Abstract
The genome of influenza A viruses is constantly changing (genetic drift) resulting in small, gradual changes in viral proteins. Alterations within antibody recognition sites of the viral membrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) result in an antigenetic drift, which requires the seasonal update of human influenza virus vaccines. Generally, virus adaptation is necessary to obtain sufficiently high virus yields in cell culture-derived vaccine manufacturing. In this study detailed HA N-glycosylation pattern analysis was combined with in-depth pyrosequencing analysis of the virus genomic RNA. Forward and backward adaptation from Madin-Darby Canine Kidney (MDCK) cells to African green monkey kidney (Vero) cells was investigated for two closely related influenza A virus PR/8/34 (H1N1) strains: from the National Institute for Biological Standards and Control (NIBSC) or the Robert Koch Institute (RKI). Furthermore, stability of HA N-glycosylation patterns over ten consecutive passages and different harvest time points is demonstrated. Adaptation to Vero cells finally allowed efficient influenza A virus replication in Vero cells. In contrast, during back-adaptation the virus replicated well from the very beginning. HA N-glycosylation patterns were cell line dependent and stabilized fast within one (NIBSC-derived virus) or two (RKI-derived virus) successive passages during adaptation processes. However, during adaptation new virus variants were detected. These variants carried “rescue” mutations on the genomic level within the HA stem region, which result in amino acid substitutions. These substitutions finally allowed sufficient virus replication in the new host system. According to adaptation pressure the composition of the virus populations varied. In Vero cells a selection for “rescue” variants was characteristic. After back-adaptation to MDCK cells some variants persisted at indifferent frequencies, others slowly diminished and even dropped below the detection limit.
Highlights
Influenza A virus is a highly virulent human and animal pathogen, due to its ability to cause severe disease
In this study we investigated the stability of the HA Nglycosylation pattern over 10 successive virus passages in MadinDarby canine kidney (MDCK) cell culture
For infection and virus production the serum-free growth medium was supplemented with a final concentration of 5 U/mL trypsin (Invitrogen/Gibco, #27250018, Darmstadt, Germany), which was prepared in phosphate buffered saline (PBS) according to the activity given by the supplier
Summary
Influenza A virus is a highly virulent human and animal pathogen, due to its ability to cause severe disease. During this virus adaptation to Vero cells, viral fitness of the RKI-strain improved: The time required to achieve specific HAU-values ($ 1.4 HAU) decreased, whereas maximum HA-titers increased from passage 2 to 4.
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