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Development of a standardized quantitative real time PCR panel for respiratory viral diagnosis Utokaparch, Soraya 2006

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DEVELOPMENT OF A STANDARDIZED QUANTITATIVE R E A L TIME PCR P A N E L FOR RESPIRATORY V I R A L DIAGNOSIS by S O R A Y A U T O K A P A R C H B.Sc, Simon Fraser University, 2001 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH C O L U M B I A December 2006 © Soraya Utokaparch, 2006 ABSTRACT Traditional viral diagnostics such as viral culture and various serological techniques tend to be slow, insensitive and labour-intensive. A large proportion of viral pathogens still go undetected using these techniques. This thesis concerns the development of a rapid and sensitive technique - standardized real-time quantitative P C R . Individual q P C R assays and synthetic plasmid controls were developed for 12 common respiratory viruses including influenza types A and B, parainfluenza (P IV) - l , -2 and -3, respiratory syncytial virus (RSV) A and B, metapneumovirus ( M P V ) , human coronavirus (HCoV) 229E and OC43 , human rhinovirus (HRV) and adenovirus. A reference gene assay using hypoxanfhine phosphoribosyl transferase (HPRT) was also developed. A retrospective analysis on nasopharyngeal aspirates from patients previously diagnosed was conducted. The results demonstrated that the respiratory viral q P C R panel was sensitive, efficient, and had a large dynamic range of detection. Some cross-reactivity was noted for H R V with an enterovirus (coxsackievirus B3). H P R T proved to be a stable reference gene with the additional benefit that q P C R viral loads could be interpreted based on copy number per unit volume of specimen. One hundred culture negative specimens were examined and viral nucleic acid was amplified in 43 of them. There was a statistically significant relationship between viral load and whether or not the same specimen was positive by culture for influenza A , P IV-3 , R S V A and B, H R V and adenovirus. Mean viral load was highest in patients with lower respiratory tract infections (LRTI) compared to those with fever or upper respiratory tract infections (URTI) and 95% confidence interval (CI) between these patients did not overlap. These results suggest that patients with more severe cl inical disease had higher viral loads. This study highlights the developmental phase of a technique that has the potential to increase the detection rate of viral pathogens involved in respiratory illnesses. TABLE OF CONTENTS A B S T R A C T i i T A B L E OF C O N T E N T S iv L IST O F T A B L E S vi i i L IST OF F I G U R E S ix A B B R E V I A T I O N S x 1. RESPIRATORY VIRUSES 1 1.1 Historical Perspectives 1 1.2 Discovery of Respiratory Viruses 2 1.3 Classification 4 1.3.1 Single Stranded Negative Polarity R N A Viruses 4 1.3.1.1 Influenza Virus 5 1.3.1.2 Paramyxoviruses 8 1.3.1.2.1 Parainfluenza Virus (PIV) 8 1.3.1.2.2 Respiratory Syncytial Virus (RSV) 8 1.3.1.2.3 Metapneumovirus ( M P V ) 9 1.3.2 Single Stranded Positive Polarity R N A Viruses 9 1.3.2.1 Human Coronavirus (HCoV) 10 1.3.2.2 Human Rhinovirus (HRV) 10 1.3.3 D N A Viruses 11 1.3.3.1 Adenovirus 11 iv 1.4 Epidemiology 11 1.4.1 Influenza Virus 11 1.4.2 Parainfluenza Virus 12 1.4.3 Respiratory Syncytial Virus 13 1.4.4 Metapneumovirus 13 1.4.5 Human Coronavirus 14 1.4.6 Human Rhinovirus 15 1.4.7 Adenovirus 16 1.5 Cl in ical Manifestations and Therapy 16 1.5.1 Influenza Virus 17 1.5.2 Parainfluenza Virus 18 1.5.3 Respiratory Syncytial Virus 19 1.5.4 Metapneumovirus 21 1.5.5 Human Coronavirus 22 1.5.6 Human Rhinovirus 22 1.5.7 Adenovirus 23 1.6 Summary 25 2. VIRAL DIAGNOSTIC TECHNIQUES 27 2.1 Introduction 27 2.2 Culture : 28 2.3 Immunostaining 30 2.4 Serology 3 1 v 2.4.1 Complement Fixation Test 31 2.4.2 Hemagglutinin-Inhibition Test 32 2.4.3 Hemadsorption Test 33 2.4.4 Neutralization Test 33 2.4.5 Enzyme Linked Immunosorbent Assay 34 2.5 Conventional Polymerase Chain Reaction 34 2.6 Quantitative Real-Time Polymerase Chain Reaction 35 2.7 Summary 37 3. RESEARCH OVERVIEW 38 3.1 Rationale 38 3.2 Specific A ims 39 4. METHODS AND MATERIALS 41 4.1 Case Selection of Nasopharyngeal Aspirates (NPAs) 41 4.2 Nucleic A c i d Extraction from Nasopharyngeal Aspirates 41 4.3 Selection of Viruses for Inclusion in q P C R Panel 42 4.4 Design of Primer and Probe Sequences for Quantitative Real-Time P C R 43 4.5 Host Reference Gene Selection 45 4.6 Standards for Quantitative Real-Time P C R 45 4.6.1 Annealing, Ligation and Transformation 46 4.6.2 Overnight Culturing of Bacterial Plasmids 48 4.6.3 Isolation of D N A Plasmids from Culture 49 v i 4.6.4 Sequencing and Re-culturing of Bacterial Plasmid 50 4.6.5 Linearization of D N A Plasmids by Restriction Enzymes 50 4.6.6 In vitro Transcription of Digested D N A Plasmids 50 4.7 Reverse Transcription 51 4.8 Quantitative Real-Time P C R on the A B I 7000 52 4.9 Data Analysis 55 5 RESULTS 58 5.1 Eff iciency and Dynamic Range of Detection of V i ra l Assays 58 5.2 Cross-reactivity of q P C R Primers and Probes 58 5.3 Assessment o f Ampli f iable Material by Host Gene H P R T 59 5.4 Relationship o f q P C R Results to Positive Vi ra l Culture Results 60 5.5 q P C R on Culture Negative Specimens 61 5.6 Virus Culture Negative versus Virus Culture Positive Specimens 61 5.7 Relationship of V i ra l Load and Cl inical Diagnosis 62 6 DISCUSSION 71 7 REFERENCES 82 vi i LIST OF TABLES Page Table 1. Summary and comparison of respiratory viruses 26 Table 2. Summary of viral assay primers, probe and amplicon length 56 Table 3. Efficiencies and R-squared values of q P C R protocols as performed on two different instruments 63 Table 4. Range of detection of viral assays on the A B I Prism 7000 64 Table 5. Sensitivity o f q P C R viral assays 67 Table 6. Summary of the types of respiratory viruses identified using q P C R screen in the culture negative specimens 68 Table 7. Logarithmically transformed q P C R loads and their relation to previously confirmed culture negative and culture positive specimens 69 Table 8. Logarithmically transformed qPCR loads and their relation to clinical diagnosis 70 vi i i LIST OF FIGURES Page Figure 1. Schematic cartoon of synthetic control preparation 57 Figure 2. Representative real-time q P C R viral amplification curve for Influenza A 65 Figure 3. H P R T amplification curve 66 ix A B B R E V I A T I O N S A C E 2 angiotensin converting enzyme 2 C A R coxsackie adenovirus receptor C D cluster of differentiation C F T complement fixation test C P E cytopathic effect C V B 3 coxsackie virus 3 D N A deoxyribonucleic acid D T T dithiothreitol E L I S A enzyme linked immunosorbent assay F A M 6-carboxyfluorescein F fusion (protein) F R E T Forster resonance energy transfer G glycosylated (protein) G A P D H glyceraldehyde-3-phosphate dehydrogenase H A hemagglutinin H A I hemagglutinin inhibition assay h A P N human aminopeptidase N H C o V human coronavirus H E hemagglutinin-esterase H E K human embryonic kidney H L A I human leukocyte antigen I H N hemagglutinin-neuraminidase H R V human rhinovirus H P R T hypoxanthine phophoribosyl transferase ICAM -1 intracellular adhesion molecule-1 L B media Luria-Bertani media IF immunofluorescence L D L - R low density lipoprotein receptor L R T I lower respiratory tract infection M D C K Madin-Darby canine kidney M H C - I major histocompatibility complex-I M K monkey kidney M P V metapneumovirus M membrane (protein) N A neuraminidase N C B I National Center for Biotechnology Information N S non-structural (protein) N P A nasopharyngeal aspirate N P S nasopharyngeal swab OPS oropharyngeal swab P C R polymerase chain reaction P M K primary rhesus monkey kidney P IV parainfluenza virus R N A ribonucleic acid x i R O X carboxy-X-rhodamine R S V respiratory syncytial virus R T - P C R reverse transcriptase polymerase chain reaction S spike (protein) S A R S severe acute respiratory syndrome S H small hydrophobic (protein) SURE® cell Stop Unwanted Rearrangement Events cell T A M R A 6-carboxy-tetramethyl-rhodamine U R T I upper respiratory tract infection V P viral protein 1. RESPIRATORY VIRUSES 1.1 Historical Perspectives Viruses are obligate intracellular parasites that are dependent on the metabolic and genetic functions of l iv ing cells for survival. As they do not encode genes for ribosomal proteins or enzymes involved in energy metabolism, they must utilize the host cells' machinery to reproduce. The ancestry of viruses is ambiguous since they have only been discerned and categorized in the last century. The first clue of the existence of viruses was uncovered in 1879 by Ado l f Mayer, a German scientist, who was credited as being the first person to transmit a plant pathogen from liquid extracts o f a diseased plant to a healthy one (1). In 1892, Russian scientist Dmitr i Ivanowski showed that tobacco plants, which were susceptible to what is now known as tobacco mosaic disease, became infected and died after exposure to a filtered substance which he thought caused the plant infection (2). In 1898, Dutch scientist Martinus Beijerinck refined Ivanowski's experiments by filtering out this infectious agent and heating it. Through his experiments, he was able to rule out chemical toxins because the agent reproduced and proliferated in plant cells (3). Bacteria were also excluded because the filter used was fine enough to prevent passage of bacteria as the disease causing agent. Ivanowski instead named the agent "contagious l iv ing f lu id" which was later renamed "virus", the Latin word for poison. Shortly after, the first animal virus, known as foot and mouth disease (4), was identified in cattle by German bacteriologists Friedrich Loffler and Paul Frosch, two trainees o f Robert Koch , a German physician who is considered one of the forefathers of bacteriology. The discovery o f the first human virus was that of yellow fever in 1900 by American bacteriologist Walter Reed and colleagues (5). 1 Scientists were only able to describe a virus by the symptoms or disease it caused; structurally, viruses were an unknown entity. In fact, dissent amongst scientists remained for wel l over twenty years as to whether viruses were particles, liquids or something else. Various findings in the fol lowing years helped to finally elucidate the nature of viruses. In 1917, Canadian scientist Fel ix d'Herelle discovered viruses that could infect bacteria, which he termed bacteriophages (6). He demonstrated that these bacteriophages could make holes ("plaques") in a bacterial culture. Since each plaque was cultivated from a single bacteriophage, this provided a method of counting the number of infectious viruses and was the forerunner of the plaque assay. Further analyses of bacteriophages in the 1940s and 1950s established certain properties (i.e., size, shape, and morphology) due to the ease of growing these bacterial viruses in the culture (7, 8). In 1935, American scientist Wendell Stanley crystallized the tobacco mosaic virus and demonstrated the virus had a definite, consistent shape (9). Only in the late 1930s, after the invention o f the electron microscope, were viruses able to be visualized. 1.2 Discovery of Respiratory Viruses Concerning viruses that have a propensity to infect the respiratory tract, influenza was first characterized in swine in 1931 by American bacteriologist Richard Shope (10). What eventually came to be known as influenza type A , was subsequently isolated (a purified sample containing no other particulates) from a human in 1933 by English physicians Wi lson Smith, Christopher Andrewes and Patrick Laidlaw (11). Both viruses were antigenically similar and serologically related to the influenza virus of the 1918 pandemic. Type B (12) and C (13) influenza viruses were isolated by Thomas Francis Jr. and Thomas Magi l l in 1940 and by Richard Taylor in 1949, respectively. 2 The 1950s and 1960s saw a rapid increase in the number of human respiratory viruses discovered. The four human parainfluenza viruses (PTV) were first identified in the late 1950s by Robert Chanock (14-19). He also discovered respiratory syncytial virus (RSV) in 1956 in infants with lower respiratory tract infections (LRTI) (20, 21). Adenovirus was accidentally isolated in 1953 by Wallace Rowe and colleagues who were trying to establish adenoidal cell lines from infected tissue of children who had tonsillectomies (22, 23). Human coronaviruses (HCoV) were discovered in 1965 by David Tyrrell and M L Bynoe in cultures of human ciliated embryonic trachea (24). In 1966, Dorothy Hamre and John Procknow characterized the 229E strain o f H C o V which was grown successfully in WI-38 cells (25). In 1967, Kenneth Mcintosh isolated six morphologically similar viruses and grew them in organ cultures; one of these was H C o V -OC43 (26). Human rhinovirus (HRV) was discovered in the 1950s by growing a strain of the virus in tissue culture and described separately by the Common Cold Unit in Great Britain and laboratories in the United States (27-29). It was not until 2001 that human metapneumovirus ( M P V ) was first isolated in cultures from hospitalized children in the Netherlands by Albert Osterhaus and colleagues (30). There is much that is still unknown and left to be discovered as the underlying cause of a significant proportion of respiratory infections are still unaccounted for. A study by Stockton and colleagues determined that in patients who presented with influenza-like illness, approximately 50% of the specimens had no identifiable virus (31). Another recent study found that no pathogen could be detected in 30-40% of hospitalized patients exhibiting a respiratory tract infection (32). Therefore, it is quite possible that there are many more, as yet, undiscovered viruses circulating that cause serious respiratory disease. Current molecular techniques such as 3 microarrays, large scale sequencing, and bioinformatics have facilitated in the discovery o f novel viruses, such as avian influenza virus in 1997 (33), H C o V - H K U l (34) and -NL63 (35) in 2004, and bocavirus (36) as of early 2006. H C o V - N L 6 3 has been reported to cause severe lower respiratory disease similar to croup in young children and in immunocompromised patients (37, 38). It is unclear at this point, how much of a worldwide impact these recently discovered respiratory viruses may have as their clinical manifestations and epidemiologies have yet to be elucidated. They may be common causes of respiratory infections as early epidemiological studies into M P V (39) have shown, or alternatively may be a rare infection l ike the severe acute respiratory syndrome (SARS) outbreak in 2003 - only time wi l l tell. 1.3 Classification Vira l classification is generally based on five main criteria. These include: (1) the nature o f nucleic acid genome ( R N A or D N A ) ; (2) number of strands of nucleic acid and their physical construction (single- or double-stranded, segmented or not); (3) polarity of the genome (negative (-) or positive (+) polarity); (4) presence or absence of a l ipid envelope; and (5) shape (icosahedral, helical or complex). Respiratory viral infections are generally caused by single-stranded negative polarity R N A , single-stranded positive polarity R N A , and double stranded D N A pathogens. 1.3.1 Single Stranded Negative Polarity RNA Viruses Orthomyxoviridae (influenza virus) and paramyxoviridae (PIV, R S V , and M P V ) are the two family of viruses included in this group. They are all enveloped, helical viruses. Their names are derived from the word myxo meaning "an affinity for mucus", while ortho and para stand for 4 "true" and "closely resembling", respectively. The negative polarity of the R N A genome prevents the virus from being immediately infectious, in contrast to the positive strand R N A viruses. Positive strand viruses are o f the same polarity as m R N A and are directly able to serve as templates for protein translation of viral proteins involved in genomic replication. Negative strand viruses go through the extra step of transcribing complementary full-length positive polarity R N A first. 1.3.1.1 Influenza Virus Influenza virus is a medium-sized (80-120 nm diameter), enveloped, helical virus that is segmented into eight sections (seven for type C) that code for ten proteins (40). Segmented viruses have multiple R N A molecules enclosed in a single particle. The R N A is protected by the nucleocapsid which contains type-specific antigen that differentiates between strains, A , B, and C. The two genes that recombine to produce new strains of influenza are hemagglutinin (HA) and neuraminidase (NA) , both found on the surface of the virion. The H A glycoprotein attaches to host sialic acid residues and allows the virus to bind and enter the cel l , while N A cleaves those sialic acid residues and assists in releasing viral progeny from infected cells. These two antigenically recognized glycoproteins are used for determining the different serotypes of influenza A virus. Fifteen subtypes of hemagglutinin and nine subtypes of neuraminidase have been described, o f which three subtypes of H A (1-3) and two subtypes of N A (1, 2) are endemic human influenza viruses. H A 5, 7, 9 and N A 7 have also been documented to infect humans (41). The prototype virus is named according to the type of virus, where the virus was isolated, specimen number and year (eg. A /HK/1 /68 = type A , isolated in Hong Kong from specimen number 1 in 1968). 5 The genetic changes in nucleic acids that occur in a segmented virus l ike influenza is the root cause of re-infection and these antigenic changes determine the extent and severity of epidemics. These changes occur primarily in type A influenza and to a lesser extent, type B. B y contrast, type C influenza is relatively stable and causes little to no illness in humans. A recent study by Matsuzaki and colleagues found only 187/84,946 (0.22%) influenza C infections over 14 years time (42). Two categories of genetic variation are involved. Antigenic drift arises when an accumulation o f point mutations in the viral genome take place to generate H A and N A glycoproteins with minute differences that gives the virus new antigenic markers which evade the host's immune system. This leads to re-infection of the same virus but of a different strain and is sufficiently distinctive enough to cause an epidemic. The severity of the epidemic is dependent on the extent of antigenic variation. The more severe type of variation, antigenic shift, occurs less frequently (43) and involves the mixing or reassortment of R N A segments o f two different influenza viruses or human segments with those of avian or porcine fragments, creating a new serotype. Birds, humans and pigs possess receptors that recognize influenza. Migratory water fowl, in particular wi ld ducks, are the primary reservoirs of antigenically stable influenza viruses. In birds, influenza virus multiplies in the epithelial cells o f the gastrointestinal tract and rarely causes illness. Virus gets excreted through feces which contaminate the water supply and other birds, such as chickens, may become infected through contact with contaminated water. Pigs are permissive, intermediate hosts to influenza; possessing both avian and human flu receptors and may become infected through contact with infected birds, humans or contaminated water. If avian and human influenza viruses infect a pig simultaneously, each independently replicating its nucleic acids and 6 proteins, segments from each may recombine into a chimeric virus with novel antigenic markers that are whol ly unrecognized by the host's immune system. Antigenic shifts are therefore the cause of global pandemics since the majority o f the population lack any immunity to fight these new strains, and are left vulnerable. Four pandemics involving influenza A have been documented in the twentieth century based on serological studies. The 1918 Spanish flu pandemic was caused by H1N1 and was replaced in 1957 by H2N2, the Asian flu pandemic (44). In 1968, H3N2 emerged to cause the Hong Kong flu pandemic (45). Most recently, H1N1 re-emerged in 1977 as the Russian flu and circulates concurrently with H3N2 (46). The current concern is the H5N1 strain ("avian influenza") which is being closely monitored by the Wor ld Health Organization and the Centers for Disease Control and Prevention. It was first detected in Hong Kong in 1997 where 6 out o f 18 patients died (33). Presently, the avian strain does not transmit easily from fowl-to-human or human-to-human. Recent studies elucidated why the virus is so lethal yet difficult to transmit (47, 48). The cellular receptor for influenza is a sialic acid linked to galactose by either an alpha-2, 3 or alpha-2, 6 linkage (49). It was recently determined that avian strains bind preferentially to an alpha-2, 3 conformation while human strains bind to an alpha-2, 6 linkage (48). These studies found that humans have both of these types of receptors; alpha-2, 6 receptors dominating the upper respiratory tract and alpha-2, 3 receptors found only in the lower respiratory tract in alveoli. This may explain the damage that occurs in the lower lungs in patients who have died (50). The current concern with most leading experts is that, over time, this avian strain wi l l mutate to gain the ability to spread easily between humans. Since the population has no immunity to H5 , it is 7 very l ikely that this strain w i l l be extremely lethal i f it becomes capable of person-to-person transmission (51). 1.3.1.2 Paramyxoviruses Paramyxoviruses are enveloped and have a non-segmented and helical genome containing six basic genes and a subset of accessory genes that are unique to each genus (52). Paramyxoviruses transport their own R N A polymerase into the host cell , as these viruses cannot use the cellular machinery to replicate. 1.3.1.2.1 Parainfluenza Virus (PIV) PIV is a large (120-300 nm diameter), non-segmented, enveloped, helical virus (52). It is differentiated antigenically into four subtypes and PIV-4 can be further subdivided into A and B strains. There are two antigenic proteins: hemagglutinin-neuraminidase (HN), a viral attachment protein which also allows the virus to adsorb and agglutinate erythrocytes, and fusion (F) which fuses the vir ion to the host cell. 1.3.1.2.2 Respiratory Syncytial Virus (RSV) R S V is a large (120-300 nm diameter), non-segmented, enveloped, helical virus that has ten genes with code for eleven polypeptides (53). The envelope contains two antigenically recognized glycoproteins, fusion (F) and glycosylated (G), that stimulate the production of neutralizing antibodies. The F protein assists in viral entry and facilitates viral fusion to form syncytia while the G protein is believed to be responsible for the initial attachment of viruses to the host cell . Unl ike other members in the family, R S V lacks H N proteins. R S V has two major 8 subtypes, A and B, which circulate concurrently in the population and are detected in similar ratios (54). Outbreaks with one subtype predominating can occur but vary depending on geographic range and year (55). 1.3.1.2.3 Metapneumovirus (MPV) M P V is a recently discovered virus that has been implicated in human respiratory disease. M P V , unlike other viruses, was originally detected using reverse transcriptase polymerase chain reaction (RT-PCR) and assigned to the metapneumovirus genera based on clinical data, sequence homology and gene alignment (30). M P V is a large (200 nm diameter), non-segmented, enveloped, helical virus. Genetically it is similar to avian pneumovirus, hence the name, metapneumovirus. Two major strains, A and B have been elucidated with further subtypes discovered (56). The genome is thought to contain eight genes that encode nine proteins (57, 58). Similar to R S V , it is predicted that the F and G glycoproteins of M P V are the major antigenic portions of the vir ion. Unl ike R S V , M P V lacks non-structural proteins which counteract interferon-alpha induction/production in the host (59). What this means for the pathogenicity of M P V and the resultant host immune response remains unclear. 1.3.2 Single Stranded Positive Polarity RNA Viruses The families of viruses included in this group are coronaviridae and picornaviridae (rhinovirus/enterovirus) which cause the majority of human upper respiratory tract infections (URTI). Since viral R N A is of positive polarity, it is able to function directly as m R N A and immediately start infecting and reproducing within its host. 9 1.3.2.1 Human Coronavirus (HCoV) H C o V is a large (80-160 nm diameter), non-segmented, enveloped, helical virus (60). The family is named after the Latin word for crown due to the large widely spaced spikes on the surface o f the vir ion when viewed by electron microscopy (61). The antigenic portion of the virus consists o f a glycoprotein found on the envelope of the virus. For all H C o V , this is the spike (S) glycoprotein and is involved in receptor binding and cell fusion (60). The membrane (M) glycoprotein which is involved in transmembrane budding and envelope formation is mostly internal. Some H C o V , such as OC43 , also have a third glycoprotein known as hemagglutinin-esterase (HE) and like the S glycoprotein, can be antigenic and neutralized by host antibodies (40). 1.3.2.2 Human Rhinovirus (HRV) H R V is a small (27-30 nm diameter), non-segmented, non-enveloped, icosahedral virus (62). The capsid is comprised of 60 copies each of the four capsid proteins (VP1-VP4) of which the first three form the external surface (40). Under the electron microscope, the virus appears smooth and round. H R V consists of over one hundred different serotypes and only five o f these have been fully sequenced (62). H R V can be grouped based on the attachment of the virus to host cell receptor. Over 90 serotypes bind to intercellular adhesion molecule-1 ( ICAM-1) while the remaining serotypes bind to low density lipoprotein receptor ( L D L - R ) (63). The one exception is H R V 87 which appears to require a sialic acid moiety on the host cell to attach (64). 10 1.3.3 DNA Viruses 1.3.3.1 Adenovirus Adenoviridae makes up a large component of this family o f viruses comprising o f six subgroups (A-F) and over 50 serotypes (65). Adenovirus is a double-stranded, medium-sized (70-80 nm diameter), non-segmented, non-enveloped, icosahedral virus (40). Antigenic portions of the virus include the penton and fibre proteins which have type-specific antigenic epitopes and hexon protein which has both type-specific and group-specific antigenic epitopes (66, 67). Adenovirus has many targets in the human body, including the respiratory tract, gastrointestinal tract and eye. Three subgroups of adenovirus are implicated in the majority of human respiratory infections: B, C and E (65). Other D N A viruses including those from the human herpes viral family w i l l not be emphasized in this thesis because they are not common causes of respiratory infection unless in specialized settings such as immunocompromised patients or those with underlying chronic disease, in which herpes viruses can cause serious respiratory infections. 1.4 Epidemiology 1.4.1 Influenza Virus Influenza is one o f the most important emerging and re-emerging infectious diseases, having caused epidemics and pandemics for centuries and with high morbidity and mortality. Influenza has certain distinctive epidemiologic features. In temperate zones, the influenza season occurs during late fall and winter. Epidemics occur every few years. The incidence o f clinical influenza illness, based on symptoms and observable signs (see section 1.4), can be as high as 40% in 11 children (68). In the United States alone, the number of influenza related deaths total between 20,000 to 40,000 per year (69). In terms of leading causes of death, mortality due to influenza, particularly influenza-associated pneumonia, ranks seventh overall in the United States as of 2002 (70). Schools and industry both display high rates of absenteeism and excess mortality is evident in the elderly and individuals with chronic health problems. The most wel l known influenza pandemic is the Spanish influenza o f 1918 which caused approximately 20-50 mi l l ion deaths worldwide (71). This strain was exceptionally lethal in that the morbidity and mortality rates in healthy adults, 20 to 40 years o f age, were extremely high. The hallmark of the 1918 H1N1 strain was its ability to penetrate deep into the lung parenchyma, causing pneumonia (72). 1.4.2 Parainfluenza Virus Epidemiological studies show that P IV generates disease year round; PIV-1 peaks during autumn, PIV-2 in winter and PIV-3 in spring/summer (52). P IV is thought to be only second to R S V in hospitalizations in children due to viral LRT I . PIV-3 achieves an infection rate o f close to 50% in children by the age o f one and by six years of age, virtually all children have been infected. P IV -1 , -2, and -4 infect most children by the age of ten years (73, 74). P IV is estimated to cause 50 to 75% of cases of croup, 10 to 15% of cases of bronchiolitis, and 10% of cases of pneumonia in children (52, 75). Immunity to P IV infection is transient and re-infection occurs regularly. Persistent infections may develop in patients who are immunocompromised (76). P IV is a significant nosocomial pathogen in the pediatric wards. Control measures may be taken to minimize spread o f infection and this may involve designating certain areas for those infants with croup. Other precautions that may be undertaken include ward closures, proper cleaning and 12 disinfection of affected areas, proper personal hygiene, proper personal protective clothing for staff, and minimizing or excluding visitors. 1.4.3 Respiratory Syncytial Virus R S V infections peak during the winter months. R S V circulates globally and predictably generates a substantial outbreak every year as the infectious season runs from fall to late spring (77). Highly contagious, R S V is thought to infect nearly all children by the age o f five years (78-80). R S V is the most common cause of severe L R T I in children and is estimated to cause approximately 50 to 90% of cases of acute bronchiolitis, 20 to 40% of cases of pneumonia and about 10%o of cases o f croup (80-82). Mortality rates are around 5% for hospitalized children and up to 40% for those hospitalized with underlying medical conditions (83). Overall mortality rate is approximately 1% (84). Hospital pediatric wards are a common source of infant R S V infections. Infected infants who are handled by hospital staff or infected staff who have mi ld symptoms may easily spread the virus to other infants by contact of the eyes and nose. L ike P IV , immunity to R S V is temporary as an incomplete host immune response allows repeated infections to occur throughout life (85). 1.4.4 Metapneumovirus Analyses conducted thus far have revealed that M P V can be found globally (39, 86-93). M P V strikes more heavily during the winter months in temperate climates l ike influenza. Peak infection rates occur during winter and early spring, which overlaps the R S V season. M P V infection tends to culminate during the latter half of the R S V season (93, 94). Results of serological studies indicate that most children have been exposed to M P V by the age of 5 years 13 and virtually all by the age o f 10 years (30, 95). Overall, M P V has been found to account for 3 to 12% of " f lu- l ike" illness (39, 91, 93, 96) . M P V , while only recently discovered, has been found to have existed in the human population for at least 50 years based on retrospective serological studies documenting the presence of antibodies against the virus (30). 1.4.5 Human Coronavirus Very few epidemiological studies have been conducted into H C o V infections. What is known is that infection rates are found to be uniform across all age groups which is markedly different from many other respiratory viruses (which have a tendency to decrease as age increases) (97). Infections are greatest during the winter months and early spring (98). In any one year, the majority o f coronaviral infections can be ascribed to one group of viruses and this interchanges from year to year (99). This pattern is found worldwide. Re-infections by H C o V of the same strain occur frequently as immunity has been found not to be permanent (100). Approximately 20 to 30% of the "common co ld" is believed to be attributed to H C o V infection (101). Coronaviruses infect not only humans, but mammals and birds as wel l . In late 2002, the Guangdong province o f China had an outbreak of a new respiratory disease termed S A R S , characterized by a high fever, headache, fatigue and myalgia with the development of a dry cough and dyspnea (102). Gastrointestinal symptoms such as diarrhea developed in close to 40% of patients (103). Respiratory distress eventually led to death in about 10% of those infected. B y June 2003, the outbreak had subsided with 774 deaths out of approximately 8000 cases worldwide (104). Preliminary assessments were conducted on clinical specimens from patients infected with S A R S . A partial sequence recovered from a PCR-based random-amplification 14 procedure placed the novel virus into the coronaviridae family; electron microscopy revealed structural features characteristic o f H C o V and immunofluorescence showed reactivity to group I C o V antibodies (104, 105). Sequencing of the S A R S - C o V genome was determined by Centers for Disease Control in Atlanta and the Genome Science Centre of the B C Cancer Agency (106, 107). It was originally believed that civet cats and a few other species of animals were natural reservoirs for S A R S - C o V (108). Further research conducted ascertained that the Chinese horseshoe bat is the natural reservoir for S A R S - C o V (109,110). L ike wi ld ducks and influenza, Chinese horseshoe bats are largely unaffected by S A R S - C o V . Presumably, civet cats were infected by these bats and virus was shed through the cats' feces. It is thought that in the food markets where these animals are considered a delicacy, people became infected because they were in such close quarters with the diseased cats. In fact, approximately one third of original S A R S cases were attributed to food handlers in these markets (108, 111). 1.4.6 Human Rhinovirus H R V infection may occur year round with peaks in the fall and spring months. H R V has been implicated in up to 80% of all respiratory infections (112, 113) and around 90% of children under two years o f age have had a rhinoviral infection (114). A s there are over 100 known serotypes, re-infection with a different serotype occurs regularly. Infections are most common during childhood and commonly decline as age increases. This is mainly due to the presence of neutralizing antibodies against previously encountered serotypes. However, l ifelong immunity is not necessarily acquired since antibody concentrations in the blood decline over time (63). Transmission of H R V is mainly through inhalation of aerosols (115). Close contact in a high density population also increases the chance of viral transmission. 15 1.4.7 Adenovirus Adenoviral infections occur throughout the year. Adenovirus infection is endemic not only to the human population but also mammals, birds and amphibians (116). Approximately 80% of children by the age of five, has been infected by one or more subgroups o f adenovirus (117). Approximately 2 to 10% of respiratory infections in children under the age o f four can be attributed to adenovirus (118). Out of that, 10% are hospitalized, generally with croup or pneumonia. Adenoviral serotypes 1 through 7 are the most prevalent (118, 119) and virtually all adults w i l l have antibodies against these 7. Once infected with a certain serotype, l ifelong immunity is conferred. Whi le neutralizing antibodies protect against re-infection with the same serotype, virus may be shed for months after the primary infection due to the host's carrier state (66). A s with most respiratory viruses, the frequency of infection is higher in children than adults. Control and prevention include implementing more stringent hygienic strategies since adenovirus is a resilient, stable virus that can survive various environments and can spread easily through the population. 1.5 Clinical Manifestations and Therapy Infection by many of the respiratory viruses share several of the same clinical consequences that are generally referred to as " f lu- l ike" or "cold- l ike" illness. These can include but are not limited to: fever, sore throat, sneezing, chills, headache, myalgia, coryza, rhinorrhea, fatigue, nasal congestion and cough. Most viral infections generate mi ld and self-limited clinical disease; however, infants, the elderly, those with underlying chronic or medical conditions, and immunocompromised individuals may develop more severe and prolonged disease. 16 Transmission of these pathogens tends to be identical as most are spread through person-to-person contact through respiratory aerosols produced by coughing, sneezing and speaking. The viruses may also be spread by contaminated fomites or through contact from hands to the nose and/or eyes. Distribution o f infection may be restricted by observing good hygienic practices. 1.5.1 Influenza Virus Concerning influenza viruses, the incubation period lasts from one to three days while the duration of illness lasts about a week (120). Symptoms such as fatigue and cough may persist for an additional seven to fourteen days. Infants, elderly and immunocompromised patients are also prone to acquire a secondary infection, commonly bacterial pneumonia, which accounts for the high mortality rates seen with influenza infection (121). Children are apt to have higher fevers that may be accompanied by febrile convulsions and may develop otitis media and gastrointestinal difficulties such as diarrhea and abdominal pains (122). Control measures include prophylaxis with vaccine, or prophylaxis and/or therapy by antiviral drugs. There are two types of influenza vaccines, inactivated and live-attenuated. Inactivated vaccine consists of three strains, two type A influenza (H1N1, H3N2) and one type B influenza. The strains incorporated into the vaccine represent the most recent antigenic variants. The viruses are grown in hen eggs and inactivated using formaldehyde or beta-propiolactone rendering them non-infectious (123). The vaccine is approximately 70 to 80% effective (124) depending on the age and immunocompetence of the recipient and the similarity between the viral strains in the vaccine and those circulating in the community. Live-attenuated vaccine (FluMist®) is given intranasally and its advantage over its inactivated counterpart is that it 17 mimics natural infection which allows for a broader immune response. It is a cold-adapted strain that grows well in the upper respiratory tract but poorly in the lower respiratory tract. FluMist® has been shown to be safe and approximately 90% effective (125). Antiviral drugs are available for influenza virus prophylaxis and therapy. The key objective o f these drugs is to inhibit viral replication, thereby reducing the number o f infectious particles. Adamantane derivatives, amantadine (Symmetrel®) and rimantadine (Flumadine®) are effective for the prevention and treatment of type A influenza only while oseltamivir (Tamiflu®) and zanamivir (Relenza®) are effective for both type A and B influenza (41). Treatments must begin within forty-eight hours of the onset of symptoms to help diminish the severity and duration. The adamantane derivatives both prevent membrane fusion by binding and block the M 2 ion channel protein (126) while oseltamivir and zanamivir are N A inhibitors (69). Treatment with either class of antiviral drug is not recommended for general influenza outbreaks. The main problem with both classes o f antiviral drug, particularly the adamantane derivatives, is the ease of resistance the virus is able to form (127, 128). Antibodies against H A neutralize the infectivity o f the influenza virus while antibodies against N A reduce the reproduction of the virus and severity of disease. 1.5.2 Parainfluenza Virus The incubation period of PPV may last between two to six days (129). In addition to the general symptoms listed above, P IV infection may involve stridor (a high pitched sound heard during inhalation due to constricted airways), loss of voice and dyspnea. The hallmark clinical manifestation of P IV infection is croup (laryngotracheobronchitis) (75), which is caused by 18 strains 1 through 3. Types 1 and 2 can also cause pharyngitis, tracheobronchitis and the "common cold". U R T I , bronchitis and bronchopneumonia tend to be associated with strains 1 and 3 (52). In fact, type 3 infections sometimes manifest cl inically l ike a R S V infection in children with bronchiolitis and pneumonia as possible disease outcomes. Type 4 infection most often develops into mi ld URTI . Adults tend to only contract mi ld U R T I with any type of P IV infection. In the late 1960s, vaccines based on whole P IV-1 , -2 and -3 that were formalin-inactivated (130, 131) were administered to children. The vaccines were ineffective as children developed antibodies against all three serotypes but at a level much lower than natural infection. L ive-attenuated vaccines based on human and bovine strains are currently in development (132, 133). 1.5.3 Respiratory Syncytial Virus R S V incubates for a period of three to six days (134). Initially, infants exhibit a febrile U R T I with possible apnea as the infection takes hold in the respiratory epithelium (135). Over the course of a few days, involvement of the lower respiratory tract may become evident as the infant develops cough, dyspnea, and tachypnea and becomes hypoxemic (136). Bronchiolit is or pneumonia may follow. Otitis media is often found in conjunction with R S V infection (137). Resolution of illness is variable as it may last several weeks. Most infants are able to recover with no detectable sequelae. Those that develop a L R T I may be subject to protracted alterations in pulmonary function leading to possible chronic lung disease throughout life. Studies have shown children with respiratory sequelae due to R S V infection are prone to recurrent L R T I and wheezing (138-140). Infections in older infants generally result in U R T I or tracheobronchitis 19 (135) and resolution of the U R T I usually takes longer than the average cold. Re-infection by R S V throughout life is common due to the existence of strain variation (85). However, in older children and adults, infection tends to be restricted to the upper respiratory tract causing mi ld cold symptoms or bronchitis (135). The elderly are prone to more severe infection, such as pneumonia, as their immune systems are not as robust (141). R S V is also known as a "persistent" virus in that it continues to replicate at low-levels for a protracted amount of time long after acute symptoms have resolved. Studies of animal models (142, 143), cell cultures (144), and patients (145) suggest the ability of R S V to persist within the lung, avoiding detection and eradication by the host immune response. There is thought that this persistence may function as a reservoir o f infection between outbreaks. Management o f R S V infection has varied. A formalin-inactivated vaccine was developed in the 1960s to prevent R S V infection in infants and children. However, it was soon observed that the vaccine did not prevent infections from occurring but was actually causing more severe disease in some children who were inoculated and subsequently contracted a R S V infection (131,146). A number o f live-attenuated vaccines followed but were rendered unusable as they caused too strong a host immune response or were unstable (147). Current research has gone into manufacturing subunit vaccines based on the envelope glycoproteins, F and G (148); recombinant vaccines based on deletion of non-structural (NS2); and small hydrophobic (SH) genes (149) and a F glycoprotein inserted into a Newcastle disease virus vector (150). Which components o f the host's immune response involved in responding to R S V infection is not very wel l understood and therefore efforts to create an effective vaccine have been hindered. Virazole (Ribavirin®), an antiviral drug intended to interfere with m R N A expression, is the only drug 20 approved for R S V pharmacotherapy. However, studies conducted on ribavirin's toxicity and efficacy were found to be questionable and it has consequently fallen out o f favour for therapeutic use (151, 152). Prophylactic treatments available include R S V - I G I V (RespiGam™) and palivizumab (Synagis®) (153). RespiGam contains a comprehensive spectrum of antibodies since it is produced from an amalgamation of high titre ant i -RSV antibody from the plasma of a large number o f normal, healthy individuals. It is targeted towards the F and G glycoproteins of R S V and clinical trials have established RespiGam to be effective in reducing the severity o f infection, number of hospitalizations and time spent in hospital (154). Synagis consists of a chimeric human-murine IgG-kappa monoclonal antibody that is designed to selectively react with the A antigenic site of the F glycoprotein. Synagis is effective against both strains of R S V (155) since the F glycoprotein has been found to be highly conserved between the A and B strains. 1.5.4 Metapneumovirus A s M P V belongs to the same subfamily as R S V , it shares many o f the same clinical attributes (156) . Less frequent symptoms include wheezing, dyspnea, pneumonia, bronchitis, bronchiolitis, conjunctivitis and otitis media (157). Whi le M P V generally manifests as a mi ld U R T I , it can develop into bronchiolitis and pneumonitis in infants and bronchitis and pneumonitis in elderly and immunocompromised patients (158). L ike R S V , tissue tropism is to the respiratory epithelium. Whi le studies have demonstrated universal infection of M P V , re-infections occur in adulthood suggesting that immunity may not be permanent. This could be due to the non-initiation of a 21 specific humoral response or it may be due to antigenic differences between different strains that may not be recognized by the antibodies already produced by a previous M P V infection (157, 158). There are currently no vaccines, antiviral agents or prophylactics available specifically for the prevention or treatment of M P V infection. Development o f live-attenuated and recombinant viral vaccines are under investigation (159, 160). A recent study has shown in vitro therapy with ribavirin or intravenously administered immune globulin has a similar response in both R S V and M P V (161). This result suggests prospective usage of either antiviral for treatment of M P V infection. Tissue culture assays exposed to N M S 0 3 , a sulfated sialyl l ipid, and heparin have demonstrated replication inhibition of M P V (162). 1.5.5 Human Coronavirus H C o V incubates between one to four days (163). Infection is localized within the epithelium of the nasopharynx and subsequently spreads throughout the upper respiratory tract. Infected cells show vacuolation and damaged ci l ia which triggers the production of inflammatory mediators and causes mi ld clinical symptoms which resolve within a few days. Some evidence shows that coronaviruses are responsible for a significant proportion of lower respiratory tract infection in infants and the elderly (97, 164). N o antiviral drugs or vaccines are currently available for H C o V - 2 2 9 E or -OC43 infection; however, a recent study has established the ability of saikosaponins to significantly inhibit HCoV-229E attachment and penetration in vitro (165). 1.5.6 Human Rhinovirus H R V is best known for causing the common cold and is the main cause of the majority of upper respiratory tract infections (113). However, infections with H R V tend to be mi ld in severity with 22 no lasting sequelae with the exception of children who have underlying chronic illnesses or compromised immune systems (166). The primary site of infection and damage caused is the nasal epithelium through binding to ICAM-1 and, to a lesser extent, L D L - R (63). Incubation time is very short as viral titres are maximal by the second or third day of infection (167). B y the fifth day, the virus is virtually undetectable though the duration of illness may last for up to two weeks (62). In young children, otitis media (114) and sinusitis (168) may occur, coinciding with a bacterial infection. No vaccines are available for H R V due largely to the number o f serotypes involved. Since infection from one strain does not confer immunity to another strain, prevention of infection by vaccination is improbable (63). Some research has focused on the development o f antivirals which could be used for treatment and prophylaxis though there are no approved therapies. Pleconaril is used in treatment of picornaviral infections which blocks the attachment and uncoating function of the virus. A recent study found that pleconaril's efficacy was related to the susceptibility of the baseline virus isolate to the drug (169). Current clinical trials exhibit promising results with ruprintrivir and possibly pyridazinyl oxime ethers (170). 1.5.7 Adenovirus Adenoviral incubation time varies widely, from 2 to 14 days (66). A characteristic feature of adenoviral infection, which occurs after the acute phase of infection has elapsed, is the establishment o f a latent infection in the lung (171), tonsils (172), adenoids and lymphoid tissue (173) where the virus replicates at a low level for months thereafter. Adenovirus is a common 23 cause of bronchiolitis in children and adolescents (118,174). Sequelae include bronchiectasis and abnormal lung function (175). Certain serotypes from subgroup C have been found to persist in tonsils for years (176). There are a number o f ways that the virus is thought to persist. It may be found in lymphocytes in episomal form, integrate into the host D N A , or replicate at a low level such that it avoids detection by the host's immune system (177). Re-activation of the virus occurs through various physical and physiological factors including changes in host immunity levels or susceptibility of cells. New military recruits have a tendency to contract pneumonia (178). Common strains involved include 4 (E), 7 (B) and 21(B) (179). Overcrowding can exacerbate the severity o f symptoms as recruits are continually exposed to high viral titres. Antiviral treatments for respiratory adenoviral infections have not been manufactured. Ribavir in and cidofovir were used for treatment at one time in immunosupressed patients but recent studies questioned the efficacy of the drugs and they have since fallen out of favour (180-182). A l ive non-attenuated vaccine incorporating types 4 and 7 and sometimes type 21 has been developed for use in the military and found to be safe and effective (183). However, manufacturing of the vaccine was discontinued as the military and sole maker of the vaccine were unable to come to an agreement to continue production (184). It was given orally as an enteric coated capsule bypassing the respiratory epithelium and replicating within the gut. This allowed for mi ld asymptomatic infection yet provided the immunological response needed to prevent more severe respiratory infections from occurring. 24 1.6 Summary Each of these respiratory viruses has a global impact, affecting mil l ions of individuals annually. Table 1 summarizes and compares the respiratory viruses. A l l of the viruses can share various clinical symptoms and therefore it is difficult to differentiate for a specific virus on that basis alone. Detection techniques have only recently improved to facilitate more rapid diagnosis yet treatment for the most part is rudimentary. For a variety of reasons, including the diversity of viral l ife cycles, host cell receptors involved, number, structure, and function of viral genes and gene products, it is difficult to produce and generate any sort o f generally effective anti-viral therapies and vaccines. Further investigation and experimentation from all aspects in this field are needed, which can be facilitated by improvements in viral detection. Chapter 2 wi l l describe in more detail many of the methods used for diagnosis of human respiratory viral infections. 25 Table 1. Summary and comparison of respiratory viruses. Virus Structure Replicates in Cellular Receptors Approved Antivirals Approved Vaccines Immunity Influenza A ss(-)RNA, enveloped, helical, segmented genome nucleus Sialic acid linked to galactose amantadine, rimantadine, oseltamivir (Tamiflu), zanamivir inactivated flu shot (2 strains type A, 1 strain B), Flumist transient due to genomic antigenic drift and shift Influenza B ss(-)RNA, enveloped, helical, segmented genome nucleus Sialic acid linked to galactose oseltamivir (Tamiflu), zanamivir inactivated flu shot (2 strains type A, 1 strain B), Flumist transient PIV-1 ss(-)RNA, enveloped, helical, non-segmented genome cytoplasm possibly a sialic acid conjugate none none transient PIV-2 ss(-)RNA, enveloped, helical, non-segmented genome cytoplasm possibly a sialic acid conjugate none none transient PIV-3 ss(-)RNA, enveloped, helical, non-segmented genome cytoplasm possibly a sialic acid conjugate none none transient R S V A ss(-)RNA, enveloped, helical, non-segmented genome cytoplasm unknown ribavirin, RespiGam, Synagis none transient R S V B ss(-)RNA, enveloped, helical, non-segmented genome cytoplasm unknown ribavirin, RespiGam, Synagis none transient M P V ss(-)RNA, enveloped, helical, non-segmented genome cytoplasm ACE2 none none transient hCV 229E ss(+)RNA, enveloped, helical, non-segmented genome cytoplasm hAPN(CD13) none none transient hCV OC43 ss(+)RNA, enveloped, helical, non-segmented genome cytoplasm affinity for H L A I and 9-O-aceytlated sialic acid receptor none none transient H R V ss(+)RNA, non-enveloped, icosahedral, non-segmented genome cytoplasm ICAM-1, L D L - R pleconaril none permanent until circulating Ab levels drop Adenovirus dsDNA, non-enveloped, icosahedral, non-segmented genome nucleus CAR, MHC-I alpha 2, CD46, sialic acid containing receptor ribavirin, cidofovir live non-attenuated enteric coated capsule (military use only) permanent ON 2. VIRAL DIAGNOSTIC TECHNIQUES 2.1 Introduction A number of diagnostic techniques currently exists for accurate identification of a viral infection. This allows for viral diagnosis for the individual patient, monitoring for outbreaks of seasonal viruses and contributes to physicians' and clinical scientists' understanding o f a disease. It also helps advance epidemiological and public health studies which in turn facilitates improved disease control and prevention and community health and education. V i ra l diagnosis is accomplished through viral isolation and identification through subtyping, various serological techniques, or detection of viral proteins or nucleic acids. Each of these tests focuses on different aspects of virus and host response and as a result, there is no comprehensive test that encompasses both. Each test w i l l be discussed in further detail below. Rapid verification is desirable to initiate proper infection control and appropriate anti-viral therapy for the patient, i f available. Nasopharyngeal aspirates (NPAs) remain the best source of specimens for diagnostics mainly because the tissue tropism for the majority of common respiratory viruses is the epithelium of the nasopharynx. Use of nasopharyngeal swabs as test specimens for viral culture has shown a 30% reduction in detection sensitivity compared to N P A s (185,186). Sputum has been found to produce comparable results as N P A s and may in fact be more representative of the respiratory tract as N P A samples exclusively from the upper respiratory tract (187). However, acquiring induced sputum from patients can prove to be difficult. Bronchoalveolar lavage may also be used (188,189) but is more invasive and may not be easy to obtain from patients. 27 2.2 Culture Vira l isolation, through in vitro approaches, is the usual "gold standard" of laboratory diagnosis o f respiratory viral infections which tests for viral replication. Many respiratory viruses wi l l grow in various cell or tissue culture lines. Primary cell lines (e.g., monkey kidney (MK) ) are obtained through freshly ki l led animals and may only be passaged once or twice. Whi le they are considered to be superior cell lines as they are permissive to many viruses, they are costly and obtaining a consistent source can be problematic. Cel l lines (e.g., transformed human embryonic kidney, H E K - 2 9 3 , M R C - 5 ) obtained from embryonic tissue may only be passaged up to 50 times as the cells eventually lose their ability to divide, or senesce. They are the easiest to handle but each line is permissive to a limited number of viruses. Continuous cell lines (e.g., HeLa , Vero, HEp-2 , L L C - M K 2 ) are derived from malignant tumours and are termed "immortal ized" cells because they may be passaged indefinitely. The test specimen is added to permissive cell lines and left to incubate at a constant temperature in a rotating drum or incubation cabinet. Growth media may be changed daily to supplement the cells with nutrients. The cells should be examined frequently for the presence of cytopathic effect (CPE) . Initial identification of viral isolates may be detected through C P E or hemadsorption (cell surface changes) while confirmatory identification may use immunofluorescence (IF), neutralization, hemagglutinin-inhibition, viral antigen detection or molecular testing. The main limitation of viral isolation is that obtaining results may necessitate a lengthy incubation time ranging from a few days to a few weeks, and some viruses may not replicate in vitro (190). Culture sensitivity can be poor as a specimen with intact, viable virus is required for positive results while contamination o f cell lines, maintenance o f cell lines or the presence of inhibitory substances add to the difficultly of isolating viruses. 28 Influenza may be cultivated in primary rhesus monkey kidney ( P M K ) , L L C - M K 2 , A549, and Madin Darby canine kidney ( M D C K ) cells (191). Type B influenza wi l l produce a cytopathic effect (CPE) when grown in M D C K cells. Presence of type A influenza is further distinguished using immunostaining or a hemagglutinin-inhibition assay (HAI) as described in sections 2.2 and 2.3.2 respectively. P IV is commonly detected by viral culture using P M K , Vero, HEp-2 , M D C K , HeLa or trypsinized L L C - M K 2 cells (52). C P E occasionally occurs in PPV-2 and -3, producing large, multinucleated syncytia. HEp-2 cells are commonly used for isolating R S V , however many cells are permissible to R S V infection including HeLa, Vero, M R C - 5 and WI-38 cells (53). R S V propagated in HEp-2 , HeLa and M R C - 5 cells exhibit a characteristic C P E of syncytial formation in approximately five to seven days (192). The ability to isolate M P V from cell culture has proven to be rather difficult as only tertiary M K , as well as L L C - M K 2 , HEp-2 and some Vero cells have demonstrated the capability to do so (30,158, 193). Compounding this challenge is that C P E takes approximately two to three weeks to develop. C P E has been found to be variable as some show RSV- l i ke syncytia and others cell rounding (158). Coronaviruses are fastidious and w i l l only grow in human embryonic fibroblasts, suckling mouse brain, or organ cultured cells (194). Syncytia may form. Generally, H R V is detected by viral culture using human embryo lung fibroblasts l ike M R C - 5 and WI-38, or HeLa cells (195). C P E consists of highly refractile cell rounding which is very similar to enteroviral C P E . H R V can be differentiated from enteroviruses since H R V is acid-labile with many research laboratories using this technique to distinguish between the two. A number of cells may be used for viral isolation of adenovirus. HEK-293 cells are permissive for all respiratory strains of adenovirus (65). Other cells that the virus may be propagated in include: A549, HEp-2 , HeLa , primary M K , M R C - 5 , SF , WI-38 and K B (66). C P E occurs anywhere from two to seven days after infection. The 29 characteristic C P E o f adenovirus consist of enlarged, retractile, rounded cells that may be clustered. 2.3 Immunostaining Antigen detection assays may involve immunostaining through direct or indirect immunofluorescence (IF). IF assays involve detecting viral antigens or virus specific antibodies (i.e., IgG, IgA, IgM) and visualizing the fluorescent cells under ultraviolet light. It is currently the most common method used for initial respiratory viral detection from patient specimens. Brief ly, the direct method involves analyzing the test specimen with a primary or detection antibody that is labeled with a fluorochrome (e.g., fluorescein) against a specific viral antigen. The indirect method involves analyzing the test specimen with a non-labeled primary antibody followed by a fluorochome tagged secondary antibody. V i ra l antigen detection may be conducted with either direct or indirect IF whereas antibody detection is mostly carried out using the indirect method. Cells containing viral protein recognized by the primary antibody w i l l produce a bright green stain. Cytoplasmic staining is normally speckled with large inclusions while nuclear staining is uniform. The test is rapid and simple; however the quality o f the patient specimen is key since an insufficient number of cells w i l l yield uninterpretable findings. Sensitivity of the technique varies between viruses. IF can become labour intensive, in particular, indirect IF since it entails an extra antibody step. Highly trained technicians are also required for assay interpretation. Influenza, P IV , R S V and adenovirus may all be detected using IF (196). Detection of influenza and P IV antigen is generally achieved by direct IF. Detection of R S V using direct and indirect IF 30 may be conducted using poly- or monoclonal antibodies. Sensitivities for these assays range from 40 to 90%. Sensitivity of detection for R S V by IF is comparable to viral isolation. IF alone for adenoviral verification, using an anti-hexon monoclonal antibody, is not particularly sensitive. A direct IF assay for M P V was developed by generating monoclonal antibodies for detection of antigen in patient N P A , however it is mostly used for retrospective analysis at this time (197). 2.4 Serology 2.4.1 Complement Fixation Test Complement fixation test (CFT) uses two-fold serial dilutions of paired sera from the acute and convalescent phases of infection, essentially testing for antibody production by the host's immune system. This assay consists of two reactions; the first involves the binding and inactivation, hence ' f ixation' o f an antigen-antibody complex in the presence o f a known amount of complement, the indicator system. The second reaction involves hemolysing sensitized sheep erythrocytes coated with anti-sheep antibody. If hemolysis occurs, free complement is present, the antigen-antibody complexes did not develop and a 'negative' outcome results. If hemolysis does not occur, free complement is fixed by the antigen-antibody complexes indicating that the patient possessed antibodies against the test virus. A four-fold or greater rise in titre of total antibody is considered as indicating current infection (198). C F T has the ability to screen against a large number o f viral infections simultaneously and is relatively inexpensive. However it is not particularly sensitive and it is time consuming and cumbersome with the time to acquire acute and convalescent sera taking days or weeks. 31 C F T may be used for new influenza viral subtypes whereas hemagglutinin-inhibition (HAI) is subtype-specific. C F T uses complement-fixing antibodies against test sera; however, results take longer than H A I and is therefore less utilized. A t times it may be non-specific, as in the case of differentiating between P IV subtypes as cross-reactivity occurs and C F antibody does not develop on initial infection from some young children (52). C F T has not been found to be particularly sensitive or useful for R S V identification since seroconversion does not occur for at least a two week period (199). In young infants, the serological response can be quite poor and undetectable by some assays. C F T is the test of choice for adenovirus in diagnostic laboratories, however infants often do not respond with C F antibody after infection. Overall, sensitivity o f the C F T assay has been found to be approximately 75% (200). 2.4.2 Hemagglutinin-inhibition Test Hemagglutinin-inhibition (HAI or HI) tests involve the ability of a virus to clump together or 'agglutinate' mammalian or avian erythrocytes. Respiratory viruses that are able to hemagglutinate include influenza, P IV and adenovirus. L ike C F T , acute and convalescent sera from the patient are used. Each well in a microtitre plate is prepared with a known amount of virus and allowed to react to two-fold serial diluted sera, followed by the addition o f erythrocytes. If virus specific antibody is present in the sera, the virus wi l l be bound and the erythrocytes w i l l not agglutinate, hence hemagglutinin-inhibition. If no antibody is present, the virus remains unbound and wi l l cause the erythrocytes to clump. The highest dilution of antigen that allows for erythrocytes to completely hemagglutinate is defined as a H A antibody unit. A four-fold or greater rise in antibody titre or seroconversion between the paired sera indicates 32 recent infection (201). The test is simple, inexpensive, and more sensitive than C F T . However, the usefulness o f H A I has limitations in terms of patient care since acquiring acute and convalescent sera takes days to weeks and cross-reactivity may occur for certain viruses such as the different strains and subtypes of influenza and P IV (198, 202). L ike R S V , M P V does not display the ability to agglutinate human or mammalian erythrocytes. Some strains o f influenza react poorly to H A I testing and subsequently is not the first test of choice (203). A s with C F , subtyping of P IV by H A I is not used due to cross-reactivity (52). Further typing by H A I is generally performed for adenovirus after viral culturing. H A I with monkey (subgroup B) , or rat (subgroups A to F excluding B) erythrocytes may be used for typing (66). 2.4.3 Hemadsorption Test Hemadsorption involves the ability of virally infected cells to adhere to erythrocytes. It is generally used in detecting influenza or PIV. For P IV subtyping, this test carries the greatest specificity o f all the serological tests available (16). Both family o f viruses contain hemagglutinin as part o f their viral structure and therefore are able to adsorb guinea pig erythrocytes. The virus inoculated cell culture is added to the erythrocytes, allowed to incubate and then examined for the presence of hemadsorption under a microscope. 2.4.4 Neutralization Test Virus or serum neutralization tests involve the loss of viral infectivity due to interactions between the virus and a specific antibody forming antigen-antibody complexes. This is due to the 33 antibody interference at various points in viral entry or release. The antibody may neutralize the virus by interfering with viral attachment to the host cell receptor or it may bind the viral capsid disabling the virus' ability to uncoat and release nucleic acid. Virus and test serum are combined and inoculated into the test system. Un-neutralized virus is then detected through C P E , hemadsorption, H A I , or plaque formation. H R V may be detected using neutralization tests. Serum neutralization tests, while the most cumbersome, are the most sensitive and type-specific adenovirus tests available with sensitivities approaching 90% (15). 2.4.5 Enzyme Linked Immunosorbent Assay Enzyme linked immunosorbent assays (ELISAs) may also be used for virus detection with a number o f commercial kits available. The basic principle of an E L I S A is the detection o f certain antibodies or antigens against the virus in sera or plasma. Influenza, R S V , PPV, H C o V , H R V and adenovirus may all be detected by E L I S A . E L I S A sensitivity tends to be high but is comparable to other serological tests that are cheaper. For some viruses, l ike R S V , sensitivity can be poor with ambiguous results which necessitate further testing for confirmation. 2.5 Conventional Polymerase Chain Reaction Polymerase chain reaction (PCR) serves to amplify a specific portion of a gene, i f present, many times over. Since both strands are copied each cycle, there is an exponential increase in the number o f copies o f the template. The main advantage of this technique is that it can detect very low amounts of template. After an initial incubation step to stimulate the heat-activated D N A polymerase to function, three major steps are involved: denaturation, annealing and extension. Denaturation allows the double stranded D N A template to melt into single strands. Annealing 34 temperature permits the primers to bind to specific sequences of the single stranded D N A . Extension permits the polymerase to produce a complementary copy of the template in a 5' to 3' manner from the 3 ' end of the annealed primer. These three steps are generally repeated up to 40 cycles. The products are then visualized on an ethidium bromide stained agarose gel. The main problem with conventional P C R is contamination as even small contaminants wi l l be amplified. A s wel l , this test w i l l only give a 'positive' or 'negative' result with no indication of actual amount of template present. 2.6 Quantitative Real-Time Polymerase Chain Reaction Current techniques in use for the detection of pathogens include culture, immunostaining, various serological assays l ike C F T , H A I , hemadsorption, neutralization tests and E L I S A , and conventional P C R . Out of all of these, P C R can detect very small amounts of viral nucleic acids, assuming an intact starting template. Conventional P C R employs end-point detection for analysis of whether the target amplicon is present. It occurs during the plateau phase o f the reaction and becomes apparent once P C R thermocycling and post-PCR processing has concluded. A newer technique has been developed combining traditional P C R technology with automated detection and quantification o f a fluorescent reporter, known as quantitative real-time P C R (qPCR). The amount of fluorescence is directly proportional to the amount of P C R product in a reaction. q P C R confers many advantages over conventional P C R . For example, q P C R requires far less template, allows the user to monitor the reaction during the exponential phase of the reaction or 'real-time' which obviates the need for post-PCR electrophoretic gel analysis, which in turns reduces the possibility of cross-contamination. 35 Two types of P C R may be performed, one-step or two-step. One-step P C R combines the reverse transcription of R N A to c D N A and subsequent amplification of that c D N A , all into a one tube reaction with the required reagents and enzymes. Two-step P C R is performed in two individual reactions; reverse transcription of R N A to c D N A and then amplification of an aliquot of that c D N A . Some investigators consider two-step q P C R to be advantageous over its one-step counterpart since it is generally more reproducible and sensitive (204, 205). In addition, one-step q P C R has been characterized by a large accumulation of primer dimers that can interfere with specific amplification of the target sequence (206). A s an additional integrity check, two-step q P C R also uses c D N A as opposed to R N A which is prone to degradation. In cases where the amount o f specimen is minute, the production of c D N A helps to increase the number o f possible assay reactions (207). The small amplicon size allows for improved amplification efficiency, the dynamic range of detection is increased, and less nucleic acid is required in the reaction. To increase sensitivity, a small D N A oligonucleotide specific for the template is used as a probe. The probe consists of a fluorescent reporter dye, 6-carboxyfluorescein ( F A M ) , located at the 5 ' -end and a quencher dye, 6-carboxytetramethylrhodamine ( T A M R A ) , located at the 3'end. Each q P C R reaction util ized a F A M - T A M R A combination as they were individual reactions and not multiplexed. Mult iplex P C R allows for the simultaneous amplification of several targets of interest in a reaction by using multiple pairs of primers (208). Mult iplex is useful in the case of l imited starting material and allows for considerable savings of time, effort and money. However, multiplex assays are difficult to engineer since each reaction must be thoroughly optimized with lower sensitivity than individual P C R and the preferential amplification of certain targets, non-specific targets or quasi-species posing additional obstacles (209). Because of these 36 potential issues, the studies presented in this thesis have focused on development o f a panel o f single target reactions. The probes are all highly specific; a mismatch of two or more base pairs is enough to prevent the probe from binding to the target. If the target is present, the probe anneals between the primer sites. The 5' nuclease activity of the Ampl iTaq Gold D N A Polymerase cleaves the bound probe, separating the reporter dye from the quencher dye, during the P C R process. Released from the quenching effect of the 3' dye, the reporter is then able to fluoresce. The increase in fluorescence after each cycle and therefore the accumulation of P C R target amplicon can be monitored and detected by the thermocycler. The reaction contains a passive internal fluorescent reference dye, 6-carboxy-X-rhodamine ( R O X ) , which is pre-incorporated into the 2 X Taqman Master M i x (Applied Biosystems, Foster City, C A , U S A ) . R O X serves to normalize the target signal due to non-PCR related fluorescent fluctuations in reagent concentrations or total volume which may occur between samples over time. This is automatically calculated by the accompanying software. This differs from the normalization that is required to correct for P C R efficiency or amount of template used. 2.7 Summary This chapter has provided an overview of some of the laboratory techniques commonly used for viral diagnosis, noting their various strengths and limitations. Overall, there is a need for a single technique that contains all these strengths in a rapid and easy-to-do format. Chapter 3 explains the reasoning and justification behind the developmental stage of the real-time quantitative P C R panel for respiratory virus detection. 37 3. RESEARCH OVERVIEW 3.1 Rationale It is wel l established that traditional viral diagnostic techniques tend to be time-consuming, cumbersome or not particularly sensitive. Even conventional P C R suffers from various drawbacks as mentioned earlier in this thesis. Current q P C R protocols for common respiratory viruses also show variability in the pre-analytical and analytical phases, which can limit the successful translation to the clinical diagnostic laboratory setting (210). Consequently, many o f these traditional techniques are still in use as there is no standard protocol or set criterion for assay interpretation for q P C R assays. The goal o f this thesis was to develop a q P C R panel that is rapid, quantitative, and standardized, with the long-term goal to facilitate validation and ultimately use in the clinical laboratory for improved decision-making and intervention. In addition, extensive experience in the area of H I V infection has clearly shown that disease activity, response to therapy, and progression are related to viral load (211, 212). It is possible that quantification o f viral nucleic acid load in clinical respiratory specimens may also provide more information than merely identifying the presence of viral nucleic acid in specimens. The introduction of q P C R technology has facilitated the ability to reliably quantify target nucleic acid sequences. The central hypothesis of this project is that quantification of viral load in clinical respiratory specimens can allow for improved interpretations of viral PCR results in terms of understanding the relationship between viral nucleic acids detected by qPCR, viruses 38 documented by other laboratory methods of viral diagnosis, and concomitant clinical features. 3.2 Specific Aims The research was divided into a number of components: 1. Designing and developing individual quantitative real-time P C R assays for influenza A , influenza B, P IV-1 , P IV-2 , P IV-3 , R S V A , R S V B, M P V , HCoV-229E , H C o V - O C 4 3 , H R V , and adenovirus using standardized conditions. 2. Designing a quantitative real-time P C R for a reference gene, hypoxanthine phosphoribosyl transferase (HPRT), that is suitable for normalizing gene expression in the test specimens. 3. Manufacturing synthetic standards for each of the respiratory viruses to allow for more accurate quantification for use in quality assurance. 4. Examine the relationship/concordance of qPCR results from archival N P A s to other laboratory techniques, specifically viral culture on the same samples. 5. Using q P C R results on archival N P A s to determine a possible "threshold" viral load level that is associated with a particular specimen exhibiting a positive culture result. 39 6. Performing a retrospective analysis using qPCR results from archival N P A s of the relationship between viral load and clinical respiratory illness diagnosed at the time of patient presentation. After designing individual q P C R assays for each respiratory virus, archival N P A s obtained from symptomatic children at the time of emergency room visits were used to test the q P C R panel. Inclusion of a reference gene assay verifies that each specimen contains viral nucleic acid and permits normalization of results. Synthetic controls test the robustness of the q P C R assays and allow for the quantification o f viral loads. One goal of this study was to better understand the differences between viral loads and corresponding culture results as well as viral loads and clinical diagnosis. If the paradigm of H I V viral load is also applicable to common respiratory viruses, then quantification may prove to be clinically important in terms of evaluation and treatment. 40 4. METHODS AND MATERIALS 4.1 Case Selection of Nasopharyngeal Aspirates (NPAs) Archived N P A s from the 2003-2004 winter season, stored frozen at -80°C, were selected based on previously diagnosed laboratory results (culture and/or direct fluorescent antibody test). In total, 365 specimens were chosen (30 each positive for influenza types A and B, 40 each positive for P IV -1 , -2, -3, R S V , adenovirus, 5 for H R V , 100 culture negative) for analysis. R S V subtypes were not discriminated for. A subset o f 187 specimens from children was then selected for further examination based on availability of clinical diagnosis as noted on laboratory requisition forms. The first subgroup consisted of children with U R T I (n = 23), a second contained children who had fever (n= 98) and a third contained children who were diagnosed with L R T I (croup, bronchiolitis, or pneumonia) (n = 66). 4.2 Nucleic Acid Extraction from Nasopharyngeal Aspirates R N A from archival N P A s was extracted using the QIAamp V i ra l R N A M i n i K i t (Qiagen, Mississauga, O N , Canada) fol lowing the manufacturer's instructions. One notable change from the protocol was the elution of the R N A from the standard 60 [iL to 50 |aL to concentrate the nucleic acid. 310 | i L of carrier R N A (1 M-g/uL) was dissolved into 31 m L Buffer A V L and aliquoted 560 \iL per 1.5 m L microcentrifuge tube. Carrier R N A helped the viral R N A to bind to the QIAamp mini membrane, particularly i f the sample was of low-titre. Brief ly, 140 (j,L o f N P A underwent lysis in the pre-aliquoted Buffer A V L which contained denaturing guanidine isothiocyanate (GITC) inactivating RNases. Ethanol was added to allow the R N A to bind to the sil ica gel-based membrane. The p H and high salt buffers ensured that protein and other 41 contaminants did not bind to the membrane. After washing with two buffers, A W 1 and A W 2 , to remove any remaining contaminants and inhibitors, the R N A was eluted using Buffer A V E . Buffer A V E contains sodium azide which prevented microbial growth and subsequent contamination with RNases. The R N A was aliquoted and stored at -80°C until use. D N A from archival N P A was extracted using the QIAamp D N A M i n i K i t (Qiagen, Mississauga, O N , Canada) fol lowing the manufacturer's instructions. In a 1.5 m L microcentrifuge tube, 20 p.L of proteinase K, 200 uL of N P A and 200 u L of lysis buffer were mixed together and allowed to incubate for 10 minutes at 56°C. 200 uE of anhydrous ethanol was added to the sample and the resultant admixture was then loaded onto a QIAamp spin column which underwent centrifugation at 12,000 x g, which allowed the D N A to bind to the column membrane. The lysate buffering conditions allowed optimal binding of D N A to the QIAamp membrane. A n y contaminants and inhibitors were washed away with the supplied wash buffers A W 1 and A W 2 and the D N A was subsequently eluted in 100 uT of Buffer A E (10 m M Tr isC l ; 0.5 m M E D T A , p H 9.0). The D N A was aliquoted and stored at -80°C. 4.3 Selection of Viruses for Inclusion in qPCR Panel The fol lowing 12 respiratory viruses were chosen for inclusion into the q P C R panel as they, collectively, form a large burden of disease in the general population (213,214). These viruses include influenza types A and B, P IV-1 , -2, and -3, R S V A , R S V B, M P V , HCoV-229E and -OC43 , H R V and adenovirus. Both P IV and R S V have a limited number o f strains; therefore, individual assays for each serotype were developed. In contrast, assays for H R V and adenovirus 42 were developed to provide a broad screen since there are a large number of strains known for each virus. 4.4 Design of Primer and Probe Sequences for Quantitative Real-Time PCR Literature searches were conducted for published q P C R protocols for the respiratory viruses under study. In late 2003, these searches showed a limited number of publications reporting working two-step standardized q P C R protocols using the A B I 7900HT Sequence Detection System (Applied Biosystems, Foster City, C A , U S A ) . Published protocols for R S V A and B, M P V , influenza B and adenovirus were modified to complement the Taqman™ probe (Applied Biosystems, Foster City, C A , U S A ) detection system (65, 215-217). The original amplicon for R S V A was lengthened to include the sequence of a C la I site that was inserted within the synthetic plasmid. The assays for R S V A and B were modified from a one-step q R T - P C R to a two-step qPCR. The original M P V assay used by Children's and Women's Hospital based on the Mackay paper (217) comprised of a 68 base pair amplicon which was too small to design an accompanying probe and did not include a C la I site for discrimination between native and synthetic amplified sequences. A new M P V forward primer was slightly modified from the published sequence. The reaction mixture volume and cycling conditions for all of these previously published assays varied but were standardized to one set of parameters (see section 4.8). The influenza A and H R V assay protocols were kindly provided by Dr. Stephen Lindstrom from C D C Atlanta and Dr. Sebastian Johnston from Imperial College, London, England, respectively and adapted to the set standardized conditions of the other assays. Primers and probes for the remaining viruses (PIV-1, -2, -3, HCoV-229E , -OC43) were constructed by 43 myself with input from Dr. Nicholas A u , one of Dr. Rusung Tan's former trainees at Children's and Women's Hospital of Brit ish Columbia, Vancouver, Canada. Sequences for the primers and probes for each respiratory virus were chosen using Primer Express™ software (Applied Biosystems, Foster City, C A ) fol lowing recommended criteria. Ampl icons were kept under 250 base pairs to allow for rapid amplification, G - C content kept between 20-80%, and runs of identical nucleotides were avoided. A 5' G on the probe was avoided as it causes quenching of the reporter dye even after cleavage. For the reference gene hypoxanthine phophoribosyl transferase (HPRT) , the amplicon was designed to span an intron which prevented the amplification of pseudogenes. Each set o f primers and probe were "blasted" against the N C B I nucleotide database to ensure specificity to its particular virus. Table 2 shows the accession numbers for each virus, forward and reverse primer sequences, probe sequence, gene segment chosen, and size of amplicon fragment amplified. The fluorescent probes were designed to anneal to a sequence internal to the P C R primers. Each probe was labelled with F A M (6-carboxyfluorescein) and T A M R A (6-carboxy-tetramethyl-rhodamine) on the 5' and 3' ends, respectively. The melting temperature o f the probe is generally 10°C higher than the primers to avoid the extension of the primer when the probe has not yet bound. The Taqman probe is based on what is known as fluorescent resonance energy transfer (FRET) for quantification. When the probe is intact, the proximity of the reporter dye to the quencher dye inhibits any fluorescent signal. During the annealing phase of P C R , i f the target sequence is present, the probe wi l l bind to the template. When the 5' nuclease activity of the Taq D N A polymerase cleaves the fluorophore from the probe on the target strand, the two dyes are 44 separated and F R E T ceases. Since the reporter dye is no longer being quenched, it emits fluorescence and this signal is measured by the plate reader on the machine. The amount of fluorescence is directly proportional to the amount of target amplicon present in the assay. Since the probe is only cleaved in the presence of the target sequence, it ensures that any non-specific amplification is not detected. 4.5 Host Reference Gene Selection The choice o f human reference genes is critical, since the selection of an unsuitable gene wi l l lead to misinterpretation and skewing of any real changes in gene expression. Requirements for an optimal reference gene is based on what the gene is N O T : in particular, the gene should not be regulated, not be highly expressed, and does not have pseudogenes. H P R T has been found to be stable, does not have pseudogenes, and is expressed at much lower levels than the traditional reference genes l ike glyceraldehyde-3-phosphate dehydrogenase ( G A P D H ) orbeta-actin, making it much more suitable for normalization of gene expression over a wide range of transcript levels (218). 4.6 Standards for Quantitative Real-Time PCR Synthetic standards were produced for each virus to allow absolute quantification of c D N A from the N P A specimens. The standards were comprised of bacterial plasmids which incorporate the target amplicon o f each virus. This was advantageous for a number o f reasons: it allowed for the validation of novel assays, contained a known sequence which allowed for accurate quantification since it was made up of only the pure species, a restriction site integrated within the amplicon allowed for the detection of any cross-contamination between the clinical specimen 45 and the synthetic one, and ease in preparation permits a virtually unlimited supply. Brief ly, an R N A molecule with identical priming sites and amplification attributes of the real viral target was created from synthetic oligonucleotides. This R N A molecule had a unique feature in that it integrated a 6 nucleotide polymorphism within the target sequence to assist in discerning between genuine and manufactured amplicons in cases of possible template contamination. Dr. John Brunstein o f Children's and Women's Hospital of Brit ish Columbia kindly constructed and provided synthetic standards for R S V A and B, M P V , HCoV-229E and -OC43, and PPV-1. The design and assembly o f synthetic standards for influenza A and B, PIV-2 , P IV-3 , H R V , adenovirus, were done in-house. Figure 1 is a cartoon representation o f a general synthetic control describing the viral gene insert, its orientation, restriction enzymes involved and the vector used. 4.6.1 Annealing, Ligation and Transformation Based on the length o f the amplicon, an average o f six oligonucleotides (three forward and three reverse which were complementary) which included a C la I restriction site were synthesized (Sigma Genosys, Oakvi l le, O N , C A N ) and phosphorylated using T4 Polynucleotide Kinase (Invitrogen, Burlington, O N , C A N ) . The T4 Kinase inserted a phosphate group to a 5' hydroxyl group on the oligonucleotide which is required for ligation. A 25 \iL reaction volume consisting of 10 }AL (15 m M ) oligonucleotide, 5 u L of 5 X Forward Reaction Buffer, 1 u L of T4 Kinase, 2.5 \iL A T P and 6.5 uT water were combined together in a 0.5 m L microcentrifuge tube, vortexed briefly, pulse centrifuged and allowed to incubate at 37°C for 10 minutes. The reaction was terminated by incubating the mixture at 65°C for 10 minutes. To hybridize the oligonucleotides 46 prior to ligation, they were incubated using a step-wise decrease in temperature starting at 95°C and ending at 40°C for 1 minute each. The oligonucleotides were ligated together using T4 D N A Ligase (Invitrogen, Burlington, O N , C A N ) which catalyzed the formation of phosphodiester bonds between double-stranded D N A s with 3 ' hydroxyl and 5 ' phosphate termini in the presence of A T P . A reaction consisting of 9 [ iL oligonucleotides, 10 2 X ligase buffer, 1 \iL 10 m M A T P and 1 (iL T4 D N A ligase was left to incubate at 15°C overnight. The reaction was terminated by heating to 75°C for 10 minutes. The contents o f the reaction were run on a 2% agarose gel to separate the ligated and unligated species. The band containing the ligated species was excised and gel purified using a MinElute Gel Extraction K i t (Qiagen, Mississauga, O N , Canada). Three gel volumes of buffer Q G was added to the gel slice and incubated at 50°C for 10 minutes with vortexing of the mixture every 2-3 minutes during incubation. One gel volume of isopropanol was added and mixed by inverting the tube several times. The mixture was applied to a MinElute column and centrifuged at 12,000 x g for 1 minute. After the addition and centrifugation of 750 u L Buffer P E to wash and purify the oligonucleotide, 10 uL of Buffer E B was added to elute the oligonucleotide. The purified ligated synthetic oligonucleotide was then cut with two restriction enzymes, Pst I on the 5' end and X b a I on the 3 ' end. The cloning vector, p G E M 4 Z (Promega, Madison, WI), was digested with the same two restriction enzymes which allowed the synthetic oligonucleotide and plasmid to ligate in a specific orientation. Using a 1:3 vectoninsert ratio, a 10 (xL reaction containing 100 ng of p G E M 4 Z vector, approximately 16 ng insert D N A , 1 unit of T4 D N A ligase, 1 p,L of 1 OX ligase buffer and water were combined in a 1.5mL Eppendorf tube and 47 allowed to ligate overnight at 16°C. The ligase was heat inactivated by incubating at 65°C for 10 minutes. The recombinant vector was then heat-shocked with E. coli Stop Unwanted Rearrangement Events (SURE®) (Stratagene, L a Jolla, C A ) cells. Briefly, an aliquot of SURE® competent cells was thawed, and separated into 100 uL portions in pre-chilled polypropylene round-bottom tubes to which 1.7 uT of P-mercaptoethanol was added, blended by swirling gently before incubating on ice for 10 minutes. The tubes were gently swirled every 2 minutes during the incubation period. Two p,L of the subcloned vector was added to the SURE® cells and allowed to incubate on ice for 30 minutes. The tubes were then heat pulsed in a 42°C water bath for 45 seconds to allow for maximal transformation efficiency to occur and subsequently chilled on ice for 2 minutes. A 0.9 m L aliquot of pre-heated S O C medium was added to the mixture and placed in a 37°C shaking incubator for 1 hour. The transformed cells were plated onto LB-ampic i l l in (100 jag/mL) agar plates with 0.5 m M IPTG and 40 ng/mL X-gal for colour screening and incubated overnight at 37°C. The plasmids harbouring vectors without inserts appeared blue while those with inserts appeared white. 4.6.2 Overnight Culturing of Bacterial Plasmids Three white colonies from each plate were picked out and added to individual 15 m L Falcon tubes containing 5 m L of Luria-Bertani (LB) media supplemented with 150 |ag/mL ampicil l in. The bacterial plasmids were allowed to propagate overnight at 37°C in a shaking incubator. A sample o f the bacterial culture was removed and stored in a 30% glycerol solution at -80°C. 48 4.6.3 Isolation of DNA Plasmids from Culture The D N A plasmids were isolated using a QIAprep Spin Miniprep K i t (Qiagen, Mississauga, O N , Canada). After removing the cultures from an overnight 37°C shaking incubator, the cells were harvested by centrifugation at 3,200 x g for 15 minutes. The cells were resuspended in Buffer PI and transferred into 1.5 m L microcentrifuge tubes. Lysis buffer P2 was added to release the D N A in which time the solution became viscous and clear. It was then deactivated by the addition of neutralization buffer N3 which made the solution cloudy. After centrifugation at full speed (12,000 x g) for 10 minutes to pellet the cellular debris, the supernatant was transferred to a QIAprep spin column. The column underwent centrifugation at 12,000 x g for 1 minute to allow the D N A to bind to the column membrane and then washed by Buffers P B and P E to remove nuclease activity and any contaminants and inhibitors. The D N A was eluted with 50 u L RNase-free, DNase-free water and stored at -80°C. D N A concentration was determined by diluting 10 (iL of sample into 490 |AL of sterile water and measuring the absorbance at 260 and 280 nm in triplicate on a U V spectrophotometer (Lambda 2 Spectrometer, PerkinElmer, Boston, M A , U S A ) . The concentration and yield was calculated using Equations 1 and 2. Equation 1. [ D N A ng/uL] - [(A260)(dilution factor)(50 ^g/mL)]/1000 Equation 2. Amount o f D N A = ([DNA])(vol. of sample) 49 4.6.4 Sequencing and Re-culturing of Bacterial Plasmid A sample o f the plasmid was submitted to the University o f Brit ish Columbia's Nucleic Ac i d Protein Service (NAPS) Unit for sequencing. Once the correct sequence was confirmed, 10 uT of SURE® cells carrying viral plasmid were placed in a 50 m L Falcon tubes containing 15 m L of Luria-Bertani (LB) media supplemented with 150 p.g/mL ampicil l in to allow propagation o f the bacteria which occurred overnight at 37°C in a shaking incubator. The D N A plasmids were isolated using the technique described in Section 4.5.3 with the following changes. After the cells were resuspended in Buffer P I , they were split into 3-1.5 m L microcentrifuge tubes. The D N A that was eluted was pooled, aliquoted and stored at -80°C. 4.6.5 Linearization of DNA Plasmids by Restriction Enzymes Twenty \ig o f plasmid D N A was linearized using 40 U of X b a I for two hours at 37°C then heated at 65°C for 15 minutes to inactivate the X b a I. One )j,L each of uncut and linearized plasmid was run on an ethidium bromide enhanced 1% agarose gel to ensure complete digestion, as any residual uncut plasmid greatly decreases the yield of in vitro transcribed R N A . 4.6.6 In vitro Transcription of Digested DNA Plasmids Two ng of digested plasmid was in vitro transcribed using a MEGAshortscr ipt kit (Ambion, Austin, T X , U S A ) . Transcription buffer, dNTPs, plasmid and enzyme mix were combined to a final volume of 20 (j,L and incubated for 2 hours. RNase-free DNase I was added to the reaction and allowed to incubate at 37°C for an additional 15 minutes to remove template D N A . To recover the transcript R N A , RNase-free water and ammonium acetate precipitation solution were added to the mix to terminate the reaction. Two volumes of 100% ethanol was added to 50 precipitate the R N A . The reaction mix was chilled at -20°C and underwent centrifugation for 15 minutes at 12,000 x g. The supernatant was carefully removed and the remaining alcohol allowed to evaporate. The R N A pellet was then resuspended in 100 p L of RNase- and DNase-free water. The R N A concentration was determined by diluting 10 p L of sample into 490 p L of sterile water and measuring the absorbance at 260 and 280 nm in triplicate on a U V spectrophotometer (Lambda 2 Spectrometer, PerkinElmer, Boston, M A , U S A ) . The concentration and yield was calculated using Equations 3 and 4. Equation 3. [ R N A pg/pL] = [(A260)(dilution factor)(40 pg/mL)]/1000 Equation 4. Amount of R N A = ([RNA])(vol. of sample) The yield o f these in vitro transcribed R N A transcripts were on the order of 10 1 4 copies/pL. A working concentration of 10 7 copies/pL for each of the transcripts was prepared while the remaining amount of concentrated transcripts were aliquoted and stored at -80°C. 4.7 Reverse Transcription For synthesis of complementary D N A (cDNA) , 10 p L R N A per 20 p L reaction was reverse transcribed using random primers and Superscript II Reverse Transcriptase (Invitrogen, Burlington, O N , Canada). One p L each of random primers (2 pg/pL) and dNTPs ( lOmM) 51 (Invitrogen) were added to the R N A , incubated at 65°C for 5 minutes, and immediately placed on ice. The mixture was pulse centrifuged to recover any condensation built up on the walls and cap of the P C R tube. A master mix of 4 uL 5 X first strand buffer, 2 uL 0.1M dithiothreitol (DTT) and 1 u L RNaseOUT Recombinant Ribonuclease Inhibitor (40U) (Invitrogen, Burlington, O N , Canada) was added to the R N A mixture and incubated at 42°C for 2 minutes. The addition o f 1 uU o f Superscript II Reverse Transcriptase (200U) (Invitrogen, Burlington, O N , Canada) completed the reaction mix. This was incubated at room temperature for 15 minutes, placed in the Robocycler (Stratagene, L a Jolla, C A ) and heated to 42°C for 50 minutes and 15 minutes at 70°C to complete the R T reaction. The c D N A produced from the in vitro transcribed transcripts were serially diluted from 10 8 to 10 1 copy numbers, aliquoted and stored along with the specimen c D N A at -80°C until use. 4.8 Quantitative Real-Time PCR on the ABI 7000 Originally, the q P C R assays were developed for the A B I 7900HT (Applied Biosystems, Foster City, C A , U S A ) machine. However, the machine was upgraded during assay development to an A B I 7900HT Fast system for use in the virology laboratory at Women's and Children's Hospital of Brit ish Columbia. It was then decided that a switch would be made to the older A B I 7000 instrument to complete the remaining experiments. The A B I PRISM® 7000 Sequence Detection System (Applied Biosystems, Foster City, C A , U S A ) was the platform used for quantitative qPCR. It utilized a tungsten-halogen lamp in a 96-well format. Target amplicon detection was via a plate reader that scanned the plate during each cycle and recorded the amount of fluorescence in each well . 52 The 25 p X reactions consisted of 2 X TaqMan Universal P C R Master M i x (containing Ampl iTaq Gold D N A Polymerase, AmpErase U N G , dNTPs with dUTP, R O X passive reference and optimized buffer components), water, primer mix and probe and 5 p X c D N A template. Each viral assay had it own optimized concentration of primers and probe ranging from 300-600 n M and 100-200 n M , respectively. H P R T was used as an internal control to normalize the differences in the quantity of total c D N A in each sample. The negative control and standards with copy numbers from 10 7 to 10 1 were assayed in triplicate while the samples were analyzed in duplicate. The results were analyzed using the accompanying software, A B I Prism 7000 SDS (Applied Biosystems, Foster City, C A , U S A ) . Standardized cycling conditions were used that included an initial incubation of 2 minutes at 50°C, which allowed for AmpErase uracil N-glycosylase (UNG) to activate and decontaminate the reaction by cleaving uracil bases from D N A strands that have been synthesized in the presence of dUTP. This was followed by 10 minutes at 95°C to activate the D N A Ampl iTaq Gold polymerase. Final ly, 50 amplification cycles were carried out, each comprising of 15 seconds at 95 °C for denaturation and 1 minute at 60°C for primer annealing and extension. The amount of fluorescence in each wel l was read by the machine during the annealing/extension phase. A standard curve generated from the serially diluted synthetic control allowed for the quantification o f the unknown specimens. Ampli f icat ion early on in the assay between background and amplicon detection signals cannot be differentiated. Only after the amplicon has been replicated enough to enter its exponential phase, and crosses the threshold cycle can the assay's progress be examined. The threshold 53 cycle, Ct, is the point at which fluorescence reaches a detectable level and may be set anywhere in the log-linear phase of the reaction. Those samples with a Ct value of 50 did not have the amplification curve cross the threshold line and were considered negative. Samples with a value of less than 50 and were within the dynamic range of detection for a particular viral assay were considered positive as the amplification curve crossed the threshold line at that cycle. The starting concentration for each amplified virus in an unknown specimen was obtained from the corresponding Ct on the standard curve. The efficiency of the reaction and dynamic range of detection can also be calculated. Theoretically, a wel l designed assay should have a slope between -3.1 and -3.6 (219). This corresponds to an efficiency of between 90 to 110%. The efficiency of a P C R reaction can be calculated by using equation 5: Equation 5. Efficiency = 1 0 ( - 1 / s l o p e ) - l There are numerous variables that affect P C R efficiency. This includes amplicon length, secondary structure, and choice of primers (220). The dynamic range of detection is then based on a log l 0-fold dilution series of virus. The more dilutions the qPCR assay can detect, the larger its dynamic range. 54 4.9 Data Analysis Statistical analysis was performed using SPSS v . l 1.0 software (SPSS Corporation, Chicago, IL). Patients whose respiratory specimens yielded a negative result from the H P R T assay were excluded from further testing and analysis because it would not be possible to determine whether a negative viral q P C R result was due to true absence of virus or due to an inadequate specimen. V i ra l load data underwent a logarithmic transformation to better approximate a normal distribution. The statistical significance o f the differences between the logarithmically transformed mean q P C R viral loads between the culture negative and culture positive groups was determined using a Student's t-test. The significance of the differences between viral load and clinical diagnosis (URTI , fever, LRTI ) was determined using an analysis of variance ( A N O V A ) , followed by pair-wise Student's t-tests. In all analyses, a p-value < 0.05 was considered to be statistically significant. 55 Table 2. Summary o f viral assay primers, probe and amplicon length. Virus/Host Gene Accession # Forward Primer (5'-3') Reverse Primer (5'-3') Probe (5'-3') Gene Segment Ampiicon Length Influenza A AY210270 C A T G G A RTG GCT A A A G A C A A G A C C A G G G C A TTT TGG A C A A A K CGT C T A TGC A G T CCT CGC T C A CTG G G C A C G Matrix (M2) gene 126 Influenza B X00897 A A A T A C GGT G G A T T A A A T A A A A G C A A C C A G C A A T A GCT C C G A A G A A A C A C C C A T A T TGG G C A ATT TCC T A T G G C HA gene 171 prv-i AF117818 G A T C C A G C A GTC G C A GCT C T A A T G C C C A A T C T A G G A A G C TTG A A G A TTC A T C A A G G C A GGT C T G T T A G A T A A G C A G GT Large (L) gene 124 PIV-2 X57559 T C A C C C C T G A A C TTG T T A TTT GTT T G C A G A G A G C GGT G A C A T T C A T T A A TGG T A A GTG A C A TGT TTG A G Large (L) gene 122 PIV-3 Z11575 GCT G G A A T G T T A GAT A C G A C A A A A TC T C G C A A A G T CCT A C T T A G TGT TTC A T A A TTC G G G TTG G C A T A A Large (L) gene 132 RSV A M l 1486 GCT CTT A G C A A A GTC A A G T T G A A T G A G C C A C A T A A CTT ATT G A T GTG TTT CTG A C A CTC A A C A A A G A T C A A CTT C T G T C A T C C A G C N gene 144 RSVB D00736 G A T G G C TCT T A G C A A A G T C A A G T T A A TGT C A A T A T T A T CTC C T G T A C T A C GTT G A A T G A T A C A T T A A A T A A G G A T C A GCT GCT GTC A T C C A N gene 104 MPV AF371337 A C C G T G T A C T A A GTG A T G C A C T C A A C A T TGT TTG A C C GGC C C C A T A A CTT TGC C A T A C T C A A T G A A C A A A C N gene 213 HCoV-229E X15498 TTT A T G GTT T G A A G A TGC T T G T A C TGT T A C GGC C A T A A A A A A GCT A A A TGC A A T CTT T G A C A C C T G GGC T A Membrane (M)gene 128 HCoV-OC43 M93390 G G A C T A T C A T A C TCT G A C GGT C A C A A GGT GTG T A A CCT T A G C A A C A G T C A T A T A A A C T A G G T A C T G G C T A TTC TTG GGC A G A TTT GC Membrane (M)gene 125 HRV AY751783 GTG A A G A G C CSC RTG TGC T GCT SCA G G G T T A A G G T T A GCC T G A GTC CTC C G G C C C CTG A A T G 5' untranslated region 68 Adenovirus L19443 GCC C C A GTG GTC T T A C A T G C A C A T C GCC A C G G T G G G G TTT C T A A A C TT TGC A C C A G A C C C G G G CTC A G G T A C TCC G A Hexon gene 132 HPRT NM000194 G A A A G G GTG TTT ATT C C T C A T G G A CTT G A G C A C A C A G A G GGC T A C A A T C TCG A G C A A G A C G TTC A G T CCT GTC C A T A Exon 2-3 111 Cla I PstI Viral insert X b a l Insert ligated synthetic viral gene segment with Pst I and X b a I cut sites into (MCS) on vector. Ampr Viral insert Figure 1. A schematic cartoon of synthetic control preparation. c D N A for each respiratory virus within the panel was digested using Pst I and Xba l restriction enzymes on the 5' and 3' ends respectively. The viral insert was then cloned into a p G E M 4 Z vector that had also been digested using Pst I and X b a I. 57 5. RESULTS 5.1 Efficiency and Dynamic Range of Detection of Viral Assays The efficiency was first assessed on the A B I 7900HT using R N A isolated from reference strain o f viruses. A lso , the efficiency and dynamic range of detection for each assay was assessed using the control plasmids on the A B I Prism 7000. Table 3 compares the average efficiency and R-squared value of each assay between the A B I 7900HT and A B I Prism 7000. Overall efficiencies and reproducibility on both instruments were high and therefore the decision was made to keep the assays as developed. Table 4 illustrates the corresponding range of detection based on use o f the A B I 7000 instrument. Whi le it is possible to redesign and reconfigure the assays to eventually conform to guidelines for efficiency (219), each of the assays could detect its target 1 9 virus sequence to a low viral copy number (10 or 10 copy numbers in a specimen) as shown in Table 4. Figure 2 illustrates a typical amplification curve; this particular curve represents the synthetic control o f influenza A with a dynamic detection range of 1.96x10 2-1.96x10 7. 5.2 Cross-reactivity of qPCR Primers and Probes Each viral assay was validated for cross-reactivity by testing each specific assay against all the developed synthetic control plasmids. Prior to machine validation, every primer and probe sequence was 'blasted' through N C B I for specificity to its particular virus to ensure no cross-reactivity with published sequences to other viruses or the human genome. Initial cross-reactivity tests were performed on the A B I 7900HT and subsequently on the A B I Prism 7000. Results came back identical between machines and showed no cross-amplification between any of the viral assays, with the only exception being H R V . H R V belongs to the 58 picornaviridae family, to which enteroviruses are also categorized. It is very common to find P C R cross-amplification between the genera (221). When a reference strain o f an enterovirus, coxsackievirus B 3 , was used the H R V assay showed minor amplification, with a Ct of approximately 38. Importantly, this was very easily distinguishable from true H R V test positives as the dynamic range of detection ended at a Ct of approximately 33. 5.3 Assessment of Amplifiable Material by Host Gene HPRT Results of the reference gene assay showed that out of 100 culture negative specimens initially chosen, five specimens were actually culture positive for H R V and one specimen was negative for H P R T . These were replaced with six other test specimens to keep the total number of evaluated culture negative specimens at 100. For the culture positive specimens examined, one influenza B culture isolated specimen returned a negative result on the H P R T assay and was subsequently excluded from further examination as insufficient amplifiable material was present. Out o f the total 365 clinical specimens tested, only those two specimens had negative outcomes for H P R T such that the overall H P R T detection rate was 99.5%. The mean H P R T Ct was 34.15 with a 95% confidence interval of 33.70-34.60. Given this very narrow range, q P C R viral loads were subsequently interpreted per starting volume of nasal specimen, analogous to how H I V viral loads are interpreted as H I V copy number per m L blood. Figure 3 illustrates a typical amplification plot for H P R T . 59 5.4 Relationship of qPCR Results to Positive Viral Culture Results Sensitivity is defined as the proportion of people with disease who return a positive test result. A sensitivity o f 100% would be interpreted as all patients with a disease who are diagnosed as such, without presence of false negatives (i.e., patients with disease but for whom test results are negative). It is calculated using the following formula (222): Sensitivity = number of true positives / (number of true positives + number of false negatives) Table 5 shows sensitivity results for each of the viral assays in which "true positives" are defined by viral culture results. A l l the assays demonstrated a high degree o f sensitivity with the exception of PIV-3 which had sensitivity of 80%. In addition, one of the PIV-3 culture positive specimens that was negative by q P C R instead came up positive at a high viral load for H C o V -OC43. Wi th respect to R S V , the viral culture method did not distinguish between subtypes A and B. O f the 40 R S V culture positive specimens studied, 39 were positive for at least one subtype of R S V by q P C R , and one specimen positive for both. Specificity is defined as the proportion of people without disease who return a negative test result. A specificity of 100% is interpreted as healthy or undiseased patients being diagnosed as such. It is calculated using the following formula: Specificity = number of true negatives / (number of true negatives + number o f false positives) 60 However, since healthy individuals do not typically come to the hospital and provide respiratory specimens, the study design did not allow for determination of specificity o f the q P C R assays. Furthermore, in this particular laboratory system, the concept of specificity is problematic because viral culture is an imperfect gold standard. For example, a negative culture result cannot be interpreted as the patient having no disease since many viral pathogens do not grow well in culture or can undergo inactivation ex vivo. 5.5 qPCR on Culture Negative Specimens O f the 100 culture negative specimens examined, q P C R amplified at least one virus in 43 (43%) of the specimens. Further analysis o f these 43 specimens showed that 36 specimens amplified one virus, 5 amplified two viruses and 2 specimens amplified three viruses. The specific viruses that were isolated from these specimens are shown in Table 6. H R V was the most commonly detected virus in the screen with 20 specimens amplifying viral nucleic acid followed by H C o V -OC43 with 13 specimens positive. M P V was found in 7 specimens tested. Less commonly identified viruses were adenovirus and PIV-3 at 3 specimens each, R S V B with 2 and R S V A , influenza A , influenza B and HCoV-229E each with a lone positive identification. 5.6 Virus Culture Negative versus Virus Culture Positive Specimens Table 7 shows the relationship between viral load and culture negativity or positivity in the human respiratory specimens studied. Not surprisingly, culture-positive specimens for the most part had statistically significant higher viral loads than culture-negative specimens. Influenza A , PTV-3, R S V A , R S V B, H R V and adenovirus all had p-values < 0.05. In addition, viral copy numbers below approximately 10 4 copies/mL in nasal specimens tended to be associated with 61 negative culture results, with the notable exception of H C o V - O C 4 3 , in which cultures were ft 9 uniformly negative despite a mean viral load of 10 copies/mL. A lso, PIV-1 had a mean viral load of 10 3 5 2 copies/mL; the only virus that had a positive culture mean viral load below 10 4copies/mL. 5.7 Relationship of Viral Load and Clinical Diagnosis Table 8 outlines the relationship between viral load and clinical diagnosis, U R T I , fever, or LRT I . The 95% confidence intervals of q P C R viral loads did not overlap between the three groups. The p-value between U R T I and L R T I was <0.001, between U R T I and fever <0.009 and between fever and L R T I <0.004. A n A N O V A revealed that all three groups were statistically different from each other with a p-value < 0.001. 62 Table 3. Efficiencies and R-squared values of q P C R protocols as performed on two different instruments. ABI 7900HT ABI Prism 7000 Virus Efficiency (%) R Efficiency (%) R Influenza A 93.8 0.995 93.3 0.995 Influenza B 107.2 0.990 95.1 0.985 PIV-1 94.5 0.989 93.9 0.993 PIV-2 96.5 0.994 96.5 0.973 PIV-3 104.4 0.994 77.4 0.966 R S V A 98.0 0.958 76.8 0.990 R S V B 102.0 0.996 73.7 0.989 M P V 98.8 0.967 82.0 0.997 H C o V - 2 2 9 E 89.9 0.979 82.3 0.993 H C o V - O C 4 3 89.6 0.950 85.4 0.922 H R V N D N D 150.7 0.999 Adenovirus 92.3 0.956 76.5 0.996 N D : not done, as t ie H R V assay was c eveloped after the switch to the A B I 7000 instrument. 63 Table 4. Range of detection of viral assays on the A B I Prism 7000. Virus Range of Detection (viral copy numbers) Influenza A 1.96xl0 2 -1.96xl0 7 Influenza B 1.69xl0 2 -1.69xl0 7 prv-i 2.34x l0 ' -2 .34x l0 7 PIV-2 2 .27x l0 ' -2 .27x l0 7 prv-3 1.82xl0 2 -1.82xl0 7 R S V A 2.23x l0 2 -2 .23x l0 7 R S V B 3.44x10 2-3.44x10' M P V 1.49x10^-1.49x10' HCoV-229E 2 .17x l0 2 -2 .17x l0 7 H C o V - O C 4 3 2 .55x l0 2 -2 .55x l0 7 H R V 2.32x l0 ' -2 .32x l0 7 Adenovirus 1.60xl0 2 -1.60xl0 7 64 o voe+001 1 Oe+000 l.Oe-001 1.0e-002 1 Oe-003 1 Oe-004 1 2 3 4 5 8 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 38 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Cycle NumbGr Figure 2. Representative real-time qPCR viral amplification curve for Influenza A . A ten-fold dilution series (107-102) o f Influenza A synthetic control was amplified in triplicate. The horizontal green line represents the default set threshold. The point at which the amplification curve crosses the threshold is called the threshold cycle (Ct) and is the point at which fluorescence is detectable above background levels. Delta Rn is the magnitude of the signal generated during the qPCR. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 3 5 36 37 38 39 40 41 42 43 44 4 5 46 47 48 49 50 Figure 3. H P R T amplification curve. Real-time qPCR amplification plot of H P R T from c D N A produced from the isolated R N A of archival N P A specimens. Each specimen was run in duplicate. 0> Table 5. Sensitivity o f q P C R viral assays. Virus n True Positives (viral culture) False Negatives Sensitivity (%) Influenza A 30 29 1 96.7 Influenza B 29 28 1 96.6 PIV-1 40 36 4 90 PIV-2 40 40 0 100 PIV-3 40 32 8 80 R S V * 40 39 1 97.5 M P V 0 - - n/a H C o V - 2 2 9 E 0 - - n/a H C o V - O C 4 3 0 - - n/a H R V 5 5 0 100 Adenovirus 40 40 0 100 * : consisted o f R S V A (n = 21), R S V B (n = 17) and R S V A + R S V B (n = 1) by q P C R n/a: not applicable 67 Table 6. Summary o f the types of respiratory viruses identified using q P C R screen in the culture negative specimens. Virus Number of Culture Negative Specimens that were Positive by qPCR Influenza A 1 Influenza B 1 PIV-1 0 PIV-2 0 PIV-3 3 R S V A 1 R S V B 2 M P V 7 HCoV-229E 1 H C o V - O C 4 3 13 H R V 20 Adenovirus 3 68 Table 7. Logarithmically transformed qPCR loads and their relation to previously confirmed culture negative and culture positive specimens. Virus n Log mean qPCR load (95% CI) p-value Influenza A culture (-) 1 4.06 0.004 culture (+) 29 6.38 (6.10-6.66) Influenza B culture (-) 1 3.06 0.25 culture (+) 28 4.54 (4.05-5.03) PIV-1 culture (-) 0 - n/a culture (+) 36 3.52 (3.22-3.82) PIV-2 culture (-) 0 - n/a culture (+) 40 6.09 (5.52-6.66) PIV-3 culture (-) 3 2.73 (2.57-2.90) O.001 culture (+) 32 7.65 (7.18-8.12) R S V A culture (-) 1 3.78 0.002 culture (+) 22 7.49 (7.02-7.96) R S V B culture (-) 2 3.28 (2.40-4.15) O.001 culture (+) 18 7.63 (7.21-8.05) M P V culture (-) 7 4.20 (3.30-5.11) n/a culture (+) 0 -H C o V - 2 2 9 E culture (-) 1 3.09 n/a culture (+) 0 -H C o V - O C 4 3 culture (-) 13 6.20 (4.94-7.47) n/a culture (+) 0 -H R V culture (-) 19 2.43 (1.30-3.56) 0.032 culture (+) 5 5.21 (1.86-8.55) Adenovirus culture (-) 3 3.38(0.61-6.14) 0.002 culture (+) 40 7.42 (6.76-8.09) 69 Table 8. Logarithmically transformed q P C R loads and their relation to clinical diagnosis. Diagnosis n Log mean qPCR load (95% CI) U R T I 23 2.49(1.03-3.94) Fever 98 4.62 (4.03-5.22) L R T I 66 5.90 (5.28-6.53) p-values: U R T I vs. fever: <0.009 U R T I vs. L R T I : <0.001 fever vs. L R T I : <0.004 70 6. DISCUSSION This thesis looked to accomplish a number of aims. The development o f a quantitative q P C R respiratory panel along with the selection of a suitable reference gene that used standardized conditions was successfully achieved. Synthetic controls for each respiratory virus o f interest were created and provided accurate quantification of viral loads. Using archival respiratory specimens, we were able to determine the sensitivity of our assays as compared to viral culture and determine viral load ranges associated with positive or negative culture results. Lastly, we tried to ascertain whether or not there was a significant relationship between a patient's clinical diagnosis and viral loads detected by qPCR. A s noted in the results section, some interesting findings were uncovered and these wi l l be discussed further. Wi th respect to the ability to use the qPCR protocols on different instruments, there was, in general, good performance in terms of efficiency and reproducibility on both the A B I 7900HT and A B I Prism 7000. The variations may be attributed to reasons unrelated to instrumentation such as variance in pipetting technique or the type of template used. The initial efficiency assessment on the A B I 7900HT was conducted using R N A isolated from reference strains of virus which contained not only viral R N A but also cell genomic D N A and its R N A , which is more representative o f the test specimens collected from patients. Synthetic controls were not used in the initial efficiency tests on the A B I 7900HT since the primer and probe set for each viral assay needed to be validated first. Only after the viral assay was shown to be in working order, were the corresponding synthetic plasmids produced. The efficiency tests on the A B I 7000 were performed using synthetic templates which contained only the pure target and are therefore 71 more accurate for quantification. Overall, the results demonstrate the ability to readily transfer q P C R assays onto a different instrument with good outcomes. In general, there was good linearity over a minimum of six log units for each assay. Theoretically, efficiency should be between 90 to 110% while the R value should be above 0.99; however val id data may be obtained from values outside this range. Influenza A and PIV-1 were the only 2 assays to stay within the theoretical criteria on the 7000 instrument. The reproducibility o f influenza B, R S V A , R S V B, M P V , HCoV-229E , H R V and adenovirus assays were all greater than 0.99 which was taken as the deciding factor to keep the assays as they were. The PIV-2 , P IV-3 , and H C o V - O C 4 3 assays may be considered for re-design in future prospective studies. This may be due to differences in instrumentation detection as these assays would have been acceptable based on results on the A B I 7900HT. Despite these limitations, all o f the assays consistently had a wide range of detection and were reproducible. Each assay was assessed against all synthetic controls made for each of the viruses in the q P C R panel. N o cross-reactivity between the viral assays was found amongst the synthetic controls as highly conserved regions of the viral genome were utilized in preparation of the plasmids. A s mentioned in section 5.2, H R V had some minor cross-amplification with a reference strain o f C V B 3 , an enterovirus which is in the same family, picornaviridae. As only five o f over 100 H R V serotypes have been sequenced, it is difficult to design an assay that wi l l detect as many rhinoviral serotypes as possible without amplifying the occasional enterovirus. A B L A S T search of the H R V primers and probe revealed that the C V B 3 sequence could be detected by the probe but the E (Expect) value was greater than that o f numerous H R V serotypes. The E value 72 describes the number o f hits that may be expected by chance when searching a database of a particular size. The closer the E value is to zero, the more significant the match is. H R V serotypes have an E = le-04 while C V B has an E = 5e-04. This study clearly demonstrated that amplification of H P R T for N P A specimens was consistent and showed little variation. This finding is in contrast to results o f other studies (223-225) in which the reference genes chosen have been found to be regulated by the experimental conditions or are so highly expressed that normalization is incorrect and unsuitable. In addition, the Ct values of H P R T were expressed within the range of detection o f the N P A test specimens and therefore allowed for detection of any minute changes in gene expression. This study also established the ability to interpret qPCR viral loads per starting volume of nasal specimen. Further studies are required to determine i f use o f H P R T as a reference gene can be extended to other types of respiratory specimens including human lung tissue. The sensitivity of all the assays compared to culture was high with values greater than 96% with the exception of the PIV-1 and PIV-3 assays. This could be due to the viral load of the false negative specimens is between the threshold levels of culturing negative or positive isolates. Whi le this was an unknown for PIV-1 as none of the 100 culture negative specimens gave a positive result; the mean log q P C R load between the negative and positive cultures for PIV-3 were 2.73 and 7.65 respectively. The large disparity in mean viral loads was an interesting result as perhaps only a swab taken at peak infection wi l l return a positive result on culture. Development of another assay may provide further insights into this issue. 73 The effect o f freeze-thaw cycles on viral R N A content in archival N P A was not investigated in this study. However, other studies have reported similar values in viral concentration between freshly processed specimens and those that have undergone freeze-thaw cycles after being stored for 16 months (226, 227). V i ra l R N A exhibited a decrease in viral load with an increase in the number of freeze-thaw cycles while viral c D N A that was freeze-thawed multiple times returned similar load values to that of freshly prepared c D N A except at lower copy numbers (i.e., 10 -10 ) as viral titres tend to fall with repeated freeze-thawing. Repeated freezing and thawing of specimens is a technical consideration in use of qPCR for quantitative viral load measurement. Overall , in seven specimens, nucleic acid from more than one respiratory virus was amplified. A s these test specimens were only taken at one particular time point, it was not possible to determine at which time the patient was exposed to a specific virus. It is interesting to note in that out of the 43 culture negative specimens in which viral nucleic acid was amplified by q P C R , 11 were positive for viruses that routinely undergo tests by culture and serology. This indicates that potentially a significant number of viral infections go undetected by "gold standard" methodology. However, literature has shown that viral culture may not necessarily be suitable as a "gold standard" for certain respiratory viruses. Johnston and colleagues found that a number of H R V detected by P C R went undetected using culture because some strains were very slow growing (228). Because H C o V s tend to grow slowly on specialized cells and are not wel l adapted to conventional cell culture methods, they do not undergo routine culturing in the majority of diagnostic or virology laboratories (229). This has potential implications interpreting the epidemiology of other studies of respiratory viruses. A greater 74 percentage of illness and disease may in fact be attributable to viral infections and therefore is currently under-reported. The New Vaccine Surveillance Network, as reported by Gri f f in, Iwane and colleagues, found that using both viral culture and R T - P C R , 61% of children who are hospitalized have a respiratory viral infection leaving 39% unassociated with a detectable pathogen (214, 230). Concerning M P V , the percent of M P V positive specimens (7%) found in the culture negative clinical specimens mirror the percentages found in some published epidemiological studies conducted thus far (39, 93, 96). However the assay modified from Mackay et al. (217) is not based on the reference strain of M P V found in the National Center for Biotechnology Information (NCBI) database A current study uti l izing an assay based on the reference strain of M P V showed a detection rate of 16.2% (231) while Mackay et al. found M P V in 9.7% of specimens tested. Therefore, this study may underestimate the number of M P V positive specimens. For the 57 culture negative specimens in which no viral nucleic acid was detected by q P C R , infection might have involved a pathogen not tested by the q P C R panel, or the test specimen had a viral load that was below the sensitivity of detection by the technique. The presence of P C R inhibitors may also contribute to a false negative result and this may be tested by adding a known amount o f virus to an aliquot o f the culture negative specimen to see i f it amplifies as expected. For the first time, a correlation between viral load and concomitant positive culture results for several viruses has been presented in this thesis. A range of viral loads was confirmed for 6 viruses that included influenza A , PIV-3 , R S V A , R S V B, H R V and adenovirus. Based on the ranges established for each virus, a viral load with a value below the determined threshold is 75 l ikely to return a negative culture result. This result builds upon the results of the culture negative specimens discussed earlier, in which a number of viral infections go undiagnosed using traditional methods of detection. Further studies are necessary to definitively address this issue. The results of this thesis also show for the first time a relationship between viral load as determined by the q P C R panel and a patient's clinical diagnosis as determined by requisition forms. The results suggest that patients with a more severe disease have higher viral loads but further large-scale studies are required to confirm this possibility. There are certain potential problems that may arise using N P A s to determine viral load. In blood and serum, nucleic acid extraction tends to be straight-forward as the specimen is relatively homogeneous. N P A s and other body fluids tend to be heterogeneous with potentially large variability in specimen composition. A cytomegalovirus ( C M V ) study found that positive B A L viral culture results could not determine between viral shedding without disease and C M V pneumonitis (232). This study also found that viral loads determined in blood had some clinical value versus B A L which needs further study. In addition, blood and serum may be easily stabilized prior to transport and processing while N P A s are not. This may potentially lead to purified nucleic acid that is degraded or subject to degradation or may co-purify inhibitors o f reverse transcription or P C R . Sample quality plays a large part in generating valid data as suboptimal nucleic acid increases the possibility o f false-negative results. However, our results with using H P R T as a reference value, indicated that these potential issues were of little concern in this particular thesis. 76 The results o f this study also suggest that q P C R is a robust, multi-faceted and sensitive technique. Once it is carefully standardized and validated, q P C R may in the future be used in conjunction with or as a replacement for other laboratory methods that are not as sensitive or unable to identify certain pathogens as other studies have reported (233, 234), particularly as virus-specific treatments are developed. Current detection methods based on culture are sometimes of limited clinical use as the time necessary to generate results takes far longer than the duration of illness itself. We have developed a respiratory viral screen that not only quantifies viral copy numbers but has resulted in new information concerning the correlation of viral load to culture results and viral load to clinical diagnosis. Whi le our studies looked at a subset of specimens based only on diagnoses from requisition forms, it would be interesting to consider any associations between infections based on chart reviews or discharge diagnoses. Addit ionally, further studies conducted in a prospective manner, rather than retrospective, are required to validate the current findings. The reported prevalence of respiratory virus infection varies in the literature. This can be attributed to a number of factors such as: type of test specimen used for diagnostic testing, type of diagnostic test used, patient population studied, and time of year. A recent study compared N P A , nasopharyngeal swabs (NPS), and oropharyngeal swabs (OPS) and the ability of these specimens to detect respiratory pathogens (235). The findings illustrated that N P A were better than N P S and were superior to OPS. A comparison between real-time R T - P C R versus culture results demonstrated that real-time R T - P C R detected a respiratory pathogen in 63% of the RTI episodes versus 2 1 % by culture (236). For L R T I , these numbers differed even more with real-time R T - P C R detecting a respiratory pathogen in 73% of the specimens versus 9% by culture. A 77 look at the distribution of respiratory viral infections by age showed that there was a higher number of infections in patients who were under the age of 1 compared to those over the age of 21 (237). In terms of patients who were immunocompromised, the opposite result was found as the percent o f respiratory infections increased from 12.5 in those 2-5 years of age to 25.6 in those 6-20 years of age. This particular study also revealed that HCoV-229E was detected solely in one season while H C o V - N L 6 3 was detected only in another, with H C o V - O C 4 3 distributed in both. Another study fol lowing a cohort of children in determining picornaviral infection with illness and effect of season found infection rates highest in the fall with similar rates found between the winter, spring and summer seasons (238). Clearly, interpretation o f results in literature requires careful appraisal to elucidate the true prevalence of respiratory infection. Some issues concerning the interpretations of these results arise with the lack of a healthy population of children for comparison. Future validation studies o f the q P C R panel w i l l need to include a group of children who are asymptomatic (healthy). A recent study by Falsey and colleagues matched symptomatic and asymptomatic patients in the diagnosis o f R S V and M P V (239). They found a significantly higher rate of detection of respiratory infection in those with respiratory illness versus those without. Since healthy children are usually not brought to the emergency room, a study recruiting volunteers or a longitudinal study where children are brought in every week/month and followed for a number o f years is needed - though a study of this magnitude would be particularly difficult to accomplish. Another possibility would be to have "matched" asymptomatic children in the population with those children who have N P A taken during symptomatic illness. A more comprehensive look at the epidemiology of respiratory 78 illness and disease may now be undertaken with the increase in sensitivity and quantification of qPCR. Another point o f interest is clinical relevance of real-time q P C R at low viral loads and the detection o f multiple infections. For respiratory viruses like R S V and adenovirus, it is well known that these pathogens may persist in the lung and replicate at low levels long after the primary infection has subsided (145, 171). They are able to evade the host immune system and can act as a conduit for subsequent cl inically symptomatic infection. Based on the viral loads, there is the ability to distinguish between current infection - those with an elevated viral titre, versus persistent infection - those that languish at a low level, through consecutive test specimens. A longitudinal study involving repeated sampling would help to discern any potential significance of multiple viruses documented by qPCR. Whi le real-time q P C R may have some advantages over conventional methods of viral diagnosis in terms of time and effort, cost is currently a large hindrance; therefore, q P C R may not yet be feasible for many laboratories. This is mainly due to the fact that most specimens are run in either duplicate or triplicate, standard curves are run in every plate, the development of probes does not always work, reagents, plastic consumables and the machine are costly. Appl ied Biosystems (Applied Biosystems, Foster City, C A , U S A ) is the industry leader in real-time P C R technology, with thermocyclers, reagents, plastic consumables, assay-by-design as some of their offered goods and services, and with this comes a high cost. However, alternative reagents, consumables, and platforms being offered by competing companies have helped drop the overall cost per specimen making the use of q P C R more affordable and attainable to smaller budget 79 laboratories. Experiments conducted within our laboratories have found comparable i f not better results with these alternative reagents. Wi th all this taken into consideration, q P C R , for the time being, is probably best left to dedicated virology laboratories for use in research, assay development, and validation. Another diagnostic method which shows promise in further advancing multiple respiratory pathogen detection includes the Luminex® microsphere system (Luminex Corporation, Austin, T X ) . Up to 100 separate sets of colour-coded beads, "carboxylated microspheres", are each coated with a specific probe and the median fluorescent intensity (MFI) is measured using the Luminex compact analyzer. A low M F I (<500) is considered negative while a high M F I (>1000) is interpreted as a positive result. The advantages of the Luminex technology is the ability to multiplex a large number of reactions with a reduction of cost as less reagent, test specimen, time and labour are needed. Further studies are needed to validate this platform toward translation into the cl inical diagnostic laboratory setting. The simplicity in design of this respiratory q P C R panel allows for assays o f new, relevant pathogens to be added and analyzed with ease. For example, a recent study in Hong Kong investigated the types of coronaviral infections found in patients (240). Out of 4,181 specimens tested, 87 were positive for H C o V , 53 positive for H C o V - O C 4 3 , 17 positive for H C o V - N L 6 3 , 13 positive for C o V - H K U l , and 4 positive for HCoV-229E. The occurrence of infection mirrors the results in this thesis as 13 specimens were positive for H C o V - O C 4 3 and only 1 H C o V - 2 2 9 E specimen was positive in all the specimens examined. The number o f H C o V - N L 6 3 positive results is enough to warrant further examination and perhaps insertion into our current 80 respiratory q P C R screen as it is clearly found in greater numbers than HCoV-229E positives which are generally tested for when investigating coronaviral infections. Other advantages of this q P C R panel are that it can be adapted to a 384-well plate format, can be multiplexed, can be modified to a real-time q R T - P C R screen and can be transferred onto another detection platform i f so desired. In conclusion, we have developed a panel of real-time qPCR protocols for the detection and quantification o f nucleic acids for 12 common human respiratory viruses. W e have successfully established a q P C R respiratory viral panel using standardized conditions across detection platforms, and developed synthetic plasmids for each respiratory virus. Our results provide new information regarding the relationship of viral load to culture positivity and clinical symtomatology. Overall , the real-time q P C R panel described in this thesis contributes to the rapidly growing field of various molecular techniques involved in the diagnosis of viral infections. 81 7. R E F E R E N C E S 1. Mayer A . Concerning the mosaic disease of tobacco. (Translation published in English as Phytopathological Classics No . 7 [1942], American Phytopathological Society, St. Paul, MN . ) . Die Landwirtschaftliche Versuchsstationen 1886;32:451-67. 2. Ivanowski D. Ueber die Mosaikkrankheit der Tabakspflanze. 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