Određivanje virusnog titra u staničnoj kulturi upotrebom fluorescentnog virusa mumpsa

master's thesis

• Title (english): Determination of virus potency using fluorescent mumps virus
• Author: Sara Drk
• Mentor: Tanja Košutić Gulija (mentor)
• Mentor: Silvija Černi (sumentor)
• Committee member: Petra Peharec Štefanić (predsjednik povjerenstva)
• Committee member: Renata Matoničkin Kepčija (član povjerenstva)
• Committee member: Renata Šoštarić (član povjerenstva)
• Committee member: Silvija Černi (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-02-24, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Virus mumpsa uzrokuje istoimenu bolest ljudi koja se prevenira cijepljenjem. Poznavanje titra
virusa važan je parametar za njegovu karakterizaciju u virologiji i vakcinologiji. Najčešće se
koriste testovi plakiranja (citopatogeni efekt u obliku plakova) i 50% infektivne doze stanične
kulture (CCID50) (detektiranje citopatogenog efekta u 50% inokuliranih stanica). U ovom
istraživanju korišten je rekombinantni virus mumpsa MRV3, izvedenica soja L-Zagreb s
ugrađenim genom za zeleni fluorescentni protein koji uzrokuje fluorescenciju inficiranih
stanica. Očitavanje ovih testova standardno se radi makroskopski ili svjetlosnim mikroskopom,
a rezultati ovise o objektivnosti i iskustvu operatera. Vrijednosti titra u ovim testovima razlikuju
se za iste sojeve mumpsa, a za konverziju vrijednosti iz testa CCID50 u vrijednosti iz testa
plakiranja ne postoji jedinstveni konverzijski koeficijent. Cilj istraživanja je bio očitati titar u
oba testa standardnim načinom te fluorescencijskom mikroskopijom, usporediti rezultate,
definirati utjecaj načina očitavanja na rezultat testa te odrediti eksperimentalni konverzijski
koeficijent za MRV3. Upotrebom fluorescencije u očitavanju testa plakiranja utvrđeno je
postojanje makroskopski nevidljivih plakova te razlika vrijednosti titra od 0.4 log. Na
očitavanje testova CCID50 upotreba fluorescencije utjecala je neznatno. Eksperimentalni
konverzijski koeficijenti za uzorke virusa MRV3 bili su u rasponu 0.04-0.15. Upotreba
fluorescentnog virusa utjecala je na titar, ali nije bitno promijenila konverzijski koeficijent.

Abstract (english)

Mumps virus causes a human disease of the same name which can be prevented by vaccine.
Determination of virus titre is an important parameter for its characterisation in virology and
vaccinology. Most frequently used methods are plaque assay, based on cytopathogenic effect
in a form of plaques, and 50% cell culture infective dose (CCID50) assay, based on detection
cytopathic effect of the virus in 50% of the cells. In this research, a recombinant virus MRV3
was used, prepared from L-Zagreb strain with an inserted gene for green fluorescent protein
which causes fluorescence of infected cells. Reading of these assays is standardly done
macroscopically or using light microscope and the results depend on objectivity and experience
of the operator. Titre values in these two tests differ for same virus strains and conversion of
obtained values presents a problem because a conversion value for all strains of mumps does
not exist. The aim of this research was to determine virus titres in both assays standardly and
using fluorescence microscope, compare the results, define the effect of the way of reading on
assays results and determine experimental conversion coefficient for MRV3 virus. Using
fluorescence in reading of plaque assay determined the existence of macroscopically invisible
plaques and a difference of titre values of 0.4 log. Using fluorescence unnotably affected
CCID50. The experimental conversion coefficient for samples of MRV3 virus was calculated to
be in the range 0.04-0.15. Using fluorescent virus effected virus titre, but it did not change the
conversion coefficient significantly.

• Keywords: mumps; fluorescencija; titar; plakiranje; CCID50; konverzijski koeficijent
• Keywords (english): mumps; fluorescence; titre; plaque assay; CCID50; conversion coefficient
• Language: croatian
• URN:NBN: urn:nbn:hr:217:254727
• Study programme: Title: Experimental Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra eksperimentalne biologije (magistar/magistra eksperimentalne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2022-02-24
• Created on: 2021-03-02 11:46:37