dc.contributor.author
Xing, Na
dc.date.accessioned
2022-12-05T10:56:38Z
dc.date.available
2022-12-05T10:56:38Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/36996
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-36709
dc.description.abstract
Avian coronavirus, also known as infectious bronchitis virus (IBV), belongs to the genus Gammacoronavirus and is the causative agent of infectious bronchitis, a highly contagious respiratory disease in the poultry industry.
In virology studies, reverse genetics systems based on BACs are extremely valuable because they allow us to manipulate viral genes. In our study, we assembled the complete genome of the IBV strain Beaudette-FUB into an artificial bacterial chromosome (BAC), producing an infectious BAC clone. From this constructed IBV BAC clone, we successfully rescued infectious viruses with identical growth characteristics to the parental viruses. To establish genetically stable EGFP viruses, we then inserted the EGFP ORF into 11 putative cleavage sites of 3CLpro. Of these, we identified three insertion sites located at the outermost 3’ end of the replicase gene– between the coding sequences of Nsp13 (helicase), Nsp14 (RNA exonuclease), Nsp15 (RNA endonuclease), and Nsp16 (RNA methyltransferase) could tolerate heterologous genes in the IBV genome. Additionally, we found that fluorescent proteins expressed by the replicase gene can be efficiently cleaved by the 3CLpro and released from the replicase polyprotein. Furthermore, we also determined the genetic stability of these three EGFP-replicase viruses. Among them, the engineered Nsp13-EGFP-Nsp14 virus still exhibited high stability in DF-1 cells after 20 serial passages. The colocalization results showed that EGFP, together with dsRNA or RdRp, accumulated in the well-defined foci at the early stage of infection. When the infection progressed, EGFP proteins were produced and distributed throughout the cytoplasm.
Our studies have shown that the replicase-EGFP viruses could be used to study viral replication and transcription, to screen antiviral drugs on a large scale, to develop multivalent vaccines, and even that the potential positions could be applied to other coronaviruses.
en
dc.format.extent
VII, 83 Seiten
dc.rights.uri
http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen
dc.subject
avian coronavirus
en
dc.subject
infectious bronchitis virus
en
dc.subject
infectious clone
en
dc.subject
3CL protease
en
dc.subject.ddc
600 Technology, Medicine, Applied sciences::630 Agriculture, Veterinary medicine::630 Agriculture, Veterinary medicine
dc.title
Engineering and characterization of avian coronavirus mutants expressing reporter proteins from the replicase gene
dc.contributor.gender
female
dc.contributor.firstReferee
Osterrieder, Klaus
dc.contributor.furtherReferee
Veit, Michael
dc.contributor.furtherReferee
Blome, Sandra
dc.date.accepted
2022-11-21
dc.identifier.urn
urn:nbn:de:kobv:188-refubium-36996-7
refubium.affiliation
Veterinärmedizin
dcterms.accessRights.dnb
free
dcterms.accessRights.openaire
open access
dcterms.accessRights.proquest
accept