2. 2
List of Abbreviations:
ACE-2: angiotensin-converting enzyme two
ARDS: acute respiratory distress syndrome
AUG: codon for methionine and site of translation initiation
BAC2: bacterial artificial chromosome number two, a plasmid vector
BAC: bacterial artificial chromosome
BCV: bovine coronavirus
BGT: rabbit beta-globin terminator sequence, stops the transcription of RNA-dependent
RNA polymerase
BHK-21: baby hamster kidney cells
BtCoV: bat coronavirus
CD26: cluster of differentiation twenty-six, also identified as dipeptidyl peptidase IV
cDNA: complementary DNA, synthesized from RNA by reverse transcriptase PCR
CL-3: containment level 3 laboratory
CMV: cytomegalovirus promoter
CPE: cytopathic effect or cytopathogenic effect
CSCHAH: Canadian Science Center for Human and Animal Health
CSM-Trp: complete supplement medium without tryptophan
DI: defective interfering; may refer to a viral particle or genome
DMEM: Dulbecco’s modified Eagle medium
DNA: deoxyribonucleic acid
dNTPs: deoxynucleotide triphosphates
DPP4: dipeptidyl peptidase IV also known as CD26
3. 3
E: coronavirus envelope protein
ECDC: European Center for Disease prevention and Control
ECMO: extracorporeal membrane oxygenation
EMC: Erasmus Medical Center
EMC/2012: an isolate of the HCoV-MERS virus
ER: endoplasmic reticulum
EVD: Ebola virus disease
GFP: green fluorescent protein
Gpt: guanine phosphoribosyltransferase
HA: hemagglutinin, influenza glycoprotein
HCoV: human coronavirus
HCW: health care worker
HDVr: hepatitis delta virus ribozyme
HE: hemagglutinin esterase
HKU4: bat coronavirus Hong-Kong university isolate four
HKU5: bat coronavirus Hong-Kong university isolate five
ICTV: International Committee on Taxonomy of Viruses
IFN: interferon
Kb: kilo-base pairs of DNA
KSA: Kingdom of Saudi Arabia
LB: Luria-Bertani medium for the growth of bacteria
M: coronavirus membrane protein
mBAC4: plasmid construct containing the same mutation in HCoV-MERS genome as
4. 4
pYES1L14
mBAC6: final plasmid construct of the HCoV-MERS genome
mBACL23: sub-cloning plasmid construct used to correct mutation in mBAC4
MEM: Minimal Essential Medium for mammalian cell culture
MERS: Middle East respiratory syndrome
MF1: MERS fragment one
MF2: MERS fragment two
MF3: MERS fragment three
MHV: murine hepatitis virus
MODS: multiple organ distress syndrome
mRNA: messenger ribonucleic acid
N: coronavirus nucleoprotein
NS6: HCoV-MERS non-structural protein number six
Nsp: viral non-structural protein
ORF: open reading frame
ORF1a/b: first open reading frame in a coronavirus genome
ORF1b: a RT-qPCR assay used to detect the HCoV-MERS genome
PCR: polymerase chain reaction
pYES1L: a bacterial artificial chromosome plasmid used for in vivo homologous
assembly of the HCoV-MERS fragments (MF1, MF2 and MF3).
pYES1L14: plasmid construct containing the same mutation as mBAC4
RBD: receptor binding domain
RDRP: RNA-dependent RNA polymerase
5. 5
RecA: essential protein for the repair and maintenance of DNA in bacteria
RFP: red fluorescent protein
RGS: reverse genetic system
RNA: ribonucleic acid
RT-PCR: reverse-transcriptase polymerase chain reaction
RT-qPCR: real-time quantitative polymerase chain reaction
RTC: coronavirus replication transcription complex
S: coronavirus glycoprotein
SARS: severe acute respiratory syndrome
sBAC: HCoV-SARS infectious cDNA clone system
SDS: sodium dodecyl sulfate
sgRNA: sub-genomic ribonucleic acid
SOC: super optimal catabolite repression broth
SOE-PCR: splice by overlap extension polymerase chain reaction
SSA: single-strand annealing
T7: bacteriophage promoter
TAQ: thermus aquaticus polymerase
TGEV: transmissible gastroenteritis coronavirus
TRS: coronavirus transcription regulatory sequence
upE: a RT-qPCR assay used to detect the HCoV-MERS genome
UTR: un-translated region
vvHCoV: vaccinia virus based coronavirus reverse genetics system
6. 6
Abstract
Coronaviruses have caused high pathogenic epidemics within the human population on
two occasions; in 2003 a coronavirus (HCoV-SARS) caused severe acute respiratory syndrome
and in 2012 a novel coronavirus emerged named Middle East respiratory syndrome (HCoV-
MERS). Four other species of coronavirus circulate endemically in the human population
(HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1), which cause more benign
respiratory disease than either HCoV-SARS or HCoV-MERS. The emergence of HCoV-MERS
provides an additional opportunity to study the characteristics of coronaviruses. Reverse genetics
can be used to study an organism’s phenotype by logical mutation of its genotype. Construction
of an infectious clone construct provides a means to investigate the nature of HCoV-MERS by
reverse genetics. An HCoV-MERS infectious cDNA clone system was constructed to use for
reverse genetics by homologous recombination of bacterial artificial chromosomes (BACs). This
system should aid in answering remaining questions of coronavirus genetics and evolution as
well as expedite the development of vaccines and prophylactic treatments for HCoV-MERS.
7. 7
Acknowledgements
This work would not have been possible without the support of my family, mentors,
colleagues and friends. I would like to particularly acknowledge Dr. Steven Theriault, Dr.
Deborah Court, Dr. Darwyn Kobasa and Dr. Karen Brassinga for acting as mentors, providing
me with guidance and the opportunity to learn. To Bradley Cook I would like to extend gratitude
for teaching me many laboratory fundamentals and being willing to discuss ideas and results on
any Sunday. Todd Cutts worked to keep the lab running smoothly and I would like to thank him
for training me to work in containment level 3. Anders Leung introduced me to the project,
providing much technical knowledge and “encouragement”. Finally, I would like to mention Dr.
Charlene Ranadeherra and Mable “wing-sum” Hagan because they played music in the lab, were
always keen to share pens and kept Anders in check.
8. 8
Dedication
I would like to dedicate this work to all of the people who contributed to my education in
school and more importantly in life. Foremost, my parents Doreen Docherty and Andrew
Nikiforuk deserve recognition for their sacrifice and love. I would also like to acknowledge
Allison Black, Dimitar Kashchiev and the lovely Veronica Izydorcyzk ()..(). I hope that I can
contribute to your lives, as you have to mine.
9. 9
Table of Contents
List of Abbreviations: …………………………………………………………………………II
Abstract: ……………………………………………………………………………………….III
Acknowledgments: …………………………………………………………………………….IV
Dedication: ……………………………………………………………………………………..V
List of Figure and Tables: ……………………………………………………………………XII
Copyright Permissions:………………………………………………………………………XIII
Chapter One: Literature Review.......................................................................................................14
1.0.0.0: The Nature of Zoonotic Disease..................................................................................................... 14
1.1.1.0 Introduction......................................................................................................................................................15
1.1.2.0 Order: Nidovirales..........................................................................................................................................16
1.1.3.0 Families of the Nidovirales: Arteriviridae, Coronaviridae, Mesoniviridae and Roniviridae .17
1.1.4.0 Subfamily: Coronavirinae...........................................................................................................................17
1.1.5.0 Genus and Lineage: Betacoronavirus, Lineage C................................................................................18
1.2.0.0: Epidemiology of the Middle East Respiratory Syndrome (MERS) ...................................... 19
1.2.1.0: Advent of the HCoV-MERS Epidemic: ................................................................................................19
1.2.2.0 Zoonotic Epidemiology:...............................................................................................................................20
1.2.3.0 Human-to-Human Transmission of HCoV-MERS..............................................................................26
1.4.0.0: Biological Structure of Coronaviruses ......................................................................................... 34
1.4.1.0 Genome Organization...................................................................................................................................36
1.4.2.0 Translated Regions.........................................................................................................................................39
1.4.3.0 ORF 1a/b Proteins..........................................................................................................................................39
1.4.4.0 Glycoprotein (Spike Protein)......................................................................................................................40
1.4.5.0 Envelope and Membrane Proteins ............................................................................................................43
1.4.6.0 Nucleocapsid Protein.....................................................................................................................................43
1.4.7.0 Accessory Proteins.........................................................................................................................................44
1.4.8.0 Un-Translated Regions.................................................................................................................................45
1.4.9.0 The Coronavirus Leader Sequence, 5’ Un-translated Region (UTR)............................................45
1.4.10.0 Coronavirus 3’ Un-translated Region (UTR)......................................................................................48
1.4.11.0 Coronavirus Transcription Regulatory Sequence (TRS).................................................................48
1.5.0.0: Coronavirus Life Cycle.................................................................................................................... 51
1.5.1.0 Viral Entry........................................................................................................................................................51
1.5.2.0 Transcription and Genome Replication...................................................................................................53
1.5.3.0 Discontinuous Negative-Strand Transcription......................................................................................56
1.5.4.0 Coronavirus Genome Replication.............................................................................................................59
1.5.5.0 Virus Assembly and Release ......................................................................................................................60
1.5.6.0 Overview of Coronavirus Replication.....................................................................................................61
1.6.0.0: Reverse Genetics Systems................................................................................................................ 64
1.6.1.0 Coronavirus Reverse Genetic Systems....................................................................................................66
1.6.2.0 Homologous Recombination of DNA for Assembly of Plasmid Vectors....................................80
1.6.3.0 In vivo Homologous Recombination........................................................................................................80
1.6.4.0 In vitro Homologous Recombination.......................................................................................................82
1.6.5.0 Conclusion........................................................................................................................................................84
1.7.0.0 Declaration of Research Intent and Hypothesis.......................................................................... 84
1.7.0.1 Research Intent................................................................................................................................................84
1.7.0.2 Hypothesis........................................................................................................................................................85
10. 10
1.7.0.3 Significance of Research..............................................................................................................................85
Chapter Two: Materials and Methods.............................................................................................86
2.1.0.0 Materials and Methods...........................................................................................................86
2.1.1.0 General Techniques............................................................................................................................ 86
2.1.1.1 Cell Culture......................................................................................................................................................86
2.1.1.2 Gel-Electrophoresis and Extraction of PCR Products........................................................................87
2.1.1.3 Enzymatic Restriction Digestion of DNA..............................................................................................88
2.1.1.4 Amplification of DNA with TAQ Polymerase for TOPO-TA Cloning....................................88
2.1.1.5 Screening of Escherichia coli (E.coli) Colonies by Polymerase Chain Reaction ......................89
2.1.1.6 Screening of Saccharomyces cerevisiae (S.cerevisiae) Colonies by Polymerase Chain
Reaction...........................................................................................................................................................................89
2.1.1.7 Electroporation of E.coli cells with Plasmid DNA..............................................................................90
2.1.1.8 Glycerol Stock Preparation of E.coli cells..............................................................................................91
2.1.1.9 Isolation of Plasmid Vectors from E.coli cells......................................................................................91
2.1.1.10 Sequencing of Polymerase Chain Reaction Amplicons and Plasmid Constructs....................92
2.1.2.0 Strategy for Molecular Cloning of the HCoV-MERS cDNA Infectious Clone System ..... 92
2.1.2.1 Overview...........................................................................................................................................................92
2.1.2.2 Synthesis of HCoV-MERS Synthetic Gene Fragments .....................................................................93
2.1.2.3 Selection of Bacterial Artificial Chromosome Vector........................................................................93
2.1.2.4 Site Directed Mutagenesis of the TrueBlue-BAC2 Vector...............................................................94
2.1.2.5 Primer Design..................................................................................................................................................97
2.1.2.6 Splice-by-Overlap Extension Polymerase Chain Reaction...............................................................97
2.1.2.7 High-Order Genetic Assembly of the HCoV-MERS Genome .....................................................101
2.1.2.8 Sub-cloning the HCoV-MERS Genome..............................................................................................103
2.1.2.9 Correction of the HCoV-MERS NS6 Mutation in the mBAC4 Vector.....................................106
2.1.2.10 Summary of HCoV-MERS cDNA Infectious Clone System Construction ...........................109
2.1.3.0 Rescue of the HCoV-MERS virus from the cDNA Infectious Clone Construct................111
2.1.3.1 Rescue of Coronavirus Infectious cDNA Clone Systems in Mammalian Cell Culture........111
2.1.3.2 Viral RNA Isolation....................................................................................................................................112
2.1.3.3 Production of HCoV-MERS cDNA From Viral Genomes Extracted by RNA Isolation.....112
2.1.3.4 Confirmation of HCoV-MERS Virus Rescue by PCR Amplification of
the Viral RDRP Gene...............................................................................................................................................113
2.1.3.5 Phylogenetic Analysis of the HCoV-MERS RDRP Gene Fragment..........................................113
2.2.0.0 Results...................................................................................................................................... 116
2.2.1.0 Objective One ....................................................................................................................................116
2.2.1.1 Construction of the HCoV-MERS cDNA Genome From Synthesized Gene Fragments.....116
2.2.2.0 Objective Two....................................................................................................................................119
2.2.2.1 Assembly of the HCov-MERS cDNA Genome Fragments Using S.cerevisiae......................119
2.2.2.2 Electroporation of HCoV-MERS pYES1L Constructs into E. coli Cells..................................123
2.2.2.2 Restriction Digest of HCoV-MERS pYES1L Maxi-Preparations...............................................125
2.2.2.3 Site Directed Mutagenesis of the True Blue-BAC2 Vector to Remove PvuI and BstEII
Restriction Enzyme Digest Sites...........................................................................................................................127
2.2.2.4 Sub-Cloning of the HCoV-MERS Genome Between pYES1L20 and TrueBlue-BAC2
ΔPvuI/ΔBstEII Vectors............................................................................................................................................129
2.2.2.5 In-vitro Homologous Recombination of the HCoV-MERS mBAC4 Plasmid to Correct The
NS6 G11194A Mutation..........................................................................................................................................132
2.2.3.0 Objective 3..........................................................................................................................................134
11. 11
2.2.3.1 Rescue of the HCoV-MERS Virus From the Infectious cDNA Clone System Construct
mBAC6 .........................................................................................................................................................................134
2.2.3.2 Confirmation of HCoV-MERS Rescue................................................................................................138
2.2.3.3 Phylogenetic Comparison of the HCoV-MERS Virus With Other Coronaviruses by a Partial
RDRP Gene Sequence..............................................................................................................................................141
2.3.0.0 Review and Discussion of HCoV-MERS Reverse Genetic .......................................... 143
2.3.1.0 Summary.............................................................................................................................................143
2.3.2.0 Discussion ...........................................................................................................................................144
2.3.2.1 Use of Reverse Genetics to Construct HCoV-MERS Mutant or Recombinant Viruses.......144
2.3.2.2 Use of Reverse Genetics to Screen HCoV-MERS Antiviral Compounds ................................146
2.3.2.3 Use of Reverse Genetics to Characterize Point Mutations Within the HCoV-MERS Genome
.........................................................................................................................................................................................147
2.3.2.4 Benefit of Homologous Recombination to Coronavirus Reverse Genetics..............................148
Appendix A: Measurements and Calculations............................................................................ 150
Table A.1 Measurements of Tissue Culture Plates................................................................................150
Table A.2 Tissue Culture Splitting Calculations....................................................................................150
Appendix B: Recipes and Protocols............................................................................................... 151
1.0 Cell Culture Media Recipes..................................................................................................................151
2.0 Polymerase Chain Reaction Recipes...................................................................................................151
4.0 T4 Ligation Procedure ...........................................................................................................................152
Table B.1 Polymerase Chain Reaction Protocol for PrimeSTAR GXL DNA Polymerase..........153
Table B.2: Splice-by-Overlap Extension Polymerase Chain Reaction Protocol for PrimeSTAR
GXL DNA Polymerase..................................................................................................................................153
Table B.3: Polymerase Chain Reaction Screening for Plasmid Vectors using the GoTaq Green
DNA Polymerase............................................................................................................................................154
Appendix C: Molecular Genetics Primers, Vectors and Sequence ........................................ 154
Table C.1: Description of synthetic gene fragments produced for the first four- thousand
nucleotides of the HCoV-MERS EMC/2012 genome............................................................................154
Table C.2: Description of synthetic gene fragments produced for the last four-thousand
nucleotides of the HCoV-MERS EMC/2012 genome............................................................................155
Table C.3: Description of oligonucleotide primers used for the construction of the HCoV-MERS
infectious cDNA clone system .....................................................................................................................155
Table C.4: Description of Oligonucleotide Primers Used to Sequence the HCoV-MERS Infectious
cDNA Clone System. .....................................................................................................................................159
Sequence 1: HCOV-MERS Genome.............................................................................................. 161
Sequence 4: True Blue-BACΔPvuIΔBstEII ................................................................................. 179
Cytomegalovirus Promoter Sequence: Multi-host Donor and Expression Vector pFlpBtM-II.182
Hepatitis D Virus Ribozyme: Hepatitis D Virus Complete Genome.................................................182
Sequence Six: Synthetic HCoV-MERS Gene Fragments ......................................................... 184
Plasmid Constructs:........................................................................................................................... 198
Works Cited......................................................................................................................................... 202
12. 12
List of Figures and Tables:
FIGURE 1.4.0.0 MOLECULAR STRUCTURE OF THE CORONAVIRUS VIRION:............. 35
FIGURE 1.4.1.0 GENETIC ORGANIZATION OF THE CORONAVIRUS GENOME:........... 38
FIGURE 1.4.4.0 SYNCYTIA DEVELOPMENT IN VIRAL INFECTED CELL CULTURE: 42
FIGURE 1.4.9.0 THE UN-TRANSLATED REGIONS OF THE CORONAVIRUS GENOME:
............................................................................................................................................... 47
FIGURE 1.4.11.0 CORONAVIRUS TRANSCRIPTION REGULATORY SEQUENCES
INFLUENCE THE FORMATION OF SGRNA:................................................................. 50
FIGURE 1.5.2.0 CORONAVIRUS-INDUCED MEMBRANE ALTERATIONS AS
PLATFORMS FOR VIRAL REPLICATION:..................................................................... 55
FIGURE 1.5.3.0 DISCONTINOUS NEGATIVE STRAND TRANSCRIPTION:...................... 57
TABLE 1.6.0.0 REVERSE GENETICS HAS BEEN USED TO CHARACTERIZE VIRUSES
FROM MULTIPLE FAMILIES:.......................................................................................... 65
FIGURE 1.6.1.0A THE COPY CHOICE MECHANISM OF RNA RECOMBINATION:. ....... 69
FIGURE 1.6.1.0B VACCINIA VIRUS BASED CORONAVIRUS REVERSE GENETICS
SYSTEMS: ........................................................................................................................... 77
FIGURE 1.6.1.0C HOMOLOGOUS RECOMBINATION BETWEEN A VACCINIA VIRUS
BASED, CORONAVIRUS REVERSE GENETICS SYSTEM AND A PLASMID WITH
HOMOLOGOUS REGIONS:............................................................................................... 78
FIGURE 1.6.4.0 THE SINGLE-STRAND ANNEALING PROCESS THOUGHT TO
PRODUCE RECOMBINANT HOMOLOGOUS DNA MOLECULES DURING
VACCINIA VIRUS REPLICATION:.................................................................................. 83
TABLE 2.1.1.9 ANTIBIOTIC CONCENTRATIONS FOR POSITIVE SELECTION OF
PLASMID VECTORS IN E. COLI CELLS:........................................................................ 91
FIGURE 2.1.2.4 STRATEGY FOR SITE DIRECTED MUTAGENESIS:................................ 96
FIGURE 2.1.2.6A THREE HCOV-MERS CDNA GENOME FRAGMENTS WERE MADE BY
SOE-PCR AND USED AS TEMPLATES FOR IN VIVO HOMOLOGOUS
RECOMBINATION:............................................................................................................ 99
FIGURE 2.1.2.6B ASSEMBLY OF HCOV-MERS SYNTHETIC GENE FRAGMENTS: ..... 100
FIGURE 2.1.2.8 CLONING OF THE HCOV-MERS GENOME BETWEEN THE BAC
VECTORS PYES1L AND TRUEBLUE-BAC2:............................................................... 105
FIGURE 2.1.2.9 CONSTRUCTION OF THE MBACL23 VECTOR FOR CORRECTION OF
THE NS6 MUTATION IN MBAC4: ................................................................................. 108
FIGURE 2.1.2.10 STRATEGY FOR THE CONSTRUCTION OF THE HCOV-MERS CDNA
INFECTIOUS CLONE SYSTEM (MBAC6):.................................................................... 110
TABLE 2.1.3.5 THE GENOMES OF SIX OTHER CORONAVIRUSES WERE
DOWNLOADED OFF OF GENBANK AND USED IN A PHYLOGENETIC
COMPARISON WITH THE HCOV-MERS RT AMPLICON: ........................................ 115
FIGURE 2.2.1A ASSEMBLY OF SMALL HCOV-MERS GENE FRAGMENTS BY SOE-
PCR:.................................................................................................................................... 117
FIGURE 2.2.1B ASSEMBLY OF LARGE HCOV-MERS GENE FRAGMENTS BY SOE-PCR:
............................................................................................................................................. 118
FIGURE 2.2.2.1A GROWTH OF S.CEREVISIAE COLONIES ON CSM-TRP PLATES:. ... 120
FIGURE 2.2.2.1B SCREENING OF LYSED S.CEREVISIAE COLONIES BY PCR:............. 121
13. 13
FIGURE 2.2.2.1C UNDILUTED SDS PREVENTED EFFECTIVE SCREENING OF
S.CEREVISIAE COLONIES BY PCR:............................................................................... 122
FIGURE 2.2.2.1 PCR SCREENING OF E.COLI COLONIES ELECTROPORATED WITH
PYES1L20 PLASMID:....................................................................................................... 124
FIGURE 2.2.2.2 RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF
THREE HCOV- MERS PYES1L VECTORS WITH THE RESTRICTION DIGEST
ENZYMES KPN1 AND NHE1:......................................................................................... 126
FIGURE 2.2.2.3 THE PVUI AND BSTEII SITES OF TRUEBLUE-BAC2 VECTOR WERE
REMOVED BY SITE DIRECTED MUTAGENESIS:...................................................... 128
FIGURE 2.2.2.4A THE SECOND FRAGMENT OF THE HCOV-MERS GENOME (PVUI-
MLUI) WAS INSERTED INTO THE INTERMEDIATE TRUEBLUE-BAC2 ΔPVUI AND
ΔBSTEII VECTOR: ........................................................................................................... 130
FIGURE 2.2.2.4B E.COLI CELLS WERE SCREENED FOR SUCCESSFUL PYES1L TO
TRUEBLUE-BAC2 SUB-CLONE CONSTRUCTS:......................................................... 131
FIGURE 2.2.2.5 E.COLI COLONIES WERE PCR SCREENED FOR THE HCOV-MERS
GENOME FOLLOWING THE SUB-CLONING PROCEDURE TO CORRECT THE
G11194A MUTATION OF MBAC4: ................................................................................ 133
FIGURE 2.2.3.1A RESCUE OF THE HCOV-MERS AND HCOV-SARS CORONAVIRUSES
FROM CDNA INFECTIOUS CLONE SYSTEMS:.......................................................... 135
FIGURE 2.2.3.1B THE NS6 MUTATION G11194A MAY HAVE PREVENTED RESCUE OF
THE HCOV-MERS VIRUS FROM THE CDNA INFECTIOUS CLONE SYSTEM: ..... 136
FIGURE 2.2.3.1C REPRESENTATION OF THE FOLDED STATE OF THE HCOV-MERS
NS6 MUTANT G11194A:.................................................................................................. 137
FIGURE 2.2.3.2A AMPLIFICATION OF A PARTIAL HCOV-MERS RDRP FRAGMENT BY
PCR:.................................................................................................................................... 139
FIGURE 2.2.3.2B A PORTION OF THE CORONAVIRUS RDRP GENE WAS PCR
AMPLIFIED FROM INFECTED VERO CELL SUPERNATANT FOR PHYLOGENETIC
ANALYSIS:........................................................................................................................ 140
FIGURE 2.2.3.3 THE PHYLOGENETIC TREE CONSTRUCTED TO CONFIRM THE
EVOLUTIONARY IDENTITY OF THE CORONAVIRUS RESCUED FROM THE
MBAC6 CONSTRUCT:..................................................................................................... 142
Copyright Permission
Figure 1.5.2.0 CORONAVIRUS-INDUCED MEMBRANE ALTERATIONS AS PLATFORMS
FOR VIRAL REPLICATION:
Perlman, S., & Netland, J. (2009). Coronaviruses post-SARS: update on replication and
pathogenesis. Nature reviews. Microbiology, 7(6), 439–450……………………………..216
14. 14
Chapter One: Literature Review
1.0.0.0: The Nature of Zoonotic Disease
Emerging infectious diseases mainly enter the human population by the zoonotic route
meaning that they transfer from an infected animal population (Wolfe, Dunavan, & Diamond,
2012). Three types of zoonotic introductions exist. A disease can naturally “spill over” from an
infected wild animal population into a domestic animal population or directly into the human
population. Alternatively, human interaction or disruption of the environment can lead to the
shift of a disease from animals to humans. A zoonotic event can also occur in an isolated manner
when an animal coincidentally infects a single human. In the last two decades, diseases have
entered into the human population by one these three mechanisms and caused significant cases of
morbidity and mortality (Daszak, Cunningham, & Hyatt, 2000). In 2002 and 2003, the severe
acute respiratory syndrome (HCoV-SARS) coronavirus “spilled over” into the human population
causing a limited pandemic (Holmes, 2009). Retrospective epidemiological and molecular
studies of HCoV-SARS have demonstrated that the virus likely originated in wild bat
populations before being transmitted into domesticated palm civet cats and eventually humans
(Hilgenfeld & Peiris, 2013). Over the course of the 2002/2003 pandemic, the HCoV-SARS virus
caused approximately 800 deaths in 30 countries (Centers for Disease Control and Prevention,
2012). The emergence of Zaire ebolavirus in human populations of Equatorial and West Africa
has occurred by environmental disruption and coincidental infection (Baize et al., 2014;
Feldmann, Wahl-Jensen, Jones, & Stroher, 2004). Population growth and economic disparity
drive Africans to hunt bush meat, a practice repeatedly linked to cases of Ebola virus disease
15. 15
(EVD) in rural villages (Feldmann & Geisbert, 2011). The infection of the index patient in the
large West Africa epidemic of 2013-2015 was an isolated incident where the subject contracted
the virus from the environment in an unknown way (Baize et al., 2014). This single zoonotic
transmission event caused the largest recorded EVD epidemic in history with ≥ 26, 933 cases and
11, 120 deaths to date (WHO, 2015). Virologists have yet to understand the specific mechanisms
that allow a virus to transfer from animals to humans, because of the complex relationship
between a pathogen, its host and the environment (Jones et al., 2008; Wolfe et al., 2012). Any
zoonotic transmission event involves numerous factors including: the type of animal host, the
type of pathogen, the genetics of the pathogen, the health of the human and the length and type
of animal to human contact (Daszak et al., 2000). Advances in molecular diagnostics and the
establishment of comprehensive surveillance programs provide the means to observe disease in
human and animal populations identifying signals of zoonotic transmission. Increasing global
population, environmental disruption for economic growth and amount of international travel
and trade, make the allocation of resources into understanding zoonotic transmission paramount
as all these factors increase the opportunity for zoonotic introduction of disease (Jones et al.,
2008).
Viruses belonging to the Coronaviridae family present a good model to study the
zoonotic introduction of disease because they infect both animals and humans.
1.1.0.0: Coronavirus Taxonomy
1.1.1.0 Introduction
A group called the International Committee on the Taxonomy of Viruses (ICTV) has the
responsibility of classifying novel and emerging viruses into specific groupings that reflect our
understanding of a virus’s origin and its relationship to other viruses (King, Lefkowitz, Adams,
16. 16
& Carstens, 2011). Rapid advances in our ability to isolate and sequence viruses has made the
work of ICTV important as every year virologists isolate novel viruses and improve their
knowledge of identified ones. The volume of knowledge that virologists have procured heightens
the need for a universal system of classifying and naming viruses so that new viruses are not
incorrectly placed into viral families (Büchen-Osmond, 2003). The ICTV has developed criteria
they use to organize viruses by the Linnaen system of biological classification. Before the
ubiquitous availability of genomic sequencing viruses were clustered by their clinical and
pathogenic properties, transmission characteristics, morphology and antigenic type. Modern
taxonomy still uses these characteristics to organize viruses on macro levels (i.e.: order and
family); however, the micro levels (i.e.: genus and lineage) are now primarily organized by
genetic differences (Büchen-Osmond, 2003). The ICTV assigned the recently emerged virus
HCoV-MERS to the order Nidovirales due to the virus’s phenotypic and genomic characteristics
(Zaki, van Boheemen, Bestebroer, Osterhaus, & Fouchier, 2012).
1.1.2.0 Order: Nidovirales
The ICTV categorized the HCoV-MERS virus to the order Nidovirales which contains
four families of viruses that differ in their morphology, genome size and host range (King et al.,
2011). The order Nidovirales belongs to the fourth group of the Baltimore classification system
because its members possess positive sense, single-stranded ribonucleic acid (RNA) genomes
(King et al., 2011). The name Nidovirales derives from the Latin word nidus-, meaning nest,
which refers to a nested set of mRNAs that the viruses produce during their replication cycle
(Gorbalenya, Enjuanes, Ziebuhr, & Snijder, 2006). In addition to producing attenuated mRNA,
Nidovirales share common genome organization. The 5’ end of the viral genome contains non-
structural genes while the structural genes reside on the 3’ side. The Nidovirales also exhibit a
17. 17
constellation of seven conserved domains in their genome within genes encoding for:
transmembrane proteins, the RNA dependent RNA polymerase (RDRP), a zinc finger-like
protein, helicase, 3C-like protease and uridylate-specific endonuclease (Weiss & Navas-Martin,
2005).
1.1.3.0 Families of the Nidovirales: Arteriviridae, Coronaviridae, Mesoniviridae and
Roniviridae
The order Nidovirales contains four families the Arteriviridae, Coronaviridae,
Mesoniviridae and Roniviridae. The families Coronaviridae and Roniviridae have large genomes
ranging from approximately 27 to 32 kilo-base (kb) pairs of nucleic acid, making these viruses
the largest known RNA viruses (King et al., 2011). The genome sizes of the small nidoviruses
(Arteriviridae and Mesoniviridae) range from thirteen to sixteen kilo-base pairs of nucleic acid
(Lauber et al., 2012). The four families are further segregated into sub-families by genome
identity, virion structure and antigenicity. This review of coronavirus taxonomy will only
examine the sub-family coronavirinae as it contains all of the six known human coronaviruses
(Gorbalenya et al., 2006).
1.1.4.0 Subfamily: Coronavirinae
The name coronavirinae or coronavirus stems from the Latin prefix corona- meaning
crown. Coronaviruses project a pattern of glycoproteins on the surface of their envelope that
gives them a crown-like structure (Gorbalenya et al., 2006). Further organization of
coronavirinae viral species depends on sequence analysis of their RDRP genes and antigenic
cross-reactivity. The highly conserved RDRP region (approximately 992 nts) of all coronaviruses
allows for identification of novel coronavirus species as well as the clustering of known
coronaviruses into phylogenetic groups (Stephensen, Casebolt, & Gangopadhyay, 1999). Four
18. 18
phylogenetic groupings termed “genera” compose the sub-family coronavirinae the:
Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus (King et al.,
2011). The alpha- and beta- coronavirus genera evolved from an ancestral bat virus, the gamma-
and delta- coronaviruses descended from avian ancestors (Drexler, Corman, & Drosten, 2014;
Lau et al., 2013) . Many of the clinically important human coronaviruses, including HCoV-
SARS and HCoV-MERS, belong to the Betacoronavirus genus (Hilgenfeld & Peiris, 2013).
1.1.5.0 Genus and Lineage: Betacoronavirus, Lineage C
The Betacoronavirus genus contains four distinct lineages (A, B, C and D). Lineage A
differs from the others to the greatest extent, as lineage A viruses possess a secondary, shorter
glycoprotein gene called hemagglutinin esterase (HE) (Chan et al., 2015). The addition of HE
projections on the coronavirus envelope slightly changes the morphology of lineage A viruses to
a halo shape (de Groot, 2006). The other lineages (B, C and D) differ from each other by
polymorphisms in their genetic sequences. HCoV-SARS belongs to the B lineage while HCoV-
MERS and bat coronaviruses BtCoV-HKU4-1 and BtCoV-HKU5-1 occupy lineage C, the D
lineage contains a variety of other bat coronaviruses (Drexler et al., 2014; Lau et al., 2013; Woo,
Lau, Li, Tsang, & Yuen, 2012). No other identified human coronavirus except HCoV-MERS
belongs to lineage C of the betacoronavirus genus (Zaki et al., 2012). The HCoV-MERS virus
requires further study to understand its evolutionary uniqueness and the properties that allowed
for its zoonotic transmission into the human population.
19. 19
1.2.0.0: Epidemiology of the Middle East Respiratory Syndrome
(MERS)
1.2.1.0: Advent of the HCoV-MERS Epidemic:
In June of 2012, a sixty-year-old male suffering from symptoms of respiratory disease
was admitted to hospital in Jeddah the second largest city of the Kingdom of Saudi Arabia
(KSA). He died eleven days into treatment from acute respiratory distress syndrome (ARDS) and
renal failure. Analysis of the patient’s blood sample showed a decreased count of lymphocytes
indicative of viral infection (Zaki et al., 2012). In an attempt to isolate a virus, a post mortem
sputum sample of the patient was added to monolayers of Vero and LLC-MK2 cells. Cytopathic
effect (CPE) was observed in the Vero cells after fifteen days of incubation (Zaki et al., 2012).
The supernatant was harvested from the infected cell culture and screened for common
respiratory viruses by a reverse transcription polymerase chain reaction (RT-PCR) assay (Zaki et
al., 2012). The RT-PCR diagnostic showed positive for a virus of the family coronaviridae; this
virus, now known as the Middle East respiratory syndrome human coronavirus (HCoV-MERS)
was named after its endemic region (Enserink, 2013).
In the year 2012, nine more confirmed cases of HCoV-MERS occurred (European Centre
for Disease Prevention and Control, 2015a). On the 22nd
of September a forty-nine year old man
suffering from respiratory distress was hospitalized in Doha, the capital city of Qatar (Roos,
2013). There was no evidence of contact between this man and the index case. The second
patient was medically transported by air ambulance to London where before his passing he was
diagnosed with HCoV-MERS. The third case of MERS occurred on the 4th
of November 2012,
when the KSA health ministry reported that a 45-year-old man was diagnosed with the virus.
20. 20
Although patient three suffered from several chronic health conditions and was a long time
smoker he eventually recovered from HCoV-MERS infection. The subsequent case (case four)
was diagnosed the 19th of November and died from renal failure. Four days later multiple cases
of HCoV-MERS occurred when two people were infected in KSA and one in Qatar. This marked
the first identified epidemiological chain of human-to-human transmission because cases six and
seven lived in the same residence as case four. Case six died of multiple organ failure, while case
seven survived HCoV-MERS infection (Khan, 2013). At the end of November 2012, the World
Health Organization (WHO) retrospectively tested samples collected April 2012 (two months
before the index HCoV-MERS case) from eleven patients in Jordan who suffered from
respiratory disease for HCoV-MERS. The testing showed that two of the eleven patients from
the Jordan cluster were infected with HCoV-MERS (Hijawi et al., 2013). As HCoV-MERS cases
continued to occur into 2013, virologists attempted to identify how the virus was entering the
human population and the extent of human-to-human transmission.
1.2.2.0 Zoonotic Epidemiology:
The first theory of HCoV-MERS transmission speculated that bats host the virus and
serve as a transmission vector to the human population (Reusken et al., 2013). Next generation
sequencing of early HCoV-MERS isolates showed that the virus phylogenetically clusters with
two bat coronaviruses (BtCoV-HKU5 and BtCoV-HKU4) in lineage C of the betacoronaviridae
(Lau et al., 2013).
The isolation of many other coronavirus species including a direct ancestor of HCoV-
SARS from bats supports the hypothesis that HCoV-MERS originated in bats and underwent an
interspecies transmission event to cross into humans (Hon et al., 2008). In late 2013, the closest
relative to HCoV-MERS (Neoromicia-PML) was isolated from a Neoromicia zulensis bat native
21. 21
to South Africa. The relatedness of Neoromicia-PML to HCoV-MERS was considered
controversial because only a portion of the RDRP gene was phylogenetically compared (Ithete et
al., 2013). Virologists could not expand the analysis to other genes because only a partial
sequence of Neoromicia-PML was isolated from bats. Molecular clock analysis of the two
viruses revealed that they shared a most recent common ancestor approximately forty-four years
ago (Cotten et al., 2013). In comparison, the most common recent ancestor between HCoV-
MERS and fully sequenced lineage C betacoronaviridae existed approximately one hundred and
thirty-four years ago (Lau et al., 2013). A similar analysis of HCoV-SARS and a virus from civet
cats showed that the two highly similar viruses (genome identity of >90%) also shared a
common ancestor of forty-four years ago (Hon et al., 2008). The molecular evolution of HCoV-
MERS from a bat coronavirus and similarity to current bat coronaviruses suggests that an HCoV-
MERS like virus probably circulates within the bat population (Ithete et al., 2013). A study of bat
cell line permissibility to HCoV-MERS virus infection confirmed that the virus infects bat cells
in vitro (Caì et al., 2014). Although a full-length MERS-like coronavirus has not yet been
identified in a bat, enough evidence suggests that the HCoV-MERS virus like the HCoV-SARS
originated and evolved in bats before becoming pathogenic to humans (Falzarano et al., 2013).
The question of how HCoV-MERS transmits from bats to humans remains; two theories exist.
Bats either directly transmit HCoV-MERS to humans or, bats transfer the virus to an
intermediate animal species capable of more efficiently infecting humans.
The HCoV-SARS epidemic of 2002 and 2003 and studies of bat ecology support the
second theory of staggered HCoV-MERS transmission as known coronaviruses rarely transmit
directly from bats to humans (Drexler et al., 2014). Instead, bats typically infect domestic
animals that then carry the infection to a human host. Chinese researchers found during the
22. 22
HCoV-SARS epidemic that civet cats and not bats served as the primary vector for HCoV-SARS
into the human population (Hon et al., 2008). Although bats could have also contributed to the
zoonotic transmission of HCoV-SARS, the ecology of bat species makes the sustained
transmission of a coronavirus into a human population unlikely as human to bat contact does not
occur frequently (Drexler et al., 2014). Bats have large home ranges and move constantly to
satisfy their complex networks of social interaction (Hayman et al., 2013). The isolation of
highly identical HCoV-MERS species from the first and second patients suggests that the victims
contracted the virus from the same source probably not a bat but a less transient, domesticated
animal (Cotten et al., 2013). As the HCoV-MERS epidemic continued to spread in KSA during
2013 and 2014, genetic sequencing of 21 HCoV-MERS isolates confirmed that the virus was
being introduced into the human population on multiple occasions across the spatiotemporal
scale (Cotten et al., 2013). The intra-genetic variety of the isolates was unexplainable by errors
occurring in viral replication (~1.12 x10-3
substitutions/per year); therefore, human-to-human
transmission was deemed a secondary method of HCoV-MERS spread, independent of zoonotic
transmission (Cotten, Watson, & Zumla, 2014). The HCoV-MERS outbreak in Riyadh- a
southeastern city within the KSA- contained three identified viral genotypes (Assiri, Al-Tawfiq,
et al., 2013). Observations of multiple zoonotic introductions of HCoV-MERS drove research on
identifying the domestic animal responsible for conducting HCoV-MERS between bats and
humans. The initial similarity and sudden development of genomic variety among HCoV-MERS
isolates indicates that the primary animal reservoir of HCoV-MERS has a high population
density or migrates in the KSA (Cotten et al., 2013). Migration of the animal reservoir affects
viral genome identity by allowing for transmission of viral genotypes between otherwise
geographically distinct populations. In this scenario, co-infection of a single animal may
23. 23
accelerate viral evolution by providing an opportunity for two genotypically distinct viruses to
recombine (Holmes, 2009).
To identify the animal primarily responsible for zoonotic transmission of HCoV-MERS,
virologists screened a number of ungulate species endemic to the Middle East. Blood samples
were taken from cattle, goats, sheep and camels to search for neutralizing antibodies targeting
HCoV-MERS (Reusken et al., 2013). All of the animals tested negative except for dromedary
camels (Camelus dromedaries); fifty camel blood samples from Oman exhibited high
neutralizing antibody titres against HCoV-MERS (Nowotny & Kolodziejek, 2014; Reusken et
al., 2013). Subsequent studies concluded that camels from other countries also possessed
neutralizing antibodies against HCoV-MERS. The presence of neutralizing HCoV-MERS
antibodies within camels geographically spread across the Middle East (Oman, Egypt and Qatar)
warranted an attempt to isolate an HCoV-MERS-like virus from camels (Alagaili et al., 2014;
Azhar et al., 2014; Haagmans et al., 2014; Memish et al., 2014; Nowotny & Kolodziejek, 2014;
Reusken et al., 2013). A 2014, study collected nasal and conjuctival swabs from seventy-six
camels in Oman and used quantitative and standard reverse transcriptase polymerase chain
reaction (RT-qPCR and RT-PCR) to detect coronavirus genome copies and amplify genes for
phylogenetic analysis (Nowotny & Kolodziejek, 2014). Seven percent of the tested camels were
PCR positive for coronavirus genome segments. The five-targeted regions exhibited a ninety-
nine percent nucleic acid identity with the corresponding areas of the HCoV-MERS genome.
Phylogenetic analysis of three camel coronaviruses and thirty-three HCoV-MERS isolates
indicated that the camel coronaviruses clustered independently of each other but with
geographically related human isolates (Nowotny & Kolodziejek, 2014). In the Qatari cluster,
only a single nucleotide of the 3,754 analyzed nucleotides differed between camel and human
24. 24
isolates (Nowotny & Kolodziejek, 2014). The relatedness of HCoV-MERS to an HCoV-MERS-
like camel coronavirus implies that domestic camels, which abundantly populate and migrate
through the Middle East, may serve as the primary source of HCoV-MERS zoonotic
transmission (Haagmans et al., 2014). Shortly after the isolation of HCoV-MERS-like camel
coronaviruses, two studies of the same case concluded possible transmission of the virus between
camels and humans.
Less than a year after the initial cluster of HCoV-MERS cases, a forty-three year old man
was hospitalized in Jeddah KSA with an HCoV-MERS infection. The patient owned a herd of 9
camels and said that he interacted with them for about 3 hours, 3 times a week (Memish et al.,
2014). He also drank unpasteurized camel milk produced by his herd. Two competing research
groups within the KSA investigated the man’s infection and attempted to isolate HCoV-MERS
virus from his camel herd (Azhar et al., 2014; Memish et al., 2014). The two teams collected
their samples from the camel farmer’s herd two days apart (November, 7nd
, and November 9th
,
2014). The team that took the earlier samples used a serological assay and real-time quantitative
polymerase chain reaction (RT-qPCR) to determine the camels’ exposure and infection with
HCoV-MERS. One of the nine camel samples was positive for HCoV-MERS virus by RT-qPCR
targeting 3 separate loci; all of the other camels tested negative (Azhar et al., 2014). The
immunofluorescence assay detected anti-HCoV-MERS antibodies within all of the camel
samples suggesting that they were at some time infected with HCoV-MERS or a virus with
serological cross reactivity. A low volume of nasal swab sample from the patient and the RT-
qPCR positive camel were used to infect fully confluent monolayers of Vero cells, after three
days the cells showed CPE suggestive of viral infection (Azhar et al., 2014). The virologists
sequenced the viruses grown in cell culture and those isolated directly from the subjects. The
25. 25
cultured human and camel viruses were 100% identical to each other, as were the directly
sequenced samples. Mysteriously, the directly sequenced genome was not identical to the
sequence of the cultured virus; two mutations were present in both cell culture propagated
viruses (Azhar et al., 2014). The presence of these mutations in both tissue culture samples
suggests that the researchers may have cross-contaminated their work (Kupferschmidt, 2014).
The conclusions of this study were scrutinized by another group, which challenged these findings
and claimed that no definitive proof supports the transmission of HCoV-MERS virus from
camels to humans (Memish et al., 2014).
The second study conducted similar molecular and serological tests on the samples that
they collected from the patient and his herd of camels. RT-qPCR confirmed the presence of
HCoV-MERS RNA in 2 of the 9 tested camels. The resulting DNA amplicons were sequenced
and aligned with the HCoV-MERS isolate (EMC/2012) (Memish et al., 2014). The camel virus
sequences covered 4,608 nucleotides or 15% of the full-length HCoV-MERS genome.
Serological test confirmed the presence of HCoV-MERS reactive antibodies in serum samples
from all 9 of the camels (Memish et al., 2014). The antibody titer was observed highest in
juvenile camels, which agrees with other studies that suggest increased susceptibility of young
camels to HCoV-MERS infection (Hemida et al., 2013). The infection of young camels with
HCoV-MERS may partially explain the spike of human cases observed in the spring of 2014
(European Centre for Disease Prevention and Control, 2015b). The parturition cycle of camels
increases the population of susceptible young camels during the spring which correlates with the
high occurrence of disease in the human population (Memish et al., 2014). Alignment of the
partially recovered camel virus and the human isolate located multiple shared single nucleotide
polymorphism not present in any previously identified HCoV-MERS virus (Memish et al.,
26. 26
2014). These molecular fingerprints support the hypothesis that the virus spread between camels
and humans; however, they do not allow for inference of the direction of transmission or rule out
the role of a third species in infecting both hosts (Memish et al., 2014). These two studies along
with others confirming the presence of anti-HCoV-MERS antibodies in camel herds or
populations support the general hypothesis that the CoV-MERS virus or a highly similar
coronavirus circulates in the camel population of the Middle East. Isolation of a full-length
HCoV-MERS virus from a camel would confirm viral infection and the status of camels as a
potential vector of zoonotic transmission. In February 2014, a full length HCoV-MERS virus
was isolated from the nasal secretions of a camel; the virus showed great phylogenetic identity to
a human isolate from England in 2012. In summary, although epidemiologists and virologists
have not observed direct transmission of the HCoV-MERS virus from a camel to a human,
present evidence suggests camels represent a zoonotic reservoir of the virus and that “spill over”
of HCoV-MERS occurs from camel to human populations.
The early identification of camels as a probable vector of HCoV-MERS into human
populations during the fall of 2013 prompted policy makers of the WHO and KSA public health
ministry to warn the public of the risks of camel exposure. This warning, intended to restrict the
occasions of HCoV-MERS zoonotic transmission, did not decrease the human caseload. The
next spring (2014) had the greatest incidence of HCoV-MERS cases since the virus’s emergence
in 2012.
1.2.3.0 Human-to-Human Transmission of HCoV-MERS
The European Center for Disease Prevention and Control (ECDC) has published
monthly updates on the HCoV-MERS epidemic since the virus emerged. In April 2014, they
reported that the number of HCoV-MERS cases in the Middle East had increased dramatically.
27. 27
From March 2013 to March 2014, HCoV-MERS caused an average of 15 cases per month,
totaling 199 cases by the 25th
, March 2014. In April 2014, the HCoV-MERS caseload spiked to
261 cases (European Centre for Disease Prevention and Control, 2015a, 2015b).Virologists
presented a variety of hypotheses to explain this swell including: mutation of the viral genome,
increase in young camel population and widespread human- to- human transmission (Drosten,
Muth, et al., 2014). Retrospective studies have shown that the increase of HCoV-MERS cases
during the spring of 2014 likely occurred because human-to-human transmission overtook
zoonotic transmission as the primary method of spread (Assiri, Al-Tawfiq, et al., 2013; Azhar et
al., 2014; Drosten, Meyer, et al., 2014).
Researchers first challenged the hypothesis that viral mutations were responsible for the
increase of HCoV-MERS cases. Genomic comparison of HCoV-MERS viruses isolated during
April 2014 from four regions in the KSA with the original virus isolate (EMC/2012) showed that
no significant mutations had occurred. Furthermore, April 2014 viruses presented similar
replication kinetics, escape of interferon response and serum neutralization as EMC/2012
(Drosten, Muth, et al., 2014). These investigations led to the conclusion that the biology of the
HCoV-MERS virus did not change in a manner responsible for the exponential transmission
observed in the spring of 2014 (Drosten, Muth, et al., 2014). Alternative hypotheses to explain
the severity of HCoV-MERS disease in April 2014 were then examined. Increased zoonotic
transmission of the virus, and laboratory contamination of clinical samples were precluded from
contributing as no evidence supported their role (Drosten, Muth, et al., 2014; European Centre
for Disease Prevention and Control, 2015b). A possibility of surveillance bias did exist as
knowledge of a disease and capacity to accurately diagnose it ameliorate with the duration of an
outbreak (Corman, Eckerle, et al., 2012; Corman, Müller, et al., 2012; Drosten, Muth, et al.,
28. 28
2014). Retrospective analysis of diagnostic requests from April showed that little surveillance
bias occurred because the number of test requests increased, while the percentage of positive
diagnoses stayed constant (Drosten, Muth, et al., 2014). Geographical inspection of HCoV-
MERS diagnostic requests revealed that nosocomial infection amplified transmission of the
virus. The majority of diagnostic samples were received from hospitals in Riyadh and Jeddah,
two of the largest cities in the Kingdom of Saudi Arabia (Drosten, Muth, et al., 2014).
HCoV-MERS cases from Jeddah possessed two characteristics indicative of nosocomial
transmission. Forty-nine percent of all HCoV-MERS cases in Jeddah and thirty-one percent of
the total cases in the month of April 2014 were reported at King Fadh hospital. All of the
sequenced HCoV-MERS genomes from Jeddah shared a unique set of five SNPs that were not
present in HCoV-MERS viruses from other geographical locations. Viruses native to Jeddah one
and five months before the King Fadh hospital cluster lacked the five SNPs (Drosten, Muth, et
al., 2014). By May 2014, the King Fadh HCoV-MERS genotype spread as far as Riyadh where it
was isolated from a man who visited his son, an admitted MERS patient at King Fadh hospital.
The temporal progression of the King Fadh HCoV-MERS genotype from the King Fadh hospital
to the community of Jeddah and eventually Riyadh shows that nosocomial infections within the
hospital occurred so abundantly that they increased the incidence of community transmission
(Drosten, Muth, et al., 2014).
The capacity for hospital environments to amplify the transmission of coronaviruses was
also observed during the HCoV-SARS pandemic (Weiss & Navas-Martin, 2005). The first two
clusters of HCoV-SARS cases occurred in Vietnam and Hong Kong where the virus spread
indiscriminately through hospital environments infecting 105 health care workers (HCW) (Lee &
Sung, 2003). The infection of HCWs impacted the global proliferation of HCoV-SARS cases. In
29. 29
5/6 examined countries the infection of HCW superseded the infection of patients or visitors
within hospitals (Lee & Sung, 2003). In the community, the HCoV-SARS virus spread most
effectively by the phenomenon of super spreading. Super spreading occurs when a single index
case produces 10 or more secondary cases (Stein, 2011). Multiple super spreading events
accelerated the transmission of HCoV-SARS in Singapore where 5 index cases infected 103
other persons (Lee & Sung, 2003). Thus far epidemiologists have not observed super spreading
events in the HCoV-MERS epidemic; however, they could transpire in the future likely
increasing the incidence and prevalence of transmission. Reports of super spreading events in
outbreaks of rubella, tuberculosis and Ebola virus indicate that they occur independently of
pathogen type and mode of transmission, the precise mechanism of super spreading remains
undefined (Stein, 2011). Several factors may determine a case of super spreading from a case of
average transmission including: viral phenotype, patient immune-type, presence of comorbidities
and pathogen titer. The reinforcement of infection control procedures in Saudi Arabian hospitals
lowered the rate of HCoV-MERS transmission through the summer of 2014 (European Centre
for Disease Prevention and Control, 2015b). In late 2014 and early 2015 sporadic cases of
HCoV-MERS were reported in the KSA, adhering to the mean monthly average of
approximately 30 cases/month (European Centre for Disease Prevention and Control, 2015a).
The caseload increased in February and March 2015 (~54 cases/month) suggesting that a similar
trend to last year may occur, with a spike of cases in the springtime (European Centre for
Disease Prevention and Control, 2015b).
Since the emergence of HCoV-MERS a cohort of scientific studies and epidemiological
analyses have contributed to our knowledge of the virus, its pathogenicity, transmission and
source. Despite these efforts mystery perpetuates the HCoV-MERS virus and some important
30. 30
questions remain unanswered. The current statistics of HCoV-MERS infection suggest that 40%
of patients infected with the virus die, a 4-fold increase in mortality from HCoV-SARS (Centers
for Disease Control and Prevention, 2015). Future studies of HCoV-MERS, should try to further
define its mortality rate and observe if 40% of HCoV-MERS patients do succumb to their
disease. The prevalence of HCoV-MERS asymptomatic infections within the human population
also remains unidentified. Work on determining the frequency of asymptomatic infections has
found that a surprising number of persons may have suffered from recent HCoV-MERS
infections (Kupferschmidt, 2015). Researchers collected over 10,000 human blood samples from
citizens of the KSA in 2012 and 2013 for serological testing. Fifteen of those samples tested
positive for HCoV-MERS antibodies, indicating a recent infection. Extrapolating the
asymptomatic infection rate of 0.15% over the entire population of KSA shows that HCoV-
MERS may have caused a recent infection in up to 40,000 persons (Kupferschmidt, 2015). Risk
assessments of the HCoV-MERS virus have stated that the virus possesses little pandemic
potential because limited human-to-human transmission has occurred outside of the endemic
region (European Centre for Disease Prevention and Control, 2015b). Many virologists have
avoided making such speculation until conducting further research on HCoV-MERS.
Specifically, the transmission characteristics of the virus require more investigation to
understand why it seemingly does not transmit effectively by chains of human infection
(Drosten, Meyer, et al., 2014; Raj, Osterhaus, Fouchier, & Haagmans, 2014).
1.3.0.0 Transmission and Clinical Symptoms of Coronavirus Infections
The highest incidence of coronavirus infection occurs within humans and domestic
livestock, virologists have isolated coronaviruses from a variety of species including cows, pigs,
31. 31
turkeys and cats (Hilgenfeld & Peiris, 2013). The type of virus and host define the pathogenesis
of a coronavirus. The majority of coronaviruses show tropism to epithelial cells, causing
infection and disease in the respiratory and gastrointestinal processes ( Li & Lin, 2013). Study of
coronavirus evolution has described a direct correlation between viral tropism- the specificity of
the viral glycoprotein gene- and the location of viral infection within a host (Jackwood et al.,
2010). Substitution of the viral glycoprotein gene between coronavirus species has resulted in
altered viral tropism and transmission of coronaviruses (Jackwood et al., 2010; Sánchez et al.,
1999). Identified species of coronavirus transmit via aerosol, contaminated fomites and the fecal-
to-oral route. Coronaviruses often cause mild chronic infections; extensive viral shedding occurs
later in the disease allowing for further intra-host transmission (Guery et al., 2013). In 2002 and
2012 the HCoV-SARS and HCoV-MERS viruses entered into the human population, causing
more severe respiratory disease than the other human coronaviruses resulting in high mortality
rates of 10% and 40%, respectively (Centers for Disease Control and Prevention, 2012, 2015). A
noticeable increase in the transmission of HCoV-SARS and HCoV-MERS was noted within
health care environments (Drosten, Muth, et al., 2014). The high rates of observed nosocomial
transmission for each virus were likely driven by the old age and comorbidities of hospital
admitted patients (Khan, 2013). The two viruses also share similar incubation periods; HCoV-
SARS infection develops over approximately 7 days, while HCoV- MERS infection becomes
established at approximately 12 days post-exposure (Assiri, McGeer, et al., 2013; Cai et al.,
2006). At the onset of disease the HCoV-MERS and HCoV-SARS viruses produce related
symptoms.
32. 32
1.3.1.0 General Symptoms of Disease for the Severe Human Coronaviruses MERS and
SARS
Although the HCoV-SARS and HCoV-MERS viruses infect different cells and tissues of
the respiratory system, they cause similar symptoms of disease (Hilgenfeld & Peiris, 2013). The
HCoV-SARS glycoprotein binds to the cellular receptor Angiotensin-converting enzyme 2
(ACE-2); cells of the ACE-2 phenotype predominately exist in the lower regions of the human
respiratory tract (Ding et al., 2003; Gu & Korteweg, 2007). HCoV-SARS infection has been
shown to occur in ciliated epithelial cells and type two alveolar cells of the upper human lungs
(Jia et al., 2005; Mossel et al., 2008). Through binding to the cellular receptor dipeptidyl
peptidase 4 (CD26) the HCoV-MERS virus establishes infection of the lower regions of the
human lungs in type two aveolar cells, non-ciliated lung epithelial cells and endothelial cells
(Coleman & Frieman, 2013; Raj et al., 2013). The restriction of infection to the lower or upper
lungs does not distinguish the early symptoms of either viral infection. The inability of HCoV-
MERS to cause upper respiratory tract infections has likely restrained its human-to-human
transmission. HCoV-MERS and HCoV-SARS infection manifests as a fever, cough and
shortness of breath (Gu & Korteweg, 2007; Zaki et al., 2012). During the later stages of each
disease, the symptoms may worsen to include pneumonia, ARDS and gastrointestinal discomfort
(diarrhea) (van den Brand, Smits, & Haagmans, 2015). In severe cases of disease, HCoV-MERS
but not HCoV-SARS causes a more systemic infection by replicating in the kidney, which may
lead to multiple organ distress syndrome (MODS) (Zaki et al., 2012). Study of both viruses has
indicated that host cells do not respond with a type I interferon (IFN) response during infection,
suggesting that the viruses possess a means to suppress the function of innate cellular immunity
(Zhou et al., 2014). Overall, the HCoV-MERS and HCoV-SARS viruses cause dangerous
33. 33
infection within humans; their high mortality rate, penchant for nosocomial transmission and
ability to transmit via aerosol makes them constant threats to public health (Hilgenfeld & Peiris,
2013).
1.3.2.0 Clinical Knowledge of HCoV-MERS
Close observation of two early HCoV-MERS patients in France provided in depth
clinical knowledge of related disease (Guery et al., 2013). Both patients were admitted to the
hospital with fever, chills and myalgia. Patient one but not patient two also suffered from
diarrhea. Acute respiratory distress quickly became the predominant symptom of each patient;
both required mechanical ventilation and extracorporeal membrane oxygenation (ECMO). In the
later stages of disease viral infection spread to include the kidneys of each patient; patient 1
succumbed to MODS while patient 2 survived (Guery et al., 2013). The common initial
symptoms of HCoV-MERS and the likelihood of increasing future infections make identification
of the virus within hospitals important. A combination of two quantitative PCR assays has been
approved for reliable and robust detection of the HCoV-MERS virus within clinical samples
(Corman, Eckerle, et al., 2012; Corman, Müller, et al., 2012). The real-time reactions target two
regions of the HCoV-MERS genome (upE and ORF1b) that occur approximately ten kilo-bases
apart (Corman, Eckerle, et al., 2012). Use of this diagnostic method on the lower respiratory
lavage of both patients showed high viral titres. Testing of nasopharyngeal samples from both
patients was inconclusive, reaffirming the hypothesis that HCoV-MERS replicates lower in the
respiratory tract (Guery et al., 2013).
34. 34
1.4.0.0: Biological Structure of Coronaviruses
Coronaviruses exhibit a unique morphology from other viruses in the order Nidovirales,
and were named by their appearance from the Latin word corona- meaning crown (Gorbalenya
et al., 2006). Electron microscopy has depicted them as spherical enveloped particles
approximately 120-160 nm in diameter (Masters, 2006). A characteristic series of projections
(12-20 nm), formed by the glycoprotein, extend from the envelope to give the fringe of the viral
particle a crown like appearance (King et al., 2011) (Figure 1.4.0.0). The A lineage of the
betacoronavirus genus possesses a secondary glycoprotein (HE) that also expresses on the
outside of the envelope, giving these viruses the appearance of being ringed by a halo, not a
crown (de Groot, 2006). The thickness of the viral envelope also distinguishes coronavirinae
from other viruses. Examination of coronavirus envelopes by cryogenic electron tomography
showed that their width measures ~7.8 nm making them twice as thick as related biological
membranes (Masters, 2006). In addition to anchoring the glycoprotein, the coronavirus envelope
houses the viral nucleocapsid and genome. The coronavirus nucleocapsid protein (N) folds into a
tight helix, an uncommon design in viruses with positive sense RNA genomes (Gorbalenya et al.,
2006). The N protein serves a dual role in viral processes as a structural and nonstructural protein
because it protects the viral RNA and aids in viral replication (Lai, 1990). Supplementation of
excess N protein in trans benefits the efficiency of coronavirus replication (Enjuanes, 2005).
Coronavirus RNA contains all of the necessary structural regulatory elements (i.e.: methylated
guanidine cap and polyadenylated tail) for direct translation of viral proteins to occur from the
genome in an infected cell’s cytoplasm (Gorbalenya et al., 2006).
35. 35
Figure 1.4.0.0 Molecular Structure of the Coronavirus Virion:
An infectious coronavirus virion is composed of many parts shown above. The outer morphology
of the molecule may change due to the presence of one or two glycoproteins on envelope
surface. Modified from (Gorbalenya et al., 2006).
36. 36
1.4.1.0 Genome Organization
The components of the coronavirus genome cluster in two general categories: genes and
intragenic or un-translated regions (Enjuanes, 2005). This section will discuss the genomic
organization of coronaviruses and explain the differences amongst coronavirus genomes.
Coronaviruses possess the largest identified positive-sense RNA genomes; the genome
length ranges from 27-32 kb (Gorbalenya et al., 2006) . All coronaviruses share a conserved
number of organized canonical genes (Figure 1.4.1.0). The 5’ end of the genome has a
methylated guanidine cap and a short leader sequence followed by the open reading frame (ORF)
1a/b that expresses the replicase polyprotein (Lai, 1990). The middle and 3’ end of the genome
harbor the coding sequences for structural and accessory proteins (Weiss & Navas-Martin,
2005). Reverse genetics studies were responsible for identifying and delineating the differences
between coronavirus non-structural, structural and accessory genes (Enjuanes, 2005). The non-
structural genes encoded by ORF 1a/b express proteins used exclusively for viral replication. The
structural genes: glycoprotein (S), envelope (E), membrane (M) and nucleocapsid (N) combine
after expression in an infected cell to form nascent viral particles (Gorbalenya et al., 2006).
Coronavirus non-structural genes consistently follow the order of S-E-M-N in coronavirus
genomes with the S gene positioned immediately after ORF 1a/b (Enjuanes, 2005; Snijder et al.,
2003). Downstream of the N gene, a coronavirus genome may contain a number of accessory
proteins not vital to viral replication in vitro. The HCoV-SARS virus possesses a unique genome
organization with the greatest number of accessory genes (n=8); some of them intervene the
37. 37
structural genes instead of being positioned posteriorly (Snijder et al., 2003). Serial passaging of
coronaviruses in mammalian cell culture seems to cause the deletion of the accessory genes from
the viral genome (Almazán et al., 2013; Gorbalenya et al., 2006; Scobey et al., 2013). Studies of
coronavirus accessory proteins are ongoing; virologists believe that their negative selection in
cell culture indicates that they contribute to in vivo pathogenesis. In rare instances the proteins
expressed by accessory genes have been isolated from extracellular coronavirus virions.
However, their role in coronavirus structure and the impact of their inclusion or omission from a
viral particle remains unclear (Gorbalenya et al., 2006). Although coronavirus follow the same
genomic organization, the size of their genomes and the complexity of their genes warrant
further study.
38. 38
Figure 1.4.1.0 Genetic Organization of the Coronavirus Genome:
Organization of the coronavirus genome by gene name and type, the non-structural genes lay on
the 5’ side while the structural genes with the exclusion of the accessory genes occupy the 3’
region. Modified from (Enjuanes, 2005).
39. 39
1.4.2.0 Translated Regions
The coronavirus genome like that of many viruses contradicts the C-value paradox
because the genome’s size translates proportionally to complexity. The C-value paradox refers to
the uncertain relationship between genome size and coding ability. The study of a variety of
genomes has shown that complex organisms may possess smaller genomes than simpler ones
(Eddy, 2012). Coronavirus proteins vary in their amino acid variance and size amongst
coronavirus species (Gorbalenya et al., 2006). The following synopsis of coronavirus protein
structure and function aims to explain how the proteins serve the virus and importantly highlight
the lack of knowledge we possess of their secondary and possibly tertiary roles in viral infection.
1.4.3.0 ORF 1a/b Proteins
The first gene in the coronavirus genome, ubiquitously named ORF 1a/b across viral
species, encodes proteins critical for coronavirus replication such as the viral RNA-dependent
RNA polymerase (RDRP) (Lai, 1990). The ORF 1a/b gene spans approximately two thirds
(20,000 bps) of the coronavirus genome meaning that it has twice the coding capacity of the
average positive-sense RNA virus genome (Gorbalenya et al., 2006). The large polyprotein ORF
1a/b divides at the sequence UUUAAAC; near this position the ribosomal context changes
slipping between reading frames by a programmed -1 shift (Enjuanes, 2005). The ribosomal
frame shift divides the transcriptional output of the sequence producing the 1a protein and 1b
protein. Post-transcriptional processing by viral proteinases (papain-like proteinase and 3C-like
cysteine proteinase) cuts the 1a protein into sixteen non-structural proteins and divides the 1b
protein into five. The products of 1b protein production include an RDRP, putative 3’ to 5’
exonuclease, suspected poly (U)-specific endoribonuclease, hypothesized 2’-O-ribose
methytransferase and superfamily 1 helicase (Enjuanes, 2005). Thus successful viral replication
40. 40
would not occur in the absence of the -1 ribosomal frame shifting mechanism. The programmed
ribosomal frame shifting of coronaviruses involves mRNA secondary structures forcing
ribosomes to change reading frames during translation. The shift typically allows for avoidance
of a downstream stop codon or the fusion of two genes into one (Dos Ramos, Carrasco, Doyle, &
Brierley, 2004). Ribosomal frameshifting either occurs in the 5’(-1) or 3’(+1) direction; for
coronaviruses the ribosome slips (-1) in the midst of ORF 1a/b (Gorbalenya et al., 2006).
Research on (-1) ribosomal frameshifts in viruses, bacterial insertion sequences and eukaryotes
has identified two components of the mechanism (Dinman, 2012). Deletion analysis has found
ribosomal frameshifting dependent on a “slippery” heptamer sequence (XXXYYYZ), which in
coronaviruses is UUUAAAC (Gorbalenya et al., 2006; L. Li, Wang, & Wang, 2001). In the
generic heptamer sequence X represents any three identical nucleotides, Y codes for either U or
A and Z can be either U, A or C (L. Li et al., 2001). At this slippery sequence tRNAs likely
dissociate from the mRNA and bind to a codon in an alternative reading frame (Enjuanes, 2005).
The second structure an mRNA pseudoknot beginning 5-9 nts to the 3’ side of the heptamer
sequence, stimulates the slippage by temporarily delaying the ribosome (Dinman, 2012). The
ribosomal frameshift of the HCoV-SARS virus works at 27% efficiency in vitro (Dos Ramos et
al., 2004). The coronavirus ORF 1a/b polyprotein gene encodes numerous proteins necessary for
viral transcription and genome replication and it differs from replicase genes of other positive-
sense RNA viruses in size and employment of ribosomal frameshifting.
1.4.4.0 Glycoprotein (Spike Protein)
The coronavirus glycoprotein serves as the primary determinant of viral tropism and
binds with a receptor on target cells to facilitate viral entry (Qian, Dominguez, & Holmes, 2013).
Coronavirus glycoproteins assemble inside an infected cell as trimers with short cytoplasmic tails
41. 41
and hydrophobic transmembrane domains responsible for anchoring them to the virion (Masters,
2006). The coronavirus glycoprotein contains two regions S1 and S2. The receptor-binding
domain (RBD), which determines the specificity of coronavirus attachment to cellular receptors
lies within S1. The S2 region contains 5 domains including the fusion peptide that enables
coronaviruses to pass through cellular membranes (Masters, 2006). The RBD domains and their
respective cellular targets have been identified for several coronaviruses. The HCoV-MERS
virus binds to the cellular receptor CD26 also known as dipeptidyl peptidase-4 (DPP4) (Raj et
al., 2013). The HCoV-SARS and HCoV-NL63 viruses target the same cellular receptor
angiotensin-converting enzyme 2 (ACE2) (Gu & Korteweg, 2007).
In addition to determining cellular tropism and inducing viral replication, the coronavirus
glycoprotein maintains a role in the later stages of viral infection. Coronavirus infected cells
present glycoproteins on their surface that promote fusion with nearby cells and the development
of syncytia (Weiss & Navas-Martin, 2005) (Figure 1.4.4.0).
42. 42
Figure 1.4.4.0 Syncytia Development in Viral Infected Cell Culture:
Early development of syncytia in Vero cells (CCL-81) infected with HCoV-MERS, 12 hours-
post infection (Experimental Data, Nikiforuk, 2015).
43. 43
1.4.5.0 Envelope and Membrane Proteins
The coronavirus envelope (E) and membrane (M) proteins form the structural foundation
of coronavirus particles (Masters, 2006). The M protein possesses three transmembrane
segments referred to as tm1, tm2 and tm3, and its N-terminus contains an ectodomain, while the
endodomain lays proximal the C-terminus (Weiss & Navas-Martin, 2005). The endodomain
interacts with the nucleocapsid and therefore M likely helps package the coronavirus genome
into nascent virion particles (Enjuanes, 2005). The M protein’s multiple transmembrane domains
means that it spans the E protein several times, joining the outer and inner components of the
virion (Lai, 1990). The M protein occurs abundantly within a coronavirus virion; its
concentration exceeds that of all other structural proteins which makes it a promising target of
viral prophylaxis (Weiss & Navas-Martin, 2005). Fewer copies of the E protein exist within a
coronavirus virion however it still plays a crucial role in coronavirus assembly. Independent or
simultaneous intracellular expression of E with M forms virus like particles (Weiss & Navas-
Martin, 2005). Some researchers have speculated that E proteins also contribute to coronavirus
pathogenesis by forming ion channels in host cell membranes (Nieto-Torres et al., 2014). The
purpose of ion channel formation in the viral replication cycle remains unknown; however, it
could factor in a variety of processes (Nieto-Torres et al., 2014).
1.4.6.0 Nucleocapsid Protein
The coronavirus nucleocapsid protein binds RNA within the virion and acts as an RNA
chaperone protein during the viral life cycle. The N protein possesses three conserved domains
separated by highly variable regions. A richness of arginine and lysine residues characterizes the
first and second domains. The presence of an arginine island in a peptide designates the potential
for binding to nucleic acid (Gorbalenya et al., 2006). The positively charged guanidine group of
44. 44
arginine allows for close binding to nucleic acid. The third domain differs in function from the
first two: it binds to the M protein serving as a link between the capsid and the viral genome
(Gorbalenya et al., 2006).
The rescue of early generation coronavirus reverse genetics systems depended on the
presence of trans-acting N protein constitutively expressed in mammalian cell culture. RNA
synthesis requires the N protein because it affects the ability of the viral polymerase to template
switch (Enjuanes, 2005). Discussion of the coronavirus life cycle (Section 1.5.0.0) will elaborate
on the phenomena of template switching and discontinuous negative-strand synthesis. The
coronavirus N protein serves a wider range of purpose than the other viral structural proteins as it
performs in all stages of the viral life cycle.
1.4.7.0 Accessory Proteins
The observed range of coronavirus accessory proteins spans from 1-8 per genome
(Gorbalenya et al., 2006). The HCoV-MERS virus possesses four accessory proteins 3, 4a, 4b
and 5. Study of HCoV-MERS accessory proteins suggests that they do not fundamentally impact
viral replication and accordingly lack selection pressure in vitro (Scobey et al., 2013). Accessory
proteins contribute to either viral tropism or pathogenesis two viral characteristics important in
vivo (van den Brand et al., 2015). The hemagglutinin esterase (HE) gene present in several
coronavirus species enhances virulence in infected animals by giving the viruses an increased
range of tropism (de Groot, 2006). The coronavirus genome most likely acquired the HE gene
and other accessory genes through RNA recombination with cellular RNA or viral RNA in
conditions of co-infection (i.e.: with influenza C) (Weiss & Navas-Martin, 2005). Some
experiments have successfully utilized reverse genetics to determine the function of accessory
proteins. The HCoV-SARS 6 accessory protein enhanced virulence in mice when expressed in a
45. 45
murine CoV virus background (Liu, Fung, Chong, Shukla, & Hilgenfeld, 2014). The HCoV-
MERS accessory protein ORF-4b and the homologous protein in genetically related viruses
BtCoV-HKU4 and BtCoV-HKU5 inhibit the innate immune response (Matthews, Coleman, van
der Meer, Snijder, & Frieman, 2014). Overall, coronavirus accessory proteins seem to contribute
to multiple dimensions of viral pathogenesis such as increased tropism, or evasion of the host
immune response.
1.4.8.0 Un-Translated Regions
Coronaviruses have three un-translated regions (UTR) in their genomes: a 5’ UTR,
transcription regulatory sequence (TRS) and 3’UTR. The naming of the coronavirus 5’UTR
region has been complicated by the identification of a short ORF at the 3’ terminus of the
sequence. It has been argued that the location of this ORF in the 5’ end of the coronavirus
genome classifies the coronavirus 5’UTR as a leader sequence (Enjuanes, 2005). This review
will refer to the 5’ end of the coronavirus genome as the leader sequence because this
terminology simplifies explanation of the coronavirus replication cycle.
1.4.9.0 The Coronavirus Leader Sequence, 5’ Un-translated Region (UTR)
The coronavirus leader sequence occurs on the 5’ end of the genome following the cap
and prior to ORF1a/b (Figure 1.4.9.0) ranging in length from 209-528 nucleotides in coronavirus
species (Enjuanes, 2005). Coronavirus leader sequences characteristically contain an AUG start
codon in sub-optimal Kozak context. Translation from this position produces a peptide of 3-11
amino acids. Coronavirologists have yet to isolate peptides of this description (Enjuanes, 2005).
However, the leader sequence AUG may still affect downstream translation, as studies on the
equine arterivirus have shown that upstream ORFs have profound regulatory effect on
downstream ORFs. For example, the leader AUG may block translation from ORF1a/b at some
46. 46
point in the viral life cycle freeing up ribosomes for the translation of sub-genomic RNAs
(Morris & Geballe, 2000). Another structure of the 5’ leader sequence further complicates its
role in initiating translation. A TRS sequence (Refer to section: 1.4.11) nests within the leader
sequence and is recognized by the motif UCUAAAC. The coronavirus N protein (an identified
transcriptional regulator) may bind to the TRS sequence increasing the efficiency of coronavirus
rescue from an infectious clone system (Enjuanes, 2005; Zúñiga et al., 2010). In theory,
translation should begin at the 5’ side of the coronavirus genome once it has entered into the
cytoplasm of an infected cell. In the cytoplasm, a ribosome recognizes the 7-methyguanosine-cap
structure and translates viral replicase proteins responsible for nascent genome production. The
reality of coronavirus translation from the leader sequence does not align with this model.
Defective interfering coronavirus genomes undergo translation without possessing a 7-
methyguanosine cap, suggesting that the leader sequence can initiate ribosomal entry by another
mechanism. This mechanism probably involves the AUG start codon and TRS (Enjuanes, 2005).
The method that coronaviruses utilize to initiate translation of their genome and the function of
the cis-acting elements in the leader sequence remain a mystery.
47. 47
Figure 1.4.9.0 The Un-Translated Regions of the Coronavirus Genome:
Molecular map of the coronavirus genome showing the location of the 5’ leader, 3’ UTR and
transcription regulatory sequences. Four types of coronavirus genome are produced during the
viral replication cycle; they all possess the three un-translated regions.
48. 48
1.4.10.0 Coronavirus 3’ Un-translated Region (UTR)
Reverse genetics experiments involving the deletion and substitution of the coronavirus
3’UTR have characterized some of its regions and described their purposes. The coronavirus
3’UTR sequences diverge by species; some share less than 70% identity (Enjuanes, 2005). The
region likely contains important sequences for initiating negative-strand synthesis and signals
necessary for the production of positive-sense full genome and sub-genomic RNA. Substitution
of partial 3’ UTR regions between mouse hepatitis virus (MHV) and bovine coronavirus (BCV)
prevented rescue of either virus, indicating that the region’s function resides in sequence identity.
Complete substitution of one 3’ UTR with the other returned viral replicative function. From
these experiments, virologists believe that a large RNA secondary structure exists in the
coronavirus 3’UTR directly downstream of the N gene stop codon (Enjuanes, 2005). Further
reverse genetics experiments along with analysis of protein binding sites in the structure will
help identify if it recruits non-structural proteins during viral replication.
1.4.11 Coronavirus Transcription Regulatory Sequence (TRS)
As in the case for the 5’ leader and 3’ UTR, successful coronavirus replication requires
the transcription regulatory sequence (TRS). The TRS sequence repeats within the coronavirus
genome positioned within the leader sequence and intragenic to every ORF (Figure 1.4.9.0). The
TRS sequences share a highly conserved core heptamer (Betacoronavirus: 5’-UCUAAAC-3’);
homology between the TRS sequences allows them to base pair with each other during RNA
synthesis (refer to section 1.5.3.0)(Masters, 2006). Transcripts of the 5’ leader region and a TRS-
ORF fuse during sub-genomic (sg) RNA synthesis. Expression of coronavirus ORFS does not
proceed without joining of 5’ leader to the TRS-ORF transcripts to form sgRNA (Figure 1.4.9.0
and 1.4.11.0). Virologists suspect that TRS bind molecules other than the 5’ leader including
49. 49
viral proteins (i.e.: N) and cellular factors necessary for the formation of the replication
transcription complex (RTC) (Sawicki, Sawicki, & Siddell, 2007). In a revolutionary reverse
genetics study, coronavirus researchers altered all the TRS sequences of the HCoV-SARS
genome by 3 nucleotides. The “re-wired” HCoV-SARS coronaviruses replicated to similar
levels, indicating that homology between TRS sequences and not an intrinsic structure of the
specific sequence dictates coronavirus replication (Yount, Roberts, Lindesmith, & Baric, 2006).
The study continued with the production of a chimeric HCoV-SARS virus with a wild type TRS
core sequence in 5’ leader and critical genes S-E-M-N; a synthetic TRS sequence was installed
upstream of the accessory genes. The chimeric virus rescued in cell culture; however, the
accessory genes were not expressed properly and attenuated coronavirus RNA was detected. The
attenuated transcripts resulted from missed recognition between the WT and synthetic TRS
sequences. In the absence of homologous sequences, the 5’ leader region joined with
homologous noncanonical sequences; the transcription network of the virus seemed to select the
best available sequence match (Yount et al., 2006). The re-wiring of the HCoV-SARS TRS
system demonstrated that coronaviruses rely on TRS sequences for efficient replication and that
such interactions show strong base pairing preferences. Description of the coronavirus life cycle
will further explain the role and purpose of TRS sequences in viral replication.
50. 50
Figure 1.4.11.0 Coronavirus Transcription Regulatory Sequences Influence the formation
of sgRNA:
A) The TRS sequences of the coronavirus genome are categorized by their location. The
intragenic TRS sequences are positioned between ORFs while the leader TRS sequence occurs in
the leader sequence. B) During coronavirus replication creation of the antisense genome by the
viral replicase allows for base pairing of antisense and sense TRS sequences. Pairing of non-
contiguous TRS sequences causes template switching. C) Base pairing between an antisense
body TRS and sense leader TRS can lead to a template switch joining an anti-leader sequence to
the 3’ terminus of an antisense transcript.
51. 51
1.5.0.0: Coronavirus Life Cycle
The coronavirus life cycle begins with attachment of a virion to the surface of a
permissive cell and finishes with the release of infectious virions. The coronavirus life cycle
consists of three processes: viral entry, genome replication and particle release. Knowledge of
the coronavirus life cycle has significantly increased since the emergence of HCoV-SARS. The
prominence of HCoV-SARS directed virologists to better understand the mechanisms of
coronavirus replication while searching for therapeutic options (Hilgenfeld & Peiris, 2013). The
coronavirus glycoproteins (S and HE) initiate the viral life cycle by allowing virus particles to
infiltrate cells.
1.5.1.0 Viral Entry
A virus’ glycoprotein only allows it to enter cells of a certain type; virologists refer to this
restriction as tropism. The tropism of a virus depends on the distribution of cellular receptors
within or amongst hosts. The HCoV-MERS coronavirus enters mammalian cells by a similar
mechanism to other coronaviruses. The coronavirus glycoprotein, a type I fusion protein,
determines the binding of a virus to a host cell and facilitates fusion of the host and viral
membranes (Masters, 2006). Various viruses like HIV and influenza utilize type I glycoproteins
to bind to receptors, mediate membrane fusion and enter cells. Type I glycoproteins also elicit
strong neutralizing antibody responses, which makes them important targets of viral prophylaxis
(Weiss & Navas-Martin, 2005).
The HCoV-MERS glycoprotein preferentially targets the cellular receptor dipetidyl-
peptidase 4 or cluster of differentiation CD26 (Raj et al., 2013). On the surface of cells, CD26
serves as a serine exopeptidase that cleaves polypeptides including chemokines, neuropeptides
and peptide hormones. The activity of CD26 has been implicated in maturation of lymphocytes
52. 52
and inhibition of the enzyme seems a promising therapy for type 2 diabetes mellitus (Matteucci
& Giampietro, 2009) . The CD26 cellular receptor exists on a variety of cell types including
epithelial cells from the lung, liver, kidney and intestine (Schmiedl et al., 2010). How the
HCoV-MERS virus enters cells expressing CD26 depends on the presence of proteases capable
of cleaving the virus’s glycoprotein. When treated with trypsin protease outside of cells a
pseudo-typed HCoV-MERS virus gained entry through the plasma membrane. When the
glycoprotein was not cleaved in the extracellular space the virus entered cells via endocytosis
(Qian et al., 2013). Study of the HCoV-SARS virus has speculated that entry at the plasma
membrane exceeds the efficiency of endosomal-mediated entry by 100-1000% (Qian et al.,
2013). Therefore, the distribution of extracellular proteases also determines viral tropism,
because coronaviruses more efficiently infect tissue rich in proteases (Belouzard, Millet, Licitra,
& Whittaker, 2012). In protease deficient environments, coronaviruses enter cells via the well-
understood endosomal pathway.
Endocytosis of HCoV-MERS virions begins with the viral glycoprotein binding to CD26.
Once the virus becomes engulfed within an endosome, the glycoprotein undergoes a
conformational change brought on by low pH exposure and proteolytic activation (Belouzard,
Chu, & Whittaker, 2009). Only preliminary studies have been conducted on how coronavirus
glycoproteins change confirmation however, the process likely follows that of influenza’s
glycoprotein hemagglutinin (HA) (Navas-Martín & Weiss, 2004).
Extensive experiments have characterized the influenza HA protein making it the model
type I fusion protein (White, Delos, Brecher, & Schornberg, 2008). The influenza virus
synthesizes the HA protein as a HA0 precursor that assembles into a trimer. The HA0 protein
becomes fusion-competent by processing into two sub-units (HA1 and HA2). The fusion
53. 53
property of the peptide resides in a hydrophobic region of HA2. Prior to exposure to low pH
conditions, the fusion peptide lays protected by a set of long and short helices. Once an influenza
virion has entered into a cell, endosomal acidification initiates a conformational change of the
HA protein. Under low pH conditions, an unstructured linker assumes a helical structure that
causes the fusion protein to project towards the cellular membrane. A secondary conformational
change brings the transmembrane region of the HA protein closer to the cellular membrane
causing fusion of the two molecules (White et al., 2008). Study of the HCoV-SARS virus
showed that the viral glycoprotein contains two-short heptad repeats (abcdefg) characteristic of
forming the six-helix bundle necessary for the secondary conformational change. Deletion of
residues in these heptad repeats prevented the fusion of HCoV-SARS with cellular membranes
confirming that the viral glycoprotein partly behaves like HA (Enjuanes, 2005). Theoretically,
the HCoV-MERS glycoprotein also works similarly to HA. In the low pH environment of the
endosome the HCoV-MERS glycoprotein undergoes a series of conformational changes,
resulting in fusion of the viral envelope with the cellular membrane and escape of the viral
genome into the cytoplasm (Qian et al., 2013). In the cytoplasm, the viral life cycle continues as
the virus begins to replicate its genome and synthesize proteins.
1.5.2.0 Transcription and Genome Replication
The transcription and genome replication of coronaviruses exceeds the strategies used by
other plus-stranded viruses in complexity. The creation of numerous sizes and types of RNA
molecules by a not fully understood mechanism of discontinuous negative strand transcription is
the hallmark of coronavirus replication (Enjuanes, 2005; Masters, 2006). In the context of the
coronavirus, the term transcription refers to the production of sense or antisense RNA while
replication defines the synthesis of full-length sense RNA from antisense templates. Translation
54. 54
maintains its traditional meaning of synthesizing proteins from genomic messenger RNA
(mRNA) (Sawicki et al., 2007). Like other positive -sense RNA viruses, the coronavirus genome
serves as mRNA in the cytoplasm where translation from the 5’ end produces the replicase
proteins pp1a and pp1ab. A programmed ribosomal frame shift of 25-30% efficiency determines
the translation of pp1a or pp1ab, meaning that pp1ab gets produced at a rate of approximately
≤30% (Enjuanes, 2005). During or after translation the ORF1a/b polyprotein undergoes
cleavage by either a viral encoded papain-like (PLPro
) or 3C-like coronavirus protease (MPro
) into
sixteen proteins. Polyprotein 1a encodes for the non-structural proteins one to eleven, while
cleavage of polyprotein 1ab yields non-structural proteins twelve to sixteen. The sixteen non-
structural proteins assemble with other viral proteins and cellular membrane proteins to form
replication-transcription complexes (RTC) (Weiss & Navas-Martin, 2005). The enzymatic
machinery of the RTC serves to conduct both transcription and replication. A preliminary
understanding of coronavirus RTC has come from studies on MHV and TGEV coronaviruses
(Enjuanes, 2005). The coronavirus RTC seems to associate with cellular membranes and the
involved membrane differs amongst viral species. The MHV coronavirus replicates in proximity
with membranes of either the endoplasmic reticulum or the Golgi apparatus (Perlman & Netland,
2009). The membrane association of HCoV-MERS has thus far been localized to the perinuclear
space where viral replication occurs within double-membrane vesicles and convoluted
membranes (Figure 1.5.2.0) (de Wilde et al., 2013) . Coronaviruses begin their transcription and
replication within these membranous structures.
