Generation and characterization of DENV- 4/2 chimeric virus-like particles

การสร้างและศึกษาคุณลักษณะของอนุภาคคล้ายไวรัสลูกผสมระหว่างเชื้อไวรัสเด็งกี่ชนิดที่ 4 และ ชนิดที่ 2

Name: Tawanchai Punyakom

LCSH: Virology

Classification :.DDC: 576.64

LCSH: Microbiology

Abstract: Dengue virus (DENV), an important mosquito-borne RNA virus, belongs to the genus Flavivirus in the family Flaviviridae. There are four serotypes (DENV-1, -2, -3, and -4) of dengue virus, all of which can cause potentially fatal diseases in human. The prevalence of dengue virus infection has dramatically increased in several countries in the world, becoming a severe public health problem. Currently, a tetravalent, live attenuated vaccine has been licensed for use in children older than nine years and adults in a number of countries although it provides efficient protection only against two dengue serotypes. Moreover, this type of vaccine carries an inherent risk related to a possibility of reversion to virulence. Virus-like particles (VLPs) have shown considerable promise for use as vaccine candidate. VLPs resemble native virus particles and are able to induce immune responses against native viruses. They are safer than live attenuated vaccine as they do not contain viral genetic materials. This study attempted to generate DENV-4/2 EDIII chimeric VLPs by substituting the EDIII domain within a DENV-2-Japanese encephalitis virus VLPs with the DENV-4 counterpart. A mosquito cell line, C6/36, was transfected with a chimeric plasmid encoding the prM and EDI+EDII coding regions of DENV-2, EDIII of DENV-4 E protein, and the stem anchor coding region of Japanese encephalitis virus. Culture media from selected transfected clones with high proportion of E-expressing cells (80-90%) were concentrated and extracellular particles purified by ultracentrifugation. Partially purified preparations were characterized by SDS-PAGE analysis and transmission electron microscopy. Three major protein bands corresponding in size to the viral structural proteins E, prM and M were detected by SDS-PAGE analysis and silver staining. Comparison of the band density of prM and M indicates that the extensive prM cleavage that was observed previously in the DENV-2 parental VLPs was minimally affected by the substitution with EDIII of DENV-4. Electron microscopic study revealed spherical particles of various sizes (range of diameter, 21-76 nm). Frequency distribution analysis of particle size was consistent with the presence of a major group of particles with two peak bin centers of 36.2 nm and 42.5 nm, which is smaller than infectious dengue particles but larger than previously described subviral particles. This group of medium size particles may derive from the shrinkage of coreless, large particles during specimen processing, or they may represent a new size class of recombinant dengue virus particles. These results indicate that DENV-4/2 EDIII chimeric VLPs can be generated in transfected C6/36 cells with minimal change in the maturation level. These chimeric VLPs may be useful as an alternative immunogen in the development of dengue vaccine in the future.

Chiang Mai University Library

Address: Chiang Mai

Email: cmulibref@cmu.ac.th

Name: Arunee Thitithanyanont, M.D.

Role: Chairman

Name: Nopporn Sittisombut, M.D.

Role: Member

Name: Rungtawan Sriburi

Role: Member

Created: 2016

Modified: 2020-01-10

Issued: 2020-01-10

วิทยานิพนธ์/Thesis

application/pdf

Th 576.64 T234G

eng

DegreeName: Master of Science

Level: Master's Degree

Descipline: Microbiology

Grantor: Chiang Mai University

©copyrights Chiang Mai University

ลำดับที่.	ชื่อแฟ้มข้อมูล	ขนาดแฟ้มข้อมูล	จำนวนเข้าถึง	วัน-เวลาเข้าถึงล่าสุด
1	micro80716tpyk_full.pdf	4.2 MB

ใช้เวลา

0.038698 วินาที