Madin-Darby canine kidney cell sialic acid receptor modulation induced by culture medium conditions: Implications for the isolation of influenza A virus.

Sarah W Nelson,Andrew S Bowman,Jacqueline M Nolting,Ian C Davis,Daral J Jackwood,Jason W Stull,Joshua N Lorbach

doi:10.1111/irv.12671

Sarah W Nelson, Andrew S Bowman + Show 5 more

Open Access

https://doi.org/10.1111/irv.12671

Copy DOI

Abstract

BackgroundThe influenza A virus (IAV) binds to α‐2,3‐ and α‐2,6‐linked sialic acid (SA) receptors expressed by Madin‐Darby canine kidney (MDCK) cells. The receptor distribution may therefore be important in regulating IAV propagation. Serum‐free medium (SFM) avoids variability in conventional culture medium containing fetal bovine serum (FBS), which can have variable composition and may contain endotoxins. However, little is known about the distribution of SA receptors on cells maintained in SFM.ObjectivesWe assessed the influence of culture media on MDCK cell SA receptor distribution along with the effect of SA receptor distribution on IAV recovery. We hypothesized that SFM would increase the proportion of α‐2,6‐linked SA receptors present and alter isolate recovery.MethodsMadin‐Darby canine kidney cells were cultured in medium containing FBS and two SFMs. Cell surface distribution of α‐2,6‐ and α‐2,3‐linked receptors was determined using flow cytometry. Recovery of swine‐ and avian‐lineage IAVs from MDCK cells maintained in each medium was quantified as TCID50.ResultsMadin‐Darby canine kidney cells cultured in UltraMDCK SFM expressed both SA receptors and supported the growth of both swine‐ and avian‐lineage IAVs. Cells maintained in other medium inconsistently expressed each receptor and the avian IAV grew to lower titers in cells cultured with FBS.ConclusionsMedium conditions altered the distribution of SA receptors present on MDCK cells and affected IAV recovery. Culture in UltraMDCK SFM resulted in cells expressing both receptors and IAVs grew to higher titers than in the other culture condition, indicating that this medium may be useful for culturing IAV from multiple species.

Highlights

Influenza A virus (IAV) is a common pathogen that infects numerous species and adversely affects both public and animal health
Culture in medium B resulted in cells commonly expressing both receptors, indicating that this brand of Serum‐free medium (SFM) might be useful for the culture of Madin‐Darby canine kidney (MDCK) cells to isolate influenza A virus (IAV) from diverse host species
Variable receptor expression was observed when cells were cultured in medium A and medium C, making them less predictable and less suitable for IAV isolation, at later time points post‐seeding: Our data indicate that cells cultured in medium A may only be effective if isolating IAV 1 or 2 days post‐seeding while cells cultured in medium B could be useful for up to 7 days post‐seeding. 7‐amino‐actinomycin D was used to verify that

Summary

| INTRODUCTION

Influenza A virus (IAV) is a common pathogen that infects numerous species and adversely affects both public and animal health. Thousands of people become infected with seasonally circulating IAVs each year resulting in substantial morbidity and mortality.[1] Outbreaks of influenza in commercial poultry and swine operations result in large economic losses and can facilitate reassortment events leading to zoonotic transmission with pandemic potential.[2,3] Continued surveillance and characterization of IAV from animal host species are needed to moderate the risk to public health. We hypothesized that culture in SFM would increase the proportion of MDCK cells expressing α‐2,6‐linked SAs and subsequently increase the recovery of mammalian origin IAV. We assessed MDCK cell SA receptor distributions over serial passages in commercially available SFM using flow cytometric analysis and quantified IAV recovery

| MATERIALS AND METHODS

Findings

| DISCUSSION