Abstract Title
Infectious cDNA Clone of Human Norovirus GII.4 Sydney: A Comparative Analysis with Murine Norovirus
Presenter
Marit Lingemann, Heidelberg University
Co-Author(s)
M. Lingemann1, F. Meichsner1, L. Wöllner1, V. Lohmann1
1University of Heidelberg, Molecular Virology, Heidelberg, Germany
Abstract Category
Entry and Replication
Abstract
Background
Human noroviruses (HuNoVs) are the primary cause of acute non-bacterial gastroenteritis but an efficient reverse genetics system for the predominant GII.4 strain is still lacking. Here, we aimed to establish infectious clones for based on GII.4 New Orleans and Sydney, using murine norovirus (MNV) as a control.
Methods
Capped, in vitro transcribed (IVT) RNA was transfected into cell lines lacking entry receptors. Initial translation was analysed using a GFP-based reporter system and NS-protein-specific antibodies. Viral replication in transfected cells was assessed by detection of replication intermediates and capsid protein and by transfer of supernatants to cells susceptible for infection.
Results
IVT RNA of both HuNoV GII.4 clones and MNV was efficiently translated in transfected cells. However, replication intermediates were found in only a few cells up to 24h after transfection, resulting in case of MNV in a viral titer of 103 TCID50/mL. For HuNoV, a low number of infection events in salivary gland cells were detectable only for the Sydney strain. In contrast, transfection of VPg-lated RNA isolated from MNV infected cells was several orders of magnitude more efficient in producing infectious virus, suggesting that initiation of RNA replication is the limiting factor for capped IVT RNAs.
Conclusions
Our work provides evidence for a bona fide infectious cDNA clone of GII.4 Sydney, paving the road towards a reverse genetics model. However, the system is very inefficient, since the function of VPg in initiation of RNA replication cannot be replaced effectively by a cap.
Human noroviruses (HuNoVs) are the primary cause of acute non-bacterial gastroenteritis but an efficient reverse genetics system for the predominant GII.4 strain is still lacking. Here, we aimed to establish infectious clones for based on GII.4 New Orleans and Sydney, using murine norovirus (MNV) as a control.
Methods
Capped, in vitro transcribed (IVT) RNA was transfected into cell lines lacking entry receptors. Initial translation was analysed using a GFP-based reporter system and NS-protein-specific antibodies. Viral replication in transfected cells was assessed by detection of replication intermediates and capsid protein and by transfer of supernatants to cells susceptible for infection.
Results
IVT RNA of both HuNoV GII.4 clones and MNV was efficiently translated in transfected cells. However, replication intermediates were found in only a few cells up to 24h after transfection, resulting in case of MNV in a viral titer of 103 TCID50/mL. For HuNoV, a low number of infection events in salivary gland cells were detectable only for the Sydney strain. In contrast, transfection of VPg-lated RNA isolated from MNV infected cells was several orders of magnitude more efficient in producing infectious virus, suggesting that initiation of RNA replication is the limiting factor for capped IVT RNAs.
Conclusions
Our work provides evidence for a bona fide infectious cDNA clone of GII.4 Sydney, paving the road towards a reverse genetics model. However, the system is very inefficient, since the function of VPg in initiation of RNA replication cannot be replaced effectively by a cap.