Student Major/Year in School
Microbiology, Medical Biochemistry, third year
Faculty Mentor Information
Dr. Bonto Faburay, Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine
Abstract
Lassa virus is an arenavirus causing a disseminated systemic primary viral infection. This virus causes Lassa fever which is a viral hemorrhagic fever endemic in West Africa and is responsible for the deaths of thousands of people each year. There is a possibility for the Lassa virus to be introduced into the US and used as a biological weapon with the potential to harm a large-scale population. Because of increasing international travel, a sizeable burden from the disease, and its potential use for biological warfare, it is necessary to develop sensitive diagnostic assays to accurately detect virus infections and mitigate against potential introduction and spread. The objective of this project was to produce recombinant Lassa virus nucleoprotein (N), the most abundant and immunogenic structural protein of the virus, using a recombinant baculovirus expression system and asses its use as an antigen to detect Lassa antibodies in infected hosts. Using the eukaryotic expression system, the gene encoding the N protein was cloned in donor plasmid, which was used to create a recombinant bacmid. The latter was used to transfect Spodoptera frugiperda (SF9) cells to rescue the recombinant baculovirus and express the target protein. The recombinant proteins were purified by affinity chromatography using nickel columns, and specific reactivity was assessed by western blot using anti-Lassa virus hyperimmune mouse ascitic fluid, mouse anti-Lassa monoclonal (ABE419) and mouse anti-Lass monoclonal (ABE420) antibodies. An estimated 63kDa recombinant N protein was overexpressed and authenticated via specific reactivity with the anti-Lassa virus hyperimmune mouse ascetic fluid but not with the monoclonal antibodies. Manifestation of specific immunoreactivity with the Lassa antiserum suggests the ability of the N protein to be recognized by host-specific Lassa antibodies and thus potentially serve as a serodiagnostic antigen for detection and seroepidemio-surveillance of Lassa fever in endemic and non-endemic regions. Future studies would entail evaluating the use of the recombinant antigen in ELISA to investigate the seroepidemiology of Lassa fever among febrile patients in endemic countries in West Africa.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License
Recommended Citation
Grover, Sahiba (2019). "Assessment of the Potential Use of Recombinant Baculovirus-expressed Lassa Virus Nucleoprotein as a serodiagnostic antigen," Kansas State University Undergraduate Research Conference. https://newprairiepress.org/ksuugradresearch/2019/posters/25
Included in
Animal Diseases Commons, Diagnosis Commons, Other Analytical, Diagnostic and Therapeutic Techniques and Equipment Commons, Pathogenic Microbiology Commons, Virology Commons, Virus Diseases Commons
Assessment of the Potential Use of Recombinant Baculovirus-expressed Lassa Virus Nucleoprotein as a serodiagnostic antigen
Lassa virus is an arenavirus causing a disseminated systemic primary viral infection. This virus causes Lassa fever which is a viral hemorrhagic fever endemic in West Africa and is responsible for the deaths of thousands of people each year. There is a possibility for the Lassa virus to be introduced into the US and used as a biological weapon with the potential to harm a large-scale population. Because of increasing international travel, a sizeable burden from the disease, and its potential use for biological warfare, it is necessary to develop sensitive diagnostic assays to accurately detect virus infections and mitigate against potential introduction and spread. The objective of this project was to produce recombinant Lassa virus nucleoprotein (N), the most abundant and immunogenic structural protein of the virus, using a recombinant baculovirus expression system and asses its use as an antigen to detect Lassa antibodies in infected hosts. Using the eukaryotic expression system, the gene encoding the N protein was cloned in donor plasmid, which was used to create a recombinant bacmid. The latter was used to transfect Spodoptera frugiperda (SF9) cells to rescue the recombinant baculovirus and express the target protein. The recombinant proteins were purified by affinity chromatography using nickel columns, and specific reactivity was assessed by western blot using anti-Lassa virus hyperimmune mouse ascitic fluid, mouse anti-Lassa monoclonal (ABE419) and mouse anti-Lass monoclonal (ABE420) antibodies. An estimated 63kDa recombinant N protein was overexpressed and authenticated via specific reactivity with the anti-Lassa virus hyperimmune mouse ascetic fluid but not with the monoclonal antibodies. Manifestation of specific immunoreactivity with the Lassa antiserum suggests the ability of the N protein to be recognized by host-specific Lassa antibodies and thus potentially serve as a serodiagnostic antigen for detection and seroepidemio-surveillance of Lassa fever in endemic and non-endemic regions. Future studies would entail evaluating the use of the recombinant antigen in ELISA to investigate the seroepidemiology of Lassa fever among febrile patients in endemic countries in West Africa.
