Diagnosis of Lassa fever
This is supplemenary data for the study on the development and evaluation of a recombinant antigen-based indirect immunofluorescence assay for diagnosis of Lassa fever. Several figures and tables were used to illustrate data gathered. One figure shows timeline for manifiestation of Lassa virus symptoms and another one shows the timeline for when various diagnostic assays can be used to detect Lassa virus. A representative figure of the pCI-neo mammalian expression vector and the recombinant LASV S segment plasmid used at the start of the project is shown. This figure also shows where the LASV S segment was inserted in the expression vector.
Two tables show the formulations used for the PCR reactios to amplify the LASV NP and LASV GP genes.A table shows the ratios used to optimise the transfection reaction using Lipofectamine 2000.Various figures showing the different PCR reactions results are included. The PCR reactions included are: an initial PCR reaction of the LASV NP and LASV GP genes, Optimisation PCR reactions (Dilution series PCR and Temperature gradient PCR) results, and colony PCR results for the LASV NP and LASV GP. A purification gel showing the purified LASV NP and LASV GP PCR products is included.Two restriction enzyme digestion gels are included. These are digestions done on the LASV NP and LASV GP plasmids. This was done to verify the orientation of the gene inserts in the plasmid backbone and ensure there was no contamination.
The sequencing results are included for both the LASV NP and LASV GP genes. The figures only sections of the full sequence. The sections include middle sections or where mutations had occured. The ends of the sequences are include to ensure the Kozak sequence, His tag and stop codon were present. The amino acid sequences were also included to ensure if any mutations occurred they did not alter the amino acid sequence or the orientation.
The full length sequences for both the LASV NP and LASV GP inserts are included as well. All the sequencing results were aligned to the full length codon-optimised LASV S segment. Multiple IFA results are shown. The purpose of these varies. Some are used to confirm initial expression of the LASV NP and LASV GP proteins in mammalian cells compared to control cells. Some are showing the expression of the LASV NP in stable cells lines. One is showing how the expression of the LASV GP decreased over time in stable cell lines. Two IFA results show the various ratios used to develop the LASV NP and LASV GP IFA slides. This was done to determine the optimal ratio for the IFA slides.Wetsern blot images are shown for both the LASV NP and LASV GP proteins. These were used to confirm expression of both the LASV NP and LASV GP proteins in both transient transfections and stable cell lines.
IFA slide layouts are shown to indicate what was added to each well on the IFA slides.A figure showing the results for the different IFA slides using serial dilutions of a known LASV positive serum samples is shown.A table showing the clinical performance results for the LASV NP and LASV GP IFA slides is included. These were compared to a virus-infected IFA slide.
A scatterplot showing the consensus IFA results for the 3 developed IFA slides is shown. This shows the number of discordant and negative results for the tested samples.A table showing the accesion numbers for the sequences used to generate the phylogentic tree are included.A table showing the raw results for the 3 IFA slides is included. This shows were which samples had discordant results and how we came to the final number of discordant samples for the IFAs.A 2x2 table is included to show how our final accuracy values were determined and the final results for the IFAs.
