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.

Development, validation and clinical evaluation of a broad-range pan-filovirus RT-qPCR.

BACKGROUND: During the five decades since their discovery, filoviruses of four species have caused human hemorrhagic fever outbreaks: Marburg (MARV) marburgvirus, and Zaire (EBOV), Sudan (SUDV) and Bundybugyo (BDBV) ebolaviruses. The largest, devastating EBOV epidemic in West Africa in 2014-16, has been followed by outbreaks of MARV in Uganda, 2017, and EBOV in Democratic Republic of Congo, 2018, emphasizing the need to develop preparedness to diagnose all filoviruses. OBJECTIVES: The aim of this study was to optimize a new filovirus RT-qPCR to detect all filoviruses, define its limits of detection (LOD) and perform a field evaluation with outbreak samples. STUDY DESIGN: A pan-filovirus RT-qPCR targeting the L gene was developed and evaluated within the EbolaMoDRAD (Ebola virus: modern approaches for developing bedside rapid diagnostics) project. Specificity and sensitivity were determined and the effect of inactivation and PCR reagents (liquid and lyophilized format) were tested. RESULTS: The LODs for the lyophilized pan-filovirus L-RT-qPCR assay were 9.4 copies per PCR reaction for EBOV, 9.9 for MARV, 1151 for SUDV, 65 for BDBV and 289 for Taï Forest virus. The test was set at the Pasteur Institute, Dakar, Senegal, and 83 Ebola patient samples, with viral load ranging from 5 to 5 million copies of EBOV per reaction, were screened. The results for the patient samples were in 100% concordance with the reference EBOV-specific assay. DISCUSSION: Overall, the assay showed good sensitivity and specificity, covered all filoviruses known to be human pathogens, performed well both in lyophilized and liquid-phase formats and with EBOV outbreak clinical samples.

Pathogenesis of experimental salmonid alphavirus infection in vivo: an ultrastructural insight.

Salmonid alphavirus (SAV) is an enveloped, single-stranded, positive sense RNA virus belonging to the family Togaviridae. It causes economically devastating disease in cultured salmonids. The characteristic features of SAV infection include severe histopathological changes in the heart, pancreas and skeletal muscles of diseased fish. Although the presence of virus has been reported in a wider range of tissues, the mechanisms responsible for viral tissue tropism and for lesion development during the disease are not clearly described or understood. Previously, we have described membrane-dependent morphogenesis of SAV and associated apoptosis-mediated cell death in vitro. The aims of the present study were to explore ultrastructural changes associated with SAV infection in vivo. Cytolytic changes were observed in heart, but not in gill and head-kidney of virus-infected fish, although they still exhibited signs of SAV morphogenesis. Ultrastructural changes associated with virus replication were also noted in leukocytes in the head kidney of virus-infected fish. These results further describe the presence of degenerative lesions in the heart as expected, but not in the gills and in the kidney.