Mutations conferring zanamivir resistance in human influenza virus N2 neuraminidases compromise virus fitness and are not stably maintained in vitro.

BACKGROUND: Viruses resistant to zanamivir have been generated in vitro, but no resistant virus has yet been isolated from a zanamivir-treated immunocompetent patient. In contrast most resistant viruses isolated from oseltamivir-treated patients correspond to those selected in vitro. However, despite mutations being in conserved residues in the neuraminidase (NA) they do not confer resistance in all NA subtypes. OBJECTIVES AND METHODS: We have used reverse genetics and the recombinant baculovirus expression system for investigating reasons for the lack of isolation of zanamivir-resistant H3N2 viruses and for further exploring subtype-specific oseltamivir resistance. RESULTS: H3N2 viruses generated by reverse genetics with H274Y, R292K E119V and E119D mutations were rescued. Those with E119G, E119A or R152K mutations could only be rescued in the presence of exogenous NA and after passage in the absence of exogenous NA only isolates that had reverted to the wild-type NA or, surprisingly, E119G/A to E119V NA were isolated. Mutations conferring zanamivir resistance significantly affected enzyme activity, virus replication or NA thermal stability. E119V viruses were stable and grew to similar titres as wild-type virus, consistent with their isolation from oseltamivir-treated patients. Mutations conferring oseltamivir resistance in N1 (H274Y) and B (R152K) NAs also conferred resistance in recombinant G70C N9 NA expressed in insect cells. CONCLUSIONS: These data suggest that zanamivir-resistant H3N2 viruses may not readily arise in vivo due to their poor viability. The G70C N9 NA may also provide a useful model for understanding the structural basis of subtype-specific drug resistance.

Mouse respiratory epithelial cells support efficient replication of human rhinovirus.

Human rhinoviruses (HRV) are responsible for the majority of virus infections of the upper respiratory tract. Furthermore, HRV infection is associated with acute exacerbation of asthma and other chronic respiratory diseases of the lower respiratory tract. A small animal model of HRV-induced disease is required for the development of new therapies. However, existing mouse models of HRV infection are difficult to work with and until recently mouse cell lines were thought to be generally non-permissive for HRV replication in vitro. In this report we demonstrate that a virus of the minor receptor group, HRV1B, can infect and replicate in a mouse respiratory epithelial cell line (LA-4) more efficiently than in a mouse fibroblast cell line (L). The major receptor group virus HRV16 requires human intercellular adhesion molecule-1 (ICAM-1) for cell entry and therefore cannot infect LA-4 cells. However, transfection of in vitro-transcribed HRV16 RNA resulted in the replication of viral RNA and production of infectious virus. Expression of a chimeric ICAM-1 molecule, comprising mouse ICAM-1 with extracellular domains 1 and 2 replaced by the equivalent human domains, rendered the otherwise non-permissive mouse respiratory epithelial cell line susceptible to entry and efficient replication of HRV16. These observations suggest that the development of mouse models of respiratory tract infection by major as well as minor group HRV should be pursued.