Detection of Influenza A Virus. THESIS. Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in

Transcription

1 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

2 Copyrighted by Sarah W. Nelson 2016

3 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

4 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

5 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

6 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

7 Vita Granville High School B. S. Biochemistry, The Ohio State University Laboratory Technician, Idexx laboratories 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

8 Table of Contents Abstract... ii Acknowledgements... v Vita... vi Fields of Study... vi List of figures... x Chapter 1: Literature Review Influenza A virus biology The HA protein IAV replication IAV subtypes Human influenza viruses The 1918 Spanish influenza pandemic The 1957 Asian influenza pandemic The 1968 Hong Kong Influenza pandemic The 1977 Russian Influenza pandemic... 8 vii

9 1.10 The 2009 swine influenza pandemic Avian IAV Swine IAV Sialic acids Cell lines Medium for culture of cells Virus isolation techniques Binding mutants Other IAV receptor possibilities Summary Chapter 2: Project Paper Abstract Introduction Tissue culture media MDCK cell culture Transitioning cells to SFM Staining for flow cytometry BCA protein assay protocol Solid-phase binging assay of receptor-binding specificity Flow images, controls and samples viii

10 2.10 Experiment 1: Monitoring MDCK cells for 25 passages, cell lot 09D023, FBS lot Experiment 2 methods Experiment 2 : Monitoring sialic acid receptors each day, cell lot 09J020, FBS lot Experiment 3 methods Experiment 3: Tissue culture infectious dose 50% (TCID 50 ) experiment, cell lot 14A025, FBS1 lot , FBS2 lot Discussion Chapter 3: Conclusion Conclusion Declaration of conflict of interests Funding References ix

11 List of Figures Figure 1. A simplified IAV structure diagram Figure 2. Genesis of the 2009 H1N1 pandemic influenza A virus Figure 3. Sialic acid linkage types Figure 4. Sialic acid biosynthetic pathway Figure 5. Diagram that illustrates how the MDCK cells were propagated from the stock Figure 6. Flow cytometry controls and example samples Figure 7. Graph of sialic acid distributions on MDCK cells cultured in MEM containing 10% FBS for 25 passages Figure 8. Graph of sialic acid distributions on MDCK cells cultured in UltraMDCK SFM for 25 passages Figure 9. Graph of sialic acid distributions on MDCK cells cultured in OptiPro SFM for 25 passages Figure T25 flasks were seeded with different concentrations of cells on day 0 from each media type Figure 11. Graph of sialic acid distributions on MDCK cells cultured in UltraMDCK SFM seeded at 8x10 6 cells per T25 flask x

12 Figure 12. Graph of sialic acid distributions on MDCK cells cultured in UltraMDCK SFM seeded at 1.5x10 6 cells per T25 flask Figure 13. Graph of sialic acid distributions on MDCK cells cultured in UltraMDCK SFM seeded at 9.25x10 5 cells per T25 flask Figure 14. Graph of sialic acid distributions on MDCK cells cultured in UltraMDCK SFM seeded at 1x10 5 cells per T25 flask Figure 15. Graph of sialic acid distributions on MDCK cells cultured in MEM containing 10% FBS seeded at 8x10 6 cells per T25 flask Figure 16. Graph of sialic acid distributions on MDCK cells cultured in MEM containing 10% FBS seeded at 1.5x10 6 cells per T25 flask Figure 17. Graph of sialic acid distributions on MDCK cells cultured in MEM containing 10% FBS seeded at 9.25x10 5 cells per T25 flask Figure 18. Graph of sialic acid distributions on MDCK cells cultured in MEM containing 10% FBS seeded at 1x10 5 cells per T25 flask Figure 19. Graph of supernatant protein concentrations Figure 20. Graph of log transformed TCID 50 /ml results Figure 21. Graph of sialic acid distributions on MDCK cells confluent the day following passing the cells xi

13 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

14 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

15 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

16 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 spillover 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

17 [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

18 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 preexisting immunity to this virus. The H1 gene from this virus persisted in swine populations and re-emerged in human populations in 2009 [18]. 6

19 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 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

20 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] 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

21 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

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

23 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 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

24 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

25 Figure 3. Sialic acid linkage types [48]. 13

26 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

27 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

28 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] 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

29 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 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

30 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 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

31 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 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

32 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

33 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

34 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

35 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 Q) was obtained from Lonza Bioscience (Basel, Switzerland). OptiPRO SFM (cat. no ) was obtained from Life Technologies (Grand 23

36 Island, NY, USA). MDCK cells (cat. no VL) 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 ; 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% CO 2. 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 Cl) from Corning a division of Thermo Fisher. Approximately 8x10 6 cells were passed into new T150 flasks every Monday. Each Thursday cells were trypsinized and approximately 1x10 7 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 14A

37 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

38 2.6 Staining for flow cytometry -PBS containing 5% FBS and 0.02% sodium azide (PBS/azide) was used to wash 1x10 6 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 ) 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 ) 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

39 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 ) 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, peroxidaselabeled 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

40 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 streptavidinphycoerytherin. Panel (G) cells maintained in UltraMDCK SFM and dual stained with biotinylated SNA lectin, MAA lectin, and streptavidin-phycoerytherin. 28

41 The manufacturer s recommendations for the concentrations of SNA (5 to 20µg/ml for 1x10 6 cells) and MAA (100µg/ml for 1x10 6 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 1x10 6 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