Sequence dynamics of three influenza A virus strains grown in different MDCK cell lines, including those expressing different sialic acid receptors

Abstract

Viruses are often cultured in cell lines for research and vaccine development, and those often differ from the natural hosts or tissues. Cell lines can also differ in the presence of virus receptors, such as the sialic acid (Sia) receptors used by influenza A viruses (IAV), which can vary in linkage (α2,3‐ or α2,6‐linkage) and form (N‐glycolylneuraminic acid [Neu5Gc] or N‐acetylneuraminic acid [Neu5Ac]). The selective pressures resulting from passaging viruses in cell types with host‐specific variations in viral receptors are still only partially understood. IAV are commonly cultured in MDCK cells which are both derived from canine kidney tubule epithelium and inherently heterogeneous. MDCK cells naturally present Neu5Ac and α2,3‐linked Sia forms. Here, we examine natural MDCK variant lineages, as well as engineered variants that synthesize Neu5Gc and/or α2,6‐linkages. We determined how viral genetic variation occurred within human H3N2, H1N1 pandemic and canine H3N2 IAV populations when serially passaged in MDCK cell lines that vary in cell type (MDCK‐Type I or MDCK‐Type II clones) and in Sia display. Deep sequencing of viral genomes showed small numbers of consensus‐level mutations, mostly within the hemagglutinin (HA) gene. Both human IAV showed variants in the HA stem and the HA receptor‐binding site of populations passaged in cells displaying Neu5Gc. Canine H3N2 showed variants near the receptor‐binding site when passaged in cells displaying Neu5Gc or α2,6‐linkages. Viruses replicated to low titres in MDCK‐Type II cells, suggesting that not all cell types in heterogeneous MDCK cell populations are equally permissive to infection.

Neu5Gc binding loss of subtype H7 influenza A virus facilitates adaptation to gallinaceous poultry following transmission from waterbirds but restricts spillback

“…3b). These findings are consistent with a prior study that demonstrated human seasonal H1N1 and H3N2 IAVs, which bind exclusively to Neu5Ac, developed adaptive HA mutations when passaged in MDCK cells expressing Neu5Gc (17). Conversely, an enzootic canine H3N2 IAV, which binds to both Neu5Gc and Neu5Ac, did not exhibit significant HA mutations under the same conditions.…”

“…We hypothesized that the expression of Neu5Gc hinders replication in H7 viruses that exclusively bind to Neu5Ac but do not affect those that bind to both Neu5Ac and Neu5Gc. To test this, we compared the growth kinetics of two mutants (rgCk/WX13-V179I and rgCk/WX13-A122N) and their parent virus, rgCk/WX13, on MDCK-Gc, a cell line expressing Neu5Gc (17), and MDCK-wt, the wild-type cell line without Neu5Gc expression. We also included A/mallard/New Jersey/A00926089/2010 (H7N3) (HA)×PR8 (H7N1) (rgMall/NJ10), with the HA of an H7 virus from a mallard ( Anas platyrhynchos ), and A/chicken/Heinan/ZZ01/2017(H7N9)(HA)×PR8 (H7N1) (rgCk/HN17), with the HA of another AH/13- lineage H7N9 virus from chicken (Table 1).…”

“…The wild type MDCK NBL-2 cells (MDCK-wt) and the MDCK cells expressing Cytidine monophospho-N- acetylneuraminic acid hydroxylase (CMAH) (MDCK-Gc) were adapted from another study (17). Limited Neu5Gc (<1%) was detected in MDCK-wt whereas approximately 40% of their total Sia in MDCK-Gc as Neu5Gc (17). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, New York, USA) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA, USA) at 37°C under 5% CO 2 .…”

Low Pathogenicity H7N3 Avian Influenza Viruses Have Higher Within-Host Genetic Diversity Than a Closely Related High Pathogenicity H7N3 Virus in Infected Turkeys and Chickens

“…Furthermore, experimental infections of a turkey-derived H9N2 virus has also shown that the highest number of nonsynonymous changes occurred in samples from infected ducks, most likely reflecting shifts in host species [ 11 ]. Similarly, experimental passage of avian, canine, or seasonal human influenza A viruses in embryonating eggs or immortalized cell lines have shown the preponderance of nonsynonymous changes that has been attributed to shifts in viral replication from its natural host to an experimental culture system [ 7 , 74 , 75 ]. This increase in nonsynonymous changes coinciding with a change in host has been further documented for a variety of viruses including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) [ 20 ], Newcastle disease virus [ 13 ], bovine viral diarrhea virus [ 76 ].…”

Can we eradicate viral pathogens?

“…Eradication currently seems unrealistic given the vaccine efficiency in blocking infections and the possibility for presence in animal reservoirs, which could differentially impact virus evolution. For instance, differences in host‐cell receptors can drive divergence among influenza virus lineages as they adapt in vitro (Barnard et al). The host itself is only part of the equation and for an ssRNA plant virus the habitat in which these plants are located also shapes evolutionary dynamics (Peláez et al).…”