RESUMEN
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.
Asunto(s)
Ebolavirus/aislamiento & purificación , Fiebre Hemorrágica Ebola/diagnóstico , Técnicas de Diagnóstico Molecular , Proteínas de la Nucleocápside/genética , Reacción en Cadena de la Polimerasa , Ebolavirus/genética , Humanos , Límite de Detección , ARN Viral/genética , Sensibilidad y EspecificidadRESUMEN
Here we report full-length sequencing of the first large set of influenza A(H1N1)pdm09 virus genomes isolated in Finland between the years 2009 and 2013 and discuss the advantages and needs of influenza virus sequencing efforts.
RESUMEN
The nonstructural protein NS1 of influenza A virus blocks the development of host antiviral responses by inhibiting polyadenylation of cellular pre-mRNA. NS1 also promotes the synthesis of viral proteins by stimulating mRNA translation. Here, we show that recombinant NS1 proteins of human pandemic H1N1/2009, avian highly pathogenic H5N1, and low pathogenic H5N2 influenza strains differentially affected these two cellular processes: NS1 of the two avian strains, in contrast to NS1 of H1N1/2009, stimulated translation of reporter mRNA in cell-free translation system; NS1 of H5N1 was an effective inhibitor of cellular pre-mRNA polyadenylation in A549 cells, unlike NS1 of H5N2 and H1N1/2009. We identified key amino acids in NS1 that contribute to its activity in these two basic cellular processes. Thus, we identified strain-specific differences between influenza virus NS1 proteins in pre-mRNA polyadenylation and mRNA translation.
