Characterization of Sialic Acid Receptors on MDCK Cells Maintained Under

Different Media Conditions by Flow Cytometric Analysis and Implications for

Detection of Influenza A Virus.

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in

the Graduate School of The Ohio State University

By

Sarah W. Nelson, B.S.

Graduate Program in Comparative and Veterinary Medicine

The Ohio State University

2016

Thesis Committee:

Dr. Ian Davis, Advisor

Dr. Jason Stull

Dr. Daral Jackwood

Copyrighted by

Sarah W. Nelson

2016

Abstract

The study of the history, ecology, and evolution of influenza A virus infections in many species is instrumental to protecting human and animal health. Thousands of people become infected with seasonally circulating influenza A virus (IAV) each year and outbreaks of influenza in commercial poultry and swine operations lead to heavy economic losses. The initial step in IAV infection is binding of the virus to host cellular receptors. In order to subtype and perform diagnostic experiments on IAVs, samples are commonly cultured in embryonating chicken eggs or Madin-Darby Canine Kidney

(MDCK) cells. Other cell lines have also been used to isolate IAV. The best culture system to use may depend on the experimental or diagnostic needs and the receptors present on the cells to be infected. For the current work, it was hypothesized that media conditions would modulate the distribution of receptors on the surface of MDCK cells, resulting in alterations in the amount of virus produced by the culture system. To test this hypothesis, MDCK cells were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) or two different commercially available serum free media (SFM) types. Distributions of α-2,6 linked and α-2,3 linked sialic acid cell surface receptors were compared by flow cytometric analysis over time.

MEM supplemented with 10% FBS resulted in a cycling between high percentages of cells expressing both α-2,6 linked and α-2,3 linked sialic acids following one passage, ii

then high percentages of cells expressing only α-2,3 linked sialic acids the next passage.

The two different SFM brands also altered the α-2,6 linked and α-2,3 linked sialic acid distributions of MDCK cells. Culture in Lonza’s UltraMDCK SFM resulted in a higher percentage of cells expressing both α-2,6 linked and α-2,3 linked sialic acid receptors over 25 passages. Culture in Life Technologies’ OptiPro SFM resulted in a more variable distribution of each receptor over 25 passages. To determine if the cells were using media nutrients in a manner that might influence the α-2,6 linked and α-2,3 linked sialic acid distributions on the cells, MDCK cells were seeded into flasks containing SFM from

Lonza or MEM supplemented with 10% FBS and cultured for seven days without changing media. Three flasks from each media group were analyzed each day for seven days by flow cytometric analysis. Culture in UltraMDCK SFM caused a higher percentage of MDCK cells to express both receptors, while culture in MEM with 10%

FBS showed variability in the α-2,6 linked and α-2,3 linked sialic acid receptor expression. In the final experiment, effects of media conditions on the amount of IAV recovered from each culture system were determined. Cells were maintained in

UltraMDCK SFM or MEM supplemented with 10% FBS, the α-2,6 linked and α-2,3 linked sialic acid receptor distributions on the cells were determined, and tissue culture infective dose 50% experiments were conducted. Cells were plated at a high density so they would be confluent the next day. MDCK cells maintained in SFM expressed predominantly α-2,6 linked sialic acids, while cells maintained in MEM supplemented with 10% FBS expressed more α-2,3 linked sialic acids. The swine origin IAV isolate grew to similar titers in MDCK cells maintained in both SFM and MEM supplemented with 10% FBS. The avian origin IAV isolate grew to significantly lower titers in MDCK iii cells maintained in MEM supplemented with 10% FBS when compared to growth in cells maintained in SFM. There may be additional factors besides the distribution of α-2,6 linked and α-2,3 linked sialic acid receptors on cells that influence the growth of IAV in culture. The effects of culture media on the distributions of sialic acids present on MDCK cells should be studied further to better understand the limitations and effects of IAV isolation in these cells.

iv

Acknowledgements

This project would not have been possible without the advice and support from

Dr. Andrew S. Bowman of the Animal Ecology and Epidemiology Research Program.

v

Vita

2003...... Granville High School

2006...... B. S. Biochemistry, The Ohio State

University

2006-2009 ...... Laboratory Technician, Idexx laboratories

2009-2012 ...... Research Assistant, Department of

Veterinary Biological Sciences, The Ohio State University

2012 to present ...... Research Assistant, Department of

Veterinary Preventive Medicine, The Ohio State University

Fields of Study

Major Field: Comparative and Veterinary Medicine

vi

Table of Contents

Abstract ...... ii

Acknowledgements ...... v

Vita...... vi

Fields of Study ...... vi

List of figures ...... x

Chapter 1: Literature Review ...... 1

1.1 Influenza A virus biology ...... 1

1.2 The HA protein ...... 2

1.3 IAV replication ...... 3

1.4 IAV subtypes ...... 4

1.5 Human influenza viruses ...... 4

1.6 The 1918 Spanish influenza pandemic ...... 6

1.7 The 1957 Asian influenza pandemic ...... 7

1.8 The 1968 Hong Kong Influenza pandemic ...... 7

1.9 The 1977 Russian Influenza pandemic ...... 8 vii

1.10 The 2009 swine influenza pandemic ...... 8

1.11 Avian IAV ...... 10

1.12 Swine IAV ...... 11

1.13 Sialic acids ...... 12

1.14 Cell lines ...... 15

1.15 Medium for culture of cells ...... 16

1.16 Virus isolation techniques ...... 17

1.17 Binding mutants ...... 18

1.18 Other IAV receptor possibilities ...... 19

1.19 Summary ...... 20

Chapter 2: Project Paper ...... 21

2.1 Abstract ...... 21

2.2 Introduction ...... 22

2.3 Tissue culture media ...... 23

2.4 MDCK cell culture ...... 24

2.5 Transitioning cells to SFM ...... 25

2.6 Staining for flow cytometry ...... 26

2.7 BCA protein assay protocol ...... 27

2.8 Solid-phase binging assay of receptor-binding specificity ...... 27

2.9 Flow images, controls and samples...... 28

viii

2.10 Experiment 1: Monitoring MDCK cells for 25 passages, cell lot 09D023, FBS lot 1515313

...... 31

2.11 Experiment 2 methods ...... 34

2.12 Experiment 2 : Monitoring sialic acid receptors each day, cell lot 09J020, FBS lot 1645629

...... 36

2.13 Experiment 3 methods ...... 44

2.14 Experiment 3: Tissue culture infectious dose 50% (TCID50) experiment, cell lot 14A025,

FBS1 lot 1723625, FBS2 lot 1743512 ...... 46

2.15 Discussion ...... 51

Chapter 3: Conclusion...... 55

3.1 Conclusion ...... 55

Declaration of conflict of interests ...... 57

Funding ...... 58

References ...... 59

ix

List of Figures

Figure 1. A simplified IAV structure diagram...... 2

Figure 2. Genesis of the 2009 H1N1 pandemic influenza A virus ...... 10

Figure 3. Sialic acid linkage types ...... 13

Figure 4. Sialic acid biosynthetic pathway...... 14

Figure 5. Diagram that illustrates how the MDCK cells were propagated from the stock ...... 25

Figure 6. Flow cytometry controls and example samples...... 28

Figure 7. Graph of sialic acid distributions on MDCK cells cultured in MEM containing 10%

FBS for 25 passages ...... 31

Figure 8. Graph of sialic acid distributions on MDCK cells cultured in UltraMDCK SFM for 25

passages...... 32

Figure 9. Graph of sialic acid distributions on MDCK cells cultured in OptiPro SFM for 25

passages...... 33

Figure 10. 45 T25 flasks were seeded with different concentrations of cells on day 0 from each

media type...... 35

Figure 11. Graph of sialic acid distributions on MDCK cells cultured in UltraMDCK SFM seeded

at 8x106 cells per T25 flask...... 36

x

Figure 12. Graph of sialic acid distributions on MDCK cells cultured in UltraMDCK SFM seeded

at 1.5x106 cells per T25 flask...... 37

Figure 13. Graph of sialic acid distributions on MDCK cells cultured in UltraMDCK SFM seeded

at 9.25x105 cells per T25 flask...... 37

Figure 14. Graph of sialic acid distributions on MDCK cells cultured in UltraMDCK SFM seeded

at 1x105 cells per T25 flask ...... 38

Figure 15. Graph of sialic acid distributions on MDCK cells cultured in MEM containing 10%

FBS seeded at 8x106 cells per T25 flask...... 38

Figure 16. Graph of sialic acid distributions on MDCK cells cultured in MEM containing 10%

FBS seeded at 1.5x106 cells per T25 flask...... 39

Figure 17. Graph of sialic acid distributions on MDCK cells cultured in MEM containing 10%

FBS seeded at 9.25x105 cells per T25 flask ...... 40

Figure 18. Graph of sialic acid distributions on MDCK cells cultured in MEM containing 10%

FBS seeded at 1x105 cells per T25 flask ...... 41

Figure 19. Graph of supernatant protein concentrations...... 43

Figure 20. Graph of log transformed TCID50/ml results...... 46

Figure 21. Graph of sialic acid distributions on MDCK cells confluent the day following passing

the cells...... 47

xi

Chapter 1: Literature Review

1.1 Influenza A virus biology- Influenza viruses are part of the Orthomyxoviridae family of viruses. IAV virions are enveloped, range from 100 nm to 300 nm in size, and can be spherical or filamentous in shape. The IAV RNA genome is negative sense, single stranded, and separated into eight segments [1]. The eight RNA segments encode eleven

viral proteins [1]. The ends of each RNA segment are formed into hairpin structures

bound by the polymerase complex, while the rest of the segment is coated with

nucleocapsid protein. Noncoding regions occur at both the 3’ and 5’ ends of each

segment and include the mRNA polyadenylation signal and a part of the packaging signal

for viral assembly. The extreme ends of all segments are highly conserved because they

function as promoters for viral replication and transcription by the polymerase complex

[1]. Virions are studded with two surface glycoproteins, hemagglutinin (HA) and

neuraminidase (NA), in approximately a four to one ratio. The M2 matrix protein

functions as an ion channel and traverses the envelope. The M1 matrix protein is directly

beneath the lipid envelope. The nuclear export protein (NEP), also called non-structural

protein 2 (NS2) is interior to the M1 matrix protein. The ribonucleoprotein complex is

comprised of genomic RNA coated with nucleoprotein (NP) and the RNA dependent

RNA polymerase complex. The polymerase complex consists of three subunits, two basic

1

and one acidic, termed PB1, PB2, and PA. The non-structural protein one (NS1) is a host

interferon antagonist protein that helps the virus evade the hosts immune system [1].

Figure 1. A simplified IAV structure diagram. [2]

1.2 The HA protein -The HA viral glycoprotein is the major surface antigen and is

involved with binding to N-acetylneuraminic (sialic) acid cell surface receptors and

fusion of the virus to the cell. Sialic acids are carbon monosaccharide cell surface

proteins that can differ within the monosaccharide chain and in how they bind to the HA protein. They are common on many cell types in many animal species. The carbon-2 of the final sialic acid on host cells can bind to the carbon-3 or the carbon-6 of galactose, creating α-2,3 or α-2,6 linkages, resulting in different conformations. HA proteins have a binding specificity for either α-2,3 or α-2,6 linkages [1]. In humans and swine α-2,6 sialic acids predominate in the trachea, nasopharynx, bronchi, and paranasal sinuses, but α-2,3 sialic acids are found in the lower respiratory tract [1] [3]. In birds α-2,3 linked sialic acids are common in the intestine, trachea, bronchi, and kidney [4]. The mortality rate for

2

humans infected with avian lineage IAV is greater than 60% [1]. The distribution of α-2,6 linked sialic acids and α-2,3 linked sialic acids may explain why avian IAV strains have a low infectivity rate in humans but a high mortality rate. In humans the α-2,3 linked sialic acid receptors are found deep within the lung. Extensive exposure is required to get infection in this area and can lead to pneumonia and other serious complications.

The HA protein must be cleaved by a serine protease into HA1 and HA2 subunits

during replication for the virus to be infectious. The HA1 portion contains the binding

site and the antigenic sites. The HA2 portion helps with fusion of the envelope to cell

membranes [1]. Antibodies to the HA portion neutralize the virus so the virus allows

frequent amino acid changes to the antigenic sites. This genetic drift can lead to an

antigenically distinct virus to which the host has no pre-existing immunity [1].

1.3 IAV replication -The IAV virion is endocytosed by the host cell. Hydrogen ions

are pumped into the virus by the M2 matrix protein ion channel. The resulting decrease in pH of the endosomal compartment triggers a conformational change in the HA exposing the fusion peptide that integrates the viral envelope with the endosome. The fusion peptide opens a pore for the ribonucleoprotein complex to be released into the host cell cytoplasm and this also helps disrupt protein-protein interactions within the NP coated

RNA [1]. The ribonucleoprotein complex and the RNA-dependent RNA polymerase complex are transported to the nucleus of the infected cell where viral RNA synthesis takes place. The polymerase complex generates two positive sense RNA segments,

3

mRNA used to make viral proteins and complementary RNA used to make negative

sense genomic RNA copies. A poly A tail of viral RNA is encoded in the negative sense

RNA genome. IAV employs a “cap snatching” mechanism where PB1 and PB2 proteins steal the 5’ cap primers from host mRNA that are required for translation. The M1 matrix protein and the NS2 protein help regulate nuclear export of viral mRNA to cell ribosomes

[1]. HA, NA, and M2 matrix proteins are generated and directed to the cell membrane where viral RNA is packaged. The NA viral surface glycoprotein has sialidase activity which facilitates release of budding virus from infected cells [1].

1.4 IAV subtypes- IAVs are subtyped based on the HA and NA glycoproteins that stud

the surface of viral particles. There are 16 different hemagglutinin types and 9

neuraminidase types detected in avian species, which are thought to be the natural

reservoir for all IAVs [5]. H17N10 and H18N11 have been detected in bats in South

America, but they are distinctly different than other IAVs. The HA protein does not bind

sialic acid and the NA protein is not a sialidase [6]. Currently only H1, H2, H3, N1, and

N2 subtypes have become common in humans [5]. H5N1 and H7N9 viruses are becoming more commonly detected in humans and are thought to be the result of spill-

over from avian species [7].

1.5 Human influenza viruses -Influenza is a common seasonal illness in humans that

typically results in an acute respiratory infection, but can lead to complications and death

4

[8]. IAV infections are generally isolated to the respiratory tract with symptoms including the sudden onset of fever, cough, headache, weakness, loss of appetite, sore throat, myalgia, and nasal congestion [9]. The risk for complications, hospitalization, and death is greater in groups of people older than 65 years old, children younger than 5 years old, and persons with pre-existing medical conditions [8]. Approximately 226,000 people are hospitalized annually in the US for influenza related illness. It is estimated that

36,000 of those cases result in death [10]. These symptoms, missed work-days, hospitalizations, and deaths lead to estimated economic losses of $87.1 billion dollars in the US every year [11].

There have been five major IAV pandemics that caused widespread illness and mortality in humans and animals- the 1918 Spanish influenza pandemic, the 1957 Asian influenza pandemic, the 1968 Hong Kong influenza pandemic, the 1977 Russian influenza pandemic, and the 2009 H1N1 influenza “swine flu” pandemic. Several outbreaks have occurred but have not spread globally as with pandemic strains. Pandemic influenza strains are thought to arise when genetic shift occurs and an IAV is generated that has novel antigens to which humans are susceptible [1]. The segmented genome allows for genetic reassortment of the RNA segments when multiple IAVs infect the same cell [5]. The 1957 Asian influenza pandemic and the 1968 Hong Kong Influenza pandemic were caused by the reassortment of human IAV with avian IAV [12]. Further analysis of past pandemic strains and their epidemiology is required to help prevent future pandemics and to help understand risks to human health.

5

1.6 The 1918 Spanish influenza pandemic- The 1918 Spanish flu was one of the

most exceptional influenza pandemics on record. It was caused by IAV of the H1N1

subtype. Approximately 50% of the world’s population was infected with influenza and

25% developed significant complications during the pandemic [5]. It is estimated that 20

to 50 million people died world-wide with an unusually high mortality rate in healthy

adults aged 15 to 34 [13]. Pneumonia, secondary bacterial infections, and extensive organ

failure were common complications leading to death [5]. The specific location of origin

of the pandemic IAV strain is still debatable, but it is generally thought that the pandemic

started in March of 1918 in the United States with a second wave occurring in September

[14]. It is hypothesized that the 1918 virus was a fully avian IAV that adapted to humans

through an intermediate host, such as swine [15]. The 1918 virus contained genes that are

closely related to avian IAV (M, HA, NA, PA, PB1, PB2 [14], NP [16]). The NS gene

appeared to be related to avian, human, swine, and equine lineage IAV [17]. The

sequence of the avian-like H1 HA protein of this virus showed evidence for circulating in a mammalian host, such as swine, where it was able to recombine with human lineage

IAV [14]. Anthroponotic transmission during the pandemic to swine was wide-spread in the United States, Europe, and China [14]. The high morbidity and mortality rate observed was hypothesized to be a result of people and animals having little or no pre- existing immunity to this virus. The H1 gene from this virus persisted in swine populations and re-emerged in human populations in 2009 [18].

6

1.7 The 1957 Asian influenza pandemic- A new influenza strain emerged in China

in February of 1957 [19]. The HA and NA antigens were shown to be different from any

found in humans before [20]. The virus spread, first afflicting Hong Kong, eastern Asia,

and the Middle East then the United States, South American countries, African countries,

and Europe [19]. A reassortant influenza virus with avian surface (HA and NA) and

internal (PB1) gene segments of the H2N2 subtype and human lineage internal (PA, PB2,

NP, M, NS) segments caused the pandemic influenza outbreak in 1957. The human

lineage gene segments were preserved from the H1N1 virus strains circulating before

1957 [14]. The virus was shown to be lethal even without the complications of bacterial

super-infections [21]. The Asian influenza pandemic was responsible for 86,000 deaths in

the United States [22] and 1 to 2 million deaths worldwide [23].

1.8 The 1968 Hong Kong Influenza pandemic- The Hong Kong influenza

pandemic of 1968 originated in Southeast Asia and spread throughout Asia and then to all

continents. Many people became ill globally, with increased death rates seen particularly

in the second wave of infections in the following year [24]. The pandemic was caused by a new virus strain of the H3N2 subtype. The H3 HA gene was derived from an avian

lineage IAV and the N2 gene was retained from the 1957 pandemic IAV [14]. It is

believed that people may have had some prior immunity to the N2 portion of this virus

resulting in fewer deaths than the 1918 and 1957 pandemics [25]. The prior immunity

afforded by the N2 portion was not observed with the second wave of the virus occurring

7

the following year because of additional genetic mutations in the N2 gene. Globally,

700,000 deaths were attributed to this pandemic virus [26].

1.9 The 1977 Russian Influenza pandemic- The next IAV pandemic resulted from

the emergence of a new IAV strain in the Soviet Union in November of 1977 [24], although some reports claim that this virus originated in Northeast China in May of 1977

[27]. This virus was genetically very similar to a 1950 seasonal IAV of the H1N1 subtype. There are a few theories but it was generally assumed that it was stored frozen and accidentally released from a laboratory [28]. The virus spread quickly, generally infected people younger than 25 years old, and was characterized by symptoms typical of seasonal influenza. The 1957 IAV pandemic was thought to have displaced earlier circulating IAVs, thus younger people would have no immunity to this virus while people born before 1957 would [24]. This pandemic IAV did not displace the seasonally circulating H3N2 IAV as seen with the previous pandemics, but co-circulated with it. It was hypothesized that the variances in immunity in the human population created an

environment conducive to both strains [24]. Multiple subtypes of IAV, including H1N1,

H1N2, and H3N2, still circulate in human populations today [1].

1.10 The 2009 swine influenza pandemic- In March of 2009 an increased incidence

of influenza-like illnesses was detected in Mexico. It was determined that people were

sick with a new strain of IAV of the H1N1 subtype [29]. In April the first cases were seen

8

in the United States. Young adults were most severely stricken by this IAV and a higher

percentage of people in this age group required hospitalization. This was in contrast to

what had been seen with seasonal IAV with higher percentages of children and the

elderly becoming very ill [30]. The virus spread globally with more than 214 countries

reporting infections during the pandemic with over 18,000 deaths [31]. The pandemic

virus was isolated from a wide range of species including dogs, cats, seals [12], and pigs

[32]. This virus was determined to be a reassortant virus composed of avian, human, and swine lineage IAV gene segments. Prior to the outbreak in March, Eurasian swine lineage

IAV NA and M genes had recombined with local swine IAV in Mexico [33]. The 2009 pandemic virus was composed of these two genes, NA and M, North American swine

HA, NP, and NS genes, mixed with North American avian lineage PA and PB2 genes, and the North American human lineage PB1 gene [34]. This virus continues to persist in human populations [35].

9

Figure 2. Genesis of the 2009 H1N1 pandemic influenza A virus [36].

1.11 Avian IAV- The main natural reservoir of influenza viruses is thought to be wild

waterfowl [14]. The interhemispheric spread of IAV has been linked to the migratory

patterns of these wild bird populations [37]. Sixteen different hemagglutinin and 9 different neuraminidase subtypes have been detected in these birds [5]. In waterfowl,

IAV is generally an enteric infection. Viral amplification occurs in cells of the intestinal tract, sometimes without causing any symptoms, leading to large amounts of virus being shed in the feces of these birds [38]. These IAVs are classified as low pathogenic strains.

Highly pathogenic IAVs cause high levels of mortality in domestic poultry and can be shed via the respiratory route [39]. Spill-overs of avian IAVs into human populations are

10

generally self-limiting as they do not transmit person to person very efficiently, however

mortality is higher than in persons infected with human seasonal IAV strains [40]. Avian

lineage IAVs have been associated with disease outbreaks in humans [41], swine [40],

commercial poultry [42], and aquatic mammals [43, 44]. Avian lineage IAV of the

subtype H3N8 has become common in horses and has since transferred to dog

populations [45]. The reassortment of avian IAVs with human IAVs has resulted in pandemics in the past due to people having little or no immunity to the newly generated

viruses [14], illustrating the importance of monitoring these viruses.

1.12 Swine IAV– Swine are important hosts to monitor for IAV because they have both

α-2,6 and α-2,3 linked sialic acid receptors in their respiratory tracts, meaning that they can become infected with avian origin and human origin IAV if exposed [3]. α-2,3 linked receptors are found mostly in the lower lungs and bronchioles of the pig lung however, pigs use their snout to root for food in their environment leading them to inhale more particles from the environment than humans typically would. Pigs have been proposed to be ‘mixing vessels’ in which avian and human IAV strains can recombine [46]. The inter species transmission of IAV is termed zoonosis and can lead to increased morbidity and mortality in naive populations of pigs or people. IAV recombination events have caused pandemics in the past.

11

1.13 Sialic acids- Sialic acids, or N-acetyl-neuraminic acids, are a diverse group of nine

carbon acidic sugars [47]. More than 50 different types have been described. Sialic acids

are commonly at the end of glycoprotein chains found on the surface of numerous cell

types across many different species. Sialic acids are thought to stabilize membranes and

modulate cellular interactions with the environment [47]. There are many different

linkages the sialic acids can make to other cells or substrates including α-2,3 linked, α-2,6

linked, and α,2-8 linked [47]. An α linkage means that the carbon 1 of neuraminic acid is

in the axial position on the opposite side of the plane from the carbon 6 in the 6 carbon

ring. In an α-2,3 linkage the carbon 2 of the neuraminic acid binds to the carbon 3 of

galactose or another glycan in the alpha conformation [48]. Sialic acid biosynthesis and expression on the cell surface is a complex cellular process. N-acetymannosamine is taken up by cells, enzymatically converted over several steps into cytidine-5’- monophospho-sialic acid, then transported to the Golgi where it is used to elongate glycan chains by sialyltransferases. The sialylated glycoprotein or glycolipid is transported to the plasma membrane where it is expressed on the outside of the cell [49].

Unnatural sialic acids and small sugar complexes can be fed to cells via the culture media

and expressed on the outside of cells through this process [49, 50].

12

Figure 3. Sialic acid linkage types [48].

13

Figure 4. Sialic acid biosynthetic pathway [49].

Binding to the correct receptor on a cell is the first step in infection for any virus.

George K. Hirst was the first person to show that IAV caused hemagglutination of red blood cells in 1941 [51]. Sialic acids were first demonstrated to be possible receptors for

IAV when IAV was shown to adsorb chicken red blood cells in a sialic acid dependent manner [52]. This was further validated when neuraminidase, which cleaves sialic acids, was applied to embryonating chicken eggs, mouse embryo cells, and mouse lung cells resulting in reduced susceptibility to IAV infection for each cell type [52, 53].

14

1.14 Cell lines- Historically, many different cell lines have been used to culture IAV

from a wide range of species. Frank Macfarlane Burnet was the first person to grow IAV

in embryonating chicken eggs in a laboratory in Australia in 1935 [54]. In the 1960s

chick embryo cells were used to isolate IAV from many different sources for analysis

[55]. The MDCK cell line was derived from the kidney of a normal adult female cocker

spaniel in September of 1958 by S.H. Madin and N.B. Darby [56]. MDCK cells were

shown to efficiently replicate IAV in 1968 [57]. Rhesus monkey kidney cells were first

used to competently culture human lineage IAV in 1978 [55]. Since then many different cell lines have been tested for their ability to replicate IAV, with embryonating chicken eggs and MDCK cells being used as the standards for comparison [58]. Baby hamster kidney (BHK-21) cells were shown to be good for the isolation of α-2,3 linkage preferring IAVs [59]. Vero cells from African green monkey kidney are efficiently infected and produce high titers of IAV from a wide range of sources [58]. Vero cells express high levels of α-2,3 linked sialic acid receptors, but can still replicate IAVs from human sources that tend to prefer α-2,6 linked sialic acids [60]. Porcine intestinal epithelial cell lines are permissive to human and swine origin IAVs and some avian IAVs due to the expression of α-2,6 linked sialic acid receptors [61]. Cells derived from human adenoids have been used to culture IAV [62]. New-born swine kidney cells, swine testicle cells, and swine trachea cells have all been studied for their effectiveness as IAV culture systems [63]. Chicken and quail fibroblasts have high levels of α-2,3 linked sialic acids and were good systems for the culture of avian IAVs [64]. Primary human airway epithelial cells have α-2,6 linked sialic acid receptors and some α-2,3 linked sialic acids,

15

so are permissible for human origin IAV and limited replication of avian IAVs [65].

Mouse airway epithelial cells have α-2,3 linked sialic acid receptors and are permissive to infection with avian IAV [66].

1.15 Medium for culture of cells- In 1959 Dr. Harry Eagle developed the formulation for minimum essential medium (MEM) or Eagle’s growth medium. This medium contains all the essential components necessary to grow human and mammalian cell lines in laboratory culture systems [67]. MEM is still commonly used today and may be supplemented with additional amino acids, hormones, and metabolites that a particular cell line requires. 5% to 10% fetal bovine serum (FBS) is commonly added for this purpose. FBS is collected from the fetuses of cows at slaughter [68]. It contains many growth factors that cells need to recover from cryopreservation, but has been shown to be variable between lots and can contain endotoxins that are toxic to cells [69]. More recently serum free media (SFM) types have been developed for vaccine production systems to reduce the risk of contamination, decrease variability, and to reduce costs.

Lonza’s UltraMDCK™ Chemically Defined, Serum-free Renal Cell Medium, with L- glutamine is designed to for the growth of MDCK cells at low and high plating densities.

UltraMDCK medium contains low levels of recombinant human insulin and bovine transferrin, resulting in a very low protein culture media. Lonza states that MDCK cells grown in this media are smaller and more densely packed than cells grown in the presence of serum and cultures can stay confluent for at least two weeks without needing to change the medium [70]. Life Technologies Gibco OptiPRO™ serum free medium is 16

an animal origin-free culture medium designed for the growth of several kidney-derived

cell lines including Madin-Darby bovine kidney (MDBK), MDCK, porcine kidney (PK-

15), and Vero cells for virus or recombinant protein production. It has been used to grow

other commonly used laboratory cell lines, like human cervical cancer cells (HeLa),

BHK-21, and African green monkey kidney cells (COS-7) [71]. ThermoFisher Scientific offers SFM options designed for a variety of other cell lines. They state that SFM

supports more consistent performance, superior cell growth, and viability [72]. However

cells grown in SFM may be more sensitive to changes in pH, temperature, osmolality, mechanical forces, and enzymatic treatments than cells cultured with FBS. Antibiotics are not recommended to be used with SFM because they could cause cellular toxicity.

Adaptation to SFM over several passages may be necessary and morphology changes may be noted [72]. The exact formulation of serum free culture media used for each cell line can vary and is proprietary information not available to the public.

1.16 Virus isolation techniques- Typically IAVs are isolated from swabs taken of

nasal secretions in pigs or cloacal secretions in birds. Swabs are maintained in viral

transport media for transport to the lab. This media can be phosphate buffered saline,

brain heart infusion broth, or the media of choice that a certain lab prefers. To isolate

IAV viral transport media is inoculated into the allantoic cavity of embryonating chicken

eggs and the allantoic fluid is harvested after three days [73]. The inoculation of cells

with IAV samples is very similar, but requires a few additional considerations. In cell

culture systems trypsin must be added to ensure efficient cleavage and activation of the 17

HA protein so the virus can infect other cells. The addition of trypsin has been found to

increase the amount of virus produced by monkey kidney cells [74]. FBS inhibits the

functions of trypsin and must be removed from the cell cultures prior to inoculation. Cells

can find this switch in media hard to handle and they may die at this point. This is

another reason why the use of SFM is good for culturing cells to be used for the isolation

of IAV. Typically cells are passed the day before inoculation and plated at a high density so that the cells are 90% to 100% confluent on the day of inoculation [73]. Importantly, however little is known about the distribution of α-2,6 linked and α-2,3 linked sialic acid receptors on cells passed in this way.

1.17 Binding mutants- IAVs from various sources have been isolated in many

different culture systems. If the culture system does not possess the receptors the IAV

prefers it may not be detected, may grow to a low titer, or may require additional

passages before the virus can be detected. This process can cause amino acid changes to

accumulate in the globular head binding region of the HA protein resulting in altered

binding specificity of the recovered IAV [75]. Isolation of human origin IAVs that tend

to prefer α-2,6 linked sialic acid receptors in embryonating chicken eggs or BHK cells

leads to the selection of receptor binding mutants that bind to the α-2,3 linked sialic acid

receptors in the egg or BHK cells [59]. This effect has not been seen with isolation of

human IAVs in MDCK cell culture systems. MDCK cells have been shown to have both

α-2,3 linked sialic acids and α-2,6 linked sialic acid receptors [59, 76]. To study naturally occurring IAV from different species the culture system used to isolate the IAV should 18 have receptors that match the species of origin. If avian species have α-2,3 linked sialic acids in their intestinal and respiratory tracts, the system to culture IAV from birds should have predominantly α-2,3 linked sialic acids. Since humans have predominantly α-2,6 linked sialic acid receptors in their respiratory tracts, the system to culture these IAVs should have α-2,6 linked sialic acid receptors. Conversely, culture systems possessing predominantly α-2,6 linked sialic acid receptors could be used as a way to screen avian origin IAV for pandemic potential in humans.

1.18 Other IAV receptor possibilities- It is possible that IAV may bind to other receptors in addition to sialic acids. IAVs cultured in MDCK cells created to express low levels of sialic acids grew to lower titers than in untreated MDCK cells and lost their neuraminidase activity [77]. GM-95, mouse melanoma cells, do not make sialic acids but do express gangliosides taken up from culture media containing FBS. When cultured in

SFM the cells did not express sialic acids. However, they could still be infected with avian and human IAVs and produced infectious virus. The sensitivity of the cells to IAV infection was lower than in cells expressing sialic acids [78]. A recent paper employing a large scale glycan microarray showed that human, swine, and migratory bird IAVs might be able to bind to Neu5Acα2-8Neu5Acα2-8Neu5Ac and Neu5Gcα2-6Galβ1-4GlcNAc, which are neuraminic acid glycoproteins that do not contain sialic acid branches. Human and swine IAVs may also bind Neu5Acα2-3, a neuraminic acid like molecule. The data from this study suggest that glycan shape might be more important for IAV binding than the composition of the glycan chain [46]. 19

1.19 Summary- IAVs are segmented RNA viruses with two surface glycoproteins, HA

and NA [1]. There are many different HA and NA types present in water fowl and other

species, with only a few persisting in human populations [5]. The segmented genome

allows the virus to mix segments when two IAVs infect the same cell. This can result in

new HA and/or NA proteins appearing on the outside of the virus. Humans might be

susceptible to newly generated IAV strains but not have pre-existing immunity, which

has led to pandemics in the past [1]. The HA protein binds to sialic acids on host cells.

Sialic acids are acidic sugars expressed on the outside of many cells types in many

different species [52]. IAVs from birds tend to prefer to bind sialic acids in the α-2,3-

linked conformation while human IAVs tend to prefer binding α-2,6-linked sialic acids

[1]. Additional receptors for IAV may exist [46]. Many different cell lines have been evaluated for their effectiveness in isolating IAVs from a wide range of species [57] [59]

[61]. The culture of IAVs in systems that do not have the preferred receptors of that virus

leads to the generation of binding mutants [75]. MDCK cells are commonly used to isolate IAVs from multiple species and have been shown to have both receptor types [59,

76]. However, little is known about the distributions of each receptor on MDCK cells

when cultured in different media systems.

20

Chapter 2: Project Paper

2.1 Abstract- Madin Darby canine kidney (MDCK) cell culture systems are commonly

used to isolate and analyze influenza A virus (IAV) samples from a wide range of hosts.

IAVs from different hosts have been shown to have different receptor affinity depending on the receptors commonly found in each host. Culture media has been shown to affect receptor expression on the outside of the cells [49]. This longitudinal study investigates

the effects of media, with and without fetal bovine serum (FBS), on the distributions of α-

2,3-linked sialic acid cell surface receptors for IAV and α-2,6-linked sialic acid cell surface receptors on MDCK cells. Cells cultured in serum free media (SFM) generally expressed both sialic acids whereas cells cultured with FBS had varying proportions that alternated passage by passage. The difference in percentages of cells expressing each receptor was hypothesized to impact the amount of IAV recovered from MDCK cells

cultured with and without FBS. A swine IAV grew to similar titers in both culture

mediums, while an avian IAV grew to higher titers in cells maintained in SFM. The cells

maintained in SFM were shown to be expressing more α-2,6-linked sialic acids while the

cells maintained with FBS were expressing mostly α-2,3-linked sialic acids at the time of

inoculation.

21

2.2 Introduction- IAVs are common respiratory pathogens that can infect many

different species. Thousands of people become infected with seasonally circulating

influenza each year. Not only do outbreaks of influenza in commercial poultry and swine

operations leads to heavy economic losses, but they can lead to zoonotic transmission

events and recombination events with pandemic potential. IAVs use the hemagglutinin

(HA) binding protein to infect cells by binding to sialic acids on the host cells [1]. The

diverse sialic acids and linkages found on different hosts cells create a barrier for the

transmission of IAV between different species [79]. Avian and equine-origin IAV have

preferential binding of galactose on the hemagglutinin protein (HA) receptor binding site

to sialic acid cellular receptors in α-2,3-linked conformation, while mammalian-origin

IAVs preferentially bind sialic acid receptors in the α-2,6-linked conformation [80]. This difference can be significant when attempting to culture IAV samples for analysis. If the culture system does not have the receptor that the IAV prefers it may not be detected or may acquire mutations in the binding region of the HA protein.

Many different techniques have been developed to isolate and characterize IAV including isolation in embryonating chicken eggs [81], Vero cells from green monkey kidney, baby hamster kidney (BHK) cells [82], porcine intestinal epithelial cells [61],

MDCK cells [81], and various other cell lines. MDCK cell lines are commonly used for

isolating IAV from swine and other species [63, 83]. MDCK cells are widely available

and easily amplify in culture. MDCK cells are used to isolate IAV samples collected

from humans because they do not induce receptor binding variants as seen with isolation

in embryonating chicken eggs [84, 85]. Culture in embryonating chicken eggs has been

22

demonstrated to select for viruses that can bind to the terminal α-2,3-linked sialic acid cell receptors that are predominantly found on chicken egg allantoic cells [83, 86, 87].

Previous characterization of MDCK cells, maintained under traditional cell culture

practices with FBS, has shown that MDCK cells have both α-2,6-linked and α-2,3-linked

sialic acid oligosaccharide cell surface receptor types [83]. In one study 95% of MDCK cells expressed α-2,6-linked sialic acid receptors and 55% expressed α-2,3-linked sialic acid receptors [61]. FBS is beneficial for culturing cells because it contains many growth factors but each lot may be slightly different and it can harbor contaminants like endotoxins [69]. The use of serum free media (SFM) to culture cells has increased in popularity to avoid the variability and contaminants in FBS and reduce costs [82]. SFM adapted MDCK cells were reported to express both α -2,3 and α -2,6 -linked sialic acid cell surface receptors and effectively replicated human and avian origin IAV [82], but little is known about the effects of prolonged culture in SFM. The objective of this study was to assess culture with FBS and commercially available SFM on MDCK cell sialic acid distributions using flow cytometric analysis.

Materials and Methods

2.3 Tissue culture media- UltraMDCK™ chemically defined, SFM, with L-glutamine

(cat. no. 12-749Q) was obtained from Lonza Bioscience (Basel, Switzerland).

OptiPRO™ SFM (cat. no. 12309-019) was obtained from Life Technologies (Grand

23

Island, NY, USA). MDCK cells (cat. no. 85011435-1VL) were purchased from Sigma-

Aldrich (St. Louis, MO, USA).

2.4 MDCK cell culture- Purchased MDCK cells were stored in the liquid nitrogen

vapor phase until use. Prior to culture, cells were thawed quickly in a 37°C dry bead bath,

and placed in pre-warmed cell growth medium composed of HyClone Minimum

Essential Medium (MEM) with Earle's Balanced Salts (EBSS) and L-glutamine (cat. No.

SH30024; Thermo Fisher Pittsburgh, PA, USA), supplemented with 1x sodium pyruvate

(cat. no.11360; Gibco Grand Island, NY, USA), 1x non-essential amino acids (cat. no.

11140; Gibco), and 10% heat inactivated fetal bovine serum (cat. no. 1082; Gibco).

Culture medium was supplemented with MycoZap prophylactic (Lonza), to prevent

growth of Mycoplasma, and other species in the mycoplasma group like Acholeplasma,

and Spiroplasma. Cells were maintained in an incubator at 37°C with 5% CO2. Media

was removed and replaced after 24 hours to remove the cell freezing media. Cells were

dissociated from culture flasks by treating with 0.25% trypsin, 0.1% EDTA (cat. no. 25-

053-Cl) from Corning a division of Thermo Fisher. Approximately 8x106 cells were

passed into new T150 flasks every Monday. Each Thursday cells were trypsinized and

approximately 1x107 cells were passed into new T300 flasks. MycoZap supplementation

was discontinued after four passages. Three different lots of Sigma MDCK cells were

used during the course of this study: 09D023, 09J020, and 14A025.

24

2.5 Transitioning cells to SFM- At each passage, MDCK cells were recovered from

each flask by treating with 0.25% trypsin, 0.1% EDTA. Once cells were detached from

the flask the trypsin was inhibited by washing the MDCK cells in MEM containing 10%

FBS. MDCK cells were transitioned to UltraMDCK™ SFM or OptiPRO SFM as

previously described beginning at passage 4 [88] [89] [90]. Briefly, the concentration of

FBS was decreased in a stepwise fashion over four passages. At each passage 25% of the media was switched to SFM, resulting in a 100% SFM culture system after four passages.

The SFM transitioned cells were washed in SFM and resuspended in SFM after trypsin was inhibited.

Figure 5. Diagram that illustrates how the MDCK cells were propagated from the stock. This figure is a diagram that illustrates how the MDCK cells were propagated from the Sigma Aldrich stock and the process of weaning the cells onto SFM over 4 passages. Flow cytometry was performed at each passage to monitor the distributions of α -2,3 and α -2,6 -linked sialic acid receptors on the cells.

25

2.6 Staining for flow cytometry -PBS containing 5% FBS and 0.02% sodium azide

(PBS/azide) was used to wash 1x106 MDCK cells per sample. The cells were incubated

with biotinylated Sambucus nigra (SNA) lectin (10µg/ml, cat. no. B-1305) from Vector laboratories (Burlingame, CA, USA) and/or fluorescein isothiocyanate (FITC)- conjugated Maackia amurensis (MAA) lectin (100µg/ml, cat. no. F-7801-2) from EY laboratories (San Mateo, CA, USA). The SNA lectin is specific for α-2,6 linked sialic

acids on the cell surface while the MAA lectin is specific for α-2,3 linked sialic acids.

PBS/azide was added to the unstained control and the streptavidin-phycoerythin only

control. Samples were incubated at 4°C in the dark for 30 minutes then washed with

PBS/azide. Wash was removed and streptavidin-phycoerythin (100µg/ml, cat. no. F0040)

from R&D systems (Minneapolis, MN, USA) was added to all samples and controls

except the unstained control. The streptavidin-phycoerythin conjugate allows detection of

biotinylated SNA. Samples and controls were mixed by vortexing and incubated in the

dark for 30 mins at 4°C, then centrifuged and washed with 300µl PBS/azide. Wash was

removed and cytofix (250µl, cat. no. 554655) from BD Biosciences (San Jose, CA, USA)

was added to all samples and controls. Cells were fixed for 30 mins at 4°C in the dark,

then washed, resuspended, and stored in the dark in 500µl PBS/azide until flow

cytometric analysis could be performed.

26

2.7 BCA protein assay protocol -The Pierce bicinchoninic acid (BCA) protein assay

was run in accordance with the manufacturer’s microplate protocol (cat. no. 23227) from

Life Technologies.

2.8 Solid-phase binging assay of receptor-binding specificity –The solid-phase

binding assay was performed as stated in a previously published protocol [91]. Briefly,

96 well plates were coated with fetuin, IAV was added to the plates to bind to the fetuin, dilutions of biotinylated 3’ or 6’ sialyl-glycoproteins were bound to the IAV, peroxidase- labeled streptavidin was added to bind the biotin, 3,3’,5,5’-tetramethylbenzidine substrate

solution was added to cause a visual color change in wells that had sialyl-glycoprotein

binding to IAV, and finally sulfuric acid was added to stop the reaction and the

absorbance was read on a microplate reader. Incubations of different times were required

between each step of the assay, sometimes running overnight followed by washing with a

detergent.

27

Results

2.9 Flow images, controls and samples

Figure 6. Flow cytometry controls and example samples. Flow cytometric analysis of the stained and fixed MDCK cells. Panel (A) forward scatter versus side scatter gate. The same gate was used for all samples. Panel (B) unstained negative control. Panel (C) single stained biotinylated SNA with streptavidin-phycoerytherin control, which binds to α-2,6 linked sialic acids. Panel (D) single stained MAA with streptavidin-phycoerytherin, which binds to α-2,3 linked sialic acids. Panel (E) streptavidin-phycoerytherin negative control. The controls were used to set the quadrants to determine the percentages of cells positive or negative for each lectin. Cells positive for the SNA stain are in the upper left quadrant. Cells positive for the MAA stain are in the lower right quadrant. Cells positive for both receptors are in the upper right quadrant. Panel (F) cells maintained in MEM containing 10% FBS and dual stained with biotinylated SNA lectin, MAA lectin, and streptavidin- phycoerytherin. Panel (G) cells maintained in UltraMDCK SFM and dual stained with biotinylated SNA lectin, MAA lectin, and streptavidin-phycoerytherin. 28

The manufacturer’s recommendations for the concentrations of SNA (5 to

20µg/ml for 1x106 cells) and MAA (100µg/ml for 1x106 cells) lectin were used. To optimize staining, cells were singly stained with 400µg/ml, 200µg/ml, 100µg/ml,

50µg/ml, 25µg/ml, 15µg/ml, 10µg/ml, and 5µg/ml. In the same experiment fixing the

cells prior to staining was investigated. It was hypothesized that this would have no effect

on cell staining. However, fixing the MDCK cells prior to staining resulted in the cells

stained with only the streptavidin-phycoerytherin to show non-specific staining and

caused this experiment to fail. The cells appeared to be strongly SNA positive when they

should not have stained at all. While it would have been more convenient to fix the cells

prior to staining, it was determined that this non-specific staining was unacceptable. The

directions for the streptavidin-phycoerytherin state that 10µl be added to 1x106 cells in

100µl of buffer that have been optimally stained.

Neuraminidases specific for α-2,3 linked sialic acids or specific for α-2,3, 6, 8,

and 9 linked sialic acids were incubated with MDCK cells in an attempt to make control

cells to further test the specificity of the SNA and MAA lectins. MDCK cells were

treated with either neuraminidase that would remove only the α-2,3 linked sialic acids or

remove α-2,3, 6, 8, and 9 linked sialic acids from the MDCK cells. If MDCK cells had

only the α-2,3 linked sialic acids removed then only the SNA lectin should bind to and

stain the cells. If MDCK cells had α-2,3, 6, 8, and 9 linked sialic acids removed then

neither stain should bind to the cells. The neuraminidases did not remove all the sialic

acids they were specific for from the MDCK cells. Longer incubation with the

neuraminidases degraded the cells to an extent that the staining did not work at all.

29

It was decided that the use of the manufacturer’s stain concentration recommendations was acceptable because when looking at the flow cytometry histograms the stains did not appear to be competing with one another. In the control cells singly stained with SNA and streptavidin-phycoerytherin 98.58% of the cells were positive and

1.42% of the cells were negative. The control cells singly stained with MAA and streptavidin-phycoerytherin had 98.69% of the cells staining positive, 0.39% SNA positive, 0.13% double positive, and 0.79% negative. As an example one sample of cells maintained in UltraMDCK SFM was 94.06% double positive, 0.07% SNA positive, 0.2%

MAA positive, and 5.67% negative. The control cells singly stained had only about 4% more cells staining positive in either control.

30

2.10 Experiment 1: Monitoring MDCK cells for 25 passages, cell lot 09D023, FBS lot 1515313

Figure 7. Graph of sialic acid distributions on MDCK cells cultured in MEM containing 10% FBS for 25 passages. This graph shows the average distribution of MDCK cells maintained in MEM containing 10% FBS that were expressing only α-2,3 linked sialic acids, only α-2,6 linked sialic acids, and cells expressing both receptors from passage 4 to passage 25. The error bars show the standard deviation of the average of 3 flasks. The percentage of cells expressing both receptors was high one passage and the next passage the percentage of cells expressing only α-2,3 linked sialic acids was high.

Culture of MDCK cells in MEM containing 10% FBS resulted in variable percentages of cells expressing each sialic acid receptor. Cells were passed with cell concentrations sufficient to make them confluent on Mondays after 4 days or on

Thursdays after 3 days. Cells stained on Mondays had higher percentages of cells expressing both receptors over the course of this experiment. Cells stained on Thursdays had one less day in culture before being confluent, passed, and stained and had higher percentages of cells expressing α-2,3 linked sialic acid receptors.

31

Figure 8. Graph of sialic acid distributions on MDCK cells cultured in UltraMDCK SFM for 25 passages. This graph shows the average distribution of MDCK cells maintained in UltraMDCK SFM that were expressing only α-2,3 linked sialic acids, only α-2,6 linked sialic acids, and cells expressing both receptors from passage 4 to passage 25. The error bars show the standard deviation of the average of 3 flasks. The majority of cells maintained in SFM were expressing both receptors.

Lonza UltraMDCK SFM increased the percentage of MDCK cells expressing both sialic acid receptors. The number of cells in culture had to be expanded during passages 1, 2, and 3 in order to pass and stain the cells. The cells were weaned onto the

UltraMDCK SFM from passage 4 to passage 7. Beginning at passage 8 the cells were maintained in SFM.

32

Figure 9. Graph of sialic acid distributions on MDCK cells cultured in OptiPro SFM for 25 passages. This graph shows the average distribution of MDCK cells maintained in OptiPro SFM that were expressing only α-2,3 linked sialic acids, only α-2,6 linked sialic acids, and cells expressing both receptors from passage 4 to passage 25. The error bars show the standard deviation of the average of 3 flasks. The percentage of cells expressing the different receptors was variable. Cells were either expressing both receptors or only α-2,3 linked sialic acids.

Culture in OptiPro SFM resulted in variable sialic acid expression by MDCK cells

over 25 passages. The cells were weaned onto the OptiPro SFM from passage 4 to

passage 7. Beginning at passage 8 the cells were maintained in SFM. Unlike MDCK cells

cultured in MEM with 10% FBS, cycling in the distributions of receptors from passage to

passage did not occur. From passage 4 to 9 the sialic acid expression on the cells was

variable with cells either expressing both receptors or α-2,3 linked sialic acids. Cells

expressed higher percentages of α-2,3 linked sialic acid receptors from passage 9 to 13.

From passage 13 to passage 25 the majority of cells expressed both receptors.

33

2.11 Experiment 2-The results from experiment 1 generated a very important question.

Were the cells using components in the MEM containing 10% FBS in a way that was influencing the sialic acid distributions on the cells? Cells maintained in MEM containing

10% FBS or SFM were passed to be confluent after 3 days. At this time the MDCK cells cultured in MEM with 10% FBS expressed mostly α-2,3 linked sialic acid receptors. The remaining cells were re-seeded to be confluent after 4 days. After 4 days most of the cells cultured in MEM with 10% FBS expressed both receptors. Cells maintained in

UltraMDCK SFM were more consistent and generally expressed both receptors regardless of passage or time in culture. Cells maintained in OptiPro SFM had variable sialic acid distributions that did not seem to change in a passage-related pattern. For this reason the OptiPro SFM was not included in the remainder of the study.

The MDCK cell sialic acid distributions were next measured following passage densities that ensured confluence at 1 day, 3 days, 4 days, or 7 days. The cells maintained in SFM were hypothesized to express high levels of both receptors regardless of seeding density. When inoculating samples onto cells they are generally seeded at a high density to be 90% to 100% confluent on the inoculation day. Cells seeded to be confluent after 7 days were hypothesized to show the sialic acid distributions change gradually over time when the MEM containing 10% FBS media was not changed.

MDCK cells with the lot number 09J020 were cultured as described in section

2.4. At passage 2, half of the cells began the transition to UltraMDCK SFM in the same way as described in section 2.5. Cells were maintained in SFM from passages 5 to 9. At

34 passage 9, 45 T25 flasks were made from cells maintained in each media type,

UltraMDCK SFM and MEM with 10% FBS, with different concentrations of cells.

Figure 10. 45 T25 flasks were seeded with different concentrations of cells on day 0 from each media type.

The concentration of cells used to make the T25 flasks was calculated based on the number of cells required to make T150 or T300 flasks confluent after 3 or 4 days respectively. Media was not changed or removed in these flasks until they were ready to be stained. The day following seeding the flasks, three flasks from each concentration and media group had 1ml aliquots of the culture media removed and stored at -80°C for determination of protein concentration. These flasks were trypsinized to dissociate cells, cells were washed, stained as in section 2.6, and the α2-3 linked and α2-6 linked sialic acids on the cell surface were measured by flow cytometry. On day 2, three flasks from

35

each concentration and media group again had 1 ml aliquots of the media removed and

stored at -80°C. These flasks were trypsinized, stained, and monitored by flow cytometry.

The 3 flasks seeded with 8x106 cells were confluent after 1 day, were stained on day 1,

and were not present on day 2. Each day until day 7 three more flasks from each

concentration and media group were pulled and monitored in the same way. By day 3 all

the flasks seeded with 1.5x106 cells were stained and no longer present. On day 4 all the

flasks seeded with 9.25x105 cells were stained and no longer present.

2.12 Experiment 2: Monitoring sialic acid receptors each day, cell lot 09J020, FBS lot 1645629

Figure 11. Graph of sialic acid distributions on MDCK cells cultured in UltraMDCK SFM seeded at 8x106 cells per T25 flask .This graph shows the distributions of α-2,6 linked and α-2,3 linked sialic acids on SFM maintained cells when they were plated at a high concentration, with 8x106 cells, and confluent the following day. The error bars are the standard deviation around the mean of 3 flasks. Approximately half of the cells were expressing both receptors the following day after seeding.

36

Figure 12. Graph of sialic acid distributions on MDCK cells cultured in UltraMDCK SFM seeded at 1.5x106 cells per T25 flask. This graph shows the distributions of α-2,6 linked and α-2,3 linked sialic acids on SFM maintained cells when they were plated with 1.5x106 cells to be confluent after 3 days. The error bars are the standard deviation around the mean of 3 flasks. MDCK cells were expressing high percentages of both receptors each day.

Figure 13. Graph of sialic acid distributions on MDCK cells cultured in UltraMDCK SFM seeded at 9.25x105 cells per T25 flask. This graph shows the distributions of α-2,6 linked and α-2,3 linked sialic acids on SFM maintained cells when they were plated with 9.25x105 cells to be confluent after 4 days. The error bars are the standard deviation around the mean of 3 flasks. MDCK cells were expressing high percentages of both receptors each day.

37

Figure 14. Graph of sialic acid distributions on MDCK cells cultured in UltraMDCK SFM seeded at 1x105 cells per T25 flask. This graph shows the distributions of α-2,6 linked and α-2,3 linked sialic acids on SFM maintained cells when they were plated with 1x105 cells to be confluent after 7 days. The error bars are the standard deviation around the mean of 3 flasks. MDCK ells were generally expressing high percentages of both receptors each day. On day 3 there was an increased percentage of cells expressing α-2,6 linked sialic acids.

Figure 15. Graph of sialic acid distributions on MDCK cells cultured in MEM containing 10% FBS seeded at 8x106 cells per T25 flask. This graph shows the distributions of α-2,6 linked and α-2,3 linked sialic acids on MEM containing 10% FBS maintained cells when they were plated with 8x106 cells to be confluent after 1 day. The error bars are the standard deviation around the mean of 3 flasks. MDCK cells were expressing high percentages of both receptors. 38

Figure 16. Graph of sialic acid distributions on MDCK cells cultured in MEM containing 10% FBS seeded at 1.5x106 cells per T25 flask. This graph shows the distributions of α-2,6 linked and α-2,3 linked sialic acids on MEM containing 10% FBS maintained cells when they were plated with 1.5x106 cells to be confluent after 3 days. The error bars are the standard deviation around the mean of 3 flasks. MDCK cells were expressing high percentages of both receptors on the first and second day with lower percentages of cells expressing each sialic acid receptor on day 3.

39

Figure 17. Graph of sialic acid distributions on MDCK cells cultured in MEM containing 10% FBS seeded at 9.25x105 cells per T25 flask. This graph shows the distributions of α-2,6 linked and α-2,3 linked sialic acids on MEM containing 10% FBS maintained cells when they were plated with 9.25x105 cells to be confluent after 4 days. The error bars are the standard deviation around the mean of 3 flasks. MDCK cells were expressing high percentages of both receptors on the first and second day. On day 3 more cells were expressing α-2,6 linked sialic acids.

40

Figure 18. Graph of sialic acid distributions on MDCK cells cultured in MEM containing 10% FBS seeded at 1x105 cells per T25 flask. This graph shows the distributions of α-2,6 linked and α-2,3 linked sialic acids on MEM containing 10% FBS maintained cells when they were plated with 1x105 cells to be confluent after 4 days. The error bars are the standard deviation around the mean of 3 flasks. MDCK ells were expressing high percentages of both receptors on the first and second day. Again on days 3 and 4 lower percentages of cells were expressing either receptor. On days 5 and 6 higher percentages of cells were expressing α-2,6 linked sialic acids. On day 7 lower numbers of cells were expressing either receptor.

MDCK cells maintained in SFM generally had high percentages of cells

expressing both receptors regardless of seeding density. These results support the use of

UltraMDCK SFM for the culture of MDCK cells used to isolate IAV samples. Culture in

SFM resulted in more consistent and predictable sialic acid distributions. The percentages

of cells that stained were lower than in experiment 1. This could be due to the different

lot of MDCK cells used.

MDCK cells maintained in MEM containing 10% FBS showed variable percentages of cells expressing α-2,6 linked and α-2,3 linked sialic acid receptors after 7

days in culture when compared to cells maintained in SFM. Flasks seeded with different

amounts of cells had different sialic acid expression ratios on each day. Cells seeded to be

41

confluent after 1 day expressed high percentages of both receptors, similar to the SFM

maintained cells. Cells seeded to be confluent after 3 and 4 days had similar results that

showed cells expressing high percentages of both receptors for the first 2 days and then

the percentages of cells expressing either receptor dropped. More cells were expressing

α-2,6 linked sialic acids than α-2,3 linked sialic acids, which is in contrast to the results

seen in experiment 1. Cells seeded with 1x105 cells started out expressing high

percentages of both receptors and then switched to expressing more α-2,6 linked sialic acids on days 5 and 6 and had lower percentages of cells expressing either receptor on day 7. The switching of receptors that was seen in experiment 1 between cells confluent after 3 or 4 days was not seen in this experiment. Flasks seeded to be confluent after 3 days had percentages of cells expressing each receptor that were similar to the flasks seeded to be confluent after 4 days. The differences between experiment 1 and 2 could be due to the different lot of MDCK cells or the different lot of FBS used in each experiment.

In conclusion UltraMDCK SFM caused MDCK cells to express high percentages of both sialic acids without media change. MEM with 10% FBS resulted in variable sialic acid expression on MDCK cells without media being changed, although different from experiment 1.

42

Figure 19. Graph of supernatant protein concentrations. This graph shows the supernatant protein concentrations in each group of 3 flasks each day of the experiment. The error bars are the standard deviation about the mean protein concentration. There was greater variability in the flasks of cells maintained in MEM containing 10% FBS than in the cells maintained in SFM. The protein concentrations of the flasks maintained in MEM with 10% FBS did not differ greatly from the stock MEM with 10% FBS media. The protein concentrations of the stock medias are shown in orange arbitrarily on day 7. After 7 days in culture the average supernatant protein concentration of SFM maintained cells was lower than the stock SFM.

The range of protein concentration of the supernatant of MDCK cells maintained in MEM with 10% FBS was from an average of 3802.5 µg/ml to an average of 4235.9

µg/ml with the stock MEM containing 10% FBS testing at 3855.3 µg/ml. The supernatant protein concentration varied modestly from day to day and did not consistently decrease over time. The average supernatant protein concentrations in these flasks did not appear to be influencing the distributions of α-2,6-linked and α-2,3-linked sialic acid receptors on MDCK cells. The sialic acid distributions on the cells varied and the protein concentrations remained high during the course of this experiment.

43

The average supernatant protein concentrations of the UltraMDCK SFM

maintained flasks ranged from 833.8 µg/ml on day 1 to 639.7 µg/ml on day 7. The stock

SFM tested at 767.4 µg/ml. When 8x106 cells were seeded into flasks and supernatant

was tested the following day the supernatant protein concentration was similar to the

stock SFM. When 1x105 cells were seeded into flasks after 7 days in culture the average

protein concentration of the supernatant was lower than the stock SFM. The slight

decrease in media protein concentration in the SFM maintained flasks did not appear to be influencing the distributions of α-2,6-linked and α-2,3-linked sialic acid receptors on

MDCK cells as the cells maintained in SFM were generally expressing both receptors over the course of this experiment.

2.13 Experiment 3 - MDCK cells with the lot number 14A025 were cultured as

described in section 2.4. The MDCK cells were split into 3 groups, two different FBS lots

and UltraMDCK SFM. At passage 4 cells began the transition to UltraMDCK SFM in the

same way as described in section 2.5. Cells were cultured in each media for 4 additional

passages until they were all at passage 11 and there were enough cells required to make 1

T25 flask and 6 96 well plates of each media group to be confluent the following day.

The T25 flasks were used for staining the cells to determine the distributions of α-2,6- linked and α-2,3-linked sialic acids with the cells confluent 1 day after seeding the flask

as described in section 2.6. Three plates from each media group were used for TCID50

testing with a swine origin IAV (A/swine/Ohio/12TOSU447/2012(H3N2)) in triplicate

and an avian origin IAV (A/green-winged teal/Ohio/175/1986(H2N1)) in triplicate. The

44

swine IAV was previously passed 4 times in SFM adapted MDCK cells. The avian IAV

was previously passed once in specific pathogen free embryonating chicken eggs. In this

experiment 8 duplicate wells (1 column) of a 96 well plate with confluent monolayers of

MDCK cells were inoculated with the same dilution of IAV. The IAV dilutions ranged

from 10-4 in column 1 to 10-14 in column 11 leaving column 12 wells as negative controls.

Trypsin is important in the viral growth media to activate the HA protein so FBS was not

included in any of the inoculation medias. Approximately 72 hours post inoculation cell

monolayers in each well of each dilution were evaluated for cytopathic effects indicative

of IAV infection by light microscopy. TCID50/ml values were calculated based on the

cytopathic effects seen and the volume of each dilution inoculated into each well [92].

After 3 days 1 T25 flask and 6 96 well plates were again made from each media group

and tested the following day in the same way as the passage 11 cells.

Data from experiment 1 showed that after 4 passages post transitioning the cells

to SFM the cells were more consistently expressing both α-2,6-linked and α-2,3-linked sialic acid receptors. This lot of MDCK cells had the α-2,6-linked and α-2,3-linked sialic acid distributions followed by flow cytometry at each passage and the percentage of cells expressing both receptors was shown to be increasing, but a high percentage of cells were expressing only α-2,6-linked sialic acids. In experiment 1 it was also seen that the cells cultured in MEM containing 10% FBS alternated between the high percentage of cells expressing both receptors to cells expressing high percentages of α-2,3-linked sialic acids the following passage. Plates for TCID50s were made at 2 consecutive passages in the

hopes of having cells that were high in both receptors and comparing the TCID50 value to

45 cells that were high in just one sialic acid receptor. Two different lots of FBS were used, designated FBS1 and FBS2, in this experiment because it was not known if the same alteration of receptors would be observed with each lot of FBS.

2.14 Experiment 3: Tissue culture infectious dose 50% (TCID50) experiment, cell lot 14A025, FBS1 lot 1723625, FBS2 lot 1743512

Figure 20. Graph of log transformed TCID50/ml results. These graphs show the log transformed

TCID50/ml results. Each box represents 3 plates, showing the low log transformed TCID50/ml result with the bottom whisker, the mean log transformed TCID50/ml result in the box, and the high log transformed TCID50/ml result with the top whisker. The swine IAV grew to similar titers in each media type in both trials. The avian IAV grew to lower titers in both lots of FBS maintained cells on both inoculation dates when compared to inoculation in SFM maintained cells.

46

Figure 21. Graph of sialic acid distributions on MDCK cells confluent the day following passing the cells. These graphs show the percentage of cells expressing α-2,6 linked sialic acids only, α-2,3 linked sialic acids only, and cells expressing both receptors when they were confluent the day following passing the cells in each media group. The SFM maintained cells were predominantly expressing α- 2,6 linked sialic acids. The MEM containing 10% FBS1 and FBS2 maintained cells were expressing mostly α-2,3 linked sialic acids.

The swine IAV grew to similar titers in all 3 media types within each inoculation trial. The average titer of this swine IAV in all 3 media groups in trial 1 was

8 7 approximately 1x10 TCID50/ml and 3x10 TCID50/ml in all 3 media groups in trial 2.

This virus was previously aliquotted so both trials used IAV of the same freeze thaw. The

47

SFM maintained cells were expressing mostly α-2,6-linked sialic acids when confluent the day following passing the cells. This was in contrast to the results found in

experiment 2 that showed the cells confluent after 1 day were mostly expressing both

receptors when cultured in either SFM or MEM with 10% FBS. This difference could be

due to the different lot of MDCK cells used or the different lots of FBS used in this

experiment. The MEM with 10% FBS1 and FBS2 maintained cells were predominantly

expressing α-2,3-linked sialic acids when confluent the day following passing the cells in

this experiment. The swine origin IAV might be able to bind to both receptor types which

would explain why it grew to similar titers in all media groups. There could be some

other unknown mechanisms that were not investigated that might account for these

results. Other receptors that IAV can bind to on the MDCK cells are a possibility [46].

The avian IAV grew to higher titers in SFM maintained cells in both trials when

compared to growth in MEM containing 10% FBS1 and FBS2. The avian IAV grew to

8 average titers of approximately 1x10 TCID50/ml in SFM maintained cells in both trials

5 6 but only to average titers ranging from 1x10 TCID50/ml to 5x10 TCID50/ml in cells

maintained in MEM containing 10% of either FBS1 or FBS2. Again this IAV was

previously aliquotted so both trials used IAV of the same freeze thaw. The average titers

of this avian IAV grown in MEM with 10% FBS1 and MEM with 10% FBS2 were very

similar to each other within each trial. As mentioned above the SFM maintained cells

were expressing mostly α-2,6-linked sialic acids while the MEM with 10% FBS1 and

FBS2 maintained cells were predominantly expressing α-2,3-linked sialic acids when

confluent the day following passing the cells. This same IAV was shown to grow to high

48

titers in MDCK cells and to grow poorly in mice in a previous study [93]. Mice are

known to express predominantly α-2,3-linked sialic acids in their lungs [66] like the FBS

adapted MDCK cells in this study. The sialic acid distributions of the MDCK cells used

in their study were not characterized. The avian origin IAV used in this experiment might prefer to bind the α-2,6-linked sialic acids that were present on the SFM maintained

MDCK cells. There may be something about the binding site of this particular IAV that

causes it to bind inefficiently to α-2,3-linked sialic acids.

The binding specificity of each IAV used in this experiment would have been

very useful when interpreting the TCID50/ml results. The HA sequences for both the

avian IAV and the swine IAV are available on genbank, accession numbers: CY018877

and JX565497, but the binding specificity could not be determined by looking these two

sequences. There are several amino acid positions in the HA molecule that have been

implicated as possible determinants of binding specificity, position 182, 192 and 226 [94,

95]. Neither of these IAVs have the specific amino acid determinants at these positions.

Two different assays were tested in an attempt to determine the binding specificity of

each IAV used in this experiment: a solid-phase assay of receptor-binding specificity [91]

and a modified hemagglutination assay. Unfortunately neither assay produced useful

results.

The solid-phase assay of receptor-binding specificity was similar to an ELISA

assay. Multiple dilutions of the sialyl-glycoproteins had to be tested to find the optimal

dilution for each virus to bind. The first time the assay was performed the wash solution

had formed crystals in the refrigerator that could not be removed. The wash solution was 49 re-made and did not have crystals in the solution for the second trial but almost no color change was observed and the absorbance values obtained were too low to be used as stated in the protocol. There were many variables in this assay that could be adjusted. The assay protocol was not very clear on how concentrated to make the peroxidase-labeled streptavidin solution. The third time running the assay the concentration was increased 10 fold which caused the negative control wells to have increased absorbance values. The average absorbance of the negative wells was subtracted from the sample wells to give the sample absorbance values. In this trial the average negative absorbance was so great that it caused the sample wells to have negative absorbance values. The binding specificity of each IAV could not be determined without considerable trial and error with this assay.

A modified hemagglutination assay was tried after the solid-phase assay failed to yield useful results. Turkey red blood cells were treated with a sialidase specific for α-

2,3-linked sialic acids, leaving only α-2,6-linked sialic acids [96]. Two fold dilutions of each IAV, swine and avian origin, were made across 8 wells of a 96 well plate with phosphate buffered saline creating a range of 1:2 to 1:256. The swine IAV and avian IAV were each diluted in 3 columns so that treated turkey red blood cells, untreated turkey red blood cells, and chicken red blood cells could be added to a separate dilution series for each virus. Chicken red blood cells only have α-2,3-linked sialic acids. Phosphate buffered saline was used as negative controls. In the first trial the avian IAV hemagglutinated the treated turkey red blood cells, untreated turkey red blood cells, and chicken red blood cells all to a dilution of 1:256. The swine IAV hemagglutinated the

50

treated turkey red blood cells, untreated turkey red blood cells, and chicken red blood cells all to a dilution of 1:128. These results indicated that both viruses bound to both receptors. It was possible that the sialidase did not work to cut off the α-2,3-linked sialic acids. In the second trial turkey red blood cells and chicken red blood cells were treated with the sialidase. Another aliquot of turkey red blood cells also had double the amount of sialidase added. The enzyme treated chicken red blood cells should not have had either sialic acid and should not have hemagglutinated either IAV. However, nearly identical results to the first trial were obtained with this second trial showing that both IAVs bound to both sialic acids. The swine IAV hemagglutinated the treated and untreated chicken red blood cells slightly less than the treated and untreated turkey red blood cells. New chicken and turkey red blood cells were obtained and a third trial was run. Again it appeared that both IAVs were binding to both α-2,3-linked and α-2,6-linked sialic acids, the sialidase enzyme was not working, or the IAVs were binding to something else on the

red blood cells. The binding specificity of each IAV could not be accurately determined

with this assay.

2.15 Discussion - In experiment 1 it was shown that MDCK cells cultured in different

media formulations had different sialic expression patterns and that the two SFMs did not

result in the same distribution of sialic acids over time. MEM supplemented with 10%

FBS resulted in the cells cycling between expressing both α-2,6 linked and α-2,3 linked

sialic acids one passage to high percentages of cells expressing only α-2,3 linked sialic acids the next passage. Cells were passed twice a week, once to be confluent after 3 days 51

and then again to be confluent after 4 days. The cells could be using up nutrients in the

media in ways that would cause this passage to passage pattern. Additional testing could

be conducted to determine if media components were being depleted. Culture in Lonza’s

UltraMDCK SFM increased the percentage of MDCK cells expressing both sialic acid receptors and culture in OptiPro SFM caused cells to express high percentages of both receptors or high percentages of α-2,3 linked sialic acid receptors.

In experiment 2 MDCK cells cultured in UltraMDCK, without passing the cells or changing the media, expressed both sialic acids. When MDCK cells were cultured in

MEM with 10% FBS and media was not changed the sialic acid expression was variable, although different from experiment 1. A different lot of MDCK cells and a different lot of

FBS were used in this experiment. This could account for some of the differences seen between the two experiments. Lot to lot variability of FBS has been documented. MDCK cells in this experiment were not confluent each day. It is possible that cells in different phases of the cell cycle could express different sialic acids. The supernatant protein concentration of MDCK cells maintained in UltraMDCK SFM did decrease over time when compared to the stock SFM. The supernatant protein concentration of cells maintained in MEM with 10% FBS did not consistently decrease when compared to the stock MEM with 10% FBS. Media protein concentration did not appear to influence the sialic acid distributions on the cells.

Experiment 3 showed that a swine origin IAV grew to high titers in cells maintained in both UltraMDCK SFM and MEM containing 10% FBS. The avian origin

IAV grew to high titers in the SFM-maintained cells and grew poorly in the MEM with 52

10% FBS-maintained cells. The sialic acids present on the SFM-maintained cells were determined to be mostly α-2,6 linked sialic acids while MDCK cells maintained in MEM with 10% FBS cells mostly expressed α-2,3 linked sialic acids, differing from results obtained in experiment 2. In this final experiment a new lot of MDCK cells and two new lots of FBS were used, which could be the cause of the differing results noted. It is hypothesized that the swine IAV was able to bind to both receptor types and the avian

IAV preferred α-2,6 linked sialic acids.

These results are significant because they show that culture media can influence the sialic acid expression of MDCK cells. The distributions of α-2,6 linked and α-2,3 linked sialic acids can skew the efficiency of IAV isolation or cause the virus to adapt to the culture system.

It would have been useful to know the binding preference of each IAV used for the

TCID50 experiments when analyzing the results. Unfortunately the amino acid sequences and both of the binding assays attempted failed to provide conclusive results of the binding preference of each IAV. There were too many variable combinations to optimize in the solid-phase assay of receptor-binding specificity that the assay became impossible.

The cost and time constraints of performing trial and error experiments at each step could not be reconciled. The hemagglutination assay was not specific enough to determine the binding preference of each IAV. The sialidase enzyme may not have been working efficiently to cut off the correct sialic acids on the red blood cells. Each IAV could have been binding to other unknown receptors on the red blood cells. Other assays that have been shown to provide accurate results to determine the binding preference should be

53 investigated.

54

Chapter 3: Conclusion

3.1 Conclusion- The HA protein mediates binding of IAV to sialic acids on the surface

of cells. This is the first step in infecting a cell. IAVs in host species are usually well

adapted to their hosts and preferentially bind to the sialic acids present in the host.

Typically avian IAVs bind to α -2,3 linked sialic acids and human IAVs bind to α -2,6

linked sialic acids. The HA protein will allow amino acid changes in the binding region so that it can change binding preference, bind to different glycoproteins on cells, and

infect different hosts. When IAV is passed through embryonating chicken eggs the virus

might adapt to the egg to be able to better infect the cells available. If IAV is passed

through MDCK cells the virus may adapt to the cell culture system in a different way.

Understanding what sialic acids are present in the culture system used to isolate and

characterize different IAVs is important for the correct classification of IAV when a new

strain is discovered. If a culture system does not possess the sialic acids to which an IAV

prefers to bind it may not be recovered or may require multiple passages and adaptation

of the virus to isolate the IAV. MDCK cells expressing high levels of α -2,6 linked sialic

acids could be used as a way to screen IAV samples from different species, such as wild

birds, for the potential to transmit to humans. If an avian-origin IAV that humans had no

55 prior exposure to was able to easily bind to the sialic acid receptors commonly found in the human trachea and lung a new IAV pandemic could arise.

The experiments conducted in this study investigate the effects of culture media on the sialic acid expression of MDCK cells. The first experiment shows that the sialic acids expressed by MDCK cells is different when the cells are cultured in different media. The two different SFMs did not result in the same sialic acid distributions being expressed by MDCK cells over time. Culture in MEM with 10% FBS and OptiPro SFM resulted in variable sialic acid expression by MDCK cells. UltraMDCK SFM resulted in cells expressing both α -2,3 linked and α -2,6 linked sialic acid receptors. The second experiment attempted to determine if media components were being depleted in a way that influenced the sialic acid distribution patterns by seeding the cells at different densities and not passing the cells. Protein concentrations in the supernatant were compared to determine if media proteins influenced the sialic acid distributions. It did not appear that cells cultured in UltraMDCK SFM were dependent on media components to determine their sialic acid profile. Supernatant protein concentrations decreased but did not appear to affect the sialic acids present on the cells. MDCK cells cultured in SFM generally expressed both α -2,3 linked and α -2,6 linked sialic acid receptors. Culture in

MEM with 10% FBS resulted in variable sialic acid distributions on the cells.

Supernatant protein concentration did not decrease. Experiment 3 showed that IAV grew to different titers depending on the media the cells were cultured in. Both the avian- lineage IAV and the swine-lineage IAV grew to high titers in UltraMDCK SFM maintained cells. The swine IAV grew to high titers in the MEM with 10% FBS

56

maintained cells, but the avian IAV did not. The binding preference of each IAV is an

important component that could not be determined. Overall, the use of UltraMDCK SFM

resulted in MDCK cells consistently expressing both sialic acid receptors and both IAVs

grew to high titers in UltraMDCK maintained cells. MEM with 10% FBS was shown to

cause variable sialic acid expression by MDCK cells and caused the avian-lineage IAV to

grow to a low titer. Culture media did effect the sialic acid distributions on MDCK cells

and the isolation efficiency of IAV.

Additional experimentation is required to fully understand the extent to which

culture media effects virus isolation. It would be useful to perform binding assays on IAV

samples from a range of host species, such as human, canine, equine, avian, and swine

origin IAVs, to determine what receptor/s they prefer to bind. The sialic acids could be

determined on MDCK cells maintained in both SFM and MEM with 10% FBS and

TCID50 experiments could be repeated to compare viral titers. Repeating the TCID50

experiment with an IAV that binds to only α-2,6 linked sialic acids, an IAV that binds to only α-2,3 linked sialic acid, and an IAV that binds to both receptors would be a better way to determine if the sialic acid distributions on MDCK cells due to media conditions

effect viral growth.

Declaration of conflict of interests. The author declared no potential conflict of

interest with respect to the research, authorship, and/or publication of this article.

57

Funding. This work has been funded in part with federal funds from the Centers of

Excellence for Influenza Research and Surveillance (CEIRS), National Institute of

Allergy and Infectious Diseases, National Institutes of Health, Department of Health and

Human Services, under Contract No. HHSN272201400006C.

58

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