Elucidating the Viral and Cellular Components Of

ELUCIDATING THE VIRAL AND CELLULAR COMPONENTS OF

STRAIN-SPECIFIC ENTEROVIRUS D68 TEMPERATURE

SENSITIVITY

by Brendan D. Smith

A dissertation submitted to Johns Hopkins University in conformity with the

requirements for the degree of Doctor of Philosophy

Baltimore, Maryland

April 2020

Enterovirus D68 (EV-D68) is an emerging respiratory pathogen that has been associated with severe acute respiratory infection and acute flaccid myelitis. Prior to global outbreaks in 2014, EV-D68 was reported infrequently and believed to manifest in mild common cold-like symptoms, characteristic of infection limited to the upper airways. This dissertation examines of the replication of contemporary and historic strains of EV-D68 to delineate both viral and cellular factors that contribute to the recent increase in severe disease cases.

Acute respiratory infections are among the most common causes of death in children worldwide. Viruses including H5N1 influenza virus, respiratory syncytial virus, SARS-CoV-1 and 2, and rhinovirus C have been attributed to severe lower respiratory infections. These viruses all replicate efficiently at temperatures of the lower respiratory tract (37°C). EV-D68 replication was initially described as being attenuated at 37°C, however, my thesis work shows that contemporary strains have gained the ability to replicate at core body temperature.

The 5’ UTR of contemporary strains was found to confer a translation efficiency advantage using reporter gene assays. By developing an EV-D68 infectious clone system, chimeras between historic and contemporary strains were able to be produced which showed this translation efficiency advantage alone does not affect the temperature sensitivity of infectious virus ii

production. Generating a full panel of chimeras swapping major genomic regions (5’ and 3’ UTR, 3’ UTR, structural genes, non-structural genes), non- structural gene exchanges between historic and contemporary EV-D68 were found to reverse temperature-sensitivity phenotypes.

Studies performed in primary human nasal epithelial cell (hNEC) cultures suggest that contemporary strains are more efficient at replicating in cells of the upper respiratory tract at both 32°C and 37°C, and elicit a robust pro-inflammatory response. Historic and contemporary strains exhibited similar cell tropism in hNEC cultures, both preferentially infecting ciliated epithelial cells.

Taken together, this dissertation identifies strain-dependent effects of temperature on EV-D68 replication and provides important insights into viral sequences that contribute to the recent uptick in severe disease caused by infection.

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Thesis Readers:

Adviser: Andrew Pekosz Molecular Microbiology and Immunology – BSPH

Gary Ketner Molecular Microbiology and Immunology – BSPH

Priya Duggal Epidemiology – BSPH

Carolyn Machamer Cell Biology – SOM

Alternates:

Diane E. Griffin Molecular Microbiology and Immunology – BSPH

Kellogg Schwab Environmental Health Engineering – BSPH

PREFACE

The fairest thing we can experience is the mysterious. It is the fundamental emotion which stands at the cradle of true art and true science.

– Albert Einstein, The World As I See It

ACKNOWLEDGEMENTS

This dissertation is a product of years of support and guidance from individuals, both directly and indirectly. First off, I would like to thank Dr.

Andrew Pekosz for his continued mentorship over the years. His passion for virology has been inspiring and his willingness to lend support, sometimes with pipette in hand, was critical for the success of my research. Andy truly pushed me to think critically about my research and always kept an open door to discuss new ideas and results. His continuous support for my career endeavors provided me with internship opportunities to grow professionally and position me for success in industry. I am most appreciative of Andy’s unwavering support for me as a father. Without question, he always allowed me to put my daughter before my work, for which I am eternally grateful.

Thank you to my thesis committee members, Dr. Gary Ketner, Dr.

Diane Griffin, Dr. Carolyn Machamer, Dr. Priya Duggal and Dr. John

Nicholas. Your guidance and insight on my project over the years has been essential for its success. You all challenged me to think critically as a scientist, which shaped the concepts of this dissertation and facilitated my growth as an academic researcher.

From the early days in the Pekosz lab, I was fortunate to have fantastic colleagues to serve as mentors and friends. The original members,

Dr. Deena Blumenkrantz, Dr. Katherine Fenstermacher, Dr. Nick

Wohlgemuth and Dr. Hsuan Liu, were all instrumental to my establishment vi

in the laboratory. I had no shortage of questions for the entirety of the group and could not have grown to be the scientist I am without their guidance.

Deena served as my mentor throughout my rotation and kindly shared her wisdom and taught me the ropes of cell culture and working with viruses.

Katherine was my point person in the early days as I took on the EV-D68 project. Our projects both looked at effects of temperature on the innate immune response (Team Temperature), making her an important resource for many of the protocols in my hNEC studies. Nick employed uncanny patience in answering an infinite number of questions pertaining to protocols and the Ph.D. program, always ending with a proper answer. It was also a pleasure to share stories of the joys and difficulties of fatherhood to amazing daughters with someone on a daily basis. Hsuan has always been committed to helping others in the laboratory. Her knowledge of virology, both conceptual and technical, was vital for the immense troubleshooting that came along with my projects.

After joining the Pekosz laboratory, it seemed our roster grew exponentially. I would like to thank postdoctoral fellows Dr. Farah El Najjar,

Dr. Katy Shaw-Saliba, Dr. Jason Westerbeck and Dr. Alyssa McCoy, master’s students Eddy Ye, Emily Thompson, James Stanton, Laura Canaday, and

Siddhant Vyas, Kirsten Littlefield, and Ph.D. students Dr. Harrison Powell and Jessica Resnick for their continued feedback on my project and for always appreciating pictures of my daughter at our group lab meetings. vii

Harry joined me as a fellow Ph.D. student in the lab one year after I joined.

Given our proximity, sitting back-to-back in the lab, he was often the first person I would ask a question, field ideas on data interpretation, and simply seek distraction when seeking a break from my computer screen. Harry’s willingness to continue our discussions at some of Baltimore’s finest establishments over a Guinness and a sidecar was undoubtedly appreciated.

He was consistently a valuable resource for my project, lending his flow cytometry expertise and always willing to carry on my experiments on days I could not come in.

I would also like to thank Dr. Sabra Klein and her lab members over the years for their continuous feedback pertaining to my project. Dr. Landon vom Steeg and Dr. Olivia Hall field an infinite number of questions, both scientific and Ph.D. program related. Dr. Kristyn Sylvia and Rebecca Ursin were wonderful colleagues to share a lab space with and always provided entertaining conversation at breakroom lunches.

Within the MMI department, I would like to acknowledge my fellow

Ph.D. cohort members: Dr. Elizabeth Troisi, Dr. Hannah MacLeod, Dr. Jane

Yeh, Dr. Julia Pringle, Dr. Kristin Poti, Dr. Phil Salvatore, and Zach Stolp.

Thank you for all of the help, particularly in getting through the first couple of years. It is safe to say that jumping back into academia after my industry hiatus was not a simple endeavor. It was comforting to have your willingness to tutor me when necessary and inspire me to keep pushing when classes viii

seemed overwhelming. Our regular social events may have faded over the years, but my appreciation for you all certainly did not. Thank you to Gail

O’Connor for all of her behind-the-scenes work that went into my program experience. She was always willing to answer any and all questions to ensure my success in the program. Thank you to Dr. Alan Scott and Dr. Gary Ketner for their necessary guidance in my early days in the Ph.D. program.

There are countless friends that have supported me throughout the duration of my dissertation research. I want to thank Jamie Newbould and

Antonio King for their insurmountable support in properly celebrating my best days and for always being there on some of my worst. Tony Kendra and

Giovanni Medoro were instrumental in supporting my weekend golf addiction. There was no better stress relief and confidence booster than beating them at Baltimore’s municipal golf courses each weekend. Juergen

Schweder, Misty, Jago and Merik were wonderful friends and neighbors that taught me the unforgettable importance of ‘stooping’ in Baltimore. Even with a departure to Germany, they continued to show long distance encouragement during the final years of my research. I would like to thank

Jim and Sue Price for their support over the years. As my director at BD, Jim is credited with my introduction to molecular biology and the importance of infectious disease research. His encouragement and insight when I proposed the idea of pursuing a Ph.D. were critical to my decision to apply to the program. I would like to thank Jim Byham and Jackie Zimmerman for their ix

support with my daughter when I needed to come into the lab often at very odd times of the night to collect time points. I am forever grateful for Puja

Mody and Shivas Patel for all of the meals during my writing, caring for my daughter at times I needed to be in lab or writing, and for graciously opening their home to my daughter and me. There are truly no words to express my appreciation for all of the support they have given. Thank you to all of the friends back in hometown of Rochester. Buffalo Bills games with Greg

Burkett, Kevin Breen and Dave Prince always gave me something to look forward to throughout the year. While our times in Rochester rarely linked up, thank you to Ryan Baker for always checking in on how things were going and for lending encouragement whenever I needed it.

My family has always been a critical piece to my success, both prior to and during my time at Johns Hopkins. Thank you to Amy, Adam and Kyle for all of the phone calls to check in on how things were going. It was refreshing to see that distance had no bearing on the amount of support they could lend.

I must thank my parents for their undying support of over these years. While neither of them grasped exactly what I was doing in the lab, they were always willing to listen to me talk about it with sincere interest. It was evident the pride they took in my work, a notion I constantly drew inspiration from. While they will not hesitate to voice my path did not always seem to be going in this direction, they relish in my success more than anyone else. Thank you for teaching me the importance of hard work, dedication, and x

sacrifice. These became some of the greatest tools throughout my dissertation research.

Lastly, and undoubtedly the most important, I want to thank my daughter, Adrienne Lillie Smith, for always allowing me to put life into perspective throughout this dissertation research. There is absolutely nothing comparable to the joy I felt on the day she entered my life in my third year at Johns Hopkins. While she may not have made my dissertation research any easier, logistically, she gave me something to look forward to everyday and every night, whether my experiments worked or not. The love and inspiration she provided each day went unmatched, and for that, I cannot thank her enough.

TABLE OF CONTENTS

ABSTRACT ...... ii

PREFACE ...... v

ACKNOWLEDGEMENTS ...... vi

TABLE OF CONTENTS ...... xii

LIST OF TABLES ...... xviii

LIST OF FIGURES ...... xix

LIST OF ABBREVIATIONS ...... xxi

CHAPTER 1: INTRODUCTION ...... 1

ENTEROVIRUS D68 HISTORY, EMERGENCE, AND PUBLIC

HEALTH IMPORTANCE ...... 2

ACUTE RESPIRATORY INFECTION CLUSTERS ASSOCIATED WITH

ENTEROVIRUS D68 ...... 4

ACUTE FLACCID MYELITIS OUTBREAKS ASSOCIATED WITH

ENTEROVIRUS D68 ...... 6

ENTEROVIRUS D68 PHYLOGENY ...... 7

PICORNAVIRUS LIFE CYCLE ...... 9

INTERNAL RIBOSOMAL ENTRY SITES ...... 13

FUNCTIONAL PROTEINS OF PICORNAVIRUSES ...... 16

TEMPERATURE SENSITIVITY OF ENTEROVIRUSES ...... 20

FIGURES AND TABLES ...... 23

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CHAPTER 2: CONTEMPORARY ENTEROVIRUS D68 STRAINS SHOW

ENHANCED REPLICATION AND VIRAL TRANSLATION AT 37°C...... 31

ABSTRACT ...... 32

INTRODUCTION ...... 33

MATERIALS AND METHODS ...... 35

Cell culture ...... 35

Viruses ...... 36

50% Tissue culture infectious dose (TCID50) assay ...... 37

Plaque assays ...... 37

Growth Curves ...... 37

Flow Cytometry ...... 38

Phylogenetic Analysis and RNA secondary structure ...... 38

IRES Activity Assay ...... 39

SDS-PAGE and western blotting ...... 40

Statistical analyses ...... 41

RESULTS ...... 41

Effect of physiological temperature ranges on historic and contemporary

EV-D68 strain infectious virus production ...... 41

Translation efficiency of contemporary and historic EV-D68 strains at

physiological temperature ranges ...... 42

Assessment of IRES activity from strains ranging from 1963 to 2016 ... 43

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Genomic and structural comparison of Corn-1963 and MO-2014 and

effects on IRES activity ...... 44

Replication of Corn-1963 and MO-2014 5’ UTR chimeric viruses at 32°C

and 37°C...... 45

DISCUSSION ...... 47

ACKNOWLEDGEMENTS ...... 51

FIGURES ...... 53

CHAPTER 3: STRUCTURAL AND NON-STRUCTURAL GENES

CONTRIBUTE TO ENTEROVIRUS D68 TEMPERATURE SENSITIVITY . 63

ABSTRACT ...... 64

INTRODUCTION ...... 65

MATERIALS AND METHODS ...... 68

Cell Culture ...... 68

Infectious Clone System ...... 68

50% tissue culture infectious dose (TCID50) assay ...... 69

Statistical analyses ...... 69

RESULTS ...... 70

Sequence analysis of historic Corn-1963 and contemporary MO-2014

indicate substantial evolution that spans the entire genome ...... 70

Swapping Functional Regions of Historic and Contemporary EV-D68

Strains ...... 71

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Corn-1963 partially overcomes temperature sensitivity with genomic

swap of contemporary non-structural genes ...... 72

MO-2014 loses temperature tolerance with genomic swaps of

contemporary structural and non-structural genes ...... 73

DISCUSSION ...... 73

ACKNOWLEDGEMENTS ...... 77

FIGURES AND TABLES ...... 78

CHAPTER 4: ENTEROVIRUS D68 PRODUCTIVELY INFECTS HUMAN

NASAL EPITHELIAL CELL CULTURES AT A RANGE OF

PHYSIOLOGICAL TEMPERATURES ...... 84

ABSTRACT ...... 85

INTRODUCTION ...... 87

MATERIALS AND METHODS ...... 88

Cell culture ...... 88

Viruses ...... 89

Sequence analysis ...... 89

50% tissue culture infectious dose (TCID50) assay ...... 90

Growth Curves ...... 90

Interferon, cytokine and chemokine measurements ...... 91

Microscopy ...... 91

Statistical analyses ...... 92

RESULTS ...... 93

Contemporary EV-D68 strain replicates to higher virus titers and viruses

release persists longer on hNEC cultures ...... 93

MO-2014 elicits increased amounts of proinflammatory chemokines and

IFN-λ at both 32°C and 37...... 94

High MOI infection of hNECs with Corn-1963 and MO-2014 limited

virus growth over time with higher induction of innate immune

responses for Corn-1963 ...... 95

Corn-1963 and MO-2014 share similar tropism for ciliated epithelial cells

in primary differentiated hNECs ...... 97

DISCUSSION ...... 98

ACKNOWLEDGEMENTS ...... 102

FIGURES ...... 104

CHAPTER 5: GENERAL DISCUSSION ...... 111

Evolution of EV-D68 has resulted in increased disease severity ...... 113

Contemporary EV-D68 strains are better able to replicate at temperatures

of the lower airways ...... 114

Identification of translation efficiency advantages of contemporary EV-D68

strains ...... 115

Identification of the mechanism behind increased translation efficiency 117

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Defining the role of non-structural genes in EV-D68 temperature

sensitivity ...... 118

Contemporary EV-D68 strains replicate more efficiently generating a more

robust inflammatory response in hNECs and show similar cell tropism for

ciliated cells ...... 120

Expansion of research to vaccines efforts ...... 121

Retrospective analysis of respiratory samples ...... 123

Conclusion ...... 127

REFERENCES ...... 128

CURRICULUM VITAE ...... 173

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LIST OF TABLES

Table 1. 1 Reported number AFM cases and number of states reporting AFM in the United States, 2014-2019...... 30

Table 3. 1 Nucleotide (nt) and amino acid (AA) lengths, changes, and % homology in contemporary MO-2014 compared to Corn-1963...... 79

xviii

LIST OF FIGURES

Figure 1. 1 AFM cases reported to the CDC from August 2014 to December

2019...... 23

Figure 1. 2 Phylogenetic analysis of EV-D68 VP1 identifying ...... 24

Figure 1. 3 Picornavirus Virus Life Cycle ...... 26

Figure 1. 4 Schematic of Enterovirus D68 genome ...... 27

Figure 1. 5 Schematic of picornavirus 5’ UTR secondary structure ...... 28

Figure 2. 1 The effect of physiological temperature ranges on EV-D68 infectious virus production...... 54

Figure 2. 2 The effect of physiological temperature ranges on EV-D68 mRNA translation efficiency...... 56

Figure 2. 3 Changes in EV-D68 IRES activity from 1963 to 2016...... 58

Figure 2. 4 Comparison of Corn-1963 and MO-2014 IRES genetic features and effects on IRES activity...... 59

Figure 2. 5 Effects of Corn-1963 and MO-2014 IRES chimeras on replication and viral protein translation...... 61

Figure 3. 1 EV-D68 Infectious clone system...... 78

Figure 3. 2 Historic and Contemporary EV-D68 Chimeras...... 80

Figure 3. 3 Replication of rCorn-1963 and its functional region swaps with rMO-2014...... 82

Figure 3. 4 Replication of rMO-2014 and its functional region swaps with rCorn-1963...... 83 xix

Figure 4. 1 Effects of temperature on replication of Corn-1963 and MO-2014 in primary hNEC cultures...... 104

Figure 4. 2 Apical and basolateral secretion of chemokines and interferon by

Corn-1963 and MO-2014 over multiple replication cycles...... 106

Figure 4. 3 Replication kinetics and secreted chemokines and interferons in hNEC cultures at 32°C and 37°C for Corn-1963 and MO-2014 following high

MOI infection...... 107

Figure 4. 4 Ciliated cell tropism of Corn-1963 and MO-2014 in primary hNEC cultures...... 109

LIST OF ABBREVIATIONS

AA Amino acid AFM Acute Flaccid Myelitis ALI air-liquid interface ARI Acute Respiratory Illnes ATCC American Type Culture Collection CBV Coxsackie B Virus CDC Center for Disease Control CoV Cornonavirus CSF Cerebral Spinal Fluid DM differentiation medium DPI days post infection dsRNA Double-stranded RNA ELISA enzyme-linked immunosorbent assay EV-A71 Enterovirus A71 EV-D68 Enterovirus D68 hNEC human nasal epithelial cells HPI hours post infection HPI Hours post infection HRV Human Rhinovirus ICAM Intercellular adhesion molecule IFN Interferon IM Infection media IRES Internal Ribosomal Entry Site ITAF IRES trans-acting factors LAIV Live, attenuated influenza vaccine mAb Monoclonal antibody MEM Minimal Essential Media MERS Middle Eastern Respiratory Syndrome MFI Mean Fluorescence Intensity MOI Multiplicity of infection NCBI National Center for Biotechnology Information NESS National Enterovirus Surveillance System nt Nucleotide ORF Open reading frame PABP Poly-A binding protein PBS Phospate Buffered Saline PCBP Poly (rC) binding protein PFA Paraformaldehyde xxi

PTB Polypyrimidine tract binding protein PV Poliovirus RD Rhabdomyosarcoma RdRp RNA dependent RNA polymerase RO Replication Organelles RT Room temperature RT-qPCR Real time quantitative polymerase chain reaction SARS Severe Acute Respiratory Syndrome TCID50 50% tissue culture infectious dose UTR Untranslated region VP1-4 Viral Capsid Protein 1-4 WS working stock wt wild type

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CHAPTER 1: INTRODUCTION

ENTEROVIRUS D68 HISTORY, EMERGENCE, AND PUBLIC

HEALTH IMPORTANCE

Enterovirus D68 (EV-D68) is a respiratory pathogen that was first isolated in the United States in 1962. Four pediatric patients hospitalized in

California were being treated for respiratory disease and pneumonia. In determining the disease etiology, throat swabs from these patients were used to isolate a virus. Viruses isolated from these patients were antigenically related or identical, they were serologically distinct from other respiratory pathogens. A series of biological tests were led by Schieble et al. to identify this pathogen as a probable picornavirus, which eventually was named EV-

D68 [1].

Since its initial identification, EV-D68 went relatively undetected until the early 2000s [2-7]. Between 1970 and 2005, there were only 26 cases of respiratory illness associated with EV-D68 reported in the United States as identified by the Center for Disease Control (CDC) National Enterovirus

Surveillance System (NESS) [5, 7], but this low number also reflects a lack of surveillance for the virus. The NESS is a voluntary, passive laboratory surveillance system that has been in place since the 1960’s to track non-polio enteroviruses and paraechoviruses. While only 26 cases of EV-D68 were reported during this 35 year span, it is likely that the virus had been circulating either mildly pathogenic or asymptomatically during this time. In the 2000s, increased cases of EV-D68 began being reported globally [7, 8]. 2

Prior to 2014, there were 699 cases of EV-D68 confirmed worldwide, with the majority of these cases stemming from small outbreaks reported between

2008 and 2013 [2, 7, 9]. Most of these cases were detected in patients suffering from respiratory illness, with very few of them manifesting in neurologic disorders. However, in 2014, a global outbreak of EV-D68 took place which was associated for the first time with clustered outbreaks of severe acute respiratory illness (ARI) and acute flaccid myelitis (AFM) [3-5, 7,

10-25]. In 2014 alone, there were 2,287 confirmed cases of EV-D68 reported worldwide, nearly three times the amount of all previously reported cases. Of these cases, most came from North America, with 1,153 cases reported in the

United States and nearly 700 cases in Canada [7, 26-31]. Europe reported over 400 confirmed cases in 2014 [7, 32, 33]. While EV-D68 is mostly detected in respiratory samples, there have been some detection in cerebral spinal fluid (CSF) and stool or sewage samples [34-38]. One study done in the

United Kingdom evaluated the seroprevalence of EV-D68 neutralizing antibodies detected in age-stratified populations in 2006 compared to 2016 to determine any differences in pre-2014 prevalence. In both years, adults had nearly 100% seroprevalence of neutralizing EV-D68 antibodies, providing evidence of widespread circulation of mild EV-D68 infections prior to the

2014 outbreaks [39]. Additional prospective analyses of pre-outbreak seroprevalence studies further supported this widespread circulation [7, 33,

40-45]. Infants and young children consistently showed higher seroprevalence 3

in 2016 compared to 2006, suggesting a heightened incidence of infection in recent years [39].

Since the widespread outbreaks of EV-D68 in 2014, there has been a unique biannual pattern of severe infections occurring in both 2016 and 2018

[46-63] with limited numbers of EV-D68 detections in 2015 and 2017 [49, 64-

66]. The basis for the biannual pattern of EV-D68 is unclear, which makes it an important area of research interest from a biological standpoint and a predictive epidemiological standpoint. One confounding issue with understanding these patterns is the limited surveillance and the lack of reporting for EV-D68 in non-severe cases. EV-D68 testing is performed on those that seek medical attention, and thus, are already categorized by severe disease. While there are some surveillance programs in place for severe EV-D68 infection, quality reporting of case numbers limits our true understanding of the virus circulation.

ACUTE RESPIRATORY INFECTION CLUSTERS ASSOCIATED WITH

ENTEROVIRUS D68

Respiratory viruses such as respiratory syncytial virus (RSV), influenza viruses (H5N1, seasonal influenza), and coronaviruses (SARS,

MERS, COVID-19), are capable of inducing severe pathogenesis of the lower respiratory tract [67-74]. These viral infections invade the lower respiratory tract causing damage by inducing pro-inflammatory responses and cell death, 4

leading to pneumonia, bronchiolitis, hypoxaemia, and even respiratory failure [75]. In late summer of 2014, several hospitals in the United States were reporting heightened numbers of acute respiratory illness (ARI) to the

CDC most notably in pediatric populations [3, 4, 12, 17, 76]. These patients were reporting wheezing, bronchiolitis, and difficulty breathing, some requiring breathing assistance for their illness. Within these clustered ARI outbreaks, EV-D68 was identified as the causative agent for the illness. A comparison of EV-D68 infections to that of HRVs and other respiratory enteroviruses indicated increased pulmonary virulence for EV-D68 [77].

Pediatric populations and asthmatics are at greater risk for suffering from severe ARI [17, 78-83]. These clustered ARI outbreaks spread globally, with outbreaks reported in Canada, Europe, and Asia [7, 16, 24, 25, 30, 32, 84-86].

Since 2014, EV-D68 has continued an association with ARI [58, 60, 87-89].

Very few studies on the biological mechanisms by which contemporary strains of EV-D68 cause ARI have been reported. In both mouse and ferret models, EV-D68 has been shown to be able to penetrate the lower airways upon nasal inoculation [90, 91]. In both studies, EV-D68 elicited a proinflammatory response, a common cause of pulmonary damage in lower respiratory infections. The relevance of these animal models to EV-D68 infection in humans remains unclear and the studies neglected to look at differences between historic and contemporary strains and their respective

potential to cause ARI. The pathological factors induced by contemporary strains that leads to ARI remain poorly defined.

ACUTE FLACCID MYELITIS OUTBREAKS ASSOCIATED WITH

ENTEROVIRUS D68

Coinciding with the clustered outbreaks of ARI came an increased incidence of AFM. AFM is defined by the onset of weakness or paralysis in one or more limbs combined with the detection of spinal cord gray matter abnormalities on magnetic resonance imaging (MRI) [92-95]. While physical or occupational therapy is recommended on a case-by-case basis, there are no current therapeutic interventions to treat AFM. Enterovirus-associated AFM is not unique to EV D68, as other EVs such as poliovirus (PV), coxsackie B virus (CBV), and enterovirus A71 (EV71) have all been associated with AFM outbreaks [96-101]. In the United States, there has been a series of recent

AFM outbreaks appearing in a biennial pattern (2014, 2016, 2018) that correlate with EV-D68 outbreaks (Fig. 1.1) [102-104]. While there is certainly a temporal association with EV-D68, there has been little detection of EV-

D68 in the CSF of AFM patients making it difficult to formally establish causality. Recent studies have applied Bradford-Hill criteria and determined a causal relationship between AFM and EV-D68 [95, 105]. Every other year since 2014, the number of AFM cases and the number of states reporting cases to the CDC have increased (Table 1.1). The growing number of AFM 6

cases have led to the initiation of AFM task forces in the United States

(implemented by the Center for Disease Control) and United Kingdom [106,

107]. Continued measures of surveillance and research will be necessary for understanding the mechanisms of virally induced AFM and initiation of preventative measures.

Several groups have focused on the ability of EV-D68 to infect neuronal cell lines and to cause AFM in animal models. One group showed that a panel of contemporary EV-D68 strains could cause paralysis in mice, while historic strains did not [108]. Efforts to define the reason contemporary strains causes AFM have focused largely on their ability to infect neuronal cells. While one report suggests historic strains lack the ability to infect neuronal cells [109], another suggests the ability is common for all EV-D68 strains [110]. The basis for association of AFM with contemporary EV-D68 strains remains unclear and is most likely more than increased neuronal cell tropism alone.

ENTEROVIRUS D68 PHYLOGENY

EV-D68 is a member of the genus enteroviruses and further classified into the enterovirus D species. There are four human genotypes that share the enterovirus D species: EV-D68, enterovirus D70, enterovirus D94, and enterovirus D111 [111, 112]. Enterovirus D120 was recently detected in the stool of apes and has not yet been associated with human infections 7

[113]. EV-D68 has undergone significant evolution since first discovered in

1962. There has been divergence into 4 primary clades (A, B, C, D) with global distribution of each clade (Fig. 1.2).Clade classification is driven by nucleotide changes in the VP1 capsid protein. Due to the lack of surveillance and detection of EV-D68 prior to the early 2000s, it is difficult to pinpoint exact times of divergence for the clades. Continued divergence of clade B has resulted in a series of subclades (B1-3). Previously, clade A was characterized into subclades A1 and A2. However, with a growing number of sequences being added to NCBI, clade A2 was reclassified as clade D. Clade B1 dominated in the 2014 outbreaks while clade B3 dominated in 2016 and 2018

[79, 88, 114-119]. While VP1 is a target of the adaptive immune response, serology studies suggest no evidence of multiple serotypes across clades [39,

42].

Mutations introduced by the error-prone RNA polymerase and recombination events are the major sources of picornavirus genomic evolution

[120-123]. EV-D68 has undergone intraclade and intrapatient recombination events contributing to its evolution [119, 124]. While the emergence of different circulating clades of EV-D68 is evident, there has been no clear association of clinical symptoms to specific phylogeny. One study pointed at six polymorphisms detected in clade B1 which dominated in the United

States during the 2014 outbreaks, however, no evidence has been presented to link these mutations to ARI or AFM [89, 114, 125]. Contemporary strains 8

have of EV-D68 have all been found to have large deletion blocks at the terminal end of the 5’ UTR [124, 126]. Given the limit available sequences available prior to the 2000s, a full understanding of the temporal evolution of

EV-D68 remains incomplete.

PICORNAVIRUS LIFE CYCLE

While the virus life cycle for many picornaviruses have been well- defined and characteristics are generally conserved, EV-D68 remains understudied in this context. Enterovirus D68 (EV-D68) is a member of the

Picornaviridae family and further classified in genus Enterovirus.

Enteroviruses are among the most common human pathogens in the world.

In the United States, nearly 10 million enterovirus infections occur annually.

EV-D68 is composed of a non-enveloped icosahedral protein capsid (18-30nm) that encapsidates a single-strand positive-sense RNA genome. Generally, picornaviruses share common processes of invading a cell and producing and shedding infectious virus particles (Fig 1.3).

Attachment

After exposure of a susceptible human to EV-D68 in respiratory droplets, the virus attaches to epithelial cells in the respiratory tract. There are currently two identified receptors that EV-D68 utilizes for cellular attachment, α2,6 sialic acid and intercellular adhesion molecule 5 (ICAM-5) [127-132].

Treating cells with neuraminidase to remove surface sialic acid residues can completely abrogate the ability for EV-D68 to infect a cell and replicate [110, 9

131]. This is more applicable to historic strains of EV-D68. Contemporary strains, however, have been found to infect cells that have been treated with neuraminidase. ICAM-5 was determined to be a factor in EV-D68 entry, suggesting that α2,6 sialic acid may be an enhancer of attachment, rather than the sole receptor [127, 129, 130].

Entry

After attachment to a receptor, the virion is internalized by receptor mediated endocytosis and undergoes a conformational change resulting in destabilization. The conformational change occurs as the receptor binds a canyon in the capsid protein viral protein 1 (VP1) which contains a fatty acid like factor in a hydrophobic pocket. As this pocket factor is displaced, capsid destabilization facilitates release of the genomic RNA from the endosome into the cytoplasm [131, 133-137]. Inhibition of capsid destabilization is a target of inhibition for several picornavirus, including EV-D68 [138-140].

Translation

Once released into the cell, viral proteins are translated by host-cell machinery without the need for additional modifications. This is due to two important factors. First, the virus genome contains a highly structured 5’ untranslated region that contains an internal ribosomal entry site (IRES) that recruits translational factors and ribosomes without the need for a 5’ methylated cap. Being a major focus of this dissertation, the 5’ UTR will be described in depth in the following section. Secondly, the polarity of the 10

genomic RNA is positive-sense, which allows for and the incoming viral genome to be translated without a need for any initial RNA replication. The genome contains a single open reading frame (ORF) and is translated into one long polypeptide comprising structural and non-structural proteins (Fig.

1.4) [141, 142]. The polypeptide undergoes an autocatalytic cleavage process in which the encoded 2A and 3C proteases cleave the polypeptide into a series of functional proteins. The specific functions of these proteins will be described in further detail later in this chapter.

Replication

After polypeptide processing, EV-D68 undergoes a series of steps of genome replication. Picornaviruses form novel membrane structures known as replication organelles (ROs) to utilize as their site of replication. These ROs are derived from host intracellular membranes and house host and viral factors necessary for viral replication [143-146]. Induction of the autophagy process has been shown to be involved in formation of ROs for poliovirus and

CBV, but is thought to be non-essential for HRV [147-153]. Within the ROs, the picornavirus genome undergoes circularization in which there are RNA-

RNA interactions between the 5’ UTR and 3’ UTR, along with RNA-protein bridges [154]. Host poly-A binding protein (PABP) binds the 25-150 nt poly-A tail and interacts with the cloverleaf secondary structure in the 5’ UTR to facilitate this step. The circularization of the viral RNA facilitates efficient and continuous genome amplification. Picornaviruses encode an RNA- 11

dependent RNA-polymerase known as 3D polymerase. 3D recognizes a covalently linked protein (VpG) on the 5’end of the viral genome that serves as a transcription primer [142, 155-157]. Replication of the viral genome forms the negative-sense RNA, which serves as the template for replication of the genomic positive-sense RNA.

Capsid Assembly and Maturation

Positive-sense RNA genomes are encapsidated by the encoded structural proteins: Viral protein 1-4 (VP1, VP2, VP3, VP4). 60 copies of each of the four structural proteins form the icosahedral capsid. Prior to a maturation step,

VP2 and VP4 are collectively called VP0. 12 copies of VP0, VP1 and VP3 form a pentamer, where 5 pentamers form an icosahedral procapsid (lacking viral genome) or a provirion (encapsidating viral genome) [137, 158]. Procapsids have been found to be unstable and lack a final maturation step essential for capsid stability, while provirions undergo a final maturation step in which

VP0 is cleaved to form VP4 (inner shell) and VP2 (outershell) by RNA- induced auto-catalysis [159, 160]. Upon maturation, the virion is stable and infectious.

Egress

Picornaviruses have been shown to utilize two methods of viral egress: lytic escape and microvesicle release. Early studies of poliovirus suggested that a buildup of virus within a cell until a sudden burst, in which the virus infects neighboring cells. Protein 2B was determined to be a viroporin that 12

facilitated this lytic release [161]. These findings were supported as the mechanism of escape for other picornaviruses, such as CBV and RV. EV71 was discovered to harness non-lytic pathways of viral egress, which led to an altered view of picornavirus escape [162]. Recently, picornaviruses have been shown to utilize interactions with cell membranes to stimulate autophagy pathways in order to promote the formation of microvesicles capable of surrounding multiple mature virions [149, 151, 152, 163-166]. Upon trafficking to the cell surface, these microvesicles release the virions without disruption of the plasma membrane, maintaining cell viability and preventing detection of DAMPs (damage-associated molecular patterns).

INTERNAL RIBOSOMAL ENTRY SITES

In general, initiation of eukaryotic translation requires mRNA to have a m7G(5′)ppp(5′)N (methylated cap) on the 5’ end for translation initiation factor binding. Several negative-strand viruses, such as influenza virus and hantavirus, undergo a process of ‘cap-snatching’, in which the 5’ end of cellular mRNAs is cleaved and incorporated in the viral genome upon replication with the RNA dependent RNA polymerase (RdRp). The newly capped positive strand RNA can then be read in the canonical cap-dependent manner. Picornaviruses have bypassed the necessity for a 5’ methylated cap on their positive-sense genome to initiate translation. They utilize an internal ribosomal entry site (IRES) element located in the highly structured

5’ untranslated region (UTR) of the viral genome for the initiation of viral protein translation [167, 168].

The secondary structure that forms within the 5’ UTR is highly conserved across different picornaviruses and can be described as a series of domains, I-VI (Fig. 1.5). While picornavirus 5’ UTRs may differ significantly in nucleotide sequence, the location, structure, and function of these domains are quite similar. Domain I is involve in genome replication while Domains

II-VI are considered IRES elements and are utilized for translation initiation.

The specific function of the individual domains have been extensively researched in PV, CBV, and EV-71.

Domain I

The first secondary structure of the 5’ UTR is comprised of four stem-loops that form a cloverleaf-like structure at the immediate start of the genome.

The cloverleaf is involved in genome replication and facilitates synthesis of the negative sense RNA strand. This strand serves as the template for synthesis of genome copies. The cloverleaf is essential for the uridylation of

(viral protein genome-linked) VPg, a small protein covalently linked to the 5’ end of the genome utilized as a primer for replication initiation [169]. Viral protein intermediate 3CDpro binds the third stem-loop of the cloverleaf. Poly-

A binding protein derived from the host-cell binds the poly-A tail of the picornavirus genome and facilitates circularization by interacting with

3CDpro and the cloverleaf-structure [154, 170]. 14

Domain II-VI (IRES elements)

Domains II-VI of the 5’ UTR secondary structure of picornaviruses comprise the IRES and are associated with translation initiation. These domains recruit a series of canonical eukaryotic translation factors as well as IRES- specific translation factors involved in protein translation. The translation initiation factor, eIF4G, binds domain V, which serves as a bridge between the 43S pre-initiation complex and the IRES. Recruitment of the 43S pre- initiation tract leads to scanning of the RNA for a start codon and subsequent translation of the polypeptide [171]. Translation initiation factors eIF2, eIF3, eIF4A, eIF4G, eIF4B, eIF1A have all been shown to be essential for IRES- mediated translation [172]. Cap-dependent host translation is inhibited by the 2A protease (2Apro) which cleaves eIF4G, leading to the inactivation of eIF4F function and inhibition of cap-dependent translation [173-176]. IRES- mediated translation is enhanced by this cleavage [177, 178]. Studies suggest that this enhancement is mediated by the carboxy-terminal two-thirds of eIF4G cleavage product, which has a higher affinity for the IRES than intact eIF4G [179]. Between domains V and VI is polypyrimidine tract that recruits polypyrimidine tract binding protein (PTB). Another ITAF (IRES trans-acting factors) that is involved in translation is poly (rC) binding protein (PCBP) which binds both the IRES element in domain IV, along with the cloverleaf in domain I [180-182]. Finally, unr (upstream of N-ras) binds domains II and V has been shown to be essential for picornavirus [183-185]. These ITAFs are 15

thought to be involved in both the chaperoning of RNA to the 43S complex and also maintenance of the secondary and tertiary structure for efficient translation.

There have been various studies outlining the impacts of picornavirus 5’ UTR mutations and their impact on IRES-mediated translation efficiency, which can be linked to temperature sensitivity, and neurotropism [167, 186, 187].

While some picornaviruses have been determined to have multiple open reading frames with an additional start codon in the 5’ UTR, this is not the case for EV-D68 [188, 189]. There has been substantial evolution in the EV-

D68 5’ UTR since its initial discovery in 1962, most notably in the form of heightened variability and large deletion blocks in the spacer region flanking the AUG start codon [190, 191]. The effects of the EV-D68’s 5’ UTR evolution remain unknown, however, are a major focus of this dissertation.

FUNCTIONAL PROTEINS OF PICORNAVIRUSES

The approximately 7.5kb genome of EV-D68 contains one ORF that is read fully and translated into a single polypeptide. While some picornaviruses have been reported to have an additional ORF located in the 5’ UTR, the initial step of generating the polypeptide remains common to the virus family. A series of autocatalytic proteolytic processing steps occur to generate functional proteins from the polypeptide. The genome divided into three

functional regions P1, P2 and P3. P1 encodes four structural proteins while

P2 and P3 encode seven non-structural proteins (Fig. 1.6) [160, 192].

Structural Proteins

The structural proteins that make up the virus capsid and encapsidate the viral genome are viral protein 1 (VP1), viral protein 2 (VP2), viral protein 3

(VP3), and viral protein 4 (VP4). These proteins are located at the N terminal end of the polypeptide. VP1-VP3 are located on the outer surface the virus capsid, while VP4 is located on the inner surface. The capsid proteins are involved in receptor binding to the host cell through a “canyon” region at the junction of VP1 and VP3 [133, 134, 136, 140]. EV-D68 has been identified as utilizing α2,6 sialic acid and, more recently, ICAM-5 as receptors for binding and entry [110, 127, 131, 132, 193].

Non-Structural Proteins

The 2A protein is a trypsin-like, cysteine protease involved primarily in protein processing [142]. After the polypeptide is generated, 2A undergoes an autocatalytic process in which it cleaves itself at the amino acid junction of

P1 and P2, separating the structural from the non-structural proteins.

Nonstructural proteins are further processed by 3Cpro. Beyond the polypeptide processing, 2Apro has been found to cleave and inactivate a series of host proteins involved in transcription (TATA-box binding protein),

translation (eIF4GI), and innate immunity (MAVS, MDA5, NLRP3) [192,

194-202] .

2B/2BC

2B and its precursor 2BC have been linked to membrane association and formation of the replication organelles [203-205]. They are also involved in membrane permeability and cellular lysis, aiding in lytic egress of the virus.

The 2B protein of EV-71 is capable of suppressing the type 1 interferon response by preventing activation of interferon regulatory factor 3 (IRF-3)

[206].

3A is membrane-associated protein involved in vesicle formation for the replication complexes. 3A expression has been shown to induce membrane association of the ADP-ribosylation factor (Arf) family, which is involved in the formation of membrane vesicles [207, 208].

3AB

3AB is another membrane association protein that functions as an anchor for the replication complex to the replication organelles. 3AB has also been shown to form interaction with the 3CD precursor protein. As mentioned above, 3CD has been shown to bind one of the stem loops of the cloverleaf structure at the start of the 5’ UTR. It is proposed that this interaction anchors the circularized RNA to the membrane vesicle during replication.

As described above, 3B (VPg) is covalently bonded to the 5’ end of the RNA genome. VPg is uridylated by 3D polymerase in conjunction with the cis- acting RNA element (CRE) to form VPg-pUpU [209]. Cre elements are specialized secondary structure in the protein coding region of the viral genome that have two adenosine residues in the loop interact with 3Dpro to add two uridine residues to the tyrosine residue of VPg. The uridylated VPg functions as a primer for initiation of transcription and thus, genome replication.

3C/3CD

3C and its precursor 3CD both function as proteases involved in processing of the initial polypeptide. The initial role of 3C is to cleave the junction between

P2 and P3 [210, 211]. Both 3C and 3CD are involved in secondary cleavage steps. 3CD cleaves the junctions of VP2/VP3 and VP3/VP1 after P1 is processed by 2A. This results in the structural proteins necessary for capsid formation. 3C is subsequently involved in cleavage steps to generate all of the mature non-structural proteins: 2B, 2C, 3A, 3B, 3Cpro (though autocleavage) and 3D. 3C is capable of entering the nucleus via its precursor 3CD, which contains a nuclear localization sequence [212-215]. 3C has been shown to play a role in the cleavage and inhibition of host-cell factors involved in transcription (TATA-binding protein-associated factor 110 (TAF110), TATA box binding protein (TBP), cAMP response element-binding protein-1 (CREB- 19

1), octamer binding protein-1 (Oct-1), p53 and transcription factor IIIC

(TFIIIC), translation (eIF4I, eIF4GI, PABP, PCBP2), and innate immune response (TRIF, MAVS, IRF7)[141, 201, 216, 217].

3D polymerase (3Dpol) is an RdRp that facilitates genome replication. It utilizes uridylated VPg as a primer for the initiation of transcription. While

VPg is necessary for replication in vivo, short primers can be substituted and facilitate initiation of genome replication. The error prone 3Dpol does not have any transcription proofreading mechanism, generally resulting in a quasispecies of viruses during infection [155, 218]. This allows for rapid selection events to take place and supports the evolution of picornaviruses.

An increased fidelity in poliovirus and EV-71 RdRp has been shown to reduce viral fitness in mice [218, 219].

TEMPERATURE SENSITIVITY OF ENTEROVIRUSES

Viruses have been extensively studied with respect to temperature sensitivity [220-228]. Due to the enteric nature of the infection, most enteroviruses are required to replicate at core body temperature.

Enteroviruses viruses known to infect the gut include PV, CBV, and EV-A71 all replicate preferentially at 37°C [229-235]. On the other hand, EV-D68,

RV-A and RV-B are respiratory pathogens by nature and exhibit replication inefficiencies at temperatures greater than 33°C [1, 16, 227, 228, 236-238]. 20

RV-C is associated with severe infections of the lower respiratory tract and has shown similar replication phenotypes at both 34°C and 37°C [239]. One study proposed that increased temperature resulted in degradation of vRNA, reducing the genomic material available for encapsidation during RV-A infection [240]. Some studies have suggested that RVs are attenuated at increased temperatures as a result of cellular upregulation of innate immune factors and apoptotic pathways [227, 228]. Sensitivity to temperature is not only a biological phenomenon of wild-type (wt) viruses, but also a tool for vaccination. Cold adaptation of viruses occurs by serial passaging at low temperatures to induce genomic mutations that drive preferential selection of variants with enhanced replication at low temperatures – which often leads to a loss of replication at higher temperatures. Two widely utilized cold adapted vaccines are the live-attenuated influenza vaccine (commercially known as FluMist) and the poliovirus live-attenuated vaccine [233, 241].

There have also been live-attenuated vaccine development efforts for EV-71

[242-246]. Temperature sensitive mutants of picornaviruses, including PV,

CBV, and EV71 have given insight into genomic regions that contribute to their temperature tolerant phenotypes [247-250]. Mutations in the 5’ UTR , single amino acid changes in VP4, VP3, VP1, and an AA acid change in 3D pol were all found to be contributors to temperature sensitivity in the cold- adapted PV strains [186, 249, 251]. The 3’ UTR was shown to augment 3D pol mediated temperature sensitivity in the Sabin 1 PV vaccine strain [249]. 21

Thus, mutations spanning all regions of the of picornavirus genome have been attributed to an aspect of temperature sensitivity.

FIGURES AND TABLES

Figure 1. 1 AFM cases reported to the CDC from August 2014 to

December 2019. Graph from CDC modified to identify years of increased

EV-D68 incidence (www.cdc.gov/acute-flaccid-myelitis/cases-in- us.html#cases-by-month, accessed March 1, 2020)

Figure 1. 2 Phylogenetic analysis of EV-D68 VP1 identifying (A) the emergence of multiple circulating clades from NextStrain (March 1, 2020) and (B) the geographical distribution of clades.

Figure 1. 3 Picornavirus Virus Life Cycle Schematic taken from Baggen et al. 2018 outlining the steps of the picornarvirus life cycle.

Figure 1. 4 Schematic of Enterovirus D68 genome from Sun, et al. (2016).

Figure 1. 5 Schematic of picornavirus 5’ UTR secondary structure

Secondary structure of the picornavirus 5’ UTR identifying the different domains of conserved hairpin loops and pseudoknots. Taken from Lin, et. al

(2009).

Figure 1. 6 Enterovirus polypeptide processing steps Schematic from

Lin, et al. (2009) of the enterovirus genome, proteolytic processing steps, and the functionality of viral proteins

Year AFM Cases # of States Reporting AFM 2014 120 34 2015 22 17 2016 153 39 2017 38 17 2018 238 42 2019 42 18

Table 1. 1 Reported number AFM cases and number of states reporting AFM in the United States, 2014-2019. Data reported by the

Center for Disease Control on the total number of AFM cases and the number of states that cases were reported each year from 2014 to 2019.

(www.cdc.gov/acute-flaccid-myelitis/cases-in-us.html, accessed March 1, 2020)

CHAPTER 2: CONTEMPORARY

ENTEROVIRUS D68 STRAINS SHOW

ENHANCED REPLICATION AND VIRAL

TRANSLATION AT 37°C

ABSTRACT

Enterovirus D68 (EV-D68) emerged in 2014 as an important pathogen linked to severe lower respiratory disease and acute flaccid myelitis outbreaks.

Historically associated with mild common-cold symptoms, clusters of severe disease attributed to EV-D68 appeared during a series of outbreaks in 2014,

2016, and 2018. Previous studies of historic EV-D68 strains demonstrated attenuated replication at temperatures of the lower respiratory tract (37°C), when compared to the upper respiratory tract (32°C). By testing a panel of historic and contemporary EV-D68 strains at 32°C and 37°C, we demonstrate that contemporary strains of EV-D68 undergo little to no attenuation at increased temperatures. Contemporary strains produced higher levels of viral proteins at 32°C and 37°C than historic strains, although both strains infected similar numbers of cells and had comparable amounts of replication complexes. IRES activity assays with dual-luciferase reporter plasmids demonstrated enhanced translation in recent EV-D68 strains mapped to regions of variability in the 5’ UTR found only in contemporary strains. Using an infectious clone system, we demonstrate that the translation advantage dictated by the 5’ UTR does not solely mediate temperature sensitivity. The strain-dependent effects of temperature on the EV-D68 life cycle gives insight into the susceptibility of the lower respiratory system to contemporary strains.

INTRODUCTION

Human enterovirus D68 (EV-D68) has recently been associated with global outbreaks of severe acute respiratory illness and acute flaccid myelitis.

This positive-sense, single-stranded RNA virus of the Picornaviridae family was first isolated in California from four pediatric patients in 1962. EV-D68 was found to be remarkably similar to human rhinovirus (HRV) in that it was acid labile, temperature sensitive, and transmitted from person-to- person via respiratory droplets [1, 9, 16, 252, 253]. The biological similarities between in EV-D68 and HRV led to an initial misclassification of human rhinovirus 87, now recognized as EV-D68, corrected only by genetic analysis and serum neutralization studies [254, 255]. Between 1970 and 2005, only 26 cases were reported to the CDC and the virus was generally associated with mild upper respiratory illness [2, 256].In 2014, unprecedented outbreaks of

EV-D68 associated with severe respiratory disease and acute flaccid myelitis emerged throughout the world [3, 4, 8, 10-12, 15-17, 19, 23, 24, 28, 32, 76, 77,

114, 125, 257-260]. Since 2014, there have been biannual patterns of severe disease associated with EV-D68 [48, 49, 51, 52, 54-57, 59, 60, 62, 104, 117,

261-267]. This unique pattern of recurring outbreaks and severe pathology sheds light on the importance of understanding evolutionary changes in contemporary EV-D68 strains.

The propensity of a respiratory pathogen to induce severe respiratory distress can be partly linked to its ability to replicate in the lower respiratory 33

tract. The upper respiratory tract is approximately 32°C due to the intake of cool ambient, while the lower airways are around 37°C, closer to core body temperature [268]. When respiratory viruses replicate poorly at 37°C, their infections are often restricted to the upper airways, limiting the potential for damage to the lower respiratory tract and ultimately mitigating disease severity. It is widely accepted that the reason most HRVs cause only mild upper respiratory illness is their preferential replication at cooler physiological temperatures [227, 228, 269].Temperature sensitivity and restriction to the upper airways has been harnessed for vaccine development and is the basis behind the cold-adapted live attenuated influenza vaccine

[270, 271]. Historically, in vitro studies indicated EV-D68 replicates optimally at 33°C and is attenuated at higher physiological temperatures [1,

9, 253]. Given the recent outbreaks of lower respiratory disease associated with EV-D68, our lab sought to investigate EV-D68 temperature sensitivity in the context of historic and contemporary strains.

Picornaviruses have a positive-sense RNA genome approximately

7.5kb long that encodes a single open reading (ORF) that is translated into a single polypeptide. The genome also has 5’ and 3’ untranslated regions

(UTRs) directly adjacent to the ORF. Both UTRs form secondary structure that are functionally involved in protein translation and genome replication.

Picornaviruses utilize an internal ribosomal entry site (IRES) located in the highly structured 5’ untranslated region (UTR) of the viral genome for the 34

initiation of viral protein translation [167, 168]. The IRES facilitates recruitment of host-cell translation machinery and initiation of translation in the absence of a 5’ methylated cap. Numerous studies have identified 5’ UTR mutations that impact IRES-mediated translation efficiency, which can be linked to temperature sensitivity, and neurotropism [167, 186, 187]. While some picornaviruses have been determined to have multiple open reading frames with an additional start codon in the 5’ UTR, this is not the case for

EV-D68 [188, 189]. There has been substantial evolution in the EV-D68 5’

UTR since its initial discovery in 1962, most notably in the form of heightened variability and large deletions in the spacer region flanking the

AUG start codon [190, 191]. Our studies identified a loss of temperature sensitivity in recent EV-D68 strains which was associated with increased translation of the viral genome. While the precise viral regions responsible for temperature sensitivity remain to be delineated, our results suggest the acquisition of efficient replication at 37°C preceded the emergence of EV-D68 as a global pathogen and may help explain its recent altered ability to induce respiratory and nervous system disease.

MATERIALS AND METHODS

Cell culture

Human Rhabdomyosarcoma (RD) cells were ordered from the American Type

Culture Collection (ATCC) (CCL-136) and cultured in Gibco Minimal

Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS),

100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM GlutaMAX (Gibco).

The cells were grown at 37°C in a humidified environment supplemented to

5% CO2.

Viruses

F02-3607 Corn (Corn-1963), US/MO/14-18947 (MO-2014), US/KY/14-18953,

US/IL/14-18952 and AY426531 (Fermon-1962) were obtained from ATCC.

USA/N0051U5/2012 (TN-2012) and US/MO/14-18949 (MO-2014/2) were provided by Dr. Suman Das of Vanderbilt University. Virus stocks were generated by inoculating a confluent T75 or T150 flask of RD cells at a multiplicity of infection (MOI) of 0.01 50% tissue culture infectious doses

(TCID50) per cell for 1 hour at 32°C with rocking every 10-15 minutes. Virus inoculum was aspirated and replaced with infection media (IM- 2.5% FBS,

100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM GlutaMAX [Gibco]) followed by incubation at 32°C. After 24 hours of infection, initial IM was aspirated and replaced with fresh IM. When complete cytopathic effect (CPE) could be determined using a light microscope (3-5 days), the infected cells and supernatant were harvested, subjected to 2 freeze thaw cycles, and centrifuged at 600 x g for 10 minutes to remove all cell debris. The clarified supernatant was aliquoted and stored at -70C. Virus stock titers were determined using TCID50 assays described below.

50% Tissue culture infectious dose (TCID50) assay

RD cells were plated in 96-well plates, grown to full confluence, and washed with PBS+. Tenfold serial dilutions of the virus inoculum were made and 200

μL of dilution was added to each of 6 wells in the plate, followed by incubation at 32°C or 37°C for 5-7 days. Cells were then fixed by adding 100

μL of 4% formaldehyde in PBS per well and incubated at room temperature for at least 10 minutes before staining with Naphthol Blue Black solution.

TCID50 calculations were determined by the Reed-Muench method [272].

Plaque assays

Plaque assays were performed using 6-well plates of confluent RD cells. Serial 10-fold dilutions of the virus inoculum were generated in IM.

Cells were washed twice with PBS containing calcium and magnesium

(PBS+) and 200 μL of inoculum was added per well. The cells were incubated for 1 hour at 32°C with rocking every 10-15 minutes. The inoculum was removed and replaced with IM containing 1% agarose. Cells were then incubated at 32°C or 37°C for 5-7 days, fixed in 2% formaldehyde, and stained in a Naphthol Blue Black solution.

Growth Curves

Multistep growth curves were performed on RD cells at an MOI of

0.001. Virus stocks were diluted to the appropriate concentrations in IM.

Cells were inoculated with virus and incubated at 32°C with rocking every

10-15 minutes. Cells were then washed 2 times with PBS+ and incubated 37

with fresh IM at 32 or 37°C. At the time points indicated, all supernatant was harvested and stored at -80°C. Infectious virus quantification at each time point was determined using TCID50 assays.

Flow Cytometry

RD cells were washed with PBS+ and infected at an MOI of 5 for 1 hour at 32°C with rocking every 10-15 minutes. Infected cells were allowed to incubate for an additional 8 hours at 32°C or 37°C, at which time they were washed twice with PBS-, detached using trypsin, and fixed for 15 minutes at room temperature with 2% paraformaldehyde (PFA) in PBS. Cells were permeabilized with 0.2% Triton X-100 (Sigma) in PBS, blocked for 1 h in PBS with 3% normal goat serum and 0.5% bovine serum albumin (BSA; Sigma), and incubated for 1 h with anti-VP1 rabbit serum (1:200; GTX132313,

Genetex) or dsRNA mAb (1:200; J2 anti-dsRNA IgG2a, Scicons), followed by incubation for 1 h with goat anti-rabbit AF647 (1:400; A-21224, Molecular

Probes) or goat anti-mouse AF488 (1:200, A-11029, Molecular Probes). The cells were analyzed by flow cytometry (BD FACSCalibur) and data analyzed using FlowJo software.

Phylogenetic Analysis and RNA secondary structure

Evolutionary trees were generated with all available EV-D68 complete genome sequences (408 sequences, December 11, 2017) from NCBI using entire genomes or 5’ UTRs only. Trees were generated using the Maximum-

Likelihood tree analysis with 500 bootstrap replicates from MEGA-X software. Alignments shown were performed using Geneious 8.1.9 software.

Predicted RNA secondary structure for each domain was determined using both RNAstructure 6.1 and Geneious 8.1 software and referencing that of CBV [46]. The structures were visualized and edited using RNAstructure

Structure Editor.

IRES Activity Assay

Dual-luciferase plasmids were obtained from Genscript which used a

CMV promoter to express and mRNA with the renilla luciferase gene, followed by the EV-D68 5’ UTR and firefly luciferase. RD cells were grown to

85-90% confluence in opaque 96-well plates overnight and plasmids were transfected into the cells for 4 hours at 37°C using TransIT-LT1 transfection reagent and incubated overnight at 32°C. The cells were then left at 32°C or moved to 37°C for 9 hours before read on a luminometer using the Dual-

Luciferase Reporter Assay Step (Promega) according to manufacturer’s instructions. IRES activity was determined as the ratio of Firefly luciferase to

Renilla luciferase.

Infectious Clone System

Recombinant Corn-1963, MO-2014, and 5’ UTR chimeras were generated using sequences available in GenBank. pUC57 vectors encoding a T7 transcription site followed by the full-length genome of interest, a 30-35 base poly-A tail, and ClaI restriction site for linearization were ordered from 39

Genscript (Piscataway, NJ). Plasmids were fully linearized by incubation at

37°C for a minimum of 2 hours using ClaI restriction enzyme (New England

Biolabs). Full-length genomic RNA was transcribed from the linearized plasmids using the MegaScript T7 Kit (Ambion). 4µg of transcribed RNA was mixed with DMRIE-C (Invitrogen) transfection reagent (1:2 µg:µL ratio) in

Opti-MEM (Gibco), serial diluted in 4-fold increments (1µg to 0.016µg RNA final), and added to 85-90% confluent RD cells in 12-well plates for 2-4 hours at 37°C followed by an overlay of IM containing 1% agarose. The plates were incubated for up to 7 days with daily monitoring for plaques. Upon appearance, plaques were picked and harvested in 500 µL of IM and used to generate seed stocks on RD cells.

SDS-PAGE and western blotting

RD cells were infected at a high MOI for 1 hour at 32°C with rocking every 10-15 minutes and allowed to incubate for an additional 8 hours. Cells were washed twice with PBS and lysed using 1% SDS in PBS. Cell lysates were sonicated and clarified of any remaining cellular debris by centrifugation. Samples were mixed with 4X Laemmli buffer (Bio-Rad) containing dithiothreitol (DTT; ThermoFisher) and denatured at 100°C for 5 min. Samples were loaded alongside Precision Plus Protein Standards All

Blue protein ladder (Biorad) into a 4-20% Mini-PROTEAN TGX Gel (Biorad) and run at 100 V for approximately 1 hour. Proteins were transferred to a

PDVF membrane for 1 hour at 100V and blocked for 1 hour (5% blotting- 40

grade blocker (Bio-Rad) in PBS containing 0.05% Tween 20). The membrane was stained for 1 h with mouse anti-VP3 (1:1000; GTX633706, Genetex) or mouse anti-β-actin (1:10000; Abcam), followed by staining for 1 h with anti- mouse AF647 (1:1000; A-28181, Molecular Probes). The membrane was washed 3 times for 5 minutes with PBS containing 0.05% Tween 20 with rocking between each step. Protein expression was analyzed using a

FluorChem Q system. Cellular expression of viral proteins was normalized by

β-actin expression.

Statistical analyses

Replication kinetics of low MOI growth curves were analyzed by two- way ANOVA. TCID50 assays, FACS data, and IRES activity assays were analyzed using unpaired or multiple t-tests. All statistical analyses were performed in GraphPad Prism 8.0.1.

RESULTS

Effect of physiological temperature ranges on historic and contemporary EV-D68 strain infectious virus production

Historic strains of EV-D68 were previously shown to have optimal growth temperatures at 33°C and undergo attenuated growth at 37°C [1, 9,

253]. Given the recent rise in acute respiratory illness correlating with EV-

D68, we tested a panel of historic and contemporary strains to determine if temperature sensitivity phenotypes differ between historic and contemporary 41

strains. In agreement with the literature, historic strains of EV-D68 were attenuated at 37°C compared to 32°C (Fig. 2.1A), with TCID50 values reduced by 1000 to 100,000 fold. However, contemporary strains showed no to at most a 20 fold reduction in infectious virus titer when TCID50 assays are performed at 32°C or 37°C (Fig. 2.1A). When analyzing plaque assays incubated at 32°C or 37°C, contemporary strains showed more plaques of greater size at 37°C when compared to historic strains (Fig. 2.1B). In low

MOI growth curves on RD cells (Fig. 2.1C), the Corn-1963 strain showed reduced infectious virus production at 37°C, while the MO-2014 produced equivalent infectious virus titers at both temperatures. At 32°C, MO-2014 produced higher infectious virus titers compared to Corn-1963, suggesting

MO-2014 had improved replication at 32°C compared to Corn-1963. The data suggest contemporary strains of EV-D68 are better able to replicate at higher physiological temperatures than historic strains.

Translation efficiency of contemporary and historic EV-D68 strains at physiological temperature ranges

To investigate the mechanisms controlling temperature restriction of

EV-D68, we selected temperature sensitive Corn-1963 (historic) and temperature tolerant MO-2014 (contemporary) strains to delineate which stages of the EV-D68 life cycle were affected by temperature. After infecting

RD cells (MOI of 5) for 9 hours at 32°C or 37°C, we detected viral RNA replication complexes using an antibody specific to dsRNA and viral protein 42

using an antibody specific for EV-D68 viral capsid protein 1 (VP1). Using flow cytometry for analysis, both strains showed similar numbers of infected cells at both temperatures as detected by dsRNA positive cells (Fig. 2.2A) and nearly all of these infections were generating detectible viral protein (Fig.

2.2B). Mean fluorescence intensity was used to quantify relative amounts of dsRNA and VP1 on a per cell basis. The MFI of dsRNA was also similar between strains (Fig. 2.2C). However, there were significant differences in

VP1 expression with the MO-2014 showing higher protein production at 32°C and 37°C compared to Corn-1963 (Fig. 2.2D). The differences in translation were further confirmed using a western blot assay and a monoclonal antibody to EV-D68 viral capsid protein 3 (VP3), which demonstrated increased VP3 at both 32°C and 37°C in MO-2014 infected cells (Fig. 2.2F). Based on the data, we inferred that while Corn-1963 and MO-2014 infected similar numbers of

RD cells at either temperature, MO-2014 produced significantly more protein at both 32°C and 37°C.

Assessment of IRES activity from strains ranging from 1963 to 2016

Since MO-2014 showed enhanced translation efficiency at 32°C and

37°C compared to the Corn-1963, we wanted to determine if IRES activity differed across a historical panel of virus strains. Sequences available on

GenBank were selected that spanned all clades and were analyzed by phylogenetic analysis of either the IRES (Fig. 2.3A) or full genome (Fig.

2.3B). Representative 5’ UTR sequences from various EV-D68 strains were 43

chosen for IRES activity analysis using a dual-luciferase plasmid encoding renilla luciferase ahead of the 5’ UTR of interest, followed by firefly luciferase. These plasmids were transfected into RD cells and IRES activity levels were determined at both 32°C and 37°C for each strain. At 32°C, all strains from 1999 or later had significantly higher levels of IRES activity (Fig

2.3C). At 37°C, five of the strains were significantly higher than Corn-1963, including three clade B strains from recent outbreaks, WY-2014, MO-2014, and MO-2016 (Fig. 2.3D). The data indicate that the WY-2014, MO-2014 and

MO-2016 5’ UTRs appear to have much greater ability to mediate translation at either 32°C or 37°C when compared to many historical EV-D68 strains.

Genomic and structural comparison of Corn-1963 and MO-2014 and effects on IRES activity

We further analyzed the genomic and structural components of the 5’

UTR by aligning the corresponding sequences from Corn-1963 and MO-2014.

The 5’ UTRs of Corn-1963 and MO-2014 share 83% homology with sporadic mutations that span the entire 5’ UTR. However, (Fig. 2.4A), there is a concentrated region of variation in bases 629-733 adjacent to the start codon, including a 35 base deletion previously reported to be in all contemporary

EV-D68 strains. Of the 733 base 5’ UTR, 50% of the sequence variation is found in this variable region. The sequence variation between Corn-1963 and

MO-2014 affect the predicted secondary structure (Fig. 2.4B) by inducing the formation of a stem loop structure (VII) in MO-2014 compared to Corn-1963. 44

Using the dual-luciferase plasmid assay, we analyzed the contribution to

IRES activity of both the 35 base deletion and the entire variable region of the 5’ UTR (Fig. 2.4C). Deleting the 35 bases in Corn-1963 increased IRES activity slightly. However, replacing the entire variable region with that of

MO-2014 increased IRES activity, nearing levels of MO-2014. Adding in 35 bases to MO-2014 increased IRES activity, but replacing the entire variable region with that of Corn-1963 resulted in a significant decrease in IRES activity. While the observed trends were detected at both 32°C and 37°C, the advantage of MO-2014 and its variable region penetrated more at 37°C. In fact, the ratio of IRES activity (MO-2014:Corn-1963) was significantly higher at 37°C, suggesting an advantage to MO-2014 at 37°C (Fig. 2.4D). Taken together, the enhanced IRES activity of MO-2014 is largely mediated by the

5’ UTR variable region which is more advantageous at 37°C.

Replication of Corn-1963 and MO-2014 5’ UTR chimeric viruses at

32°C and 37°C.

Because our IRES-activity assay indicated increased translation efficiency for MO-2014 that penetrated more strongly at 37°C, we generated chimeric viruses to test the influence of the 5’ UTRs on virus replication at

32°C and 37°C. TCID50 assays were performed at 32°C and 37°C on chimeric virus working stocks. The recombinant Corn-1963 (rCorn-1963) and MO-2014

(rMO-2014) viruses showed the same temperature sensitivity patterns as the natural isolates they were derived from (Fig. 5A). Surprisingly, replacing 45

either the variable region or the entire 5’ UTR of either virus with that of the other had no effect on temperature sensitivity (Fig. 2.5A). Because TCID50 assays represent an endpoint assay, the effect of the 5’ UTR swaps were assessed in low MOI growth curves. Again, replacement of the variable region alone or the entire 5’ UTR had no effect on temperature dependent infectious virus production (Fig. 2.5B). Given that there was no change in the temperature sensitivity phenotypes involving infectious virus production, we determined the translation efficiency of the recombinant viruses using

Western blotting for VP3. When the rCorn-1963 5’ UTR was replaced with that of rMO-2014, translation was increased (Fig. 2.5C; 32 to 57 at 32°C and

17 to 31 at 37°C) and the reciprocal swap resulted in a reduction of translation (Fig. 2.5C;117 to 42 at 32°C and 73 to 45 at 37°C). Replacing only the variable regions had a minimal impact on rCorn-1963, however, rMO-

2014 lost translation efficiency when replaced with that of rCorn-1963. Taken together, the data demonstrate that that the 5’ UTR of MO-2014 increases viral protein translation in a recombinant virus when compared to the 5’ UTR of rCorn-1963. However, the 5’ UTR-mediated increase in translation efficiency is not the determining factor for EV-D68 temperature sensitive infectious virus production.

DISCUSSION

EV-D68 has previously been shown to have an optimal growth temperature of 32°C compared to 37°C [1, 9, 253]. This temperature range is important because it represents the temperature of the human upper respiratory airways (32°C), which are cooled by inhalation of ambient air, and the lower respiratory airways (37°C), closer to core body temperature.

Respiratory viruses incapable of replicating at higher physiological temperatures have correlated with mildly symptomatic infections of the upper respiratory tract [268]. HRV A and HRV B are two viruses with attenuated replication at higher physiological temperatures and are best known for causing common-cold symptoms [227, 228]. The recently discovered HRV C is not attenuated at 37°C and is associated with severe infections of the lower airways [273-277]. In EV-D68 outbreaks (2014, 2016,

2018), there have been correlations to increased acute respiratory illness

(ARI) [4, 13, 14, 24, 58, 87, 278, 279]. Our data indicate that contemporary strains of EV-D68 are better fit to replicate at 37°C than historic strains.

Extrapolation of this data suggests that temperature is no barrier for the viruses to infect both upper and lower airways of the human respiratory tract. Additionally, while recent studies have given insight into the ability of

EV-D68 to infect neuronal cell lines in vitro and in animal models as it pertains to receptors, the mechanisms by which EV-D68 infects the central nervous system to cause AFM remain unclear [95, 127-130, 280]. Other 47

picornaviruses capable of causing AFM such as poliovirus, EV-A71, and CVB all replicate at high physiological temperatures. While temperature tolerance alone will not allow neuronal infections, our data suggests that contemporary strains of EV-D68 have overcome this barrier to efficient replication.

Picornaviruses utilize an internal ribosomal entry site (IRES) element located in the 5’ UTR of the virus genome for the initiation of viral protein translation. There have been various studies outlining the impacts of picornavirus 5’ UTR mutations and their impactions of IRES-mediated translation efficiency. While some picornaviruses have been determined to have multiple start codons, this is not the case for EV-D68 [188, 189].There has been substantial evolution in the EV-D68 5’ UTR since its initial discovery in 1962. Comparing the genetic composition, there is 83% 5’ UTR sequence homology between our representative historic and contemporary strains and these differences do impact the overall predicted secondary structure (Fig. 4A and B). In addition to sporadic mutations throughout the 5’

UTR, there is a concentrated area of variability, including a 35 base deletion just prior to the translation start codon that is found in all contemporary EV-

D68 strains. Due to the lack of sequences prior to the early 2000s, it is unknown when this sequence divergence took place. There is mounting evidence of EV-D68 recombination events that could potentially explain the heightened diversity in this specific region of the 5’ UTR [124]. Our dual- luciferase plasmid system gave the ability to test the impact of the 5’ UTR 48

variability on translation by generating chimeric plasmids. Our data indicate that contemporary strains of EV-D68 have acquired increased translation efficiency driven by evolution in the 5’ UTR. The 35 base deletion did not have much of an effect on the historical strains translation efficiency, however, incorporation of the entire region of high variability increased translation significantly (Fig. 4C). Predictive secondary modeling suggest that the variable region of the contemporary strains lead to an additional stem-loop after domain VI (Fig. 4A). The effects of additional secondary structure in this region remains unknown, but could play a role in recruitment of translation initiation factors.

While our data suggest that contemporary strains of EV-D68 have a translation efficiency advantage over historic strains based on our bicistronic reporter assays, chimeric viruses were necessary to determine the impact it may have on temperature sensitivity and replication kinetics. Swapping the variable region or the entire 5’ UTRs of Corn-1963 and MO-2014 did not phenotypically change their temperature sensitivity. That is, Corn-1963 encompassing components of MO-2014 remained attenuated at 37°C, while

MO-2014 with the 5’ UTR of Corn-1963 remained temperature tolerant (Fig.

5A and 5B). We confirmed that the 5’ UTR swaps do alter translation efficiency (Fig. 5C), however, with no notable effects on temperature sensitivity or replication kinetics (Fig. 5D). There are many other benefits that may arise by increased translation. Picornaviruses, including EV-D68, 49

have been shown to utilize their encoded proteases to manipulate the host- cell immune function [216, 281]. Increased translation efficiency and the generation of more of these proteases could allow for a more robust immune evasion.

Our results suggest that determinants outside the 5’ UTR control temperature depending infectious virus production. Recent literature suggests that EV-D68 temperature sensitivity may be linked to the viral capsid protein VP1 [253]. Specifically, by taking a temperature tolerant EV-

D94 virus and swapping the VP1 with that of the prototype EV-D68 strain

(Fermon-1962), the virus becomes temperature sensitive. While interesting, the reverse swap (EV-D94 VP1 into EV-D68) was not able to be rescued, and therefore a temperature sensitivity phenotype swap for EV-D68 has not yet been successful. Generating a full panel of genomic swaps between the representative strains used in our studies investigating the role of structural and non-structural genes would lend insight into the role of their role temperature sensitivity. Additionally, while the 5’ UTR contains the IRES element, the 3’ UTR for picornaviruses also contains secondary structure and has been shown to influence translation and replication [142, 156, 282, 283].

While extensive studies have not been done previously for EV-D68 3’ UTR, one can postulate that similar interactions may occur between its 5’ and 3’

UTRs. Including an assessment of the 3’ UTR in chimeric viruses may lend

insight into its involvement in temperature sensitive infectious virus production.

The data from these studies provide a functional basis for the genetic evolution that has been reported for the 5’ UTR of contemporary EV-D68 strains. Additionally, all contemporary strains of EV-D68 tolerate replication at 37°C better than historic strains. This provides evidence that contemporary strains may have increased their ability to infect the lower respiratory tract. Continued research into the mechanism of contemporary strain temperature tolerance is essential for a full understanding of recent severe EV-D68 outbreaks. Given the continued biannual patterns associated with severe disease since 2014, understanding all potential contributors is essential.

ACKNOWLEDGEMENTS

We would like to thank Dr. Suman Das at Vanderbilt University for kindly providing TN-2012 and MO-2014/2 strains. Thank you to Dr.

Katherine Fenstermacher and Dr. Elizabeth Troisi for their initial work with our EV-D68 virus stocks and Alysha Ellison for her help with generating rMO-2014. We thank Harrison Powell of the Pekosz laboratory for his support with flow cytometry. We would like to thank members of the Pekosz laboratory, Dr. Sabra Klein and members of the Klein laboratory, and Dr.

Kimberly Davis and members of the Davis laboratory for their critical 51

discussions of the data. This work was supported by HHSN272201400007C

(AP) and T32 AI007417 (BS).

FIGURES

Figure 2. 1 The effect of physiological temperature ranges on EV-D68 infectious virus production. (A) Infectious virus titers were determined with various EV-D68 virus strains at 32°C and 37°C using (A) TCID50 or (B) plaque assays. (C) RD cells were infected at an MOI of 0.01 and infectious virus titers (TCID50/mL) in the cell supernatants were determined at various times post infection. Statistical significance determined by unpaired t-test

(TCID50) or 2-way ANOVA (growth curves) with *p<0.05, ***p<0.001,

****p<0.0001. The data shown are pooled from 3 experiments performed in triplicate for each experiment.

Figure 2. 2 The effect of physiological temperature ranges on EV-D68 mRNA translation efficiency. RD cells were infected with Corn-1963 or

MO-2014, or Mock (infection media) for 9 hours at an MOI of 5 and measured by flow cytometry for: (A) Percentage of infected cells (double-stranded RNA positive), (B) percentage of infected cells (gated to dsRNA+ cells) actively producing VP1 (VP1+), (C) relative amount of dsRNA in infected cells, measured by mean fluorescence intensity, and (D) relative amount of VP1 in infected cells. (E) EV-D68 genome copies were determined in virus infected cells by RT-qPCR. (F) Representative data of β-actin (loading control) and

VP3 protein amounts analyzed by western blot in cell lysates generated at 9 hours post infection (MOI=5). VP3 band intensity was quantified using

FluorChem Q and normalized to the intensity of the corresponding β-actin band (ND=not detected). Statistical significance determined by unpaired t- tests with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The data shown are pooled from 2-3 experiments performed in triplicate for each experiment.

Figure 2. 3 Changes in EV-D68 IRES activity from 1963 to 2016.

Phylogenetic trees generated with all available EV-D68 complete genome sequences (408 sequences, December 11, 2017) from NCBI using (A) the entire genome or (B) 5’ UTR only. Trees were generated using the Maximum-

Likelihood tree analysis with 500 bootstrap replicates from MEGA-X software. Various 5’ UTR sequences from historical EV-D68 sequences were selected (phylogeny indicated by red arrows on trees) and IRES activity analyzed at (C) 32°C or (D) 37°C using a dual-luciferase plasmid reporting system encoding the 5’ UTR EV-D68 sequences driving firefly luciferase activity (IRES mediated) compared to renilla luciferase activity (CMV- promotor mediated). Relative expression of firefly to renilla luciferase activity is graphed. Statistical significance determined by one-way ANOVA to Corn-

1963 with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The data shown are pooled from 3 experiments performed in triplicate for each experiment.

Figure 2. 4 Comparison of Corn-1963 and MO-2014 IRES genetic features and effects on IRES activity. (A) Sequence alignment of Corn-

1963 and MO-2014 5’ UTR (conserved sequence black, non-conserved in grey) identifies a 104 nt region of high variability (red box), including a 35 base deletion present in contemporary EV-D68 strains. (B) Predicted secondary structure of Corn-1963 and MO-2014 determined using RNAstructure 6.1 59

and Geneious 8.1.9 and visualized using RNAstructure StructureEditor software highlighting the region of high variability (red), point mutations

(blue), and deletion (green) of MO-2014. (C) IRES activity measured using a dual-luciferase reporter system at 32°C (left) and 37°C (right) for Corn-1963

5’ UTR (blue), MO-2014 (green) and the chimeric 5’ UTRs. (D) Ratios of MO-

2014 and Corn-1963 IRES activities were determined at 32°C (black) and

37°C (grey). Statistical significance determined by multiple or unpaired t- tests with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The data shown are pooled from 3 experiments performed in triplicate for each experiment.

Figure 2. 5 Effects of Corn-1963 and MO-2014 IRES chimeras on replication and viral protein translation. (A) Infectious virus titers were determined with Corn-1963 and MO-2014 chimeric IRES viruses at 32°C and

37°C using TCID50 assays. (B) RD cells were infected at an MOI of 0.01 and infectious virus titers (TCID50/mL) in the cell supernatants were determined at various time points post infection. (C) Representative data of β-actin

(loading control) and VP3 protein amounts analyzed by western blot in cell lysates generated at 9 hours post infection (MOI=5). VP3 band intensity was quantified using FluorChem Q and normalized to the intensity of the corresponding β-actin band. Statistical significance determined by unpaired t- tests (TCID50) or 2-way ANOVA (growth curves) with *p<0.05, **p<0.01,

***p<0.001, ****p<0.0001. The data shown are for 2-3 experiments performed in triplicate for each experiment.

CHAPTER 3: STRUCTURAL AND NON-

STRUCTURAL GENES CONTRIBUTE TO

ENTEROVIRUS D68 TEMPERATURE

SENSITIVITY

ABSTRACT

Enterovirus D68 (EV-D68) has been linked to a series of clusters of acute respiratory illness in recent years, most notably in 2014. Prior the global outbreak of 2014, EV-D68 cases were rarely reported and most often associated with mild upper respiratory infections. Previous studies of historic

EV-D68 strains demonstrated attenuated replication at temperatures of the lower respiratory tract (37°C), when compared to the upper respiratory tract

(32°C). Studies in our lab indicated that contemporary strains of EV-D68 undergo little to no attenuation at increased physiological temperatures.

Using an infectious clone system to generate chimeric viruses between the historic and contemporary strains, we performed systematic swaps of functional genomic regions to determine their role in temperature sensitivity.

From these chimeras, we found that regions of both structural and non- structural regions impacted the ability of EV-D68 to replicate at increased physiological temperatures in a strain dependent manner. While the temperature sensitive historic strain showed partial rescues at 37°C with contemporary structural or non-structural genes encoded, the temperature tolerant contemporary strain showed no sensitivity when encoding historic structural genes and complete inhibition when encoding the historic non- structural genes at 37°C. A contemporary strain with historic 5’ and 3’ UTRs or 3’ UTR alone showed no reduction in its ability to replicate at high temperatures. Taken together with work done previously in our lab on the 5’ 64

UTR alone, the structural and non-structural genes play a role in EV-D68 temperature sensitivity. This work lends insight into the biological implications of EV-D68 evolution that may contribute to expanded tropism of contemporary strains to the lower respiratory tract.

INTRODUCTION

Enterovirus D68 (EV-D68) is a respiratory pathogen in the

Picornaviridae family recently linked to outbreaks of severe acute respiratory illness (ARI) and acute flaccid myelitis (AFM). Picornaviruses are small non- enveloped viruses with icosahedral capsids and positive-sense RNA genomes.

Within the picornavirus family is the genus Enteroviruses, which includes the species enterovirus (EV) A-J and rhinovirus (RV) A-C. While EV-D68 was originally found to be biologically similar to RVs in its respiratory droplet transmission, respiratory tract tropism and illness, acid lability, and temperature sensitivity, it was found to genetically cluster with enteroviruses

A-C [9].

Previous studies in our laboratory have indicated that contemporary strains of EV-D68 are significantly less attenuated at increased physiological temperatures when compared to historic strains (described in Chapter 2). The importance of temperature sensitivity in the context of respiratory viruses is based on the respiratory tract temperature gradient of 32°C (upper respiratory) to 37°C (lower respiratory) that serves as one biological barrier 65

of virus tropism for the lower airways. Rhinovirus A (RV-A), rhinovirus B

(RV-B), and the live-attenuated influenza vaccine (LAIV) are examples of temperature sensitive respiratory viruses that cause mild or asymptomatic upper respiratory infections [227, 237, 271]. Rhinovirus C (RV-C) and avian influenza (H5N1) both replicate at 37°C and are associated with severe lower respiratory disease [239, 275, 277, 284]. While viral tropism can depend on other factors such as cellular receptors and immune suppression, temperature is one biological barrier to overcome [285-287].

Several picornaviruses, including poliovirus (PV), RVs, coxsackie B virus (CBV), and enterovirus A71 (EV-A71), have been extensively researched with respect to temperature sensitivity. PV, CBV, and EV-A71 are all fecal-orally transmitted and cause enteric infections, requiring tolerance for replication at high temperatures and low-pH. While the wild-type (wt) viruses lend little insight into the genetic contributions to their temperature tolerance, vaccine efforts to generate temperature sensitive live-attenuated viruses have given virus and strain-dependent mutations that lead to temperatures sensitivity [233, 242, 246, 288-290]. Analysis of the Sabin 1 temperature sensitive PV vaccine determined mutations in VP4, VP3, and

VP1 structural proteins and the non-structural 3Dpol all contributed to temperature sensitivity in vitro [249, 291]. An additional mutation in the 3’

UTR in combination with the 3Dpol mutations further augmented the growth defect at higher temperatures [249]. A temperature sensitive EV-A71 strain 66

was found to have a phenotype mainly derived from a mutation in VP1.

Additionally, incorporations of temperature sensitive mutations from the

Sabin 1 could also confer sensitivity in this EV-A71 strain [242].

Analysis of the contribution of individual or multiple mutations of within temperature sensitive viruses can be accomplished by employing reverse genetics to either introduce mutations into the wild-type, or generate revertants from the temperatures sensitive strains. There has been one recent study that focused on structural protein contributions of EV-D68 temperature sensitivity, however, these studies focused on genomic exchanges between a temperature sensitive EV-D68 strain and a temperature tolerant EV-D94 (another member of the EV-D species) [253].

While this group showed exchange of the historic EV-D68 strain VP1 caused temperature sensitivity for EV-D94, they were unable to rescue the opposite chimera. Thus, the genetic contributor to EV-D68 temperature sensitivity remains to be defined.

Given the temperature tolerance phenotype of contemporary strains of

EV-D68 from our previous research, these studies focus on chimeric viruses with genetic exchanges between two EV-D68 strains with opposing temperature sensitivity phenotypes. Functional region swaps of the strains including structural and non-structural gene segments and the 5’ and 3’

UTRs will address regions important to EV-D68 evolution conferring temperature tolerance in contemporary strains. Insight into the genetic 67

contributors of EV-D68 temperature sensitivity could shed light on the evolution of contemporary strains and their association with ARI.

MATERIALS AND METHODS

Cell Culture

Human Rhabdomyosarcoma (RD) cells were ordered from ATCC (CCL-136) and cultured in Gibco Minimal Essential Medium (MEM) supplemented with

10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM GlutaMAX (Gibco) at 37°C.

Infectious Clone System

Recombinant Corn-1963 (rCorn-1963), MO-2014 (rMO-2014), and functional region chimeras swapping 3’ UTR (rCornMO3’, rMOcorn3’), 5’ UTR and 3’

UTR (rCorn5’3’, rMO5’3’), structural genes (rCornStr, rMOcornStr), and non- structural genes (rCornMOstr, rMOcornNS) were generated using sequences available in GenBank. pUC57 vectors encoding a T7 transcription site followed by the full-length genome of interest, a 30-35 base poly-A tail, and

ClaI restriction site for linearization were ordered from Genscript

(Piscataway, NJ). Plasmids were fully linearized by incubation at 37°C for a minimum of 2 hours using ClaI restriction enzyme (New England Biolabs).

Full length genomic RNA was transcribed from the linearized plasmids using the MegaScript T7 Kit (Ambion). 4ug of transcribed RNA was mixed with 68

DMRIE-C (Invitrogen) transfection reagent (1:2 ug:uL ratio) in Opti-MEM

(Gibco), serial diluted in 4-fold increments (1ug to 0.016ug RNA final), and added to 85-90% confluent RD cells in 12-well plates for 2-4 hours at 37°C followed by an overlay of IM containing 1% agarose. The plates were incubated for up to 7 days with daily monitoring for plaques. Upon appearance, plaques were picked and harvested in 500 uL of IM and used to generate seed stocks on RD cells.

50% tissue culture infectious dose (TCID50) assay

RD cells were plated in 96-well plates, grown to confluence, and washed with

PBS+. Tenfold serial dilutions of the virus inoculum were made and 200 μL of dilution was added to each of 6 wells in the plate, followed by incubation at

32°C or 37°C for 5-7 days. Cells were then fixed by adding 100 μL of 4% formaldehyde in PBS per well and stained with Naphthol Blue Black. TCID50 calculations were determined by the Reed-Muench method [272].

Statistical analyses

Statistical significance for TCID50s determined by unpaired t-tests between

32°C and 37°C counterpart. All statistical analyses were performed in

GraphPad Prism 8.

RESULTS

Sequence analysis of historic Corn-1963 and contemporary MO-2014 indicate substantial evolution that spans the entire genome

Previous work in our laboratory evaluated the role of the 5’ UTR in

EV-D68 temperature sensitivity by generating chimeric viruses using our infectious clone system (Fig. 3.1) adapted from previously published poliovirus and enterovirus A71 protocols [292-294]. While contemporary strains replicated significantly better than historic strains at 37°C and were more efficient at translating viral proteins, chimeric swaps of the 5’ UTR yielded no evidence that the 5’ UTR alone impacts temperature sensitivity

(described in Chapter 2 of this dissertation). That is, replacing the 5’ UTR of

Corn-1963 (historic; temperature sensitive) with that of MO-2014

(contemporary; temperature tolerant), there was no change in the attenuation incurred at 37°C. In the opposite direction, replacing the 5’ UTR of MO-2014 with that of Corn-1963, there was no change in temperature tolerance at 37°C. Altogether, this suggested that temperature sensitivity is likely a phenotype derived downstream of the 5’ UTR or a combination of the

5’ UTR with other genomic regions.

An extensive sequence analysis of Corn-1963 and MO-2014 show 937 nucleotide (nt) changes (87.3% nt homology) conferring 88 amino acid (aa) changes (95.9% AA homology) that span all structural and non-structural proteins, with the exception of the conserved virus capsid protein 4 (VP4) and 70

3B (VPg) (Table 3.1). The virus capsid proteins that make up the outer shell

(VP1, VP2, VP3) have substantially greater AA changes than the non- structural proteins, likely owing to selection pressure from neutralizing antibodies over years of evolution. None of these structural or non-structural

AA changes have been assessed for their biological impacts on viral replication, which leave them all as candidates for contributors to temperature sensitivity. In addition to the 5’ UTR variability previously described, there are 8 nt changes in the 3’ UTR, a genomic region that interacts with the 5’ UTR and associated replication proteins upon genome circularization and a reported association with poliovirus temperature sensitivity [249]. There have been no reports focused on EV-D68 3’ UTR evolution and the potential impacts of these mutations.

Swapping Functional Regions of Historic and Contemporary EV-D68

Strains

To assess the contributions different genomic regions lend to temperature sensitivity, we developed a full panel of plasmids encoding functional genomic region swaps. Plasmids encoding full EV-D68 wild-type genomes (rCorn-1963, rMO-2014) were used to generate chimeras swapping the 3’ UTR (rCornMO3’, rMOcorn3’), both the 5’ UTR and 3’ UTR

(rCornMO5’3’, rMOcorn5’3’), structural genes (CornMOstr, MOcornStr), and non-structural genes (CornMOns, MOcornNS) (Fig. 3.2). These plasmids were used to generate recombinant virus utilizing our infectious clone system 71

described above. All viruses were rescued using our infectious clone system, with the exception of CornMO3’ and CornMO5’3’, and used for analysis of temperature sensitivity phenotypes. Multiple attempts of rescuing CornMO3’ and CornMO5’3’ yielded no detectable virus, suggesting potential genomic incompatibility of the 3’ UTR with MO-2014.

Corn-1963 partially overcomes temperature sensitivity with genomic swap of contemporary non-structural genes

rCorn-1963 chimeric viruses were analyzed for their ability to replicate at both 32°C and 37°C. TCID50s were performed using working stocks of the functional region chimeras (Fig 3.3). As previously described, rCorn1963 was significantly attenuated at 37°C compared to 32°C. At 37°C, there is no detectable infectious virus, indicating a temperature sensitive phenotype.

CornMO3’ and CornMO5’3’ was unable to rescued, thus unable to generate data at 32°C or 37°C, as indicated on the figure. CornMOstr was significantly attenuated at 37°C compared to 32°C. Similar to rCorn-1963, there was no detectable virus at 37°C. CornMOns was significantly attenuated at 37°C compared to 32°C, however, there was detectable virus at 37°C. Rather than the greater than a five log reduction of virus incurred by rCorn1963 and rCornMOstr, rCornMOns underwent a two log reduction in virus titer at

37°C. This suggests that incorporation of the non-structural region from MO-

2014 results in a partial temperature tolerance phenotype.

MO-2014 loses temperature tolerance with genomic swaps of contemporary structural and non-structural genes

To evaluate the effects that functional genomic regions of the historic

EV-D68 strain may have on rMO-2014, chimeric viruses were tested for their temperature sensitive phenotypes. TCID50 assays were performed on rMO-

2014 chimeric virus working stocks at either 32°C or 37°C (Fig 3.4). As described previously, rMO-2014 replicated similarly at both 32°C and 37°C, indicating the virus is tolerant to higher physiological temperatures. rMOcorn3’ did not display significant attenuation at 37°C, suggesting that the 3’ end of historical strain does not contribute to temperature sensitivity.

MOcorn5’3’ also showed no evidence of attenuation at 37°C. rMOcornStr was significantly attenuated at 37°C, however, infectious virus was detected.

There was a 1.5 log reduction in the virus titer at 37°C, suggesting a sub- detrimental role in temperature sensitivity. rMOcornNS was significantly attenuated at 37°C compared to 32°C, where no virus was detected at the higher physiological temperature. This suggests that structural proteins do impact temperature sensitivity to a degree, however, non-structural genes play a critical role for the maintenance of temperature tolerance for rMO2014.

DISCUSSION

Previous studies have shown historic strains of EV-D68 to have an optimal growth temperature of 32°C and undergo attenuation at 37°C [1, 9, 73

253]. This temperature range is important because it represents the gradient of the human respiratory tract, in which the upper airways are cooled by inhalation of ambient air to be around 32°C, and the lower respiratory airways are closer to the core body temperature of 37°C. In general, respiratory viruses incapable of replicating at higher temperature restrict infections to the upper respiratory tract and cause mild common-cold symptoms [268]. Evidence is mounting that the historically mild EV-D68 infections are associated with severe outcomes, such as acute flaccid myelitis

(AFM) and acute respiratory illness (ARI). Previous work done by our laboratory showed that contemporary circulating strains are better able to replicate at 37°C than historical strains. While we defined increased IRES- mediated translation as an advantage of contemporary strains, chimeric viruses between temperature tolerant and temperature sensitive strains swapping the 5’ UTRs did not alter their temperature sensitive phenotype.

Most enteroviruses are transmitted fecal-orally and cause enteric infections, requiring acid-stability and temperature tolerance at 37°C.

Temperature sensitive mutants of picornaviruses, including poliovirus (PV), coxsackie B virus (CBV), and enterovirus 71 have given insight into genomic regions that contribute to their temperature tolerant phenotypes [247-250].

The temperature sensitive PV mutants and the development of cold-adapted

PV vaccines have revealed mutations in the 5’ UTR, structural proteins, non- structural proteins, and the 3’ UTR can all play a role in picornavirus 74

temperature sensitivity [241, 249, 251]. Our sequence analysis between Corn-

1963 (temperature sensitive) and MO-2014 (temperature tolerant) revealed nucleotide differences in both 5’ and 3’ UTRs and amino acid changes in all proteins, with the exception of VP4 and 3B. Given that evolutionary mutations spanned all functional regions of the genome, our approach was to generate chimeric swap the 5’ and 3’ UTRs, 3’ UTR, structural coding region, and non-structural coding region to determine where determinants of EV-

D68 temperature sensitivity may lie.

Swapping both the 5’ and 3’ UTR or the 3’ alone did not alter the temperature tolerant phenotype for MO-2014. Unfortunately, Corn-1963 containing either the contemporary 5’ and 3’ UTR or 3’ alone was unable to be successfully rescued. Without these rescues, one cannot definitively say there is no role in temperature sensitivity for Corn-1963, however, there data for

MO-2014 chimeras suggest that these contributions may be minimal.

Chimeras swapping structural or non-structural regions of Corn-1963 and MO-2014 revealed determinants of temperature sensitivity or tolerance.

For Corn-1963, replacing structural genes with that of MO-2014 did not alter its temperature sensitive phenotype. A previous report showed that replacing

EV-D94 (temperature tolerant) VP1 with that of a historic EV-D68 conferred a temperature sensitive phenotype [253]. Our data suggests that VP1 is not the determinant for Corn-1963. Swapping non-structural genes of Corn-1963 was able to alter its temperature sensitive phenotype. While it did not 75

replicate to levels seen at 32°C, there was over a two-log increase in infectious virus detected over the rCorn-1963 (no detectable virus) at 37°C.

The partial rescue suggests that a combination of non-structural genes and additional regions may confer the full-temperature sensitive phenotype. For

MO-2014, the incorporation of Corn-1963 structural genes gave a partial temperature sensitive phenotype, however, infectious virus was still detected at 37°C five-logs above the limit of detection. Swapping the non-structural genes with those of Corn-1963 resulted in complete attenuation at 37°C, suggesting a critical importance of non-structural genes for the maintenance of MO-2014 temperature tolerance.

Taken together, our data spotlight the significance of the non- structural genes to EV-D68 sensitivity. This is the first report identifying such temperature sensitivity determinants for EV-D68. The non-structural regions of Corn-1963 and MO-2014 differ by 29 amino acids across all proteins except 3B. Further refinement of our chimeric approach should be performed focusing specifically on the non-structural region to determine which proteins are contributing the most to temperature sensitivity.

Considering the aforementioned association reported between 3D polymerase mutations and poliovirus attenuation, there is potential the 11 AA differences between Corn-1963 and MO-2014 in this protein could be impacting temperature sensitivity. Defining the determinants of temperature

sensitivity lends insights into the biological effects of EV-D68 and could explain a recent association of EV-D68 associated ARI.

ACKNOWLEDGEMENTS

We thank the members of the Pekosz laboratory, Sabra Klein, members of the Klein laboratory, Kim Davis, and members of the Davis laboratory for critical discussions of the data. The work was supported by

HHSN272201400007C (AP) and T32 AI007417 (BS).

FIGURES AND TABLES

Figure 3. 1 EV-D68 Infectious clone system. EV-D68 chimeras were generated using T7 plasmids encoding the full EV-D68 genome of interest. In vitro transcription was performed to generate positive-sense viral RNA, followed by transfection into RD cells. Upon transfection, infection takes place and infectious virus is shed.

Region Length (nt) nt changes % nt Homology Length (AA) AA changes % AA Homology 5'UTR 733 109 85.1 Structural VP4 207 20 90.3 69 0 100.0 VP2 744 100 86.6 248 13 94.8 VP3 705 107 84.8 235 14 94.0 VP1 927 129 86.1 309 33 89.3 Non-Structural 2A 441 58 86.8 147 5 96.6 2B 297 34 88.6 99 1 99.0 2C 990 121 87.8 330 4 98.8 3A 267 26 90.3 89 2 97.8 3B 66 8 87.9 22 0 100.0 3C 639 81 87.3 213 6 97.2 3D 1284 136 89.4 428 11 97.4 3'UTR 68 8 88.2 Total All 7368 937 87.3 2189 89 95.9

Table 3. 1 Nucleotide (nt) and amino acid (AA) lengths, changes, and

% homology in contemporary MO-2014 compared to Corn-1963. 5’ UTR length corresponds to that of Corn-1963, thus, nt changes account for point mutations and deletions in MO-2014.

Figure 3. 2 Historic and Contemporary EV-D68 Chimeras. Schematic outlining the functional genomic swaps between rCorn-1963 (blue) and rMO2014 (green) generated to test their role in temperature sensitivity.

Successful rescue of recombinant virus is indicated by a green check mark

(), while a red x (x) indicates no infectious virus was generated after four attempts. The effects of 5’ UTR on temperature sensitivity were previously described in Chapter 2 of this dissertation (no altered temperature sensitivity phenotype), indicated by an asterisk (*), and were excluded from this study. rCorn-1963 and rMO-2014 were also described in Chapter 2 of this dissertation to have temperature sensitivity phenotypes of their respective wt

counterparts (Corn-1963: temperature sensitive; MO-2014: temperature tolerant) and were included in these studies as controls.

Figure 3. 3 Replication of rCorn-1963 and its functional region swaps with rMO-2014. Infectious virus working stock titers were determined with rCorn-1963, rCornMOstr, rCornMOns chimeric viruses at 32°C (solid) and

37°C (dashed) using TCID50 assays. As indicated on graph, rCornMO3’ and rCornMO5’3’ from the chimera panel yielded no infectious virus from rescue.

Statistical significance determined by unpaired t-tests (TCID50) with

****p<0.0001. The data shown are for pooled for 3 experiments performed in triplicate for each experiment.

Figure 3. 4 Replication of rMO-2014 and its functional region swaps with rCorn-1963. Infectious virus working stock titers were determined with rMO-2014 and rMOcorn3’, rMOcorn5’3, rMOcornStr, rMOcornNS chimeric viruses at 32°C (solid) and 37°C (dashed) using TCID50 assays.

Statistical significance determined by unpaired t-tests (TCID50) with

***p<0.0001, ****p<0.0001. The data shown are for pooled for 3 experiments performed in triplicate for each experiment.

CHAPTER 4: ENTEROVIRUS D68

PRODUCTIVELY INFECTS HUMAN NASAL

EPITHELIAL CELL CULTURES AT A RANGE

OF PHYSIOLOGICAL TEMPERATURES

ABSTRACT

Enterovirus D68 (EV-D68) has become a significant respiratory pathogen in recent years due to its association with clusters of acute respiratory illness (ARI) and acute flaccid myelitis (AFM). Since its identification in 1962, EV-D68 was detected rarely and only associated with mild upper respiratory infections. Given the association with severe ARI outbreaks, EV-D68 seems to have expanded its tropism to the lower respiratory tract, causing pneumonia, bronchiolitis, and difficulty breathing.

Previous work determined that contemporary EV-D68 strains replicate better at 37°C than historic strains, whose optimal growth temperatures were 32°C-

33°C. To further characterize replication differences between historic and recent EV-D68 strains, we compared virus replication and the induction of innate immune response genes after infection of primary human nasal epithelial cells (hNECs). Our data indicate that contemporary strains of EV-

D68 are better suited to replicate in hNECs at both 32°C and 37°C, resulting in increased proinflammatory chemokines and interferon-λ production.

During high MOI infection, contemporary EV-D68 was detected at lower levels, as were proinflammatory chemokines and interferon-λ. Cell tropism studies determined that both contemporary and historic strains preferentially infect ciliated epithelial cells. Our studies suggest that while both historic and contemporary EV-D68 strains infect similar cells of the respiratory tract, contemporary strains are better suited to generate infection 85

virus and illicit greater innate immune factor production when presented with low levels of virus. Taken together, these findings suggest the increased severity of contemporary strain infections may be linked to enhanced replication at 37°C.

INTRODUCTION

Enterovirus D68 (EV-D68) is a member of the Picornaviridae family, a group of small non-enveloped positive-sense RNA viruses. It is further classified in the genus Enteroviruses. Other common human pathogens in this genus include poliovirus (PV), coxsackie B virus (CBV), enterovirus A71

(EV-A71), and rhinoviruses (RVs). Nearly 10 million enterovirus infections are estimated to occur annually with symptoms that include mild rhinitis, febrile illness, acute respiratory infection (ARI), encephalitis, and acute flaccid myelitis (AFM) [295, 296]. EV-D68 was first isolated from four pediatric patients in California in 1962 and historically associated with mild common cold-like symptoms [1]. In recent years, EV-D68 has been associated with severe ARI and AFM, most commonly affecting pediatric patients and asthmatics. While there is great interest in this altered disease manifestation and several groups have defined the phylogeny of EV-D68 and emergence of multiple clades [115, 119, 126, 297], little is known as to the factors that mediated this change in disease manifestation.

While much of the recent literature on EV-D68 has focused on its association with acute flaccid myelitis and infection of neuronal cells [110,

127, 128, 298, 299], less has been done to define its recent association with severe ARI. Acute respiratory infections are common illnesses in humans and one of the leading causes of death in children under five years of age [300]. 87

Similar to rhinoviruses, the initial site of infection of EV-D68 are the upper respiratory tract epithelial cells. Upon infection, respiratory cells elicit innate immune factors, such as chemokines, cytokines, and interferons, to help control virus growth and spread to neighboring cells [301]. While control of virus growth is important, a pro-inflammatory response can be damaging to epithelial cells and is often attributed to severe disease [302]. Given the association of contemporary EV-D68 outbreaks ARI, our studies investigate historical and contemporary EV-D68 strain replication, induction of the innate immune response and cell tropism in primary ciliated epithelial cultures.

MATERIALS AND METHODS

Cell culture

Human Rhabdomyosarcoma (RD) cells were obtained from the

American Type Culture Collection (ATCC) (CCL-136) and cultured in Gibco

Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM

GlutaMAX (Gibco). The cells were grown at 37°C in a humidified environment supplemented to 5% CO2.

Human nasal epithelial cell (hNEC) cultures were grown from epithelial tissue isolated during endoscopic sinus surgery for non-infection related conditions and grown at air-liquid interface (ALI) as previously 88

described [303-305]. hNEC differentiation medium and culture conditions have previously been described in detail [306].

Viruses

F02-3607 Corn (Corn-1963) and US/MO/14-18947 (MO-2014) were obtained from ATCC. Virus stocks were generated by inoculating a confluent

T75 or T150 flask of RD cells at a multiplicity of infection (MOI) of 0.01 50% tissue culture infectious doses (TCID50) per cell for 1 hour at 32°C with rocking every 10-15 minutes. Virus inoculum was aspirated and replaced with infection media (IM- 2.5% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM GlutaMAX [Gibco]) followed by incubation at 32°C. After

24 hours of infection, initial IM was aspirated and replaced with fresh IM.

When complete cytopathic effect (CPE) could be determined using a light microscope (3-5 days), the infected cells and supernatant were harvested, subjected to 2 freeze thaw cycles, and centrifuged at 600 x g for 10 minutes to remove all cell debris. The clarified supernatant was aliquoted and stored at -

70C. Virus stock titers were determined using TCID50 assays described below.

Sequence analysis

Corn-1963 and MO-2014 sequences from GenBank were aligned using

Geneious 8.0.1. Nucleotide and amino acid differences were quantified for each of the UTRs and the structural and non-structural proteins.

50% tissue culture infectious dose (TCID50) assay

RD cells were plated in 96-well plates, grown to full confluence, and washed with PBS+. Tenfold serial dilutions of the virus inoculum were made and 200 μL of dilution was added to each of 6 wells in the plate, followed by incubation at 32°C or 37°C for 5-7 days. Cells were then fixed by adding 100

μL of 4% formaldehyde in PBS per well and incubated at room temperature for at least 10 minutes before staining with Naphthol Blue Black solution.

TCID50 calculations were determined by the Reed-Muench method [272].

Growth Curves

Virus stocks were diluted to the appropriate concentrations in infection media (IM- 2.5% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM

GlutaMAX [Gibco]). hNEC cultures were washed twice (apically and basolaterally) with PBS+ and inoculated with virus at an MOI of 0.1 (low

MOI) or 5 (high MOI) for 1 hour at 32°C. Virus inoculum was aspirated, cells were washed 3 times with PBS+ and fresh basolateral media was replaced.

Infected cultures were moved to 32°C and 37°C for 7 (low MOI) or 4 (high

MOI) days. At the time points indicated, apical washes and basal media were collected and stored at -80°C. Basolateral media was replaced with fresh basolateral media at each collection step. Infectious virus quantification at each time point was determined using TCID50 assays.

Interferon, cytokine and chemokine measurements

Secreted interferons and proinflammatory cytokines and chemokines were quantified from the apical and basolateral samples from the hNEC multistep growth curves at 24, 48 and 96 hour post-infection (hpi) and the high MOI growth curve at 23 and 48 hpi. Protein quantification was performed using the V-PLEX Human Chemokine Panel 1 (Eotaxin (CCL11),

Eotaxin-3 (CCL26), IL-8, IP-10 (CXCL10), MCP-1 (CCL2), MCP-4 (CCL13),

MDC (CCL22), MIP-1α (CCL3), MIP-1β (CCL4), TARC (CCL17)), V-PLEX

Human IL-6 kit (Meso Scale Discovery), and the DIY Human IFN Lambda

1/2/3 (IL-29/28A/28B) ELISA (PBL Assay Science) according to manufacturers’ instructions. Protein concentrations were normalized to mock infected (IM only) cultures at the respective time points and temperatures.

Microscopy

hNEC cultures were washed twice (apically and basolaterally) with phosphate buffered saline containing calcium and magnesium (PBS+) and infected (apically) at an MOI of 5 for 1 hour at 32°C. Virus inoculum was removed and cells were washed three times with PBS+. Infected cells were allowed to incubate (with basolateral medium only) for an additional 8 hours at 32°C or 37°C, at which time they were washed twice with PBS and fixed for 20 minutes at room temperature with 4% paraformaldehyde (PFA) in

PBS. Cells were permeabilized with 0.2% Triton X-100 (Sigma) in PBS for 10 minutes, blocked for 1 h in PBS with 3% normal goat serum and 0.5% bovine 91

serum albumin (BSA; Sigma), and stained for 1h with rabbit polyclonal VP1

(1:200; GTX132313, Genetex) and mouse anti beta IV tubulin IV (1:200; ab11315, Abcam) in blocking solution, followed by staining for 1 h with goat anti-rabbit AF555 (1:200; A-27039, Molecular Probes) and goat anti-mouse

AF488 (1:200, A-11029, Molecular Probes) secondary antibodies in blocking solution. Cells were washed after each step in PBS with 0.2% Tween-20 wash solution. Cells were washed one final time in MilliQ water before mounting in

Prolong gold antifade with DAPI (ThermoFisher). Cells were imaged using a

Zeiss AxioImager M2 microscope with Volocity software for image acquisition. Quantification of infected cells expressing cilia was performed by analyzing multiple fields of vision at 40x and determining the number of cells expressing VP1 alone (infected, not ciliated) or both VP1 and beta IV tubulin

(infected and ciliated).

Statistical analyses

Multistep growth curves were analyzed by two-way ANOVA. Secreted cytokine, chemokine, and interferon differences were analyzed with multiple t-tests. All statistical analyses were performed in GraphPad Prism 8.0.

RESULTS

Contemporary EV-D68 strain replicates to higher virus titers and viruses release persists longer on hNEC cultures

Multistep growth curves were performed at both 32°C and 37°C at a multiplicity of infection (MOI) of 0.1 to analyze the susceptibility of hNECs to

Corn-1963 and MO-2014 infection and their replication kinetics. While respiratory viruses generally infect on the apical side of epithelial cells, infectious virus can egress either apically, basolaterally, or both in polarized epithelial cells [307-309]. Given the polarization of hNECs, both apical washes and basolateral samples were collected and tested for infectious virus.

Our apical data suggests that MO-2014 replicates significantly better than Corn-1963 at both 32°C and 37°C over multiple rounds of replication in primary hNECs. At both temperatures, MO-2014 replicated to higher peak titers and had prolonged infectious virus detection in the apical washes (Fig

4.1A and B). Both Corn-1963 and MO-2014 exhibited a degree of attenuation at 37°C compared to 32°C, with the temperature sensitive phenotype penetrating greater for Corn-1963 where no virus was detected starting at 48 hours post infection (hpi). No virus was detected above the limit of detection in the basolateral samples for Corn-1963 or MO-2014 at either temperatures

(Fig. 4.1C and D), suggesting that release of the viruses is primarily targeted to the apical surface.

MO-2014 elicits increased amounts of proinflammatory chemokines and IFN-λ at both 32°C and 37.

Pathogenesis of respiratory virus infections can largely be attributed to damage due to the pro-inflammatory innate immune response [310].

Additionally, a recent report suggests that rhinovirus attenuation at 37°C results from an overall more robust innate immune response to infection at

37°C compared to 32°C [227]. To determine if Corn-1963 and MO-2014 elicit differential pro-inflammatory profiles at either 32°C or 37°C , samples collected from our multi-step growth curves were analyzed for secreted proinflammatory chemokines (CCL2, CCL3, CXCL10, CCL11, CCL13, CCL17,

CCL22 and CCL26), cytokines (IL-6), and interferons (IFN-λ) in the apical washes and basolateral supernatants at 24, 48 and 96 hpi (Fig. 4.2). CXCL10 induction was greatest for both viruses, with 1000-fold or higher increases over mock in the 32°C apical samples. The remaining analytes were detected at levels approximately five-fold over mock or less.

In the apical samples, there were no differentially expressed analytes at 32°C between the viruses. At 37°C, Corn-1963 elicited significantly more

IL-8 secretion apically at 24 hpi, however, these levels were less than two- fold higher than mock infected cells and the differences were not found at the later time points.

In the basolateral samples, differential expression of CXCL10 (24 and

48 hpi at 32°C; all time points at 37°C), CCL17 (24 hpi at 32°C), CCL22 (24 94

hpi at 32°C) and IFN-λ (96 hpi at 32°C) was detected between the viruses, with MO-2014 at higher levels. At 37°C, the differential secretion of innate immune proteins was not detected and the relative amounts were substantially lower than 32°C, with the exception of CXCL10 at 24 hpi.

Taken together, the data suggest that over multiple rounds of replication, MO-2014 elicits a slightly stronger innate immune response, most notably with CXCL10 and IFN-λ. Detection of differential secretion of innate immune proteins was evident in the basolateral samples, suggesting targeted release of the chemokines, cytokines and interferons to the basal lamina during infection. At 37°C , the response to both Corn-1963 and MO-

2014 is dampened compared to 32°C (likely due to lesser virus production), suggesting that a heightened innate immune response at 37°C is not the cause of EV-D68 temperature sensitivity.

High MOI infection of hNECs with Corn-1963 and MO-2014 limited virus growth over time with higher induction of innate immune responses for Corn-1963

hNEC cultures were infected with Corn-1963 and MO-2014 at an MOI of 5 at and incubated at 32°C and 37°C to analyze virus growth kinetics and secreted innate immune factors following high MOI infection. Apical washes were collected at 1, 24, 48, and 72 hpi and infectious virus titers were quantified by TCID50 assay (Fig. 4.3A). Following infection, both Corn-1963 and MO-2014 were detected at relatively low numbers, both having peak 95

titers of approximately 104 TCID50/mL at 32°C and 103 TCID50/mL at 37°C .

At 32°C, Corn-1963 had detectable virus until 48 hpi, whereas MO-2014 had minimal detectable virus after 24 hpi. After 24 hpi at 37°C, no infectious virus was detected for Corn-1963 or MO-2014. The data suggest that following high MOI infection, amounts of infectious Corn-1963 and MO-2014 remain similar but at low levels compared to their peak titers in their respective low MOI growth curves (Fig 4.1A and B).

To determine if there is differential induction of innate inflammatory chemokines (CCL2, CCL3, CXCL10, CCL11, CCL13, CCL17, CCL22 and

CCL26) and interferons (IFN-λ) following high MOI infection, basolateral samples from our growth curves at 24 and 48 hpi were analyzed (Fig. 4.3B).

For CCL13 (48 hpi at 37°C), CXCL10 (all time points and temperatures),

CCL22 (48 hpi at 32°C and 37°C), and IFN-λ (48 hpi at 32°C and 37°C), cultures infected with Corn-1963 were secreting higher levels of immune proteins.

The data suggest similarities to data acquired in our low MOI growth curve samples in that CXCL10 and IFN-λ are the major factors induced from our innate immune panel. Again, induction of innate immune chemokines and interferons was not more robust at 37°C than at 32°C. Corn-1963 and

MO-2014 induced similar innate immune profiles at levels that generally correlate with their patterns of peak titer and infectious virus persistence.

Corn-1963 and MO-2014 share similar tropism for ciliated epithelial cells in primary differentiated hNECs

Primary hNEC cultures are differentiated to comprise various epithelial cell types lining the respiratory tract, including ciliated epithelial cells, goblet cells (mucus producing), club cells, and basal cells [311]. Some viruses, such as respiratory syncytial virus and rhinovirus, have been shown to preferentially infect ciliated epithelial cells [312, 313]. To determine if EV-

D68 has a specific cell tropism within the epithelium and if there was differential tropism between Corn-1963 and MO-2014, primary differentiated hNEC cultures were infected with Corn-1963 and MO-2014 at an MOI of 5 for 9 hours at 32°C and 37°C. hNEC cultures were stained for beta IV tubulin

(red; marker for ciliated cells), EV-D68 virus capsid protein VP4 (green; marker for infected cells) and DAPI (blue; marker for nucleus) and analyzed using a fluorescent microscope. Representative images of infected cells at both 32°C and 37°C indicate a preferential infection of ciliated cells.

Quantification of the number the number of infected cells expressing cilia over the total of number of infected cells indicated approximately 95% for both Corn-1963 and MO-2014 at both 32°C and 37°C. Based on this data,

Corn-1963 and MO-2014 show similar cell tropism at both 32°C and 37°C in primary differentiated hNECs, with preferential infection of ciliated cells.

DISCUSSION

Infections with EV-D68 have recently been associated with severe disease, including acute flaccid myelitis (AFM) and acute respiratory infection (ARI) [5, 38, 43, 49, 51, 57, 58, 63, 94, 95, 103, 104, 259, 279, 314-

320]. Many recent studies have focused on the biological aspects of neurotropism of contemporary strains with respect to AFM [105, 108-110,

127, 128, 321], however, there have been minimal reports focused on delineating the recent association with ARI. Mouse and ferret models have shown that EV-D68 is capable of penetrating the lower respiratory tract and eliciting a proinflammatory response upon intranasal inoculation [90, 91].

The ferret studies were performed using a historical strain of EV-D68 and mouse studies were done using interferon-knockout mice and a contemporary strain that required 30 passages in mice, resulting in a series of nucleotide changes in the 5’ UTR and amino acid changes in the structural and non- structural proteins. While useful models for vaccination and therapeutic efforts, these studies lend little insight into the recent association with ARI.

Previous work in our laboratory used primary, differentiated human nasal epithelial cells (hNECs) as a physiologically relevant in vitro model of the airway epithelium to study influenza virus replication and the innate immune response [306, 322, 323]. Using this relevant model, our studies outlined here focused on the replication of historic and contemporary EV-D68

strains (Corn-1963 and MO-2014, respectively), the innate immune response to infection, and epithelial cell tropism.

Previous studies in our laboratory determined that contemporary strains of EV-D68 replicate significantly better at 37°C than temperature sensitive historic strains [324]. Based on these findings, all of our hNEC studies were performed at 32°C and 37°C. By infecting hNEC cultures at a low multiplicity of infection, we were able to show that MO-2014 replicated significantly better and persisted at higher viral titers than Corn-1963 at both 32°C and 37°C. Both Corn-1963 and MO-2014 did replicate to lower peak titers at 37°C, however, this attenuation penetrated greater for Corn-

1963. Testing basolateral samples for both Corn-1963 and MO-2014 indicated no detectable virus, suggesting that upon apical infection, EV-D68 is released from or through the apical surface. Analysis of apical washes and basolateral supernatants for pro-inflammatory chemokines, cytokines, and type III interferons showed that both Corn-1963 and MO-2014 elicit similar innate immune profiles in the context of proteins secreted, however, the amounts produced from MO-2014 were significantly higher, most notably for CXCL10 and IFN-λ. CXCL10 is a chemotractant secreted during viral infections for the recruitment of lymphocytes expressing the CXCR3 receptor, including natural killer cells [325]. Expression of CXCL10 has also been linked to acute respiratory distress syndrome due to enhancement of neutrophil-mediated inflammation. Given the high levels of CXCL10 during MO-2014 infection, 99

particularly at 37°C, it presents a potential mechanism for pulmonary damage if the virus is capable of reaching and replicating in the lower airways. INF-λ stimulates the expression of interferon-stimulated genes

(ISGs) specifically on epithelial cells, generating a highly antiviral response without causing inflammation [326]. The persistence of MO-2014 at higher titers could suggest that it better evades the antiviral genes stimulated by

IFN-λ. One study showed for rhinovirus that the innate immune response of primary epithelial cells is enhanced at 37°C, suggesting that may be the mechanism by which rhinovirus is restricted to the upper airways [227]. In our studies, the panel of secreted innate immune proteins were generally detected at lower levels at 37°C, suggesting the innate immune response is not the cause of EV-D68 temperature sensitivity. Several chemokines in the basolateral media showed statistically significant increases for MO-2014, including CXCL17 and CXCL22. Both of these chemokines were detected approximately two-fold changes over mock or less, calling into question the biological relevance of such changes and the necessity for further investigation.

Our high MOI infection growth curves showed infectious virus of Corn-

1963 and MO-2014 was detected at relatively low numbers, both having peak titers of approximately 104 TCID50/mL at 32°C and 103 TCID50/mL at 37°C that quickly dropped after 24 hours. Corn-1963 infection induced a slightly stronger innate immune response, most notably with CXCL10 and INF-λ. In 100

conjunction with our low MOI data, CXCL10 and INF-λ seem to be major players in the general response to EV-D68, rather than a strain specific response. There were chemokines that showed significant differences between the viruses, including CCL13 and CCL22, but their relative levels to mock were three-fold or less, suggesting that further investigation into these induction levels is necessary to determine their biological relevance. Similar to the low MOI growth curve samples, at 37°C, the innate response was dampened, suggesting an alternative mechanism by which EV-D68 undergoes temperature sensitivity.

Primary hNEC cultures are well-differentiated to comprise various epithelial cell types, including ciliated cells, goblet cells (mucus producing), club cells, and basal cells [311]. Some viruses, such as respiratory syncytial virus, rhinovirus, and H1 and H3 influenza viruses have been shown to preferentially infect ciliated epithelial cells [312, 313, 327]. On the other hand, H5N1 was shown predominantly infect non-ciliated cells [327]. Given the differences in Corn-1963 and MO-2014 replication efficiency in our hNEC model, we investigated whether MO-2014 is capable of infecting more cell types that line the epithelium. Our data suggest that both Corn-1963 and

MO-2014 preferentially infect ciliated epithelial cells, where approximately

95% of infected cells were ciliated at both 32°C and 37°C. Ciliated cells are rich with α2,6-sialic acid [327], correlating with reports that α2,6-sialic acid is involved in EV-D68 attachment and entry [131, 132]. Our data indicate that 101

both strains have similar cell tropism in hNECs and is unlikely the cause for increased infectious virus detected for MO-2014 in our low MOI growth curves.

Taken together, MO-2014 replicates more efficiently in primary differentiated hNECs than Corn-1963 at both 32°C and 37°C. Corn-1963 and

MO-2014 elicit similar pro-inflammatory chemokines, cytokines, and type III interferons at levels dependent on their replication efficiency and the detection of infectious virus. Given that MO-2014 replicated more efficiently over multiple rounds of replication, it is more likely to cause increased inflammation and epithelial damage, potentially contributing to recent associations with severe ARI. At 37°C, the immune response to EV-D68 is dampened in hNECs. Both Corn-1963 and MO-2014 almost exclusively infect ciliated epithelial cells, discounting expanded cell tropism of MO-2014 as a variable for more efficient replication. Expansion of our studies to include primary, differentiated epithelial cell cultures obtained from the lower respiratory tract may lend more insight to the ability for EV-D68 to cause lower airway damage.

ACKNOWLEDGEMENTS

for providing the hNEC cultures. The work was supported by

HHSN272201400007C (AP) and T32 AI007417 (BS).

103

FIGURES

Figure 4. 1 Effects of temperature on replication of Corn-1963 and

MO-2014 in primary hNEC cultures. hNECs were infected with Corn-

1963 and MO-2014 at an MOI of 0.1 and infectious virus titers (TCID50/mL) were determined in the apical washes and basolateral supernatants (1, 12,

24, 36, 48, 72, 96, and 168 hours post-infection. Statistical significance determined by unpaired t-test (TCID50) or 2-way ANOVA (growth curves) with *p<0.05. The data shown are pooled from 2 experiments performed in quadruplicate for each experiment.

104

105

Figure 4. 2 Apical and basolateral secretion of chemokines and interferon by Corn-1963 and MO-2014 over multiple replication cycles. Apical washes and basal supernatants were collected from a low MOI infection (MOI= 0.1) at 32°C and 37°C at 24, 48, and 96 hours post infection.

Secreted innate inflammatory chemokines and IFN-λ were quantified and normalized to the concentration of secreted proteins in mock infected samples at the indicated times and temperatures. Significance between Corn-1963 and MO-2014 determined by multiple t-test with *p<0.05. The data shown are pooled from two experiments performed in quadruplicate for each experiment.

106

Figure 4. 3 Replication kinetics and secreted chemokines and interferons in hNEC cultures at 32°C and 37°C for Corn-1963 and MO-

2014 following high MOI infection. A) hNECs were infected at an MOI of

5 at 32°C and 37°C and infectious virus titers (TCID50/mL) in the apical washes were determined at 24, 48 and 72 hours post infection. B) Secreted

107

chemokines and IFN-λ in the basolateral supernatants were quantified and normalized to the concentration of secreted proteins in mock infected samples at the 24 and 48 hours. Significance between Corn-1963 and MO-2014 determined by 2-way ANOVA (growth curves) or multiple t-tests (chemokines and interferons) with *p<0.05. No statistical significance was determined for the growth curves at either temperature. The data shown are pooled from two experiments performed in triplicate for each experiment.

108

Figure 4. 4 Ciliated cell tropism of Corn-1963 and MO-2014 in primary hNEC cultures. Primary hNECs were infected at an MOI of 5 for

9 hours with Corn-1963 or MO-2014 at 32°C or 37°C. (A) Cells were stained for β-tubulin IV (ciliated cells; red), VP1 (infected cells; green), and DAPI

(nuclei; blue) and analyzed using a fluorescent microscope (scale bar= 16 µM).

(B) Percent quantification of infected cells (VP1+) with the presence of cilia

(beta IV tubulin+). No statistical differences were determined by using

109

unpaired t-test with p<0.05. The data shown are pooled from 3 independent experiments.

110

CHAPTER 5: GENERAL DISCUSSION

111

The overarching goal of the work outlined in this dissertation was to understand if and how contemporary strains of EV-D68 overcome the temperature barrier of 37°C presented by the lower respiratory tract. Using multiple historic and contemporary strains of EV-D68, I was able to show that contemporary strains replicate more efficiently at temperatures of the lower respiratory tract than historic strains. While this is not the only barrier a virus must overcome to infect the lower airways, it does play a significant role. I showed that contemporary strains more efficiently translate viral proteins due to evolutionary differences in the 5’ UTR and by developing and employing an EV-D68 infectious clone system, I generated chimeric viruses that ruled out the 5’ UTR as the sole contributor to EV-D68 temperature sensitivity. Expanding my chimeric approach, I generated chimeras that swapped all functional regions of the EV-D68 genome. Several contributors to

EV-D68 temperature sensitivity were determined, including the structural and non-structural genes. This is the first report of specific regions contributing to EV-D68 temperature sensitivity. I was also able to show that a contemporary EV-D68 strain replicated more efficiently in hNECs eliciting a more robust proinflammatory innate immune response over multiple rounds of infection, while maintaining similar traits of cell tropism as the historic strain. Taken together, the research in this dissertation expands our biological understanding of EV-D68 and provides groundwork for further

112

research into the severe acute respiratory illness associated with recent outbreaks.

Evolution of EV-D68 has resulted in increased disease severity

As described thoroughly in Chapter 1, there has been a substantial shift in the disease manifestation associated with EV-D68 in recent years.

Historically, EV-D68 was attributed to mild upper respiratory infections, however, recent outbreaks of EV-D68 have been associated with acute flaccid myelitis (AFM) and acute respiratory illness (ARI). While there is evidence of significant evolutionary changes since the virus was first isolated in 1962

[115, 125, 328], the field is limited by a temporal gap in virus isolates and sequencing data. Thus, there has been little evidence of when significant evolutionary changes took place, whether gradually or through recombination, and what biological effect these changes had on the virus.

Several groups focused on EV-D68-associated AFM have proposed that altered disease manifestation is the consequence of altered receptor selectivity. Contemporary strains have been shown to infect and propagate in cells treated with neuraminidase, which cleaves the sialic acid receptors associated with historical strain entry [127, 128, 131, 132]. Recent work has highlighted ICAM-5 as a functional receptor, suggesting that sialic acid dependence is strain specific, while ICAM-5 is necessary for all strains [127].

Other groups found that cells lacking ICAM-5 are still permissive to EV-D68 and mouse studies detecting EV-D68 in neuronal tissues have minimal 113

ICAM-5 expression, suggesting a lack of correspondence between the virus and ICAM-5 expression [110, 128]. The conflicting reports on EV-D68 infection of neuronal cell lines and discrepancies between receptor preferences have resulted in association of AFM with contemporary is most likely more than increased neuronal cell tropism alone.

The impact of EV-D68 evolution on the biological mechanisms leading to ARI has not been a large focus of the field. To date, there are two reports outlining a ferret model and mouse model that show intranasal inoculation of

EV-D68 can lead to infection of the lower airways and elicit a proinflammatory response. Neither study investigated differences between historic and contemporary strains in the context of their respective models, leaving many questions to be answered regarding the recent association of

ARI.

Contemporary EV-D68 strains are better able to replicate at temperatures of the lower airways

As described in Chapters 1-3, the major focus of my dissertation is that contemporary strains of EV-D68 replicate efficiently at both 32°C and 37°C.

While the optimal growth temperature of 32°C and attenuation at 37°C has been described for historic strains of EV-D68 previously, no research has focused on this phenotype for contemporary strains [1, 9, 253]. The hypothesis that contemporary strains have evolved to replicate efficiently at

37°C was based largely on the association of 2014 outbreaks with ARI. There 114

is a temperature gradient of the human respiratory tract of 32°C-37°C, with upper airways being cooled by ambient air and lower airways being closer to core body temperature. For respiratory viruses incapable of replicating at higher temperatures, they are often restricted to the upper airways and cause mild upper respiratory infections [231, 276]. ARI induction by viral infection corresponds with the ability of the virus to penetrate the lower respiratory tract and cause pulmonary damage, either directly by disruption of epithelial cells through cell death pathways, or indirectly by the pro-inflammatory immune response [287, 300]. Given the recent association of EV-D68 ARI, it was likely that contemporary strains evolved to replicate more efficiently at

37°C. Described in Chapter 2, using a panel of historic and contemporary EV-

D68 strains, I was able to show that contemporary strains are better able to replicate at 37°C than historic strains [324]. This finding has not been previously reported and offers a phenotype that could link contemporary strains to EV-D68-associated severe disease.

Identification of translation efficiency advantages of contemporary

EV-D68 strains

Contemporary strains have of EV-D68 have all been found to have large deletion blocks at the terminal end of the 5’ UTR [124, 126]. In Chapter

2, I was able to show that contemporary EV-D68 strains have increased translation of viral proteins compared to historic strains. Using a bicistronic 115

dual-luciferase assay, I mapped the translation advantages to regions of variability in the 5’ UTR that also comprise the aforementioned deletion blocks. By developing an EV-D68 infectious clone system by modifying protocols for poliovirus and enterovirus 71 infectious clone system, I was able to generate chimeric viruses swapping historic and contemporary 5’ UTRs and test their association with temperature sensitivity. At both 32°C and

37°C, my finding show that the 5’ UTR alone does not mediate EV-D68 temperature sensitivity.

While temperature sensitivity is not affected by 5’ UTR swaps and increased translation efficiency, there may be other advantages to generating more viral proteins. 2A protease and 3C protease have been well described for their role in picornavirus evasion of the innate immune response and host translation and transcription factors [141, 194, 329, 330]. Both EV-D68 and

EV-A71 3C protease have previously been shown to cleave IRF7 in infected cells and suppress antiviral type I interferon response [216, 331]. I hypothesize that the Corn-1963 containing the contemporary 5’ UTR would result in a decreased detection of IRF7 in cell lysates. To test this, RD cells could be infected at a high MOI with rCorn-1963, rMO-2014, and the 5’ UTR swap chimeras. Cell lysates generated every three hours and analyzed for levels of IRF7 using western blot assays would determine translation efficiency and its impact on innate immunity evasion.

116

Identification of the mechanism behind increased translation efficiency

While Chapter 2 defined regions of the contemporary strain 5’ UTR that confer increased translation efficiency, the mechanism by which this is occurring should be explored. IRES-mediated translation initiation involves the recruitment of host-factors to the RNA secondary structure, including host-specific translation factors eIF4A, eIF4G, eIF4B, eIF1A [172] and IRES trans-acting factors (ITAFs) polypyrimidine tract binding protein (PTB), poly

(rC) binding protein (PCBP) unr (upstream of N-ras) [183-185]. Given the substantial amount of evolution in the 5’ UTR and the effects on secondary structure outlined in Chapter 2, I hypothesize that MO-2014 more efficiently recruits necessary translation factors to the 5’ UTR leading to the differential translation efficiencies. To test this, RNA-protein pull down assays should be performed using biotinylated Corn-1963 and MO-2014 5’ UTR RNA and RD cell lysates. The pull down assays can be quantified by band intensity for relative amounts of translation factors.

Beyond the 5’ UTR, differentiation between overall translation efficiency and proteolytic processing should be performed. As outlined in

Chapter 3, both 2Apro and 3Cpro are involved in the proteolytic processing of the polypeptide. Performing a pulse-chase experiment to detect the processing of the polypeptide in lysates every hour following high MOI infection should be performed. This will determine if the increased amount of 117

VP1 and VP3 (fully processed) detected in cells infected with contemporary strains is exclusively due to translation advantages or if efficient processing results in final products at a faster rate.

Defining the role of non-structural genes in EV-D68 temperature sensitivity

As outlined in Chapter 2, contemporary strains of EV-D68 replicate efficiently at 37°C, while historical strains are attenuated. Using our infectious clone system to generate chimeras between Corn-1963 and MO-

2014, the 5’ UTR alone was shown to not contribute to the temperature sensitivity phenotypes [324]. In Chapter 3, I expanded my approach by generating a full panel of chimeras swapping both the 5’ UTR and 3’ UTR, 3’

UTR alone, structural genes, and non-structural genes. Testing this panel for temperature sensitive phenotypes, I showed that Corn-1963 could partly overcome temperature sensitivity with the incorporation of MO-2014 non- structural genes. In the other direction, MO-2014 became partly temperature sensitive (ten-fold reduction) by incorporating Corn-1963 structural genes and completely attenuated by incorporating Corn-1963 non-structural genes.

Thus, non-structural genes are the main contributor to EV-D68 temperature sensitivity.

Moving forward, continued refinement of the non-structural gene evolution should be investigated. Six of the seven MO-2014 non-structural 118

proteins have amino acid changes compared Corn-1963. 3Dpol has the greatest number of AA differences (11 changes) and has previously been attributed to temperature sensitivity in poliovirus and EV-71 mutants [242,

246, 249]. I hypothesize that 3Dpol is the major contributor to EV-D68 temperature sensitivity, however, exploration of all non-structural proteins may be necessary to gain a full understanding. Beyond the link of 3Dpol to temperature sensitivity in other picornaviruses, 2Apro and 3Cpro are highly involved in proteolytic processing of the single polypeptide and cleavage of host factors to facilitate immune evasion and optimal replication.

Accumulation of AA changes in these proteins could subsequently enhance these processes. Given the range of AA changes across the non-structural proteins, a panel of 12 chimeras should be generated focusing on protein swaps in the non-structural region only. This includes swaps of each non- structural protein, excluding 3B, which is 100% homologous between the two viruses. The temperature sensitive phenotypes should be evaluated by

TCID50 assays at 32°C and 37°C.

Given that Corn-1963 with MO-2014 non-structural genes at 37°C did not fully reach the infectious virus titers detected at 32°C, it suggests there may be a combination of multiple regions contributing to temperature sensitivity. This was evident in the Sabin 1 temperature sensitive PV strain, where a mutation in the 3’ UTR amplified the temperature sensitivity phenotype attributed to 3Dpol. This suggests that a Corn-1963 chimera 119

incorporating MO-2014 non-structural genes and 3’ UTR may further enhance the temperature tolerance of the chimera. This chimera should be generated and tested for temperature sensitivity by TCID50 assays at 32°C and 37°C.

At this point, there have been no confirmed mutations attributed to the

AFM associated with EV-D68. While my focus has primarily been on temperature sensitivity with respect to the respiratory tract, the average temperature of the CNS is also core body temperature. The Corn-1963 chimera conferring temperature tolerance and the MO-2014 chimera conferring temperature sensitivity should be utilized in a paralytic mouse model, described in the literature [108], for their ability to cause limb paralysis.

Contemporary EV-D68 strains replicate more efficiently generating a more robust inflammatory response in hNECs and show similar cell tropism for ciliated cells

The Pekosz lab has utilized an in vitro model of primary, differentiated human nasal epithelial cells (hNECs) to study replication of influenza virus and the innate immune response [306, 332, 333]. The hNEC cultures are well-differentiated into ciliated cells, goblet cells, club cells and basal cells. In

Chapter 4, I showed that following low MOI infection of hNECs, MO-2014 replicated more efficiently and persisted at higher titers than Corn-1963 at 120

both 32°C and 37°C. Analysis of proinflammatory chemokines, cytokines, and type III interferons identified robust secretion CXCL10 and IFN-λ for both

Corn-1963 and MO-2014, however at higher amounts for MO-2014.

CXCL10 has previously been shown to be involved in virus-induced pulmonary damage by the recruitment of inflammatory neutrophils expressing its CXCR3 receptor [334]. To expand on my finding, I would use a

CXCL10 or CXCL3 knockout mouse model described by Sun et al [298] to infect with Corn-1963 and MO-2014. Both survival curves and histopathology of lung sections should be analyzed to determine the role of CXCL10 in EV-

D68-induced ARI.

The role of IFN-λ in controlling EV-D68 in epithelial cells has not been reported. It should be determined whether Corn-1963 and MO-2014 have differential responses to similar levels of IFN-λ. To do this using our hNEC system, cultures should be pre-treated with levels of exogenous IFN-λ for 24 hours prior to low MOI infection with Corn-1963 and MO-2014. Infectious virus titers should be measured in apical washes for 7 days following infection to determine the effects of IFN-λ treatment on replication.

Expansion of research to vaccines efforts

While EV-D68 is continuing to gain global importance, there are currently no vaccines available for prophylaxis. Some groups have shown in mice and cotton rats protective immunity following vaccination with inactivated EV-D68 or virus-like particles [191, 335, 336]. Currently, there 121

are no studies outlining a live-attenuated vaccine for EV-D68. Given the success of the live-attenuated polio vaccine in conferring protection, this effort is worth pursuing.

Temperature sensitive viruses have been utilized for the development of the live-attenuated influenza vaccine (LAIV; commercially known as

FluMist) and the PV live-attenuated vaccine [233, 241]. These virus were cold adapted by serial passaging as low temperatures to select for temperature sensitive mutants. As described in Chapter 2 and 3, contemporary strains of

EV-D68 replicate similarly at both 32C and 37C, thus, cold-adaptation is unlikely to derive temperature sensitive mutants. Given the findings of

Chapter 3, I was able to generate a temperature sensitive contemporary EV-

D68 strain by incorporating non-structural genes of the temperature sensitive historic strain. This chimeric virus replicated to high titers at 32C while no detectable virus was found at 37C. This provides the groundwork for a potential live-attenuated vaccine for contemporary EV-D68 strains, encompassing the contemporary structural proteins that facilitate an antibody response but the historic non-structural proteins that facilitate temperature sensitivity.

Serology studies suggest no evidence of multiple serotypes across the different EV-D68 clades [39, 42]. Thus, one might propose that a live vaccine of a naturally temperature sensitive historic strain may be a practical candidate for a live vaccine. As noted in Chapter 4, contemporary strains of 122

EV-D68 replicate more efficiently and induce a more robust innate immune response than historic strains. In order to further credit the temperature sensitive MO-2014 chimera as a live vaccine, it should be tested in human nasal epithelial cells to determine if it still maintains a replication advantage. If this replication advantage and robust immune response is detected for the chimera, it has potential to induce a more robust adaptive immune response and further support its candidacy as a live vaccine.

Ferrets have been shown to generate neutralizing antibodies following intranasal infection with a historic EV-D68 strain [90]. Preliminary studies should be carried out infecting ferrets with Corn-1963, MO-2014, and the

MO-2014 temperature sensitive chimera followed by neutralization assays to determine neutralizing antibody titers in their respective sera.

Retrospective analysis of respiratory samples

There has been a growing focus on the surveillance of EV-D68 due to the recent recognition of an association with severe ARI and AFM disease manifestations. Prior to the global outbreaks in 2014 associated with EV-

D68, there was minimal attention directed at surveillance, owing to the relatively low case numbers between 1962 and the early 2000s. As described in Chapter 1, there are mild and asymptomatic cases of EV-D68 from the seasonal circulation of EV-D68 indicative by neutralizing antibodies convalescent serum. It is unlikely that outbreaks of this virus is a 123

phenomenon of the recent millennium. The issue with surveillance is capturing the true numbers of EV-D68 infections each year. Synonymous to

RV-A and RV-B, mild respiratory infections often do not require the attention of medical professionals. Rather, those suffering from these ‘common-cold viruses’ rely on self-care while allowing the infection to be self-resolved.

While these mild cases of EV-D68 may not be as medically important as the severe cases, their evolutionary importance should not be discounted.

Recent studies have focused on the rapid divergence of EV-D68 of into multiple clades circulating globally. There has been a focus on pinpointing mutations associated with ARI and AFM based on these evolutionary studies that have yielded no clear genetic link to severe outcome. However, while it is important to analyze the recent divergence of EV-D68 strains, severe cases have been recognized since the early 2000s albeit in lower numbers [16, 337].

While 2014 was the largest outbreak of EV-D68, it is possible that mutations associated with severe disease appeared in the genomes years prior. Similar to our temporal analysis of IRES activity in historic and contemporary strains (1999-2016), there is a necessity to begin investigating the biological phenotypes these viruses.

One challenge this necessity presents is the lack of EV-D68 virus isolates throughout this time. ATCC and BEI have made available four isolates collected during the 2014 outbreaks (contemporary) and two isolates from 1962 and 1963 (historic). Several laboratories have isolated viruses 124

independently for research, but the number of available strains prior to 2014 remains low [109, 132, 191]. Many viral RNA genomes have been detected and sequenced from patient samples without the isolation of infectious virus.

One potential focus of the field could be increasing the number of available isolates of EV-D68 by virus isolation on these positive samples. In the absence of infectious virus presence or sample, employment of our infectious clone system of developed and described in Chapters 2 and 3, we are capable of generating infectious virus for any available full genome. Given this tool, additional temporal recombinant viruses should be generated for each circulating clade beginning with the first available full genome sequence

(currently from 1997) and every available year to follow through 2014. These viruses should be tested for temperature sensitivity on RD cells using TCID50 and plaque assays as described in Chapters 2 and 3. There is conflicting evidence in the field regarding the function and necessity for ICAM-5 receptor mediated entry for contemporary EV-D68 strains to infect cells and causes neurovirulence [127, 128, 130]. Receptor mediated entry should be investigated by performing low MOI infections on RD cells in the presence of neuraminidase treatment. Neuraminidase cleaves the extracellular sialic acid, the initial receptor associated with EV-D68 attachment and entry. Next, low MOI growth curves should be performed in the presence of ICAM-5 blockers or by developing a stable ICAM-5 knockout cell line using

CRISPR/Cas-9 to fully abrogate ICAM-5 expression. If there is altered 125

receptor mediated entry, these studies will help define when this divergence occurred and which mutations facilitated the change. Finally, these viruses should be used to administer sub-lethal doses to mice to test the ability in an animal model for the outcome of AFM. Any nucleotide changes in the UTRs or AA in the coding regions discovered to contribute to temperature sensitivity, receptor selectivity, or neurovirulence should be systematically evaluated using site-directed mutagenesis on the DNA plasmid to reverse the mutations and determine their importance to the phenotype. Generating a central repository for available EV-D68 isolates or recombinant viruses could streamline progression of research in this field.

While viruses beginning in the early 2000s may lend insight to the biological evolution of EV-D68, there still remains a gap of nearly four decades of evolution unaccounted for. EV-D68 was only reported in 26 cases between initial isolation in 1970 and 2005. Determination of most of these cases relied heavily serological neutralization, thus, there remains little genomic information during this time. Retrospective analysis of available respiratory samples collected during these decades for patients suffering from respiratory could be analyzed for the detection of EV-D68. While there was no

EV-D68 surveillance program in place, surveillance efforts for respiratory pathogens such as influenza and adenoviruses could be utilized if available.

This technique was used by a team in Denmark who harnessed samples

126

collected for influenza surveillance from 1994-2010 to determine an abundance of EV-D68 positives and isolate viruses [8, 132, 337].

Conclusion

EV- D68 has undoubtedly evolved since its initial isolation in 1962 leading to an increase in pathogenicity in the form of ARI and AFM. Prior to 2014 outbreaks and the association with disease, there was little research done to address the mechanisms of EV-D68 disease manifestation. Beyond the basic characterization of temperature sensitivity, acid lability, and serum neutralization, there was little work done at the molecular level regarding the stages of infection and the cellular response. Since the 2014 outbreaks and mounting evidence of a shift in EV-D68 pathogenic severity, there has been growing interest in the impacts of EV-D68 evolution on viral replication and the cellular response. The research outlined in this dissertation provides advances in the understanding of contemporary EV-D68 strains compared to historic strains with respect to temperature sensitivity, translation efficiency, innate immune response, and cell tropism. These findings not only contribute to a biological understanding of this understudied virus, but given the shift in temperature sensitivity in contemporary strains and the pro-inflammatory response following infections, they provide a plausible hypothesis for their ability to penetrate and damage the lower respiratory tract that has not yet been reported. 127

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172

CURRICULUM VITAE

BRENDAN SMITH

615 N Wolfe Street, Rm W2104-1 Baltimore, MD 21205 443-287-8750 [email protected]

EDUCATION Johns Hopkins University, Johns Hopkins Bloomberg School of Public Health, W. Harry Feinstone Department of Molecular Microbiology and Immunology 2014 – Present

PhD Candidate in Molecular Microbiology and Immunology GPA: 3.68 Thesis: “Elucidating the Viral and Cellular Components of Enterovirus D68 Temperature Sensitivity” Laboratory of Dr. Andrew Pekosz

University of North Carolina 2006 –2011 Bachelor of Science Degree Major: Chemistry Graduated cum laude GPA: 3.52 Analytical Chemistry Research in Laboratory of Dr. Brent Dawson

PROFESSIONAL EXPERIENCE Johns Hopkins Technology Ventures, Business Development Fellow Baltimore, MD 2018-2020

Becton Dickinson Diagnostic Systems, R&D Scientist I and II Sparks, MD 2012–2014

Laboratory Corporation of America, Laboratory Technologist Burlington, NC 2009 –2012

University of North Carolina at Greensboro, Laboratory Assistant Greensboro, NC 2007-2009

173

City of Lexington Wastewater Treatment Plant, Intern Chemist Lexington, NC 2006

PUBLICATIONS Short, S.M., van Tol, S., Smith, B.D., Dong, Y., Dimopoulos, G. The mosquito adulticidal Chromobacterium sp. Panama causes transgenerational impacts on fitness parameters and elicits xenobiotic gene responses. Parasites & Vectors, Apr 2018

Klein, E.Y., Blumenkrantz, D., Serohijos, A., Shakhnovich, E., Choi, J., Rodrigues, J.V., Smith, B.D., Lane, A.P., Feldman, A., Pekosz, A. Stability of the Influenza Virus Hemagglutinin Protein Correlates with Evolutionary Dynamics. mSphere, Jan 2018

PRESENTATIONS Historic and contemporary Enterovirus D68 strains have altered replication, translation efficiency and induction of cell death across physiological ranges of temperature Keystone Symposia on Molecular and Cellular Biology Conference; Killarney, Ireland 2019 Annual Conference Poster Presentation

Strain-dependent effects of physiological temperature ranges on Enterovirus D68 translation and cell death American Society for Virology 2018 Annual Conference Oral Presentation

Effects of Physiological Temperature Ranges on Enterovirus D68 Replication in vitro American Society for Virology 2017 Annual Conference Oral Presentation

Effects of Temperature on the Replication Kinetics of Enterovirus D68 in Primary Human Nasal Epithelial Cells American Society for Virology 2016 Annual Conference Oral Presentation

174

TEACHING ASSISTANT EXPERIENCE Johns Hopkins University 2015-2019

Advanced Virology, Public Health Biology, Public Health Perspectives on Research

University of North Carolina at Greensboro 2006-2009 General Chemistry I and II, Organic Chemistry I and II

HONORS AND AWARDS Frederik B. Bang Award, Johns Hopkins University Katharine E. Welsh Fellowship, Johns Hopkins University 2018 Keerti Shah Fund Scholarship, Johns Hopkins University 2017 Keerti Shah Fund Scholarship, Johns Hopkins University 2016 Keerti Shah Fund Scholarship, Johns Hopkins University 2015 Outstanding Lab Assistantship Award, UNC Greensboro 2009 Sherri Forrester Award, UNC Greensboro 2008 Florence L. Schaeffer Memorial Scholarship, UNC Greensboro 2007

STUDENT ORGANIZATIONS Johns Hopkins Graduate Consulting Club (JHGCC) Member 2018 –2020

Johns Hopkins School of Public Health, Molecular Microbiology and Immunology Student Group Treasurer 2015 –2016

Chemistry and Biochemistry Club, University of North Carolina at Greensboro Member 2006 –2009

ASSOCIATION MEMBERSHIPS First Tee of Baltimore Young Professionals Board 2018-Present American Society for Virology 2016-Present American Society for Microbiology 2014-Present

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LEADERSHIP AND MENTORING ACTIVITIES  Serving as active member on the First Tee of Greater Baltimore Young Professionals Board organizing fundraising activities and acting as an ambassador for the First Tee of Greater Baltimore program  Functioning as a one-on-one mentor in the Baltimore City YMCA Youth Reach and Rise program, working with mentee to define and achieve actionable academic and personal goals.  Represented Becton Dickinson in tutoring workshops for local Teach for America educators and an array of STEM activities for Baltimore City middle and high schools.  Served as a Becton Dickinson representative and presenter at University of Maryland BioPark BioBlast event.  Volunteered at Smith High School of Greensboro, aiding in chemistry teaching workshops.  Served as an active member and director of Science Olympiad for high school and middle school aged children at UNC Greensboro.

ADDITIONAL SKILLS AND TRAINING Writing: Proficient in scientific writing, grant proposals, and non-technical writing Data analysis and visualization for academic and industry research Computer Skills: Microsoft Office suite, Microsoft Visio, Microsoft Project, Sharepoint, GraphPad (statistical and graphical analysis), Minitab (statistical and graphical analysis), Adobe Illustrator, CRM Software (Salesforce, Inteum) Leadership: Excellent communication, presentation, mentoring, and motivational skills

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