PhD Scientific Days 2020

Molecular Sciences IV. Posters

Investigation of the adaptation mechanism of the Crimean-Congo haemorrhagic fever virus using in vitro culture and next-generation sequencing methods

Előadó neve

Dr. Magyar, Nóra

Előadó munkahelye

National Public Health Center

Előadó e-mail címe

magyar.nora@nnk.gov.hu

Az előadás címe

Investigation of the adaptation mechanism of the Crimean-Congo haemorrhagic fever virus using in vitro culture and next-generation sequencing methods

Szerző(k) neve és munkahelye

Nóra Magyar1,2, Bernadett Pályi1, Ákos Tóth1, Judit Henczkó1,2, Zoltán Kis1
1 National Public Health Center
2 School of PhD Studies, Semmelweis University

Szekció

Molecular Sciences IV. Posters

Összefoglaló szövege

Introduction: The tick-borne Crimean Congo haemorrhagic fever virus (CCHFV) is a risk group 4 pathogen in terms of biohazard. According to domestic legislations, the living agent can only be handled under biosafety level 4 conditions. To develop and optimize diagnostic and research assays, it is necessary to propagate the viable virus in laboratory environment. The genome structure of the CCHFV consists of 3 single-stranded, negative sense RNA segments. The longest L segment encodes the RNA-dependent RNA polymerase enzyme that lacks proper proof-reading mechanism leading to mutations often developing during the replication cycle. Mutations can be stabilized in the genome leading to better viral fitness and causing phenotypic changes.
Aims: In the course of our research, we investigated the kinetics of the CCHFV replication, the genetic variability during short-term serial passages on different cell lines, and the mutations that occur during adapting to the host cells.
Methods: Different kinetics were observed in vitro in the selected three cell lines (Vero E6, SW13 and A549) under biosafety level 4 conditions using infective titration and real-time RT-PCR methods. After identifying the optimal passage conditions, the virus was serially passaged, and the viral RNA was isolated from the virus-containing cell culture supernatant and subjected to next-generation sequencing using the Illumina MiSeq platform.
Results: No mutation was found throughout the small S segment, which encodes the viral nucleoprotein. Several mutations were identified in the medium M segment encoding the surface glycoproteins (Gn and Gc) and the L segment, some of which appeared exclusively in case of certain cell lines with different allele frequencies. For the L segment, three nonsynonymous mutations also appeared in the catalytic domain of the L protein, the biological role of which needs further investigation.
Conclusions: Genome-wise mutations developed by different adaptation mechanisms of RNA viruses can lead to phenotypic changes (e.g., changes in cell susceptibility) of the virus, thereby influencing the outcome of further studies including antiviral drug testing, vaccine testing, alteration of antigen expression patterns that may cause molecular and serological diagnostic difficulties. With the advent of next-generation sequencing, it has become possible to determine and analyze the entire genetic clouds of RNA virus quasispecies, present with low allele frequencies. In the future, we plan to study the mutations evolving during long-term passages, and to study the adaptation mechanisms and quasi-species in an in vivo mouse model.

Bemutatás módja

Poszter

Kind

Szabad

Status

elfogadva

Accepted presentation method

poszter

Előadás fájl jóváhagyás

nem rendelkezett róla

Előadó

5134

Start

12:55

End

12:58

Authors (legacy)

Nóra Magyar1,2, Bernadett Pályi1, Ákos Tóth1, Judit Henczkó1,2, Zoltán Kis1
1 National Public Health Center
2 School of PhD Studies, Semmelweis University