (2012) and Weidmann et al. (2004). These assays are used regularly in diagnostic laboratories. The limit of detection (LOD), intra-assay repeatability using different matrixes, sensitivity and specificity were determined. In addition, the primers and probes were aligned with the sequences available in ongoing and past outbreaks in order to check the mismatches. The specificity of all three EBOV NP-RT-qPCRs were excellent (100 %), and LODs were under or 10 copies per PCR reaction. Intra-assay repeatability was good in all assays, however the Ct-values were bit higher using the EDTA-blood based matrix. All of the primers and probes in EBOV NP-RT-qPCR assays have one or more mismatches in the probes and primers when the 2267 Zaire EBOV NP sequences, including strains Ituri from DRC outbreak (year 2018), was aligned. The EBOV strain of Bikoro (year 2018) circulating in DRC was 100 % match in Trombley and Weidmann assay, but had one mismatch in Huang assay.Peer reviewe

Amiodarone and metabolite MDEA inhibit Ebola virus infection by interfering with the viral entry process

Ebola virus disease (EVD) is one of the most lethal transmissible infections characterized by a high fatality rate, and a treatment has not been developed yet. Recently, it has been shown that cationic amphiphiles, among them the antiarrhythmic drug amiodarone, inhibit filovirus infection. In the present work, we investigated how amiodarone interferes with Ebola virus infection. Wild-type Sudan ebolavirus and recombinant vesicular stomatitis virus, pseudotyped with the Zaire ebolavirus glycoprotein, were used to gain further insight into the ability of amiodarone to affect Ebola virus infection. We show that amiodarone decreases Ebola virus infection at concentrations close to those found in the sera of patients treated for arrhythmias. The drug acts by interfering with the fusion of the viral envelope with the endosomal membrane. We also show that MDEA, the main amiodarone metabolite, contributes to the antiviral activity. Finally, studies with amiodarone analogues indicate that the antiviral activity is correlated with drug ability to accumulate into and interfere with the endocytic pathway. Considering that it is well tolerated, especially in the acute setting, amiodarone appears to deserve consideration for clinical use in EV

Cranberry (Vaccinium macrocarpon) Extracts with a High Content of A-Type Proanthocyanidins Strongly Impair Hazara Virus Attachment to Target Cells

Data used to prepare figure

The new generation hdhodh inhibitor meds433 hinders the in vitro replication of sars-cov-2 and other human coronaviruses

Although coronaviruses (CoVs) have long been predicted to cause zoonotic diseases and pandemics with high probability, the lack of effective anti-pan-CoVs drugs rapidly usable against the emerging SARS-CoV-2 actually prevented a promptly therapeutic intervention for COVID-19. Development of host-targeting antivirals could be an alternative strategy for the control of emerging CoVs infections, as they could be quickly repositioned from one pandemic event to another. To contribute to these pandemic preparedness efforts, here we report on the broad-spectrum CoVs antiviral activity of MEDS433, a new inhibitor of the human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of the de novo pyrimidine biosynthesis pathway. MEDS433 in-hibited the in vitro replication of hCoV-OC43 and hCoV-229E, as well as of SARS-CoV-2, at low nanomolar range. Notably, the anti-SARS-CoV-2 activity of MEDS433 against SARS-CoV-2 was also observed in kidney organoids generated from human embryonic stem cells. Then, the antiviral activity of MEDS433 was reversed by the addition of exogenous uridine or the product of hDHODH, the orotate, thus confirming hDHODH as the specific target of MEDS433 in hCoVs-infected cells. Taken together, these findings suggest MEDS433 as a potential candidate to develop novel drugs for COVID-19, as well as broad-spectrum antiviral agents exploitable for future CoVs threats

Human soluble ACE2 improves the effect of remdesivir in SARS-CoV-2 infection

There is a critical need for safe and effective drugs for COVID-19. Only remdesivir has received authorization for COVID-19 and has been shown to improve outcomes but not decrease mortality. However, the dose of remdesivir is limited by hepatic and kidney toxicity. ACE2 is the critical cell surface receptor for SARS-CoV-2. Here, we investigated additive effect of combination therapy using remdesivir with recombinant soluble ACE2 (high/low dose) on Vero E6 and kidney organoids, targeting two different modalities of SARS-CoV-2 life cycle: cell entry via its receptor ACE2 and intracellular viral RNA replication. This combination treatment markedly improved their therapeutic windows against SARS-CoV-2 in both models. By using single amino-acid resolution screening in haploid ES cells, we report a singular critical pathway required for remdesivir toxicity, namely, Adenylate Kinase 2. The data provided here demonstrate that combining two therapeutic modalities with different targets, common strategy in HIV treatment, exhibit strong additive effects at sub-toxic concentrations. Our data lay the groundwork for the study of combinatorial regimens in future COVID-19 clinical trials

Experiences of outbreak laboratory management in the Ebola Disease outbreak in West-Africa 2014-2015

During the Ebola Disease outbreak in 2014-2015 West-Africa about 24 organizations operated laboratories at 40 sites in Guinea, Sierra Leone and Liberia. Representatives of ten organisations which had deployed laboratories to 16 sites across the three countries in West-Africa convened for a two day symposium in Dakar (4-5.02.16) to exchange their experiences. This article summarizes the discussion and points made during the discussion of the laboratory deployment experiences during the epidemic touching organisational and procedural issues.Additional co-authors: P Jansen van Vuren, K Stroecker, J Paweska, C Picard, H Sheeley, P Smit, AA Sal