Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants

Six ebolavirus species are reported to date, including human pathogens Bundibugyo virus (BDBV), Ebola virus (EBOV), Sudan virus (SUDV), and Taï Forest virus (TAFV); non-human pathogen Reston virus (RESTV); and the plausible Bombali virus (BOMV). Since there are differences in the disease severity ca...

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Autores principales:	Qingbo Shu, Tara Kenny, Jia Fan, Christopher J. Lyon, Lisa H. Cazares, Tony Y. Hu
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/fc015d95c0454c8cac679063bfc43269
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spelling	oai:doaj.org-article:fc015d95c0454c8cac679063bfc432692021-11-25T05:47:32ZSpecies-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants1553-73661553-7374https://doaj.org/article/fc015d95c0454c8cac679063bfc432692021-11-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8601621/?tool=EBIhttps://doaj.org/toc/1553-7366https://doaj.org/toc/1553-7374Six ebolavirus species are reported to date, including human pathogens Bundibugyo virus (BDBV), Ebola virus (EBOV), Sudan virus (SUDV), and Taï Forest virus (TAFV); non-human pathogen Reston virus (RESTV); and the plausible Bombali virus (BOMV). Since there are differences in the disease severity caused by different species, species identification and viral burden quantification are critical for treating infected patients timely and effectively. Here we developed an immunoprecipitation-coupled mass spectrometry (IP-MS) assay for VP40 antigen detection and quantification. We carefully selected two regions of VP40, designated as peptide 8 and peptide12 from the protein sequence that showed minor variations among Ebolavirus species through MS analysis of tryptic peptides and antigenicity prediction based on available bioinformatic tools, and generated high-quality capture antibodies pan-specific for these variant peptides. We applied this assay to human plasma spiked with recombinant VP40 protein from EBOV, SUDV, and BDBV and virus-like particles (VLP), as well as EBOV infected NHP plasma. Sequence substitutions between EBOV and SUDV, the two species with highest lethality, produced affinity variations of 2.6-fold for p8 and 19-fold for p12. The proposed IP-MS assay differentiates four of the six known EBV species in one assay, through a combination of p8 and p12 data. The IP-MS assay limit of detection (LOD) using multiple reaction monitoring (MRM) as signal readout was determined to be 28 ng/mL and 7 ng/mL for EBOV and SUDV respectively, equivalent to ~1.625–6.5×105 Geq/mL, and comparable to the LOD of lateral flow immunoassays currently used for Ebola surveillance. The two peptides of the IP-MS assay were also identified by their tandem MS spectra using a miniature MALDI-TOF MS instrument, greatly increasing the feasibility of high specificity assay in a decentralized laboratory. Author summary We developed a quantitative assay for carefully selected species-specific VP40 peptide variants by selecting two VP40 tryptic peptides (i.e., LGPGIPDHPLR from EBOV VP40 and LRPILLPGK from BDBV VP40) to generate antibodies pan-specific for variants of these peptides. Since species-specific structural difference may affect VP40 liberation during sample inactivation, and sequence variations may affect target peptide capture by pan-specific target antibodies, we quantified VP40 in EBOV and SUDV virus like particles (VLPs) to build species-specific standard curves to account for the greatest differences and improve the accuracy of VP40 quantitation. We also evaluated the feasibility of using miniature mass spectrometer to collect the tandem MS spectra for both target peptides.Qingbo ShuTara KennyJia FanChristopher J. LyonLisa H. CazaresTony Y. HuPublic Library of Science (PLoS)articleImmunologic diseases. AllergyRC581-607Biology (General)QH301-705.5ENPLoS Pathogens, Vol 17, Iss 11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
spellingShingle	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5 Qingbo Shu Tara Kenny Jia Fan Christopher J. Lyon Lisa H. Cazares Tony Y. Hu Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants
description	Six ebolavirus species are reported to date, including human pathogens Bundibugyo virus (BDBV), Ebola virus (EBOV), Sudan virus (SUDV), and Taï Forest virus (TAFV); non-human pathogen Reston virus (RESTV); and the plausible Bombali virus (BOMV). Since there are differences in the disease severity caused by different species, species identification and viral burden quantification are critical for treating infected patients timely and effectively. Here we developed an immunoprecipitation-coupled mass spectrometry (IP-MS) assay for VP40 antigen detection and quantification. We carefully selected two regions of VP40, designated as peptide 8 and peptide12 from the protein sequence that showed minor variations among Ebolavirus species through MS analysis of tryptic peptides and antigenicity prediction based on available bioinformatic tools, and generated high-quality capture antibodies pan-specific for these variant peptides. We applied this assay to human plasma spiked with recombinant VP40 protein from EBOV, SUDV, and BDBV and virus-like particles (VLP), as well as EBOV infected NHP plasma. Sequence substitutions between EBOV and SUDV, the two species with highest lethality, produced affinity variations of 2.6-fold for p8 and 19-fold for p12. The proposed IP-MS assay differentiates four of the six known EBV species in one assay, through a combination of p8 and p12 data. The IP-MS assay limit of detection (LOD) using multiple reaction monitoring (MRM) as signal readout was determined to be 28 ng/mL and 7 ng/mL for EBOV and SUDV respectively, equivalent to ~1.625–6.5×105 Geq/mL, and comparable to the LOD of lateral flow immunoassays currently used for Ebola surveillance. The two peptides of the IP-MS assay were also identified by their tandem MS spectra using a miniature MALDI-TOF MS instrument, greatly increasing the feasibility of high specificity assay in a decentralized laboratory. Author summary We developed a quantitative assay for carefully selected species-specific VP40 peptide variants by selecting two VP40 tryptic peptides (i.e., LGPGIPDHPLR from EBOV VP40 and LRPILLPGK from BDBV VP40) to generate antibodies pan-specific for variants of these peptides. Since species-specific structural difference may affect VP40 liberation during sample inactivation, and sequence variations may affect target peptide capture by pan-specific target antibodies, we quantified VP40 in EBOV and SUDV virus like particles (VLPs) to build species-specific standard curves to account for the greatest differences and improve the accuracy of VP40 quantitation. We also evaluated the feasibility of using miniature mass spectrometer to collect the tandem MS spectra for both target peptides.
format	article
author	Qingbo Shu Tara Kenny Jia Fan Christopher J. Lyon Lisa H. Cazares Tony Y. Hu
author_facet	Qingbo Shu Tara Kenny Jia Fan Christopher J. Lyon Lisa H. Cazares Tony Y. Hu
author_sort	Qingbo Shu
title	Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants
title_short	Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants
title_full	Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants
title_fullStr	Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants
title_full_unstemmed	Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants
title_sort	species-specific quantification of circulating ebolavirus burden using vp40-derived peptide variants
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/fc015d95c0454c8cac679063bfc43269
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Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants

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