Influenza A viruses serially passaged in different MDCK cell lines exhibit limited sequence variation across their genomes, with the exception of the hemagglutinin gene

Abstract

ABSTRACTNew methods for deep sequence analysis provide an opportunity to follow the emergence and dynamics of virus mutations in real time. Although viruses are commonly grown in cell culture for research and for vaccine development, the cells used to grow the virus are often not derived from the same tissue or even the same host that the virus naturally replicates in. The selective pressures of culturing virusin vitroare still only partially understood. MDCK cells are the standard cell for growing influenza viruses yet are derived from the epithelium of the canine kidney and are also heterogenous. We passaged human H3N2, H1N1 pandemic, and canine H3N2 influenza A viruses (IAV) in different lineages of MDCK cells, as well as lines engineered to express variant Sia receptors, including α2,3- and α2,6-linkages orN-glycolylneuraminic acid (Neu5Gc) orN-acetylneuraminic acid (Neu5Ac) forms. MDCK-Type II cells had lower infection efficiency and virus production, and infection appeared more dependent on protease activation of the virus. When viruses were passaged in the different cells, they exhibited only small numbers of consensus-level mutations, and most were within the HA gene. Both human IAVs showed selection for single nucleotide minority variants in the HA stem across cell types, as well as low frequency variants in the HA receptor binding site of virus passaged in cells expressing Neu5Gc. Canine H3N2 also showed minority variants near the receptor-binding site in cells expressing Neu5Gc and also in those expressingα2,6-linkages.IMPORTANCEThe genetic variation and adaptability of viruses are fundamental properties that allow their evolutionary success in the face of differing host environments and immune responses. The growth of viruses in cell culture is widely used for their study and for preparing vaccines. However, the selection pressures that cell passaging imposes on viruses are often poorly understood. We used deep sequence analysis to define, in detail, how three different influenza A viruses respond to passaging in different lineages of canine MDCK cells that are commonly used for their growth, as well as in variant cells engineered to express different forms of their cell surface receptor, sialic acid. This analysis revealed that most mutations occur in the HA gene and few sequence changes in the virus population reached high proportions. This is relevant for understanding the selective pressures of virus growth in cell culture and how it shapes evolutionary patterns.