PMC:4504098 / 20819-23279 JSONTXT

{"project":"2_test","denotations":[{"id":"26184657-23343532-143716478","span":{"begin":361,"end":363},"obj":"23343532"},{"id":"26184657-19785757-143716479","span":{"begin":491,"end":492},"obj":"19785757"},{"id":"26184657-24747773-143716480","span":{"begin":517,"end":518},"obj":"24747773"},{"id":"26184657-20694141-143716481","span":{"begin":601,"end":603},"obj":"20694141"},{"id":"26184657-21093485-143716482","span":{"begin":1544,"end":1546},"obj":"21093485"},{"id":"26184657-21546353-143716483","span":{"begin":2325,"end":2327},"obj":"21546353"}],"text":"Serology and molecular assays were undertaken at AAHL. Samples were handled at BSL 4 until inactivated. An indirect ELISA (using mixed recombinant RESTV and EBOV (formally ZEBOV) N antigens) was used to screen sera, with Western blot (using individually run recombinant RESTV and EBOV N antigens) performed on ELISA-positive sera as described in Olival et al. [10]. Cut-off values to determine ELISA-positive sera were determined using a statistical approach as described in Pourrut et al. [1] and Olival and Hayman [5]. Confirmatory Western blot analysis was conducted as described in Hayman et al. [24]. Molecular assays comprised quantitative (q) and conventional RT-PCR in series. Swabs of the same sample type were pooled (five per pool) and RNA extraction undertaken using a QIAamp viral mini kit according to manufacturer’s instructions. Eluates were tested using a US CDC qPCR which targeted the RESTV NP gene (P. Rollin, 2010, pers. comm.). A sample yielding a repeatable Ct value of less than 40 was regarded as positive, and the authenticity of the amplified products corroborated by melt curve analysis; a sample yielding a repeatable Ct value of 40–45, or a non-repeatable Ct value of less than 40 was regarded as potentially positive. All other samples were regarded as ‘not detected’. Positive and potentially positive pools were re-tested in the same assay, as were the component individual samples. Where adequate sample remained, positive or potentially positive individual samples were tested by a PCR targeting the NP gene [25] adapted to a hemi-nested PCR with a second forward primer (FiloNP-hnFe – TGATGGTAATCTTYAGATTGATGAGG) in an attempt to gain adequate product for direct sequencing. Purified PCR products were sequenced at the AAHL sequencing facility using a BigDye Terminator v1.0 Kit (Applied Biosystems) and an ABI PRISM 377 DNA Sequencer (Applied Biosystems). Every nucleotide was sequenced with a minimum of threefold redundancy to ensure a consensus and repeatable sequence data. The Clone Manager and Align Plus programs in the Sci Ed Central software package (Scientific and Educational Software) were used for sequence management and analysis. Phylogenetic analysis based on the 519 bp fragment of the NP genes from different Ebola virus sequences was conducted using the MEGA5 program [26]. The phylogenetic tree was constructed using the maximum likelihood algorithm with bootstrap values determined by 1,000 replicates."}