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A biological mutant of coxsackievirus B3 and its pathogenesis in mice Sadeghi, Assai Mir Mohammad 1997

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A Biological Mutant of Coxsackievirus B3 and its Pathogenesis in Mice by Assal Mir Mohammad Sadeghi B.Sc. (Microbiology), University of British Columbia, 1994. A Thesis Submitted in partial fulfilment of the requirements for the degree of Master - of Science in The Faculty of Graduate Studies Department of Pathology. We accept this thesis as conforming to the required standard. The University of British Columbia. June 1997. © Assal M.M.Sadeghi, 1997. In p resen t ing this thesis in partial fu l f i lment of the requ i remen ts fo r an advanced d e g r e e at the Universi ty o f British C o l u m b i a , I agree that t h e Library shall make it f reely available fo r re ference and study. I fu r ther agree that permiss ion fo r ex tens ive copy ing , of this thesis fo r scholar ly pu rposes may be g ran ted by t h e head o f m y d e p a r t m e n t or by his o r her representat ives. It is u n d e r s t o o d that c o p y i n g or pub l i ca t ion o f this thesis fo r f inancial gain shall n o t be a l l o w e d w i t h o u t m y w r i t t e n permiss ion . D e p a r t m e n t of i a o R ^ o V c ^ ^ V The Univers i ty o f Brit ish C o l u m b i a Vancouver , Canada Date : ^ u A t - U • c[l- . DE-6 (2/88) ABSTRACT An antibody escape-mutant of coxsackievirus B3 / CVB3 (RK strain) was isolated using a neutralising monoclonal antibody against CVB3. This escape-mutant EM1 was then compared with the wt+ strain CVB3 (RK) both in vitro and for its pathogenesis in mice. In vitro EM1 was found to be temperature sensitive at 40°C and less stable on prolonged incubation at 37°C than CVB3 (RK). Also EM1 was slightly growth-restricted in Vero cells, giving yields 2 to 5 fold lower than the parental strain and also has a small plaque phenotype. A similar reduction in viral replication was found in the macrophage cell-line, J774A.1. However growth of EM1 in the Wehi 231 B-cell-line was reduced 10-20 fold while both EM1 and wt+ were totally restricted in the T-cell-line, EL-4. In vivo. EM1 was found to be less myocarditic while producing equivalent amount of pathological damage in pancreas and liver. Correlating with this equivalent amounts of virus were detected both by plaque assay of tissue homogenates, and in situ hybridization in all tissues apart from spleen and heart. In particular in heart tissue, the ability of the EM1 to replicate and cause damage was much less than the wt+(RK) strain, i.e., EM1 was less cardiovirulent than CVB3 (RK). Sequence analysis of the NTR region and P1 structural gene regions of CVB3 (RK) and EM1 strains following RT/PCR of genomic RNA identified several mutations in EM1 including a single nucleotide change in the E loop of the NTR region and several mutations in P1. The most significant changes appear to be in VP1 where 4 point mutations and a deletion/insertion were identified. One of these is silent but the others Ill are associated with amino acid changes including T to H and F to V substitutions in the BD region of VP1 structure. These mutations most likely explain the lack of reactivity with neutralising MAb and also the reduced heat stability of EM1. iv TABLE OF CONTENTS Abstract ii Table of contents iv List of tables Xiii List of figures xiv Acknowledgements xvi;£, ABBREVIATIONS x 1. INTRODUCTION A. Historical background 1 B. Infectious process 1 C. Clinical diagnosis 2 D. Classification 2 E. Properties of the virus particles 2 F. The genome 5 1. VPg 6 2. Poly A tract 6 3. Non-translated region (NTR) 6 4. ORF 9 G. Replication 11 H. Host factors and their role in CV tissue tropism 12 V 1. Coxsackievirus cellular receptors 14 J. CVB variants and their role in pathogenesis 15 K. CVB and insulin dependent diabetes mellitus 18 L. CVB and myocarditis 19 1. Pathogenic mechanisms of myocarditis 20 a. autoimmunity and myocarditis 20 b. direct myocardial injury by CVB 21 M. Interaction of CVB with the immune system 23 1. Non-specific immune reactions 23 a. Macrophages 23 b. Natural Killer (NK) Cells 24 c. Interferons 24 2. Specific immune reaction 25 a. Role of the humoral immune response to CVB 25 b. The role of humoral response in tissue damage 27 c. The role of cell mediated immune response to CVB 27 N. Coxsackievirus B3 and persistence 29 2. OBJECTIVE 31 3. MATERIALS 32 I. Cell lines 32 II. Cell culture materials 32 vi III. Fixative and stains 33 IV. Antibodies 34 V. Virus 35 VI. Infectious clone 35 VII. Solutions for in_sjtu hybridization 35 VIII. Large scale plasmid solutions 38 IX. Reverse transcriptase PCR solutions 39 4. METHODS 41 A. Cell maintenance 41 B. Virus stock preparation 42 C. Coxsackievirus titration 42 D. Preparation of EM1 stock 43 E. Purification of virus by sucrose gradient centrifugation 44 F. Viral growth curves in Vero, J774A.1, Wehi and EL-4 cells 44 G. Virus decay curves 45 H. Temperature sensitivity of EM1 and RK strains 45 I. Large scale preparation of CVB3CR1 plasmid using PEG precipitation 46 J. RNA probe labelling by in_vjtro transcription 47 K. Quantification of DIG-labelled transcript 48 L. In situ hybridization using DIG-labelled probe 48 M. Infection of mice with CVB3 strains 49 N. Histology 51 vii 0. Histopathological interpretation 51 P. Plaque titration of the virus in organs 51 Q. Reverse transcriptase polymerase chain reaction (RT/PCR) 52 a. Preparation of viral RNA 52 b. First strand synthesis 52 c. Polymerase chain reaction (PCR) 53 R. Statistical Analysis 55 5. RESULTS AND DISCUSSION 56 1. Isolation of the escape mutant (EM1) 56 II. Pathogenesis of EM Ijnvjvo 58 A. Experiment 1 59 2.1. Histopathology 59 a. Pancreas 60 b. Heart 62 c. Other organs 64 2.2. Virus titration in tissue 64 a. Pancreas 64 b. Heart 66 c. Spleen 67 d. Liver 68 e. Lung !. 69 viii 2.3. Detection of viral genome 69 a. Pancreas 71 b. Heart 73 c. Spleen 77 d. Liver 77 2.4. Summary 79 B. Experiment 2 80 3.1. Histopathology 81 a. Pancreas 81 b. Heart 81 3.2. Virus titration in tissues . 85 a. Pancreas 86 b. Heart 87 c. Spleen 88 d. Serum 89 3.3. Detection of viral genome 91 a. Pancreas 91 b. Heart 92 c. Spleen 93 3.4. Summary 93 Conclusion 94 4. Characterization of EM1 and CVB3(RK) in vitro 96 ix 4.1. Temperature sensitivity assay 96 4.2. CVB3 decay curve 98 4.3. Virus growth curve 100 a. Vero cells 100 b. J774A.1 cells 103 c. Wehi 231 cells \ . 106 d. EL-4 cells 108 5. Sequence analysis of the two CVB3 variants 110 1. NTR region 111 2. P1 region 111 a. VP4 .111 b. VP2 111 c. VP3 112 d. VP1 112 6. CONCLUSIONS 118 7. REFERENCES 122 ABBREVIATIONS APC Antigen presenting cells bp Base pair CV Coxsackievirus CVB Coxsackievirus group B CPE Cytopathic effect CTL Cytotoxic T-Lymphocytes DAF Decay accelerating factor DEPC Diethylpyrocarbonate ddH20 Distilled. Milli-Q-filtered water DIG Digoxigenin DNA Dioxy ribonucleic acid DMEM Dulbecco's modified eagle medium DTT Dithiothreitol EDTA Ethylenediamine tetraacetic acid E M 1 Escape-mutant # 1 FBS Fetal bovine serum FLS Fetal lamb serum HCl Hydrochloric acid H&E Haematoxylin- Eosin HI FBS fetal bovine serum XI hrs IFN ISH IL ip IR IRES Kda Kb MAb mRNA NBT/BCIP NTR 0/N PBS PCR PF PFU Pi RI RNA Hours Interferon in situ hybridization Interleukin Intraperitoneal Immune response Internal ribosome entry site Kilodalton Kilobase Monoclonal antibody Messenger RNA Nitroblue-tetrazolium chloride/5-bromo-4-chloro3-indolyl- Phosphate, p toluidine salt Non Translated Region Overnight Phosphate buffered saline Polymerase chain reaction Paraformaldehyde Plaque forming units Post infection Replicative intermediate Ribonucleic acid xii RPMI Roswell Park Memorial Institute RT Room temperature RT/PCR Reverse transcription - polymerase chain reaction SCID Severe combined immunodeficiency SDS Sodium dodecyl sulphate Sec Seconds TE Tris, EDTA buffer T h 1 T helper-1 T h 2 T helper-2 TNF Tumour necrosis factor t-RNA Transfer RNA VPg Genome linked viral protein wt+ Wild type xiii LIST OF TABLES: Tables: 6.1.1. Titres from Vero cells 57 6.2.1. Myocardial scoring of Heart and Pancreas 60 (animal experiment #1) 6.2.2. Virus Titration in tissues 69 (animal experiment #1) 6.2.3. ISH scoring of Heart and Pancreas 70 (animal experiment #1) 6.3.1. Myocardial Scoring of Heart and pancreas 82 (animal experiment #2) 6.3.2. Virus Titration in tissues 85 (animal experiment #2) 6.3.3. ISH scoring of heart and pancreas 91 (animal experiment #2) xiv LIST OF FIGURES Figure 2.1. Icosohedral capsid structure of coxsackievirus 4 2.2. Coxsackievirus genome 5 2.3. The structure of IRES elements 7 2.4. The structure of IRES in coxsackievirus 8 2.5. Organisation and expression of CVB3 genome 10 3.1. Polymerase chain reaction 53 6.1.1 Plaque morphology 58 6.2.2. Histology of pancreas 61 6.2.3. Histology of heart 63 6.2.4. CVB3 Titres in Pancreas 65 6.2.5. CVB3 Titres in Heart 66 6.2.6 CVB3 Titres in Spleen 67 6.2.7. CVB3 Titres in Liver 68 6.2.8. Detection of CVB3 genome in pancreas by ISH 72 (day 3) 6.2.9. Detection of CVB3 genome in pancreas by ISH 74 (day 6) 6.2.10/ Detection of CVB3 genome in heart by ISH 75 (day 3) 6.2.11. Detection of CVB3 genome in heart by ISH 76 (day 6) XV 6.2.12. Detection of CVB3 genome in spleen by ISH 78 6.3.1. Histology of pancreas 83 6.3.2. Histology of heart 84 6.3.3. CVB3 Titres in Pancreas 86 6.3.4. CVB3 Titres in Heart 87 6.3.5. CVB3 Titres in Spleen . . 88 6.3.6. CVB3 Titres in Serum 90 6.4.1. Temperature sensitivity assay 97 6.4.2. CVB3 decay curve 99 6.4.3. Growth curve of EM1 and CVB3(RK) in Vero cells 101 (supernatant medium) 6.4.4. Growth curve of EM1 and CVB3(RK) in Vero cells 102 (Intracellular virus titre) 6.4.5. Growth curve of EM1 and CVB3(RK) in J774A.1 cells 104 (supernatant medium) 6.4.6. Growth curve of EM1 and CVB3(RK) in J774A.1 cells 105 (Intracellular virus titre) 6.4.7. Growth curve of EM1 and CVB3(RK) in Wehi 231 cells 106 (supernatant medium) 6.4.8. Growth curve of EM1 and CVB3(RK) in Wehi 231 cells 107 (Intracellular virus titre) 6.4.9. Growth curve of EM1 and CVB3(RK) in EL-4 cells 108 (supernatant medium) 6.4.10. Growth curve of EM1 and CVB3(RK) in EL-4 cells 109 (Intracellular virus titre) 6.5.1. NTR region 113 xvi 6.5.2. VP2 capsid protein 114 6.5.3. VP3 capsid protein 115 6.5.4. VP1 capsid protein 116 6.5.5. Structure of VP 1 capsid protein 118 6.5.6. Structure of CVB3 capsid proteins 119 X V ACKNOWLEDGEMENT: Foremost, I should thank God for helping me in every single step of this project. I would like to extend my thanks and appreciation to my great supervisor Dr. Janet Chantler for all her support and guidance throughout this study. Jaki, I thank you for believing in me and giving me a chance to experience the most interesting years of my life. I could not have done it with out you. I would also like to thank the members of my research committee Dr. G. Bondy, Dr. B. Bowie, Dr. R. Hegele, Dr. J. Hudson, Dr. B. Bowie, and Dr. E. Thomas for all their insights and great suggestions. I would specially like to thank Karen Lund for putting up with me and helping me with all the techniques that I have used. Karen, I admire your patience and hope that you get all the success that you deserve in life. I will never forget you. Last but not least I would like to thank my family for all their support;, my dear husband, Kamran, who changed the path of my life and taught me how to be patient love you so much. My wonderful parents Shirin and Mohammad for providing me all that I needed both emotionally and financially throughout my life. I love you both and hope that one day I would be able to return all your kindness. Also my two beloved brothers Ali and Amir and wish them both the best of luck in their lives. I would like to thank you all for supporting me to pursue this goal. 1. INTRODUCTION A. Historical Background: In 1947, the first coxsackievirus was isolated from the feces of two boys with a paralytic poliomyelitis-like syndrome in upstate New York in the village of Coxsackie by Gilbert Dalladorf and Grace Sickles 1 Since that time they have been associated with a wide range of clinical syndromes including encephalitis, hepatitis, pleurodynia, herpangina, pancreatitis, and myocarditis. In fact, coxsackievirus has been identified as the number one cause of acute viral myocarditis. Coxsackieviruses have been divided into two major subgroups based on their pathogenesis in newborn mice and their ability to be propagated in tissue culture 2. Group A strains, with 23 serotypes, usually infect only skeletal and cardiac muscle causing myositis and are more difficult to culture in vitro. Group B strains on the other hand, with 6 serotypes, infect a variety of different tissues including pancreas, spleen, liver, kidney, brown fat and heart and are easily propagated in tissue culture 3 A 5 . B. Infectious Process: Coxsackievirus infection is initiated by oral ingestion of virus followed by primary multiplication in the oropharynx and intestine (alimentary phase). The virus then multiplies extensively in the Peyer's patches and tonsils (lymphatic phase) and later moves into the deep cervical and mesenteric lymph nodes and spreads to the blood stream (viraemic phase). Once inside the blood stream, the virus is capable of 2 reaching different organs and causing a systemic infection with infection of a variety of target organs as described above. C. Clinical Diagnosis: Coxsackieviruses can be isolated from body fluids such as nasal secretions, cerebrospinal fluid, rectal swabs, throat swabs, stool and serum and can be readily cytopathology by anti-CVB serum in culture 6. Viral serology is frequently used to identify a coxsackievirus infection 7 , s . Recently, more rapid techniques for seroconversion such as enzyme linked immunosorbent assay (ELISA), and also virus detection by immunoperoxidase staining and in situ hybridization with higher sensitivity and specificity have been developed 9 , 1 0 i 1 1 . D. Classification: Coxsackieviruses belong to the family Picornaviridae, genus Enterovirus the same as poliovirus and echovirus 1 4. As the name picornavirus suggests, it is a small virus and contains a positive polarity RNA genome which can directly act as mRNA inside the host cells. E. Properties of the virus particles: Coxsackievirus virions are 24-30 nm consisting of an icosahedral capsid and containing an RNA molecule that has a length of about 2.5it 1 3 . Under the electron microscope, the virions appear almost spherical and lack a lipid envelope. Thus they 3 are not affected by organic solvents such as ether. However they lose infectivity very rapidly in the presence of 0.3% formamide. Coxsackieviruses are relatively stable at acidic pH, surviving pH 4 or less, an important feature as the virus must pass through the acidic condition of the stomach prior to initiating infection in the intestinal epithelium. Coxsackieviruses are stable at 37°C maintaining infectivity for more than a week. In the presence of divalent ions, they can survive at 50°C for up to 3 hours 1 2. The capsid structure has several important functions including: 1. Protection of the RNA genome from nucleases in the environment 2. Recognition of specific cell receptors in the plasma membrane and thus in determining host range and tissue tropism 3. Determination of antigenicity 4. Packaging of the viral genome and providing a proteinase involved in maturation of the virions 5. Delivering the RNA genome through the cell membrane and into the cytosol of the susceptible host cells. The capsid is composed of pentameric subunits. Within each of which five trimers are found that contain the capsid proteins VP1, VP2 and VP3. These proteins contain both a-helical and j8 sheet regions and have similar folding patterns which gives them a wedgelike shape needed to fit compactly. The association of the capsid proteins 4 gives a capsomer structure consisting of a central peak region (mainly VP1 protein) surrounded by a ridge (mainly VP2 & VP3) with a canyon in between (see figure 2.1). Surrounding the canyon are the epitopes for neutralizing antibodies. Therefore VP1, VP2, and VP3 form the icosohedral capsid structure, while VP4, the smallest of the capsid proteins, lies internally to the capsid in close association with the RNA genome and is the most conserved of all the capsid proteins36,37. The major viral attachment protein of the capsid is VP2 which is mostly present on the rim of the canyon, and together with VP1 shapes the antigenic character of the virus3839. Figure 2.1 The Icosohedral Capsid Structure of Coxsackievirus VP* WJ The capsid Is made of pentameric subunits containing trimers of VP1, VP2jand VP3. The association of the capsid proteins results in a structure with a central peak region (VP1) in the middle surrounded by a ridge formed by VP2 and VP3 making a central canyon. The cell surface receptor Is inserted into the canyon when a virion attaches to the cell. Surrounding the canyon on VP1.VP2 and VP3 are the epitopes for the neutralizing antibodies (Reprinted from Row* « 3rd oditioo p4a9-90) 1 4 9 . 5 F. The Genome: The coxsackievirus genome is 7390 nucleotide long with a small virally encoded protein VPg covalently linked to its 5'end and a poly A tract of 50-80 bases at its 3'end. The first 10% of the genome, up to nucleotide 742, codes for no known protein and is therefore called the non-translated region (NTR). The remaining 90% of the genome has one open reading frame and is divided into 3 coding regions P1, P2, and P3. P1 encodes the structural proteins whereas P2 and P3 encode various enzyme activities including the polymerase and several viral proteinases (See Figure 2.2). Figure 2.2 CVB Genome 6 VPg: The virally encoded VPg is believed to be involved in initiation of RNA synthesis. Antibodies against VPg were shown to inhibit RNA replication in vitro but did not affect the infectivity of the RNA in transfection assays using transcribed RNAs 1 5- i e-1 7-4 1. Poly A: The function of the poly A tract is not fully characterised except that it plays a role in stabilizing the mRNA. Experiments have shown that the RNA molecules with a long poly A tract have a higher infectivity than the ones with a short poly A tract1 8 1 9 2 0. NTR: The NTRs of the picornaviruses are unusually long and contain a lot of secondary and tertiary structure 2 1 2 2 . As with poliovirus, coxsackieviruses lack the 5' methyl cap that is present in almost all eukaryotic mRNAs and most viruses, and is important for translation initiation 2 3 2 4 2 5 . Within the 5' NTR there is a cis-acting element known as the internal ribosome entry site (IRES) to which the ribosome binds and initiates translation from a start codon 5' of the single open reading frame comprising the P1 structural region and P2 and P3 non-structural gene regions 2 6 , 2 7 . Based on different RNA sequences and proposed secondary structures, the IRES of different picornaviruses are divided into two types (See Figure 2.3). Both types share the YnXmAUG motif where Yn is a stretch of 8-10 pyrimidine bases that are separated from the down stream AUG by 18-22 non- conserved nucleotide sequences (Xm). 7 In type 1 IRES, found in Enterovirus and Rhinovirus, the AUG of the motif is silent and an AUG downstream from the motif acts as the initiating codon for protein synthesis. In the second type of the IRES, found in cardiovirus, aphthovirus, and hepatovirus, the AUG of the motif acts as the polyprotein initiating codon for protein synthesis 1 1 3 . Figure 2.3 The Structure of IRES Elements Schematic presentation of the predicted secondary structures for the poliovirus (A) and encephalomyocarditis virus (B) IRES elements. The IRES (shaded areas) enables the ribosome to bind directly to an internal site of the 5'NTR region (Reprinted from: Fields p 482 3rd edition)147 8 In the case of CVB3, the NTR contains 6-8 AUG codons of which only one is used. The mechanism by which this specific AUG is chosen as initiation site is not completely understood. It appears that the ribosome binds directly to the IRES and scans from this site to the correct AUG ^ '^ (See Figure 2.4). Figure 2.4 The Structure of IRES in Coxsackievirus A B C D E: F G H: I J K 10-34 40-81 104-180 188-208 209-481 484-514 519-560 581-624 625-641 646-659 688-741 .50' -.1 j?350 c Q. 150 250| 1 The IRES 4 5 0 550 6 0 0 L UUCAUUUU 1 0 0 2 0 0 B 5 0 7 4 2 FIG. 1. Diagram of RNA secondary structures in the 5'UTR of CVB3 D i a g r a m o f R N A s e c o n d a r y s t r u c t u r e s i n t h e 5' N T R o f CVB3. T h e s h a d e d a r e a r e p r e s e n t s t h e I R E S c o n t a i n i n g a s i l e n t A U G . T h e A U G d o w n s t r e a m o f t h e m o t i f i s r e s p o n s i b l e f o r i n i t i a t i o n o f t r a n s l a t i o n (Reprinted f r o m Yang V i r o l o g y 1997) 9 Cellular proteins such as P52, P57 and P97 participate in binding of the 40S ribosomal subunit to IRES 2 8 2 9 3 0 3 1 3 2 . These proteins could act directly as a ribosome recognition site or act to alter the RNA structure to facilitate ribosome binding. O R F : The P1 structural region codes for capsid proteins VP4, VP2, VP3 and VP1 as shown in figure 2.2 3 S . This region is more variable between CVB strains than either the P2 or P3 regions which are highly conserved. The P2 non-structural region of the genome is approximately 1700 nucleotides long. P2 encodes 3 proteins 2A, 2B and 2C. Protein 2A has been characterized to have proteinase activity and cleaves between P1 and P2 regions cotranslationally (see figure 2.4). The function of the remaining proteins of the P2 region is not yet completely understood other than the fact that both 2B and 2C are involved in protein synthesis 4 0 3 9 . The P3 region of the coxsackievirus genome is about 2400 nucleotides long and codes for four proteins: 3A, 3B, 3C and 3D. Protein 3B codes for VPg, a small protein involved in initiating RNA synthesis (see figure 2.5). Protein 3C is a proteinase which mediates most of the cleavage reactions in the polyprotein to yield both the final cleavage products (2A, 2B, 2C, 3A, 3B, 3C and 3D) and stable processing intermediates (2BC, 3AB and 3CD). Protein 3AB is a membrane bound protein that delivers VPg to the membranous 10 replication complex and protein 3CD encodes a proteinase (3C) and the RNA polymerase (3D) 3 9 4 1 . The 3' NTR region of the CVB3 genome is about 98 nucleotides long; its function is not yet known. Figure 2.5. Organization and expression of CVB genome 20C I I IA8 IB X ! HDC The genome of CVB3 is organised as shown above. A single ORF encompassing the P1,P2 and P3 regions commences at an AUG downstream of the IRES. Proteins needed for RNA synthesis and the proteinases required to cleave the polyprotein are encoded downstream from the P1 capsid proteins. The cleavage of the polyprotein is accomplished through 3 virus-coded proteinases. The first cleavage, between 1D and 2A Is accomplished by the 2A proteinase. Subsequent cleavages are preformed by 3C. The Maturation proteinase (M) cleaves VPO to VP4 and VP2 after the RNA has been packaged in the protein Shell (Modified from Raids edition, 199e)15°. 11 G. Replication: The replication process starts with attachment of the virions via their viral attachment protein to the cell surface receptors located in the plasma membrane (See next section). It is believed that the attachment site on the viruses is the canyon formed by VP1, VP2 and VP3 4 2 . Once the virion is bound to its receptor, recruitment of more cell receptors for virion binding and tighter association with the receptors occurs resulting in invagination of the membrane around the virion causing endocytosis 4 3 . Within the endosome the VP4 capsid protein is released causing the capsid to swell which results in the extension of the hydrophobic N-terminus of the VP1 protein into the membrane. RNA is then released from the capsid into the cytosol through these channels in the cell membrane 4 3. The transient intermediate that successfully transfers its genome into the cytoplasm and initiates infection is known as the infectosome44. Once inside the cell, host cell protein synthesis is shut down by viral inactivation of elF-2, which is required for Met-tRNA binding to the 43S ribosomal subunit during assembly of the 80S initiation complex 1 1 4 . elF-2 is a trimer made of three distinct subunits: a, /3, and 7 and phosphorylation of elF-2cr is responsible for the inhibition of protein synthesis 1 3 3 . Once host translation is inhibited ribosomes are released from cellular mRNA's and are available for viral translation. The positive strand viral RNA acts directly as a mRNA and as mentioned previously, the ribosome binds to an internal ribosome binding site within the 5' NTR of the genome to initiate synthesis of 12 a polyprotein. This process does not require elF-2 and therefore viral translation can proceed in the infected cell. Several other host factors do however play a crucial role in ribosome binding to the CVB genome. For example, protein p52 (also known as La autoantigen) and other cellular factors such as elF-4E, recognize the IRES and bind to it. Subsequently, elF-4A and elF-4B will recognize this complex, attach to it, and unwind the RNA secondary structures of the NTR, facilitating ribosome attachment to the IRES and translation of the polyprotein. The polyprotein is autocatalytically cleaved cotranslationally into three proteins encompassing the P1 structural region and P2 and P3 non-structural regions. It takes approximately 15 minutes for the viral proteins to be synthesized and cleaved into functional products 4 5 , 4 6 . Once the viral RNA polymerase, encoded by the P3 region, is synthesized, the sense strand RNA is used to make complementary negative strand RNA which, together with the plus-strand, is known as the Replicative Intermediate form (RI). The RI, which is associated with smooth endoplasmic reticulum, then generates an expanding pool of positive strand RNA. The sense strand can have one of three fates: It can either be used as a mRNA to make more proteins, it can be used to make more RI, or it can become encapsidated. The progeny virions are released following cell lysis in a manner not yet characterised 4 7 4 8 4 9 . H. Host factors and their role in CV tissue tropism: Different host factors are involved in tissue tropism caused by coxsackievirus infection. The first step in virus replication is recognition and binding to a cell surface 13 receptor, and the distinctive organ tropisms of many viruses can be linked to the distribution of the receptors on different tissues. The presence of a cellular receptor for the virus on the plasma membrane is clearly essential but is not necessarily sufficient for infection. Thus even if the virus gets inside the cell, it requires an optimal environment and a variety of cellular factors for translation, replication and assembly of progeny virus. As was mentioned in the previous section, host protein P52 binds to the IRES of the NTR region. This complex is then recognized by host initiation factors such as elF-4A and elF-4B that aid in unwinding the RNA complex structure and allowing for the host ribosome to bind to the IRES and start translation. Several host factors are known to be essential for viral protein synthesis and others are believed to be required for RNA replication and viral assembly. In the context of the whole organism, factors such as age, sex, genetic susceptibility, and immune deficiency determine the extent of viral spread in the infected host and therefore the ability of virus to reach its target organ(s). Coxsackievirus is known to infect neonates more severely than adults probably due to the fact that the immune system in neonates is not as well developed as in adults 9 1 , 9 2 . Also, female Balb/c mice are shown to be more resistant to CVB infection than male animals as they respond more quickly to CVB and produce a higher titre of neutralising antibody to clear the infection 9 3 . Host genetics also plays a role, for example, different stains of mice have different susceptibilty to coxsackievirus, A/J and Balb/c mice being more susceptible than C57BI6 mice. 14 I. Coxsackievirus cellular receptors: For an enterovirus such as CVB, the presence of a cell receptor is a major determinant of tissue tropism. Since cell receptors provide potential sites for selective inhibition of the virus, several attempts have been made to characterize the CVB receptor. In the 1980's, R. Crowell and his colleagues isolated a 49.5 Kda protein (Rp-a) from Hela cell extracts that was suggested to be responsible for recognition and binding to all six serotypes of CVB 5 0 . In 1984, Crowell et al. discovered that a variant of CVB, which was capable of growing in rhabdomyosarcoma (RD) cells, recognized a different human cellular receptor (HR2) on RD cells in addition to the receptor recognized by the parental virus (HR1) on Hela cells. It was postulated that Hela cells have both HR1 and HR2 whereas RD cells lack HR1 1 3 4 . Identification of HR2 on western blots using CVB as probe showed that the receptor was a 70KDa protein135. More recently, Shafren et al. (1995) characterised this 70KDa secondary receptor as Decay Accelerating Factor (DAF), a protein that protects cells from complement mediated lysis 5 1 . They also showed that DAF can only recognize three of the six CVB serotypes, CVB1, 3 and 5. Lastly, using a different approach, Kandolf and his group have characterized a 100 Kda binding protein that is able to bind to all six serotypes of Coxsackie B viruses 5 2 , 5 3 . Sequence analysis has suggested that this protein has homology with the human nucleolin protein 5 4 5 5 ' 5 6 . Thus despite the attempts by different researchers during the past 10 years, there is still some dispute as to the true receptor or receptors used by the virus. It is possible that all three identified proteins may act as the receptor in different tissues. 15 J. CVB Variants and their role in Pathogenesis: Several variants of CVB have been described and studied with respect to their role in pathogenesis in animals. It is important to note that every virus stock contains a heterogenous population, but only the dominant strain is expressed phenotypically. These variants arise by mutation or recombination influenced by both positive and negative pressures of the host or environment on the virus, at an estimated frequency of 1 per 104 nucleotides 1 4 0 . Variants of CVB which have been described include antigenic variants, receptor variants, and mutants with varying sensitivity to temperature or interferon. A receptor variant of CVB has been studied in detail by R. Crowell and his co-workers. They showed that human Rhabdomyosarcomma (RD) cells lack the primary CVB receptor, present on Hela cells. However, blind passage of CVB in RD cells resulted in selection of a new variant (CVB3-RD) that was capable of infecting both RD and Vero cells. Further experiments in mice have shown that while the parental CVB3 is myocarditic, the CVB3-RD variant is non-myocarditic 1 3 4 - 1 3 5 1 4 1 . Interferon sensitive variants were isolated from cells treated with IFN and infected with CVB4. These mutants did not induce IFN and were less virulent than the IFN resistance variants,i.e., they were not diabetogenic in mice pancreas 1 4 2 . Temperature sensitive mutants and their variation in pathogenesis was studied by Gauntt et al. 1 4 4 . These mutants were isolated from a myocarditic CVB3 parental strain using 5-flourouracil (FU). The mutants were divided into three prototypes based on their defect in: i) assembly, ii) a capsid polypeptide required for proper configuration 16 during capsid construction and iii) inability to synthesize viral RNA. None of the temperature sensitive mutants induced myocarditis in adolescent CD-1 mice. However, two temperature sensitive mutants, ts_l and ts6, that were defective in synthesizing viral RNA, resulted in brain damage in adolescent mice 1 4 3 1 4 4 1 4 5 . In another study, a natural non-myocarditic variant of CVB3 (CVB30) was isolated from a human heart in 1979 1 4 6 . Tracy et al. (1995) compared the sequence of CVB3Qwith the passage-adapted myocarditic variant (CVB3J, and showed that there is an U-C mutation at nucleotide 234 of the NTR of the genome. They concluded that this single mutation was responsible for the amyocarditic characteristic of CVB3 0 1 4 6. Moreover, Huber et al. (1994) have isolated an antibody-escape mutant of CVB3 using a neutralising MAb (10A1) against CVB3 that had cross-reactivity with heart myosin antigen 1 1 8 , 1 2 1 . Results obtained from in vitro characterization of the escape-mutant H310A1 suggested that the mortality rate and the degree of heart damage was much less with H310A1 strain than the parental H3 strain 1 1 8 . In a later study done by the same group, it was shown that the difference in pathogenicity with H3 and H310A1 correlated with the type of CD4+ T-helper cell responses induced during infection. In an infection with H3 strain, IL-1 and IL-2 (produced by Th-1, CD4+ cells ) were released in the supernatant medium of infected J774A.1 macrophage cell line cocultivated with T-helper cells. In contrast, IL-4 (produced by Th-2, CD4+ cells) was released in the supernatant medium of J774A. 1 cocultivated with T-helper cells and infected with H310A1. Exogenous treatment of animals infected with H310A1 with IL-1 and IL-2 resulted in increased pathogenicity. They concluded that since the H310A1 17 strain was capable of inducing myocarditis in the presence of exogenous cytokines, it must contain the peptide sequences that gave rise to a pathogenic T-cell response. In other words, the genetic change in this mutant was not a loss of a pathogenic T-cell epitope but failure of the virus to stimulate the pathogenic T-cell response to an existing epitope 1 1 8 . An infectious cDNA clone of each of the variants was made using RT/PCR in order to compare the genome of the mutant to the wild type. Sequence analysis revealed a mutation in nucleotide 1442 of the VP2 region of H310A1 genome which results in an asparagine to asparatate mutation in amino acid 165 of the puff region. Site-directed mutagenesis confirmed that the presence of asparatate at amino acid position 165 of VP2 could lead to a decrease in production of TNF by the mutant and consequently less inflammation and damage to the heart118. Thus a number of different mutants of CVB have been isolated by different researchers that have been found to be mostly non-virulent in comparison to the parental virus. The ability to compare the nucleotide sequences of these mutants with the wild type strain enables identification of areas of the genome that are important for pathogenesis in the host. Following this, site-directed mutagenesis may be used to confirm the importance of specific nucleotide changes in pathogenesis. These approaches are important for developing vaccines against coxsackieviruses. 18 K. CVB and insulin dependent diabetes mellitus (IDDM): Insulin dependent diabetes mellitus (IDDM) results from pathological changes in islets of Langerhans of the pancreas. The pathogenic process involves an inflammatory reaction against islet tissue leading to the destruction of j8-cells and consequently insulin deficiency 1 5 1 . For many years viruses have been suspected as providing the initial stimulus for this inflammatory reaction, and several viruses (mumps, rubella and coxsackieviruses) have been shown to infect B- cells both in vivo and in vitro 1 5 1 . CVB4 was first associated with diabetes in animal models by Yoon et al. in 1978 1 3 e . Later it was shown that some but not all strains of CVB4 are capable of directly infecting /3-cells causing hypoinsulinemia and hyperglycemia in mice 1 3 6 . Three different pathways may be involved in islet cell destruction and diabetes 1) direct lysis of the target cells by the virus and 2) virus-induced immunopathology and 3) autoimmunity. The following summarises some evidence indicating that IDDM is virus-induced. 1: The presence of viral specific antigens in the islets of Langerhans associated with the destruction of beta-cells in the pancreas of diabetic patients137. 2: Epidemiological studies showing the presence of high antibody titres against certain viruses in IDDM diagnosed patients 1 3 7 . 3: High frequency of CVB specific IgM antibodies in newly diagnosed diabetic children 1 3 8 . 1 9 4: j8-cell damage in children who died of acute viral infections with mumps, EMC, 1 3 8 . 5: Isolation of virus from patients with acute diabetes and demonstration that these viruses induce diabetes in mice 1 3 7 . However the induction of antibodies to coxsackievirus B has not been correlated with the production of autoantibodies in patients with IDDM. Also there is no convincing evidence for the presence of anti-islet cell Ab during coxsackievirus infection. Thus in an acute infection coxsackievirus it is more likely involved in a cytolytic reaction rather than in the induction of an autoimmune response. However in a chronic infection where minor viral epitopes may become dominant in the immune response, these may cross-react with islet tissue resulting in an autoimmune reaction 1 5 0 . In addition, interferon and possibly other mediators, may induce aberrant expression of MHC II on the surface of endocrine cells enabling them to present autoantigens to lymphocytes and triggering an autoimmune reaction 1 3 9 . L. CVB and Myocarditis: Coxsackieviruses have been identified as a primary cause of viral heart diseases such as myocarditis, pericarditis and dilated cardiomyopathy 5 7 by molecular genetic techniques such as in situ hybridization and immunoperoxidase staining 5 8 5 9 6 0 . Myocarditis is defined as an inflammation of the myocardium with the presence of necrotic myocardial cells associated with infiltration of immune cells. The myocyte damage may be induced by three different mechanisms. Firstly, by direct viral cytolysis; secondly by an immunopathological process directed against virus-induced 20 antigens on infected cells and thirdly by direct viral induction of pro-inflammatory mediators in heart tissue 6 9- 1 3 1- 1 3 2 Pathogenic Mechanisms of Myocarditis: I. Autoimmunity and Myocarditis The original hypothesis on the mechanism of induction of myocardial injury by CVB suggested that an autoimmune reaction mediated against both infected and normal myocytes was involved. More specifically, Woodruff and Woodruff (1977) first suggested that T-lymphocytes were primarily responsible for myocyte necrosis in their model of coxsackievirus-induced murine myocarditis 6 8 . Their studies showed that 1) athymic mice, 2) thymectamization, irradiation, and bone marrow reconstitution, or 3) administration of monoclonal Ab against T-cell marker antigens that result in decreased T-cells, developed minimal cardiac inflammation and no obvious myocyte necrosis despite high CVB titres in heart. This theory was further supported by the presence of the inflammatory cells (neutrophil, macrophage, T-cell) and also heart-reactive Ab close to the necrotic areas in the heart. Moreover, it was further shown that exogenous administration of inflammatory cells could augment pathogenesis of CVB3 infections in resistant strains of mice 6 1 •62'66'67. For instance, Rose et al. (1992) have shown that co-treatment of CVB3 infected B10.A mice with IL-1, IL-2, LPS or TNF promoted the induction of an extensive myocardial inflammation 6 7 . Huber et al. (1994) studied the effect of exogenous administration of 21 IL-1 and IL-2 in Balb/c mice infected with a less myocarditic variant of CVB3, H310A1. They showed that co-treatment of mice infected with H310A1 with and inflammatory cytokines produced a myocarditic score similar to highly myocarditic strains of the virus 6 e . II. Direct Myocardial injury by CVB: In the late 1980's, a second hypothesis was proposed suggesting that infection of myocytes by the virus directly leads to metabolic dysfunction and cell death 6 3 , 6 4 . In order to prove that the necrosis and cytolysis of myocytes was a direct consequence of the virus itself, the histopathology of CVB3 infected hearts from immune-competent and non-competent mice were studied using Masson's trichrome and H&E stains. The results suggested that most of the damage to myocytes was observed up to day 5 post-infection and prior to the presence of inflammatory infiltrates in the heart 0 4 . According to McManus et al. (1994) 6 4 the size of the myocarditic lesions did not increase with time beyond the acute injury period in immunocompetent mice which suggested an early and non-immune process was involved in the genesis of these lesions. The importance of the immune system in reduction of virus-induced damage was further shown in the studies done by McManus, (1991) in which inhibitors of the immune potentiator, interleukin 2 (cyclosporin A and FK-506) were administrated to the CVB3 infected mice. This resulted in increased myocarditis and myocyte necrosis in addition to higher mortality rates 1 1 5 . In addition, Chow et al. (1992) 6 3 have shown that the exposure of SCID (severe, combined mmunodeficiency) 22 mice that lack both T-cells and B-cells, to CVB3 can lead to severe heart pathology and a much higher mortality rate in comparison to control mice. The technique of in situ hybridization permits detection of replicating viral genome early in infection and in close proximity to the necrotic areas of the heart65. This clearly demonstrates the largely protective effect of the immune response. Although initially the hypothesis that CVB3 induced myocarditis was mediated by an autoimmune or immunopathological process was accepted, more recent evidence on the direct effect of CVB3 on cardiomyopathy has made this second hypothesis more favourable. It is probable that some collatoral damage is done to myocytes as the immune system overcomes the virus infection. However it is clear that the virus alone, in the absence of an immune response can induce widespread cardiac necrosis (e.g., in the SCID mice). Overall, the importance of the immune system in clearance and reduction of the virus is unquestionable, however, the role of an autoimmune reaction in exacerbating the damage is yet to be proven. M. Interaction of CVB with the immune system: 23 1. Non-specific immune reactions: a) Macrophages: In CVB infgction both the innate and adaptive immune responses appear to participate in the host resistance. Macrophages are one of the first immune cell types to be encountered in an infection. They are crucial both for limiting the spread of CVB to the target organs as well as clearing virus from the tissues. Moreover, they possibly play an important role in the dissemination of virus to different tissues and in some viral systems to exacerbate viral infections through "antibody enhancement" of infectivity. In addition, macrophages are important antigen presenting cells (APC) that process viral antigens and present them to T or B cells in context of the major histocompatibility complex (MHC) 1 0 7 1 0 8 . A number of preliminary studies have shown the protective effects of macrophages in CVB infections. For example treatment of macrophages with toxic substances such as silica resulted in an increase in CVB3-induced myocarditis ". Also studies done by Scanlon et al. (1985) showed that inhibition of the inflammatory mediator prostaglandin, produced by monocytes, by ibuprofen resulted in an increase in CVB3-induced myocarditis in mice 7 0 , 7 1 , 8 0 , 1 0 6 j n c o n t r a s t ) unpublished data from our laboratory has shown that there is a 10 fold increase in CVB3 titres in cultures of macrophages infected in the presence of non-neutralising (anti-CVB2) antibody, indicative of enhancement of infectivity by antibody. Subsequent infectious focus assays have shown that in the presence of non-neutralising antibody, the number of infected macrophages increased from 2% to 24 25% (approximately 10 fold) most likely due to infectious complexes of virus and Ab gaining access to macrophages by Fc- mediated uptake. These studies indicate that in the absence of non-neutralizing antibodies, macrophages play an important role in limiting viral infection; however, in their presence, macrophages can act as a host for viral amplification and also as a vehicle promoting viral dessimination to different organs. b) Natural Killer (NK) cells: Activated NK cells limit CVB replication both in vitro and in vivo by lysing the infected cells non specifically 7 2. Mice depleted of NK cells by anti-asialo GM1 were shown to have approximately 300 fold more virus in their heart, associated with larger myocarditic lesions 7 3. Both the NK cells and macrophages are among the first host defense mechanisms that protect the host by limiting the spread of the virus non-specifically. Although, their mechanism of action is through lysing the infected cells, this limits viral spread and their overall role is protective rather than damaging to the tissues. c) Interferons (a,p,y): Interferon induction by cells at the site of infection forms another major line of defence against viruses. Their importance in protecting the host from viruses has been indicated by several lines of experiments. Firstly, inhibition of IFNs a//3 in mice enhances the severity of the infection. For example, experiments done in two-month 25 old CVB3-infected Balb/c mice showed that administration of 6000 units of anti-IFN a//3 serum increased the percentage mortality, and shortened the average life span post-infection 7 5 , 7 6 . Secondly, mice with genetically defective IFN system are more susceptible to viral infection, and thirdly, substances which induce IFN, such as polyinosinic-polycytidilic acid (poly l:C) have a protective effect in viral infections. For instance, administration of the IFN inducer poly l:C to CVB3-infected mice prevented the development of myocardial lesions 7 4 J 5 J 6 . iFNs or treatment with IFN inducers was found to beneficial only if they were introduced prior to, or very early in an infection. For example, in a coxsackievirus B3 infection of mice, it was shown that IFN must be administrated at least 6 hours prior to infection in order to be able to reduce myocardial lesions 7 7 7 8 7 9 . A number of host factors such as age, gender, hormones, and stress are important in early production of IFNs and the outcome of picornaviral infections 8 1 8 2 8 3 1 1 1 . 2. Specific immune reactions: a) Role of the humoral immune response to CVB: Resistance to coxsackieviruses is believed to be mainly mediated by the humoral immune response. For example, individuals with antibody deficiency syndromes, but not T-cell deficiency, suffer more frequent and severe infections with coxsackieviruses. Studies in murine model systems has confirmed the importance of the humoral immune system in CVB3 infection. Susceptibility of adult mice to CVB is increased by treatments that interfere with general immune responsiveness including 26 corticosteroids and cyclophosphamide. Investigation of the relative importance of circulating antibody and other mediators of antiviral defenses has led to the conclusion that humoral antibody is critical in limiting CVB3 infection 8 4 8 5 8 e . In the late 1980's a number of experiments were done to study the effect of the humoral response on CVB3-induced myocarditis in different strains of mice. The results of these studies suggested that myocarditis was reduced noticeably in those strains of mice that were capable of producing neutralising antibodies by day 3 post-infection in comparison to the strains that produce antibodies on the fourth day post-infection 8 7 8 8 8 9 9 0 . The early neutralising antibodies react with extracellular virus reducing viraemia and viral spread. This in turn decreases the amount of virus reaching the heart and therefore both myocyte necrosis and the inflammatory response in the heart. In addition to mouse strain, the age and gender of the animals have been found to be important factors in the production of neutralising antibodies against coxsackievirus. For example, neonatal mice are more susceptible to CV infection probably due to the fact that their immune system is not as developed as adult mice. Administration of neutralising anti-CVB prior to or at the time of infection greatly reduced the number of CVB induced heart lesions and the mortality rate in these animals ° 1 , 9 2. Also, Wong et al. (1977) have shown that female Balb/c mice respond more quickly to CVB3 and produce a higher titre of neutralising antibody to clear the infection 9 3 . b) The role of humoral immune response in tissue damage: In addition to its protective effect, the humoral immune response may also participate in the production of heart lesions following CVB infection. Anti- CVB antibodies have been detected in the serum of patients with myocarditis that have cross-reactivity with myocytes and can cause cell lysis by interaction with the complement system 9 4 . From the second week of infection onwards, heart specific antibodies have been detected in the sera of the patients with CVB3-induced myocarditis. For example, IgM autoantibodies to myolemmal antigens can be detected in the acute phase of CVB induced myocarditis whereas IgG and IgA autoantibodies to myolemmal antigens can be detected in the chronic phase of the disease 9 5 . Since these antibodies showed specificity for cardiac cells and did not bind to viral epitopes, it was suggested that as a result of CVB infection, heart cell antigens become accessible to the immune system which therefore stimulates B cells to produce antibodies against these self epitopes. These autoantibodies have been shown to effect the function of myocytes by disturbing energy metabolism, impairing contractility, increasing Ca 2 + influx, or directly killing cardiomyocytes 9 6 9 7 9 8 . c) The role of the cell mediated immune response to CVB: The cell mediated immune response is believed to be less crucial than the humoral immune response in host resistance to CVB3 infections99. For example, when mice were depleted of either T-cells or B-cells, CVB3-induced disease was more frequently observed in the animals depleted of B-cells 1 0°. Even though the humoral immune 28 response seems to be more important in CVB3 infection, the cell mediated immune response also plays a role in host resistance 1 1 2 . For instance, studies in T-cell depleted mice showed that CVB3 can be detected in the heart of these animals for a longer period than is normally found in immunocompetent mice 1 0 1 1 0 2 . On the other hand, early studies by Woodruff and Huber suggested that myocardial damage may be mediated by activated T-cells 1 0 3 1 0 4 . Recently, in experiments done by Huber et al. (1989, 1991) in which Balb/c mice were infected with a myocarditic strain of CVB3, two distinct population of T-cells were identified after infection. Virus-specific CTL (CD8+ cells) that lysed the virus infected myocytes and were detected at later stages of the infection. In addition, an autoreactive CTL (CD4+) clone was detected in the early stages of infection which was capable of damaging uninfected heart cells 1 0 9 1 1 0 . The relative importance of these two populations of CTL is still unknown, except for the fact that the autoreactive CTL caused a more severe heart necrosis when transfused into T-lymphocyte deficient animals whether infected or uninfected 104,105,116,117,118,119,120 As was mentioned previously, studies have been carried out by Huber and her co-workers (1994) using a viral mutant, H3-10A1 which was found to be less pathogenic than the wild type myocarditic strain H3121. Even though both strains were capable of growing in myocytes to approximately the same titre, the mutant caused less cardiac inflammation and minimal mortality in comparison with the wild type. Their studies suggested that the difference between these two variants may be related to the type of T-helper response induced during infection. More specifically, in adult Balb/c mice 29 infected with both strains of the virus, production of IL-2 and IL-4 were measured. The results indicated that, with the wild type strain high levels of IL-2 were produced suggesting that a Th1 response was predominant. In contrast, with the mutant H3-10A1, IL-4 was produced suggesting that a Th2 response was predominant, associated with less myocarditis118,122. In addition, they exogenously administered IL-2 to the mice infected with the H310A1 variant and observed that the T-helper response had switched from type 2 to type 1, associated with more inflammation in heart and a higher mortality rate in animals, similar to that seen with the wild type strain. These results indicate that the pathogenic and non-pathogenic CVB3 variants demonstrate differential induction of Th1 and Th 2 cells respectively. Also production of specific cytokines during infection and activation of Th1 cells may be an important factor in pathogenesis 1 1 8 . N. Coxsackievirus B3 and Persistence: a) In vitro: CVB3 generally produces a highly cytolytic infection of susceptible cultured cells which is incompatible with viral persistence. Only in a few semi-permissive cell-lines persistent infections have been established. For example, cultured human lymphoid cell lines can be persistently infected with CVB3 for up to 2 years in the absence of CPE 1 2 4 . Also, a myocarditic strain of CVB3 was found to be capable of growing in mouse cardiac fibroblasts in the absence of CPE for up to 12 days 1 2 5 . In other systems, CVB persistence has been established by viral mutants, or ts virions, by addition of IFNs or antiviral antibodies to the culture medium, or by 30 growth in cultures composed mainly of resistant cells 1 2 6- 1 2 7- 1 2 8. Coxsackievirus persistence occurs generally when only a small portion of cells are susceptible to infection, forming carrier cultures where not all cells are infected and persistence can be terminated by addition of anti-viral antibody to the culture 1 2 f M 2 9 . b) In vivo: Coxsackieviruses are usually considered to be eliminated after the acute stage of disease. However, the development of new molecular techniques, such as in situ hybridization, has allowed detection of viral RNA in patients months after the initial acute infection suggesting that CVB persistence occurs in certain circumstances. In particular viral RNA has been detected in heart biopsies of patients with myocarditis or cardiomyopathy suggesting that virus may persist in human heart tissue. Moreover, in an acute infection there is more of the positive strand genome and less of the replicative intermediate, but, in a chronic infection, similar amounts of plus and minus strand RNA are detected by ISH 1 3 0 . It has also been shown that during a chronic infection, spleen and lymph nodes show persistent infection of lymphoid cells. This may play an important role in viral dissemination and act as a noncardiac viral reservoir. In summary aspects of the host immune system play an important factor in determining whether a persistence infection will occur but the precise mechanism determining whether virus is eliminated or not is not yet completely understood129'130'131. 31 3. OBJECTIVE The overall objective of this thesis was to map genetic changes in CVB3 genome associated with alterations in phenotypic properties. To do this, a mutant of the well-characterised CVB3 (RK) strain was isolated by selection of variants not affected by a MAb oc CVB3 which completely neutralises the dominant parental strain. As was described in the introduction section of this thesis, CVB neutralising epitopes are located around the rim of the canyon made by the capsid proteins. Thus the mutation(s) selected for would be expected to be located in one or more of the capsid proteins of the non-neutralised mutants to allow the virions to escape the effect of the neutralising MAb. Further plaque purification provided the EM1 mutant which has formed the basis of the reported studies. Its growth properties in vitro as well as its pathogenesis in vivo are described. In view of the predicted mutations in capsid proteins, the genetic mapping studies have focused on the P1 (structural) gene region of the mutant and its sequence in comparison with the CVB3 (RK) strain by RT/PCR. Further mutations in P2 and P3 may be present in EM1 but have not formed a part of the investigation reported. 32 4. Materials: I. Cell Line i. Vero (African green monkey kidney) cell line was obtained from American Type Culture Collection (ATCC). ii. J774A.1 (Balb/c mouse monocyte/macrophage) cell line was generously provided by Dr. R. Stokes' laboratory, University of British Columbia. iii. EL-4 (C57BL/6N mouse T-Cell) cell line was generously provided by Dr. F. Takei, University of British Columbia, Canada. iv. Wehi 231 (mouse immature B-cell lymphoma) cell was generously provided by Dr. M. Gold, University of British Columbia, Canada. II. Cell Culture Materials -DMEM-F12, and RPMI media was obtained from Gibco/BRL as powder and prepared according to the manufacturer's instructions. -Fetal bovine serum (FBS) and fetal lamb serum (FLS) were bought from Gibco/BRL. -Heat-inactivated fetal bovine serum (HIFBS) was made by heating the serum at 60°C for 30 minutes. 33 -Gentamicin reagent 1 mg/mL was obtained from Gibco/BRL and used at 1% (v/v). -Trypsin (0.25%) 2.5 g 1:250 Trypsin (Fisher) Dissolved in 1 L of Hank's balanced salt solution without Ca+2 or Mg+2 (Gibco) The trypsin was sterilized using 0.22 um pore diameter filter. -Phosphate buffered saline (PBS) 34.0 g NaCl 4.28 g Na,HP04 1.38 g NaH2P04.H2O Dissolved in 4L of ddH20 and autoclave. III. Fixatives and Stains: 1. Camoy's Fixative 750 ml_s 95% Ethanol 250 ml_s Glacial Acetic Acid 2. Paraformaldehyde (PF) 4% 40.5 mL 0.1 M Na^ HPC^  9.5 mL 0.1 M of NaH2P04.H20 Heated the phosphate buffer to 80°C and add 2 grams of PF (Mallinckrodt) Add 5-10 drops of 5N NaOH for pH 7.4 34 3. Coomassie Blue Stain 100 ml_s Glacial Acetic Acid (10%) 250 mLs Isopropanol (25%) 2.50 g Coomassie blue stain (Sigma) Dilute to 1 L with ddH20 4. Eosin 70 mLs Ethanol (70% v/v) 25 mLs Glacial Acetic Acid (25% v/v) 0.5 g Eosin (0.05% w/v) 5. Haematoxylin 0.2 g Haematoxylin (0.2% w/v) 0.7 g AL,(S04)3.15H20 (12.6 mM) 0.008 g Nal03 (0.4mM) 25 mLs 1,2 ethanediol (25% v/v) 2 mLs Glacial Acetic Acid (2% v/v) Dilute to 100 mLs with ddH20 IV. Antibodies -Murine monoclonal antibody (mAb) against Coxsackievirus B3 (Chemicon international Inc). -Anti-Digoxigenin antibody (Boehringer Mannheim) 35 V. Virus The RK (Reinhard Kandolf) strain of Coxsackievirus B3 was derived from an infectious clone pCVB3/T7 which was transcribed and transfected into Hela cells. The resulting virus was passaged in mice and virus obtained from heart tissue was grown into CVB3(RK) stock. VI. Infectious clone The CVB3 infectious clone pCVB3/T7 was provided kindly by Dr Reinhard Kandolf ( Germany). VII. Solutions for In Situ Hybridization (ISH) 1. DEPC treated ddH20 0.1% (VA/) Diethyl pyrocarbonate 2. 10% SDS 100 g SDS in 1L ddH20 3. 20X SSC 175.3 g NaCl 882.0 g Na Citrate Made up to 1 L with ddH20 4. 4M NaCl 233.7 g NaCl In 1 L ddH20 5. 1M Tris pH 7.4 242.2 g Tris in 1L ddH20 6. Tris/0.1M EDTA pH 7.4 121.0 g Tris 37.22 g EDTA Made up to 1 L with ddH20 7. 1M DTT 154g DTT Made up to 1 L with ddH20 8. 100% Formamide (Gibco/BRL) 9. 10mg/mL Proteinase K (Gibco/BRL) 10 mg Proteinase K in 1 ml ddH20 10. 0.2M CaCI2 22.2 g CaCI2 in 1LddH20 11. 1.8M NaCl (10% Dextran Sulfate). 105.1 g NaCl 100.0 g Dextran Sulfate Made up to 1 L with ddH20 12. 0.5M MgCI2.6H20 101.7 g MgCI2 in 1LddH20 13. 100 X Poly VinylPyrrolidone, Ficoll, BSA (PFB) (2%) 20 g Poly VinylPyrrolidone 20 g Ficoll 50 g BSA Made up to 1 L with ddH20 14. Wash Buffer 500 mL 100% Formamide 10.0 mL 1M Tris/0.1 mM EDTA (pH 7.4) 150 mL 4M NaCl Dilute to 1 L with ddH20 15. Tween 20 (Sigma Chemical Company) 0.1% in DEPC treated water 16. Buffer 1 37.5 mLs 4M NaCl 100 mLs 1M Tris (pH 7.5) Dilute to 1 L with ddH20 17. Buffer 2 37.5 mLs 4M NaCl 100 mLs 1M Tris (pH 7.5) Dissolved in 800 mLs ddH20 Add 20 mLs of Lamb Serum (Gibco) and make up to 1 L with ddH20 18. Buffer 3 26.7 mLs 4M NaCl 100 mLs IMTris 100 mLs 0.5M MgCI2 Dissolved in 800 mLs ddHzO pH to 9.5 with NaOH and made up to 1 L with ddH20 19. Colour Substrate for ISH (Sigma) Sigma Fast 229 mg tablets Containing BCIP\NBT (5-bromo-4-chloro-3-indolyl phosphate/ nitro blue tetrazolium) Dissolved 2 tablets in 200 mLs ddH20 VIII. Large Scale plasmid Solutions: 1. 2xYT broth 20 g Bacto-tryptone 10 g Bacto-Yeast extract 10 g NaCl Made up to 1 L with ddH20 2. TE pH 8.0 1.2 g Tris (10mM) 0.3 g EDTA (1mM) Dissolved in 800 mis ddH20 and adjusted the pH to 8.0 with 5N HCl Made up to 1 L with ddH20 3. Solution I 9.0 g Glucose (BDH) (50mM) 3.0 g Tris/HCI pH 8.0 (25 mM) 2.9 g EDTA (10mM) Made up to 1 L with ddH20 4. Solution II 8 g NaOH (0.2M) 10 g SDS (0.1%) Made up to 1 L with ddH20 5. Solution III 600 mL 5 M Potassium Acetate 115 mL Glacial Acetic Acid Made up to 1 L with ddH20 IX. Reverse Transcriptase PCR solutions: 1. Dulbecco's PBS 0.2 g KCI 0.2 g KH2P04 8.0 g NaCl 2.16 g Na2HP04.7H20 Made up to I L with ddH20 2. Dulbecco PBS + 30% sucrose 150 g sucrose (Beckman) Make up to 500 mLs with Dulbecco PBS 3. Trizol reagent (Gibco/BRL) 4. Isopropanol (Fisher) 5. Superscript II kit (Gibco\BRL) 6. First Strand Reaction 5/iL 5x buffer %JL DTT (0.1 M) 1/-/L hexonucleotides (250 pmoles) 1/JL Trizol purified RNA 1/JL RNAsin (Promega) 10.000 units 1/L/L Superscript II (Gibco/BRL) 200 u/fuL Make up to 25 /L/L with 14 pL DEPC treated water. 7. Enzyme mixture (100 uL) 1/L/L dNTP (25 mM) 10 iA_ 10x Taq buffer with MgCI2 (Bio/Can) 0.5 fjL Taq DNA polymerase (0.5 U/JL/L) from (Bio/Can) Dilute to 86 uL with ddH20 8. Reaction mixture for PCR (50/iL) 48 fjL enzyme mix 1 uL each primer set C1 & C2 or C3 & C4 1 pL cDNA from first strand reaction of either virus strain 9. Light Mineral oil (Fisher) 41 5. METHODS A. Cell Maintenance Vero cells: The cells were incubated in a 5% C02 humidified atmosphere at 37°C. To passage, the intact monolayer was washed with phosphate buffered saline (PBS) and incubated at 37°C in 0.25% (w/v) trypsin for approximately 15 minutes until the cells were detached. The cells were then resuspended in DMEM-F12 culture medium supplemented with 10% FBS and 1% gentamicin and distributed to new tissue culture flasks or petri dishes. J774A.1 (a murine macrophage cell line): The cells were passaged 1:2 by removing half of the old medium and scraping the monolayer into the remaining cell culture medium. The cells were resuspended gently and then distributed in new cell culture flasks or petri dishes after increasing the volume with fresh DMEM supplemented with 10% HIFBS, 1% of 200mM glutamine, and 1% gentamicin. EL-4 (a murine T-cell line): The cells were passaged 1:3 every second day by transferring 3.5 mLs of T-cell containing medium to a new flask T75 and then increasing the volume to 10.0 mL with fresh DMEM medium containing 5% FBS. Wehi 231 (a murine B- cell line): The cells were split 1:2 twice a week by transferring 5.0 mis of the cell containing medium into a new T75 flask and supplementing with 5.0 mis of fresh RPM1 1640 containing 10% FBS, 50uM 2-ME, 1% 200mM glutamine 42 and 1mM pyruvate. After infection, cells were supplemented with the appropriate medium containing 5% HIFBS. B. Virus Stock Preparation a) CVB3(RK) stock Vero cells were split 24 hrs prior to use so that the monolayer was 95% confluent at the time of infection. The cells were washed with 5 mLs of PBS and infected with 0.1-1 plaque forming unit (pfu) of CVB3(RK) per cell. The virus stock was diluted in 1.0 mL of cell culture medium without serum and allowed to absorb to the monolayer for 60 minutes. The inoculum was then removed and replaced with DMEM-F12 + 5% HIFBS + 1% gentamicin. The cells were incubated at 37°C, in an atmosphere of 5% C0 2 until 100% cytopathic effect (CPE) was observed- usually after 24 hours. The flask was then frozen at -70°C and three freeze-thaw cycles were performed to release the virus from the cells. To separate the cell debris from the virus-containing supernatant, the flask contents was centrifuged at 2000 RPM for 10 minutes at 4°C. The supernatant was collected and stored in aliquot parts at -70°C. C. Coxsackievirus Titration Virus titration was done by assaying plaque formation in Vero cells by standard techniques. The virus was titrated in duplicate by infecting 90-95% confluent Vero cells in small petri plates with serial 10 fold dilutions of virus in DMEM (without FBS). 43 The were inoculated with 0.5 ml of each serial dilution, starting with the highest dilution (i.e., lowest virus concentration) and were gently rocked several times to distribute the inoculum evenly over the monolayer. Following 1 hour adsorption at 37°C in humidified 5% C02, the inoculum was replaced with an overlay of 0.5% agarose in DMEM containing 5% HIFBS. The Vera cell monolayer was fixed, 48 hours later, with 2.0 ml of Carnoy's fixative per petri plate for 30 minutes. The overlay was then removed gently and the cells were stained with coomassie blue dye. The plates were rinsed after 10 minutes and the plaque forming units (pfu) were counted as colourless areas in the violet cell monolayer. D. Preparation of (EM1) stock An antibody-escape mutant was selected using a murine neutralising MAb against the wild type coxsackievirus B3. Stock was pretreated with a 1:10 dilution of MAb to neutralise the dominant strain of virus. Staph A protein A was then added and the culture centrifuged to remove virus/MAb complexes. The remaining supernatant was used to infect a flask of 90% confluent Vero cells. After adsorption, 20 uL of MAb (diluted 1:10) was added to the culture medium. The cells were cultured for 48 hours in the presence of neutralising MAb and were split 1:2 at which time CPE was apparent. The supernatant was serially diluted in DMEM and 0.5 ml was added to lightly confluent Vero monolayers in 35-mm tissue culture plates. After incubation for 1 hour at 37°C, the monolayers were overlaid with medium containing 1% agarose and 5% HIFBS and incubated in 37°C for 48 hours. The plates were examined with an 44 inverted microscope and separate well-differentiated plaques were marked. The medium under one single plaque was removed using a pasteur pipette and stored at -70°C in DMEM + 5% HIFBS. Ten different plaques were purified using the above technique and were later used to infect Vero cells in the presence of MAb. A single purified plaque that showed strong CPE after 24 hours was chosen to make the escape mutant EM1 stock. E. Purification of virus by sucrose gradient centrifugation Six to eight flasks of Vero were infected with the CVB3(RK) or EM1 strains of the virus at a very low multiplicity of infection (0.0025 pfu/cell). After 48 hours, when CPE was maximal, the cells were freeze-thawed three times and the supernatant virus was clarified by centrifugation. 25 mLs of virus- containing fluid were overlayed on 6.0 mis of 30% sucrose in Dulbecco's phosphate saline in Beckman Ultra-Clear centrifuge tubes. The tubes were then centrifuged at 20,000 RPM for 3 hours at 4°C. The supernatant was decanted and the virus pellet was resuspended in 1.0 mL PBS and stored in a small centrifuge tube. The suspension was then sonicated on ice for 20 seconds before being divided into aliquots. A plaque forming assay was then done to determine the titre of the sucrose purified virus. F. Virus growth curves in Vero, J774.A1, Wehi, and EL-4 cells The different cell types were set up in triplicate in small petri dishes at a known cellular density. They were then infected with 10-20 pfu of either CVB3(RK) or EM1 45 strains or mock- infected with medium alone. After 1 hour adsorption at 37°C, the viral inoculum was removed and the petri dishes were washed gently with PBS 6- 8 times. They were then cultured in 1.0 mL of DMEM + 5% HIFBS. At 2, 4, 6, 8, 10, 12, and 24 hours post infection, samples were collected. The supernatant medium was harvested and the cells were washed with PBS 3-4 times. They were then sonicated in 1.0 ml of medium to obtain intracellular virus samples. Intracellular and extracellular virus titres were then determined by plaque assay. G. Viral Decay Curves To determine the decay rate of EM1 in comparison to CVB3(RK), a sample of each virus strain was incubated at 37°C in 5% C0 2 incubator. A sample of each strain was removed every day for 1 week. The decay rate of each virus was determined by recording the reduction in viral titre with continuing viral incubation. H. Temperature Sensitivity of EM1 and RK strains Plaque assays on RK and EM1 stocks were carried out at three different temperatures. The plaque assay was done as described previously except that the two strains of virus were adsorbed at 36°C followed by a 48 hour incubation at 36°C, 39°C and 40°C. The virus titre was then compared between the wild type CVB3(RK) and the mutant CVB3(EM1) at each temperature used. 46 I. Large Scale preparation of CVB3R1 plasmid using PEG precipitation Overnight cultures of JM109 bacteria carrying the plasmid pCVB3-R1 were grown in 200 mLs of 2xYT broth containing 50 ug/ml of ampicillin at 37°C with vigorous shaking. The cells were collected by centrifugation at 6,000 rpm for 10 minutes at room temperature using a GSA rotor for a Sorval centrifuge. Solution I was added to the pellet to stabilize the sample and lysozyme was added to the pellet to digest bacterial cell walls. Solutions II and III were added for alkaline lysis and neutralisation respectively. The sample was centrifuged at 10,000 rpm and the supernatant was collected. The nucleic acid was precipitated with 50% Isopropanol and washed in 70% ethanol. The pellet was dissolved in TE pH(8.0). 5M LiCI2 (1:1) was added to precipitate large nucleic acid strands of bacterial genomic DNA. After centrifugation the supernatant was precipitated with isopropanol to collect the plasmid and washed with 70% ethanol to remove the excess salt. The pellet was dissolved in TE and precipitated with polyethylene glycol 8000 to remove smaller nucleic acids. The DNA was then extracted with phenol/chloroform followed by chloroform alone and was precipitated with 3M NaOAc and 100% ethanol. The pellet was finally washed with 70% ethanol, dried and dissolved in 100 uL TE. The concentration of the purified plasmid was determined by running a small sample on 1% agarose gel using a known quality of X DNA digested with Hind III as the standard. 47 J. RNA probe labelling by in vitro transcription Both positive and negative strand RNA probes were synthesized by in vitro transcription of CVB3(RK) DNA, cloned into the EcoR1 site of the pSPT18 plasmid, flanked by SP6 and T7 RNA promoters. Transcription of the resulting pCVB3/R1 plasmid was carried out using dideoxygenin-labeled UTP in the NTP mixture as described below. 10 fig of the purified plasmid was linearized using Sail at 37°C or Sma1 at 25°C for 1 hour. The digests were then purified using a Qiagen column. The eluted DNA was precipitated for 1 hour at -20°C with 2.5 x volume 100% ethanol and 0.1 x volume NaOAc containing 1 uL of 20mg/ml glycogen (Boehringer Mannheim). The DNA pellet was then washed twice with 70% ethanol and dissolved in 20 //L of TE. The concentration of the linearized plasmid was then determined to be 50-100 ng//iL by running a small sample of the plasmid against a known plasmid concentration on a 1% agarose gel. In vitro transcription was carried out in a final volume of 20 /il, containing 1 /il of RNAsin ribonuclease inhibitor (20u//zL), 2 /il_ of 10X DIG labeled NTP (containing 10mmol/L ATP, CTP, GTP and 3.5 mmol/L of DIG labeled UTP), 4 //L of 10X transcription buffer, 1 //L of 0.1M DTT. Finally 2 //L of either T7 or SP6 polymerase were added plus 0.05 //g///L of the linearised plasmid. The reaction was left at 37°C for 2 hours. The labeled RNA transcript was precipitated using 3M Sodium acetate and ethanol and was then stored in 20 /iL of TE pH 8.0. K. Quantification of DIG-labelled transcript DIG- labelled transcript was quantified using the procedure supplied by Boehringer Mannheim. Ten fold serial dilutions of the CVB3 transcript and the control labelled RNA were made. 1uL of each dilution of the control and the CVB3 transcript was spotted on a Hybond nitrocellulose membrane and UV cross linked for 2 minutes by exposing the membrane to UV-light (Cole-Palmer). The membrane was then placed in a petri dish and covered with wash buffer containing 100 mM maleic acid and 150mM NaCl. The membrane was then treated with blocking buffer for 20 minutes and incubated with a 1:5000 dilution of anti-digoxy- antibody conjugated to alkaline phosphatase (anti-DIG-AP) for 10 minutes. The membrane was equilibrated to pH 9.5 with buffer 3 (100 mM Tris-HCI 9.5, 100 mM NaCl, 50 mM MgCI) for 2 minutes. An insoluble blue precipitate was produced by a subsequent enzyme catalyzed colour reaction with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium salt (NBT) as substrates. This was carried out for 24 hours in the dark. The spot intensities between the probe and the labelled control RNA were compared to estimate the yield of DIG labelled RNA. L. In Situ Hybridization using DIG-Labeled probe Tissues were fixed with 4% paraformaldehyde solution overnight, rinsed with PBS, embedded in paraffin blocks, cut into 4 /jm sections and placed on silanated glass slides. The sections were baked overnight at 60°C, deparaffinized using xylene and 49 rehydrated in graded alcohols. The tissues were permeabilized using 0.2N HCl, 2 X SSC, 20mM Tris/2mM CaCI containing 1ug/ml of proteinase K, and 0.25% acetic anhydride containing 0.1 M triethanolamine. The slides were then dehydrated using graded alcohols. Once the tissues were completely dried, 25 JL/L of the hybridization solution containing 100 ng/mL of the DIG-labeled sense or anti-sense probe was added to each section. The sections were then covered with glass coverslips and placed in a sealed humidified dish at 42°C overnight. Post-hybridization washes were preformed overnight using 50% formamide, 10mM Tris/1mM EDTA and 600mM NaCl in a 56°C rocking water bath, followed by several washes in 2xSSC. The slides were equilibrated in buffer 1 containing 0.15M NaCl and 0.1M Tris-HCI and blocked with 2% lamb serum. 100 pL of anti-DIG-AP (diluted 1:500 in buffer 1 containing 1% lamb serum and 0.1% Tween-20) was added per section and incubated 45 minutes at room temperature in a humidified chamber. The slides were washed with buffer 1 and were equilibrated to pH 9.5 in buffer 3. The alkaline phosphatase linked anti-DIG antibody was detected by incubation with NBT/BCIP substrate (Sigma fast tablets) for 24 hours at room temperature. The slides were counterstained with eosin and were examined with a light microscope for a positive reaction indicated by a blue-black colour. M. Infection of mice with CVB3 strains 3-4 week old male A/J mice were obtained from Jackson laboratories, Bar Harbor, Maine. The animal were allowed to acclimatize for a week before starting the experiments. 50 In the first animal experiment, 30 A/J mice were divided into 5 groups and injected intaperitoneally as follows: Group 1, the control group: 0.5 mL of sterile phosphate buffered saline (PBS) Group 2 105 PFU of (EM1) in 0.5 mL of PBS Group 3 10s PFU of EM1 in 0.5 mL of PBS containing 1:250 dilution of neutralising anti CVB3(RK) MAb. Group 4 105 PFU of the wild type CVB3(RK) strain in 0.5 mL of PBS. Group 5 105 PFU of CVB3(RK) in 0.5 mL of PBS containing 1:250 dilution of anti CVB3(RK) MAb. On days 3 and 7 post-infection 3 animals of each group were anaesthetized with sodium pentobarbital (60 mg per kg) and heart, spleen, kidney, pancreas, liver and lung were removed aseptically. The tissues were sectioned transversely into 2 pieces, one section was fixed with 4% PF for histopathology and ISH and the other was snap frozen in liquid nitrogen for plaque titration. In the second experiment, 5 animals were used per group, and the mice were injected with CVB3(RK), (EM1), or PBS as the control as before. The animals were sacrificed on days 3,6,9, and 11 post-infection for removal of organs as described before. To obtain serum, approximately 0.5 mL of blood was obtained by cardiac puncture from each animal. The serum was separated by centrifuging at 1000 rpm. 51 Undiluted and serial dilutions of the collected serum were titred by plaque assay. Except for these few modifications, the second experiment was carried out as before. N. Histology Organs fixed in 4% PF were sent to the histology laboratory in the Department of Pathology and Laboratory Medicine of the University of British Columbia to be cut into 3 /L/m sections using a Leica microtome. These sections were stained with haemotoxylin and eosin (H&E) and Masson's trichrome for pathological assessment. O. Histopathological interpretation The myocardial necrosis scoring was done on Masson's Trichrome stained slides with the help of Dr. Bruce McManus. Using this stain, myocytes stain pinkish-red and the collagen, cartilage and basic granules will stain purplish-blue. P. Plaque titration of virus in organs Snap frozen sections were homogenized in 1 mL of DMEM with an electric homogenizer (pellet pestle motor from VWR scientific of Canada). The homogenate was then centrifuged to remove the cell debris at 12,000g. The supernatant was stored at -70°C for plaque assay. 52 Q. Reverse Transcription Polymerase Chain Reaction (RT/PCR) 1. Preparation of Viral RNA CVB3(RK) or EM1 virus was prepared by sucrose gradient centrifugation as described previously. The pellet was then resuspended in 1.0 mL of Trizol Reagent (Canadian Life Technologies) to isolate total RNA from cells. The sample was incubated at room temperature for 5 minutes to allow the complete dissociation of nucleoprotein complexes. After adding 0.2 mLs of chloroform, the tube was centrifuged to form a lower red, organic phase, an interphase, and an upper colourless aqueous phase. The RNA in the upper phase was precipitated with 1:1 volume of isopropyl alcohol and centrifuged at 8000 rpm for 10 minutes. The pellet was washed with 75% ethanol, air dried, and dissolved in 50 uL of DEPC treated water. 2. First Strand Synthesis: cDNA synthesis was carried out using the superscript kit and random hexonucleotides ( pharmacia pd(N)6) as primers. In 25 tiL total volume: 14 //L of DEPC treated water, 5 /JL of 5X buffer (Superscript kit ), 2 fjL of 0.1 M DTT, 1 /JL containing 250 pmoles of pd(N)6 and 1 juL of Trizol purified RNA from either CVB3(RK) or (EM1) were mixed and denatured at 90°C for 1 minute. After the mixture was cooled to room temperature, 1JUL of RNAsin and Superscript II RT were added and the reaction mix was transferred to a 42°C water bath for 1 hour. 53 3. Polymerase Chain Reaction (PCR) PCR carried out with Taq DNA polymerase (Biolabs, NewEngland). Two primer sets (C1,C2 & C3,C4) were designed to amplify the NTR and the P1 regions of the genome respectively ( see figure 3.1) Figure 3.1 The final concentration of the reagents used in 50 juL total volume in the PCR reaction of the NTR region was as followed 1x PCR buffer, 0.2 mM of each dATP, dGTP, dCTP, and dTTP, 1.0 fiM primers C1 & C2, 1 unit of Taq DNA Polymerase and 1 uL of first-strand NTR product. The mixture was vortexed and centrifuged briefly to collect the sample at the bottom of the tube. An overlay of 50 tiL of mineral oil was added to the mixture to prevent evaporation. The PCR reactions were carried out in a minicycler (MJ Research). The amplification reaction parameters were as follows: 54 95°C for 60 sec 2 cycles of 95°C for 30 sec Denaturation 60°C for 30 sec Annealing 72°C for 60 sec Extension 25 cycles of 95°C for 30 sec Denaturation 72°C for 70 sec Annealing & Extension The PCR reaction constituents for the P1 structural region were the same as for the NTR region except that the C3 & C4 primers were used. The amplification conditions for the P1 structural region as follows: 95°C for 60 sec 25 cycles of 95°C for 30 sec Denaturation 60°C for 180 sec Annealing & Extension The result of the PCR reactions was determined by running 1 fiL of each sample on an agarose gel beside Hindlll-digested lambda markers. Bands of approximately 750 and 2500 bp regions were expected for the NTR and P1 region respectively. Once the desired DNA fragment was separated on an agarose gel, the band was purified using Gene Clean Kit (Bio 101, CA): The desired band from the agarose gel was excised from the gel, weighed and solubilized in Nal containing buffer for 10 minutes at 60°C. Nal also aids DNA binding to the glass milk. After incubating at room 55 temperature for 5 minutes the mixture was centrifuged for 5 seconds at room temperature to remove the excess buffer. The pellet was then washed three times with the "new wash buffer" from the kit and DNA was eluted by adding dH20, centrifuging for 1 minute to remove the glass milk and collecting the supernatant. The purified DNA was sent to Biotechnology Laboratory, NAPS Unit at University of British Columbia for sequencing using ABI's AmpliTaq Dye Terminator Cycle Sequencing chemistry. All four base reactions take place in a single tube on a thermal cycler, and the reagent is supplied as a premixed, single tube formulation, reducing the number of handling and pipetting steps required. AmpliTaq DNA Polymerase, FS (FS Taq), a mutant form of Taq DNA polymerase designed and developed specifically for fluorescent cycle sequencing with both dye-labelled primers and terminators. R. Statistical Analysis Data were analyzed by the student's t-test when comparisons were made between independent groups 1 5 3 . The student t-test was done at the level of 0.95 accuracy. Results were considered statistically significant when the p-values were less than 0.05. 56 6. Results and Discussion: I) Isolation of the Escape Mutant (EM1) Isolation of the EM1 mutant is described in Methods page 44. Briefly, the wild type virus CVB3(RK) was treated with a neutralising monoclonal antibody against coxsackievirus B3. After neutralisation of the predominant population of virions, selection of low numbers of non-neutralised mutant virus was allowed to occur by two "blind" passages of the supernatant medium to fresh cells in the presence of MAb. At this stage, cytopathology was apparent and the medium was collected for plaque titration (also in the presence of MAb). Virus present in selected plaques was collected and grown in the presence of MAb and the virus preparation that showed the most extensive CPE after 24 hours was selected as the Escape Mutant 1(EM1). A plaque assay was carried out to determine the ability of EM1 to grow in vitro and to compare its yield from Vero cells with the wild type strain CVB3(RK). The result (Table 6.1.1) shows that the EM1 strain grows to approximately 1/3 of the titre of CVB3(RK), although between different stock preparations, the EM1 titre ranged between 1/2 to 1/10 of the CVB3(RK) titre. It appears therefore that EM1 is mildly growth restricted relative to CVB3(RK). In addition, the titre of EM1 was not affected by the presence of neutralising MAb while the CVB3(RK) strain completely inhibited. 57 Table 6.1.1 Titres From Vero Cells Sample PFU/ml EM1 1.5 x 10 7 EM1+ MAb 1.3 X 10 7 CVB3(RK) 5.0 x 10 7 CVB3(RK)+ MAb 0 A comparison of virus yield of EM1 and CVB3(RK) strain in Vero cells by plaque assay. Both strains were grown in the presence and absence of MAb to confirm that CVB3(RK) was completely neutralised with the MAb while the growth of EM1 was not affected. In addition the results suggest that the EM1 is slightly growth restricted in comparison to CVB3(RK) in Vero cells. Associated with its reduced replication in Vero cells, the plaque size of EM1 is routinely much smaller than that found for CVB3(RK) (see Fig 6.1.1). 58 Figure 6.1.1. Plaque Morphology A. B. A c o m p a r i s o n o f p laque m o r p h o l o g y of CVB3(RK) or EM1 s t ra ins , s t a i n e d w i t h c o o m a s s i e b lue at 48 h o u r s pos t - i n fec t i on . As can be s e e n , t h e p laque s ize assoc ia ted w i t h EM1 (B) w a s m u c h sma l le r than that f o u n d w i t h t h e CVB3(RK) s t ra in (A). II. Pathogenesis of E M 1 in vivo To determine whether the mutation(s) in E M 1 was associated with an altered pathogenesis in mice, the course of disease in A/J mice was compared with that of the parental CVB3(RK) virus. This strain of mice was chosen due to its known susceptibility to CVB3-induced disease, particularly myocarditis. 59 A. Infection of A/J mice with C V B 3 Experiment 1 In the first experiment, 3-4 week-old A/J mice were divided into five groups of six animals and were injected intraperitoneally as follows. Group 1, PBS Group 2, 105 pfu (EM1) Group 3, 105 pfu (EM1)+ neutralising MAb anti CVB3 Group 4, 105 pfu CVB3(RK) Group 5, 105 pfu CVB3(RK)+ neutralising MAb anti CVB3 The MAb against CVB3 was included in Group 3 to prevent reversion of virus to wt+ characteristics. The effect of the MAb on wt+ CVB3(RK) was also assessed in group 5. On days 3 & 7 post-infection, 3 animals in each group were sacrificed and organs were removed and processed as described in methods for viral titration, in situ hybridization and histopathological studies. 2.1. Histopathology The histopathological interpretations were done using H&E and Masson's trichrome staining. The scoring of myocardial necrosis was done using an arbitrary scale of 1-5, where 1 indicates a low amount of necrosis (1-2 foci per section) and 5 indicates high amount of necrosis (> 20 foci). The results presented in table 6.2.1 are the mean of myocardial necrosis of 3 animals. 60 Early necrosis resulting from CVB3 infection causes direct damage (i.e. cytopathic effects, coagulation necrosis, contraction bands necrosis and vacuolation). Later during the course of infection, connective tissues such as collagen replace the damaged cells and calcification is observed. a) Pancreas: On day 3 post-infection, there was wide spread necrosis of the acinar cells of the pancreas in animals injected with either strain of the virus. The islets were not affected (see figure 6.2.1). Similar results were seen on day 7 post-infection. The results obtained from the histopathological studies agree with the viral titres of each strain detected in pancreas (see next section) and confirm that the mutant strain EM1 causes a similar amount of damage to pancreatic acinar cells as the CVB3(RK) strain (See table 6.2.1). Table 6.2.1 Scoring of Heart and Pancreas Organs Day 3 EM1 Day 3 RK Day 7 EM1 Day 7 RK Pancreas 4.0 4.0 5.0 5.0 Heart 0.5 1.0 1.0 3.0 Scored on an arbitrary scale of 1 -5. 61 Figure 6.2.1 Histology of Pancreas using Masson's Trichrome Staining Histology of the pancreas is indicated using Masson Trichrome stain. On day 3 post-infection a widespread necrosis of pancreas can be detected with both CVB3(RK) and EM1 strains (C & E respectively) as compared to the control pancreas (A). The undamaged areas are the insulin producing islet cells which are not infected by the CVB3(RK) strain. Similar results were found for day 7 tissues (D&F). 62 b) Heart: Table 6.2.1 summarizes the result of the myocardial necrosis observed in the animals injected with CVB3(RK) and EM1. On day 3 post-infection, the degree of myocardial necrosis of group 2 animals (injected with 105 pfu of EM1) was estimated to be less than 0.5 indicated by 1-3 dispersed, vacuolated cells in the heart section. Also on day 3 post-infection, the damage to heart observed in group 4 animals (injected with 105 pfu of CVB3(RK)) was estimated to be 1.0 indicated by 10-15 individual cells displaying cytopathology, or an occasional small focus in the heart section. On day 7 post-infection, the damage to heart of the animals injected with EM1 was about 1.0 on the arbitrary scale. However, in CVB3(RK) injected animals the degree of myocardial necrosis was estimated to be 3 to 3.5 indicated by large areas of necrosis (i.e. 5-6 large foci) (see figure 6.2.2 D&F arrows). In the control group injected with PBS and the group injected with the wt + virus in the presence of neutralising MAb, no sign of myocardial necrosis was observed on either day post-infection. 63 Figure 6.2.2. Histology of Heart by Masson's Trichrome Staining A. c. E. B. D . F. The degree of myocardial necrosis was assessed by Masson's Trichrome staining of heart tissue. On day 3 post-infection, individual infected cells showing vacuolation could be seen in CVB3(RK) infected animals (C). No viral cytopathology was observed with EM1 strain (E). By day 7 post-infection, large necrotic lesions were apparent with the CVB3(RK) strain (D). In contrast the number and size of lesions were much smaller for the EM1 strain (F). Panels A and B represent negative control heart on days 3 and 7 respectively. 64 c) Other organs: No cytopathology was detected in any other organ examined. 2.2. Viral titration in tissues: The results of the plaque assays calculated as the mean pfu/g tissue from the five animals in each group presented in figures 6.1.2- 6 as pfu/g tissue. Each data point is the mean value determined from the tissue of 3 animals.. a) Pancreas: Titre of virus/g of tissue are shown in figure 3. High titres of both CVB3(RK) and EM1 strain (> 106 pfu/g) were detected in pancreas and no significant difference was detected in the levels of either strain (p= 0.1). The injection of MAb with the EM1 had no effect on the titre of the EM1 variant, and no virus was present in the PBS control or the group injected with CVB3(RK) plus MAb. By day 7, no virus was detected in any of the animals (see figure 6.2.3). 65 Figure 6.2.3. CVB3 Titres in Pancreas Control EM1 EM1 + Ab wt wt + Ab H Day3 B Day7 A n i m a l s were sacr i f i ced on days 3 and 7 pos t in fec t ion . Plaque t i t ra t ion w a s ca r r ied ou t us ing the superna tan t f lu id of the h o m o g e n i s e d pancreas . A c c o r d i n g t o the resu l ts s h o w n CVB3(RK) and ENI1 g r o w t o a h igh t i t re (107) in the pancreas by day 3 pos t - in fec t ion . The d i f fe rence be tween the t w o s t ra ins w e r e not s ta t is t ica l ly s ign i f i can t (p= 0.1). No v i r u s w a s de tec ted in th is t i ssue in an imals sacr i f i ced on day 7. 66 b) Heart: Titres of virus in heart are presented in figure 6.2.4. The result is shown on a logarithmic scale. On day 3 post-infection there is about a hundred fold more virus detected with the wild type CVB3(RK) strain than with the EM1 strain (870pfu/g v.s. 1.8 pfu/g). Moreover, unlike other organs, on day 7 post-infection there is more virus present in the heart than on day 3, while the difference between the titre of CVB3(RK) and the EM1 is maintained at approximately a thousand fold (2300 pfu/g v.s. 21 pfu/g) see figure 6.2.4. Figure 6.2.4 CVB3 TITRES IN HEART I Day 3 I Day 7 Plaque t i t ra t ion w a s car r ied out on the superna tan t f l u id o f the h o m o g e n i z e d hear t s a m p l e s ob ta ined f r o m an ima ls sac r i f i ced on days 3 and 7 pos t - in fec t ion . The resu l t is p resen ted on a loga r i t hm ic sca le and s h o w s that ENI1 t i t res were app rox ima te l y 100 f o l d less than those of CVB3(RK) s t ra in in heart. More over , h igher t i t res were de tec ted on day 7 t han on day 3 in th is t i ssue . 67 c) Spleen: Figure 4 presents the titre of the CVB3 strains in spleen. Very low titres of infectious virus were detected in this tissue- approximately 4.6x 102 pfu/g and 1.3 x 10 c pfu/g EM1. However, in this case the difference in titre of about 50 fold between the strains is statistically significant. Again no virus was detected in the negative control or the group injected with RK in the presence of MAb (see figure 6.2.5). By day 7, virus had been cleared from the spleen. Figure 6.2.5 CVB3 TITRES IN SPLEEN CONTROL EM1 EM1+AB WT WT+AB UDAY3 U D A Y 7 Plaque t i t ra t ion w a s ca r r ied out on h o m o g e n i z e d sp leen s a m p l e s ob ta ined on days 3 and 7 post - in fec t ion. The resu l t s u g g e s t s that there is app rox ima te l y 50 t i m e s m o r e CVB3(RK) than EM1 in sp leen by day 3 pos t - in fec t ion . The v i r u s w a s c leared f r o m th is o r g a n by day 7 post -in fec t ion . 68 d) Liver: The titres of virus detected in liver on days 3 & 7 are shown in figure 6.2.6. The level of EM1 detected was approximately 5.5x 104 pfu/g tissue as compared with 6.5x 104 pfu/g for CVB3(RK). These figures are not significantly different (p= 0.1) indicating that the titres of the virus found in liver are essentially equal for both strains. Injection of the neutralising MAb with the CVB3(RK) strain completely inhibited viral growth in vivo. However in the case of EM1, the MAb did not significantly alter viral titres in liver. On day 7, no virus was detected in liver in any of the animals (see figure 6.2.6). Figure 6.2.6 A n i m a l s w e r e sacr i f i ced on days 3 and 7 pos t - in fec t ion . P laque t i t ra t ion w a s ca r r i ed ou t us ing the superna tan t f lu id f r o m h o m o g e n i s e d t i ssue . The resu l ts s u g g e s t that the EM1 and the CVB3(RK) s t ra ins g r o w to app rox ima te l y the s a m e t i t re (p=0.1) in l iver. 69 e) Lung: No virus was detected in the lung of any of the animals by plaque assay (data not presented). Table 6.2.2. Viral Titration in Tissues Organs* Day 3 EM1 RK Day 7 EM1 RK Pancreas 7 7.2 0 0 Heart 0.3 2.9 1.3 3.4 Spleen 1.1 2.7 0 0 Liver 4.7 4.8 0 0 * E x p r e s s e d as l og p f u / g t i s s u e 2.3. Detection of viral genome: Detection of viral genome in the tissues was carried out by in situ hybridization (ISH) using RNA probes (DIG-labelled) prepared from a plasmid (CVB3R1) which contains almost the entire genome of CVB3(RK). The probe labelling and quantification techniques have been described in the methods section. Both positive (sense) and negative (anti-sense) strand probes were used in order to detect both genomic RNA 70 and the replicative intermediate (negative strand). Presence of RI is used to determine whether virus is replicating in the tissue. A positive signal is indicated by the presence of bluish-black dots in the organ which is counterstained with eosin. The results of the ISH in heart and pancreas are summarised in table 6.2.3. Table 6.2.3 ISH scoring of Heart and Pancreas Organs Day 3 EM1 Day 3 RK Day 7 EM1 Day 7 RK Pancreas 4.0 4.0 0.5 0.5 Heart 0.5 1.0 1.0 3.0 Scored on an arbitrary scale of 1-5. 71 a) Pancreas: On day 3 post-infection, using the anti-sense probe which hybridizes to genomic RNA, a large amount of CVB3 genome was detected in the pancreas of the animals injected with CVB3 (RK) strain, the mutant (EM1) strain and the mutant in the presence of the MAb in agreement with the high levels of virus detected by plaque assay (See figure 6.2.7). Using the sense probe, negative strand (replicative intermediate) was detected in both the mutant and the wt+ stains. However the signal was not as intense as found with the antisense probe since there is approximately 100 fold less of the RI present than the positive strand genome in most productive infections (figure 6.2.7). Figure 6.2.7 Detection of CVB3 Genome in Pancreas by ISH 72 ISH w a s car r ied ou t t o s h o w the p r e s e n c e of CVB3(RK) and EM1 g e n o m e s o n day 3 pos t - i n fec t i on in t h e panc reas u s i n g an an t i -sense p robe . A pos i t i ve s igna l is i nd i ca ted by t h e b lack d o t s (A&C) . A pos i t i ve s igna l w a s a lso s e e n w i t h the s e n s e p r o b e fo r b o t h CVB3(RK) a n d EM1 (B&D respec t i ve ly ) t h i s i nd i ca tes that the rep l ica t ive in te rmed ia te is p resent . On day 7 post-infection, a low level of viral genome can be detected with the anti-sense probe in the animals with CVB3(RK) but the virus is now mostly cleared from the pancreas. The replicative intermediate was not detected indicating that even in wt+ samples, the virus is no longer replicating at detectable levels by day 7 (see figure 6.2.8). b) Heart: On day 3 post-infection single cells and a few small foci containing CVB3 (RK) genome were detected in the heart while even less signal was seen in the EM1 samples (See figure 6.2.9). By day 7 post-infection, more viral genome was detected with both sense and the anti- sense probes in the animals infected with CVB(RK) or EM1. However, much less virus was detected in EM1 than the CVB3(RK) strain (Figure 6.2.10). Thus, the result of the ISH suggests that firstly, the peak of infection in heart is later than other tissues tested where virus had been totally cleared by day 7. Secondly, that the escape mutant EM1 is associated with a much more restricted infection in heart than the CVB3(RK) strain. This may in part reflect a reduction in virus reaching the heart, but the small size of occasional foci seen also suggest a. restriction in growth of EM1 in the heart. Figure 6.2.8. Detection of CVB3 genome in Pancreas by ISH 74 ISH w a s car r ied ou t t o s h o w the p r e s e n c e of CVB3(RK) a n d EM1 g e n o m e s in panc reas o n day 7 pos t - i n fec t i on . A low level o f CVB3 (RK) and E M I g e n o m e s w e r e d e t e c t e d w i t h an t i - sense p r o b e (A&C) . N o s igna l w a s d e t e c t e d u s i n g the s e n s e p r o b e w i t h t h e w t + or EM1 s t r a i n s i nd i ca t i ng that t h e RI f o r m (negat ive s t rand ) w a s no longer p resen t at de tec tab le leve ls . 75 Figure 6.2.9. Detection of CVB3 Genome in Heart by ISH ISH s h o w i n g t h e p r e s e n c e of CVB3(RK) and EM1 g e n o m e s in heart by day 3 pos t - i n fec t ion u s i n g an an t i - sense p robe . A pos i t i ve s igna l is i nd i ca ted by the b lack d o t s . A s can be s e e n ind i v idua l ce l l s or s m a l l f oc i c o n t a i n i n g h igh leve ls o f g e n o m i c RNA w e r e f o u n d in CVB3(RK) i n fec ted an ima ls (A). O n l y rare ce l l s s h o w i n g a pos i t i ve s igna l c o u l d be s e e n w i t h E M I (C) . Co- in jec t i on o f v i r us w i t h neu t ra l i z ing M A b c o m p l e t e l y i nh ib i ted the in s i tu hyb r id i za t ion s igna l o f CVB3(RK) s t ra in (B) , but had n o ef fect on the in fec t ion by EM1 (D). 76 Figure 6.2.10. Detection of CVB3 Genome in Heart by ISH ISH s h o w i n g the p resence o f CVB3(RK) and EM1 g e n o m e s o n day 7 pos t - i n fec t ion u s i n g an an t i -s e n s e p robe . A pos i t i ve s igna l is i nd i ca ted by the b lack d o t s . A w i d e s p r e a d d i s t r i b u t i o n o f CVB3(RK) v i ra l g e n o m e w a s s e e n t h r o u g h o u t the heart (A) . A nomina l i nc rease in the a m o u n t of EM1 v i ra l g e n o m e w a s a lso o b s e r v e d c o m p a r e d t o day 3 pos t - in fec t ion , a s s o c i a t e d w i t h s m a l l d i s c r e t e foc i o f n e c r o s i s (C) . Co- in jec t ion of v i rus w i t h neu t ra l i s ing M A b c o m p l e t e l y i nh ib i ted t h e in s i t u hyb r id i za t i on s igna l o f CVB3(RK) s t ra in (B) , but had n o ef fect on EM1 (D) 77 c) Spleen: On day 3 post-infection, in both CVB3(RK) and EM1 infected animals, viral genome was detected in the germinal centres of lymphoid follicles using anti-sense probe. These centres contain primarily B cells surrounded by macrophages of the marginal zone. In general there were more infected follicles in spleens of mice infected with CVB3(RK) than EM1, and the staining was more intense. Presumably related to the 50-fold difference in viral titre in this organ found between the strains. (See Figure 6.2.11). No signal was found using the sense probe in either CVB3(RK) or EM1 infected spleens, consistant with being trapped in the spleen and not replicating, (pictures not presented). By day 7 post-infection, a few positive foci were detected in both CVB3(RK) and EM1 infected animals. However, no RI was picked up with the sense probe (pictures not presented). d) Liver: On day 3 post infection, a few small positive cells were detected in the animals injected with CVB3(RK) arid EM1 using the anti-sense probe. No RI was found. By day 7 post-infection, no viral genome was able to be detected with the anti-sense probe in either the wild type or EM1 indicating that the virus is cleared from the liver by day 7 post infection (pictures not presented) 78 Figure 6.2.11. Detection of CVB3 Genome in Spleen by ISH A. B. ISH w a s ca r r i ed ou t t o s h o w the p r e s e n c e o f CVB3(RK) a n d EM1 g e n o m e s o n d a y 3 pos t - i n fec t ion u s i n g an an t i - sense p robe . A pos i t i ve s igna l w a s i nd i ca ted by b lu i sh -b lack d o t s . Foc i c o n t a i n i n g v i ra l RNA g e n o m e w e r e d e t e c t e d w i t h t h e CVB3(RK) s t ra in (A) and sma l le r less in tense foc i w e r e d e t e c t e d w i t h t h e EM1 s t ra in (B). P resence of rep l ica t ive in te rmed ia te (RI) u s i n g t h e s e n s e p r o b e w a s not d e t e c t e d in t h e s p l e e n . N o ISH s igna l w a s d e t e c t e d o n day 7 pos t - i n fec t ion i n d i c a t i n g that the v i rus is c lea red f r o m t h e sp leen by th is t ime (P ic tu res not s h o w n ) . 2.4. Summary: The results indicate that both the wt+ CVB3(RK) strain and EM1 establish a systemic infection in A/J mice following intraperitoneal inoculation. However while the titres of the virus detected in pancreas and liver were not significantly different between the two strains, the levels of EM1 found in spleen and heart tissue were reduced 50 and 100 fold respectively. Injection of neutralising MAb at the same time as the virus completely inhibited infection with the CVB3 (RK) strain but had little effect on the replication and spread of EM1. The results obtained from plaque assay of the tissues correlated well with the ISH data and also the histopathological studies. Accordingly, EM1 strain is capable of growing in pancreas and liver to the same extent as the wt+ CVB3 (RK), but the growth of EM1 appears to be restricted in the spleen and heart. 80 Experiment 2: A second experiment comparing the pathogenesis of EM1 and CVB3(RK) in A/J mice was carried out with the following objectives: 1. To confirm the findings in the first experiment using more animals per group. 2. To extend the observation to a later time post-infection in order to determine whether EM1 produced a delayed disease in heart. 3. To obtain titres of virus in serum and therefore the level of viraemia with each strain. The experiment was therefore set up with five animals per group and injections were carried out as follows: Group 1: PBS Group 2 : 105 pfu of EM1 Group 3 : 105 pfu of CVB3(RK) On days 3,6,9, and 11 post-infection, 5 animals per group were sacrificed and heart, lung, spleen, pancreas, kidney, liver, and serum samples were removed for plaque assay, in situ hybridization and histopathological studies. Injection of the two virus strains with neutralising MAb was not included this time as there did not appear to be a problem with reversion of the EM1 to wt+ characteristics in experiment 1, and this enabled fewer animals to be sacrificed. 81 3.1 Histopathology: The histopathological examinations were done on tissues stained with Masson's Trichrome and H&E stains. The results are summarised in table 6.3.1. a) Pancreas: On day 3 post-infection the pancreas shows widespread necrosis but some un-damaged cells can still be detected with both virus strains (Figure 6.3.1) indicating that the course of infection is slightly delayed relative to the first animal experiment. On days 6, 9, and 11 post-infection the pancreatic acinar cells were largely destroyed while the islets of Langerhans were preserved. b) Heart: Scoring of the degree of heart necrosis was carried out as before on a 1-5 scale. The results are summarized in table 6.3.1. On day 3 post infection the degree of myocardial necrosis in EM1 infected hearts was less than 0.5 as the hearts looked almost normal with only very occasional individual damaged cells detected. On the same day the score for myocardial necrosis in CVB3(RK) infected animals was about 1 with higher number of sporadic, damaged cells observed in the heart. On day 6 post infection, the degree of myocardial necrosis in EM1 infected animals was estimated to be 1 with a few more individual cells displaying cytopathology but 82 little virus spread from cell to cell, while the score for CVB3(RK)-infected animals was estimated to be about 3 representing 5 or 6 foci of 10-20 necrotic cells. On days 9 and 11 post-infection it is easier to detect the damage to heart as the areas of necrosis become replaced with connective tissues such as collagen and are • readily distinguished by Masson's trichrome stain. However, the actual size of the affected foci was not substantially different from that observed on day 6 post-infection (See figure 6.3.2 for day 11; pictures of days 3, 6 and 9 post infection are not presented). Table 6.3.1 Scoring of pancreas and heart Organs DAY 3 Day 6 Day 9 Day 11 EM1 RK EM1 RK EM1 RK EM1 RK Pancreas 3.0 3.0 5.0 5.0 5.0 5.0 5.0 5.0 Heart 0.5 1.0 1.0 3.0 1.0 3.0 1.0 3.0 Scored on an arbitrary scale of 1 -5. Figure 6.3.1. Histopathology of Pancreas The d e g r e e of nec ros i s in panc reas is i nd i ca ted u s i n g M a s s o n ' s T r i c h r o m e s t a i n . O n d a y 3 pos t i n fec t ion c y t o p a t h o l o g y w a s s e e n a f fec t ing approx ima te ly 7 0 % o f t h e panc rea t i c ac inar ce l l s o f an ima ls i n jec ted w i t h CVB3(RK) (C) or EM1 (E) in c o m p a r i s o n t o t h e c o n t r o l panc reas (A & B ) . O n d a y s 6,9, a n d 11 post i n fec t ion t h e r e m a i n i n g ac inar ce l l s h a d b e e n d e s t r o y e d (D&F respec t i ve ly ) . Pancrea t i c is le ts w e r e not a f fec ted . The p ic tu res of d a y s 9 a n d 11 pos t - in fec t ion is p r e s e n t e d . 84 Figure 6.3.2 Histopathology of heart tissue The d e g r e e o f M y o c a r d i a l nec ros i s w a s s h o w n u s i n g M a s s o n ' s T r i c h r o m e s t a i n i n g . O n d a y s 3 pos t -in fec t ion i nd i v idua l i n fec ted ce l l s s h o w i n g vacuo la t i on c o u l d be s e e n in CVB3(RK) i n f e c t e d an ima ls (p ic tu re not p r e s e n t e d ) . N o v i ra l c y t o p a t h o l o g y w a s o b s e r v e d w i t h EM1 s t ra in (p ic tu re no t s h o w n ) . F r o m day 6 pos t - i n fec t i on large nec ro t i c l es ions w e r e apparen t w i t h t h e C V B 3 ( R K ) s t ra in . In con t ras t t h e n u m b e r a n d s ize of l es ions w e r e m u c h sma l le r for the EM1 s t ra in . The d e g r e e o f m y o c a r d i a l d a m a g e is eas ier t o v isua l i se o n d a y s 9 a n d 11 pos t - in fec t ion as the co l l agen d e p o s i t i o n o c c u r s in t h e nec ro t i c areas d e t e c t e d by pu rp l i sh -b lue s ta in ing w i t h t h e M a s s a o n ' s t r i c h r o m e s t a i n . The above p i c tu re is t h e resu l t of day 11 post in fec t ion in CVB3(RK) a n d EM1 s t ra ins ( A & B respec t i ve l y ) . 2.2. Virus titration in tissues The results of the plaque assays calculated as the mean pfu/g of tissue from the five animals in each group is summarized in table 6.3.2. Overall the results were found to agree very well with those described in Experiment #1. Table 6.3.2 Virus Titration in Tissues Organs Day 3 EM1 RK Day 6 EM1 RK Day 9 EM1 RK Day 11 EM1 RK Pancreas* 6.8 6.9 2.5 2.5 0 0 0 0 Heart* 1.0 2.6 2.3 4.4 0 1.4 0 0 Spleen* 4.3 5.7 0 0 0 0 0 0 SerurrT 3.3 3.7 0 0 0 0 0 0 * Expressed as log pfu/g x Expressed as log pfu/mL 86 a) Pancreas: The titres of both strains in pancreas on day 3 were about the same (~ 107) and similar to that detected in the first experiment. Day 3 appeared to be the peak of CVB3 replication in pancreas with very low titres of both strains (102 pfu) detected on day 6 post-infection and no virus found on any subsequent days (see figure 6.3.3). Figure 6.3.3 CVB3 TITRES IN PANCREAS Control EM1 RK MDAY3 MDAY6 MDAY9 • DAY11 Animals we re sac r i f i ced on days 3,6,9 and 11 pos t - in fec t ion . P laque t i t ra t ion w a s ca r r ied ou t us ing the superna tan t f l u i d f r o m h o m o g e n i s e d pancreas t i ssue. The resu l ts s u g g e s t tha t the EBI1 and CVB3(RK) s t ra ins g r o w t o approx imate ly the s a m e t i t re (p=0.2) in pancreas . O n day 6 pos t - in fec t ion v e r y l o w t i t res (10 2 ) o f bo th s t ra ins were de tec ted in the pancreas and no rep l i ca t ion w a s o b s e r v e d on the subsequen t 87 b) Heart: Similar titres were detected on day 6 to those found previously for day 7 and the 100 fold difference between EM1 and CVB3(RK) was again seen. However, importantly, this appeared to be the peak of viral infection in both cases and by day 9, no EM1, and only low titres of CVB3(RK) were detected in heart. Thus EM1 does not produce a delayed infection of heart, but a limited infection is quickly brought under control (see figure 6.3.4). Figure 6.3.4. CVB3 TITRES IN HEART A n i m a l s were sac r i f i ced on days 3,6,9 and 11 pos t - in fec t ion . Plaque t i t ra t ion w a s ca r r ied out us ing the superna tan t f l u id f r o m h o m o g e n i s e d heart t i ssue. The resu l t s u g g e s t s that the h ighes t t i t res were de tec ted on day 6 pos t - in fec t ion . A l s o the EM1 t i t re is a b o u t 100 f o l d l ower than CVCB3(RK) . On day 9, no EM1 and low levels o f CVB3(RK) w e r e de tec ted in the heart. This s h o w s that EM1 does not cause a late onse t d isease, 88 c) Spleen: The titres of both strains in spleen on day 3 were much higher (>104) than in the first experiment (101-102) but the 50 fold difference in titre between CVB3(RK) and EM1 was maintained. No infectious virus was detected in this tissue in animals sacrificed on later days (see figure 6.3.5). Figure 6.3.5. 60 50 40 30 20 10 CVB3 TITRES IN SPLEEN CONTROL I DAY 3 EM1 I DAY 6 CVB3(RK) I day9 I I dayl 1 A n i m a l s were sac r i f i ced on days 3,6,9 and 11 pos t - in fec t ion . P laque t i t ra t ion w a s ca r r i ed ou t us ing the superna tan t f l u id f r o m h o m o g e n i s e d sp leen. The t i t re o f CVB3(RK) in sp leen is approx ima te l y 50 f o l d h igher than EW11. No in fec t ious v i r u s w a s de tec ted in the sp leen on later days pos t - in fec t ion . d) Serum: In animals sacrificed on day 3 post infection, the titres of EM1 found in serum were 2x103 pfu/mL while those of CVB3 (RK) were about 6x103 pfu/mL (see figure 6.3.6). Therefore there is a higher level of viraemia in the animals infected with wt+ virus probably reflecting the impaired ability of EM1 to replicate, even in cultured cells. However, the three-fold difference in serum titre does not explain either the 50-fold reduction of EM1 in spleen or the 100 fold reduction in heart tissue. No virus was detected in serum with either strain on day 6, 9, or 11 post-infection. This indicates that the virus becomes viraemic in early stages after infection and is quickly cleared from the blood stream. The peak of infection in the serum has been determined by Anderson (1996) to be day 2 or day 3 post-infection. 90 igure 6.3.6. CVB3 TITRE IN SERUM Control EM1 ! DAY3 MDAY6 M DAY9 I \ DAY 11 A n i m a l s were sacr i f i ced on days 3,6,9 and 11 pos t - in fec t ion . Plaque t i t ra t ion w a s p e r f o r m e d on s e r u m s a m p l e s as d e s c r i b e d in the m e t h o d sec t i on . On day 3 pos t - in fec t ion , the t i t re of CVB3(RK) w a s 2-3 f o l d h igher than EWI1 (p> 0.1). No in fec t ious v i r u s w a s de tec ted on s u b s e q u e n t days w h i c h means that the v i rus b e c o m e s v i r a e m i c in ear ly s tages after in fec t ion and is q u i c k l y c leared f r o m the b l o o d s t ream. 91 2.3. Detection of viral genome The amount of viral genome detected by in situ hybridization on tissues from the second experiment correlated with the results of the plaque assay done on these organs and generally confirmed the results from the first in vivo experiment. The results of the ISH can be summarised in table 6.3.3. as follows: Table 6.3.3 ISH Scoring of heart and pancreas Organs DAY 3 Day 6 Day 9 Day 11 EM1 RK EM1 RK EM1 RK EM1 RK Pancreas 3.0 3.0 1.0 1.0 0 0 0 0 Heart 0.5 1.0 1.0 3.0 0 0 0 0 S c o r e d o n an arb i t rary sca le of 1-5. a) Pancreas: On day 3 post-infection the ISH results obtained from pancreatic tissue was similar in EM1 and CVB3(RK) injected animals. Large areas of the tissue gave a strong signal indicating massive viral growth in acinar cells. However, in contrast to the first animal experiment, some areas of pancreas were still uninfected. 92 On day 6 post-infection viral genome was detected in the remaining areas of the pancreas such that the whole tissue was now involved with both stains. These results suggest that the peak of infection in the pancreas was later than day 3 in this experiment, and that the infection progressed at a slower rate than in the first experiment. No virus was detected on either day 9 or 11 with either strain in the pancreas suggesting that by day 9 the damage has been done and the virus is cleared (pictures not presented). b) Heart: On day 3 post-infection the result of the ISH in heart of the animals injected with EM1 showed a maximum of 1 or 2 positive cells detected with the anti-sense probe, and about 10-20 positive cells in the animals injected with the CVB3 (RK) strain. On day 6 post-infection 4 or 5 individual positive cells and an occasional small focus of 5-10 positive cells were detected in the EM1 injected animals. In contrast in the animals injected with CVB3 (RK) strain 5 or 6, large foci involving over 20 cells were detected in addition to individual positive cells. No virus was detected in the heart of the animals injected with EM1 on either day 9 or 11 indicating that EM1 does not cause a late-onset disease. An occasional positive cell could still be detected in the CVB3(RK) animals on day 9 post-infection, but the virus had generally been cleared by this time. 93 These results confirmed the ones obtained from the first experiment in that while the CVB3(RK) strain is cardiotropic, the growth of the EM1 strain is very restricted in heart, giving 100 fold less titres than CVB3(RK). Also, the peak of infection of heart by the virus is about day 6 or 7 post-infection and the virus is cleared from the heart on later days. c) Spleen On day 6 post infection one or two small foci were detected in the spleen of the animals injected with EM1. In contrast in the animals injected with the CVB3 (RK), 5 or 6 strong foci of positive signal were observed within the germinal centres. This result confirms the result of the plaque assay where the titre of the EM1 was approximately 50 fold lower than that of the CVB3(RK). No viral genome was detected with either strain on days 9, or 11 post-infection. 3.4 Summary The result of the second in vivo experiment confirmed and extended those found in the first. Most importantly, EM1 was found to be restricted in growth in heart tissue while growing to similar titres to CVB3(RK) in pancreas and liver. Related to this, myocardial necrosis was much reduced with smaller and fewer lesions indicated by the histological studies, and also, less viral genome and replicative intermediate detected by the ISH. In addition, EM1 was shown not to produce a late on-set disease in heart and in fact was eliminated from this tissue by day 9 when low levels 9 4 of CVB3(RK) could still be detected. Finally serum titres showed that the level of viraemia with EM1 was about one-half that found with CVB3 (RK). Conclusion: The results from the two in vivo experiments on the pathogenesis of CVB3 (RK) and EM1 in A/J mice indicate that while both strains establish a widespread infection in A/J mice there is variation in the degree of involvement of certain tissues. More specifically, viral titres in liver and pancreas are similar while those in spleen and heart are significantly different. Histopathological examination of pancreas indicates that both strains cause massive acinar cell necrosis while islets of Langerhans are preserved, a notable difference from some CVB4 strains. In the first animal experiment, levels of virus in spleen were so low (< 102 pfu/g tissue) that the 50 fold difference in titre between EM1 and CVB3(RK) should be viewed cautiously. However the repeat experiment showed a similar 50 fold reduction in EM1 although the titres were > 104 indicating that the difference is genuine and significant. Why such a large variation of infection levels of virus in spleen on day 3 is found between experiments is not clear as levels of virus in heart and pancreas were comparable. It is possible that in the first experiment, the peak of infection of spleen occurred on day 2 and had rapidly diminished by day 3 when the animals were sacrificed. In the case of heart tissue, the hundred fold reduction in titre of EM1 relative to CVB3(RK) on both days 3 and 7 is highly significant and may be due to a number of different reasons. Firstly, the amount of virus which gains access to the heart may 95 differ between the strains, related to factors such as serum titres and ability to cross the endothelial barrier, etc. Secondly, EM1 may be less able to establish an infection in heart tissue. This could be due to a number of different reasons. Firstly, the viral attachment protein in EM1 virions may have a lower affinity for the cellular receptor on the heart and spleen cells in comparison to the CVB3(RK) strain. Secondly, the restriction may be in viral replication; a number of cellular factors are required for viral replication and EM1 may have less affinity for these than CVB3(RK). This includes factors required for viral translation or RNA replication. Thirdly, EM1 replication may be restricted at the stage of assembly and egress. This would restrict the virus to only one round of replication resulting in lower viral titres and less damage in these organs. It is likely that several factors are important as the number of foci of infection of EM1 in heart is diminished, suggesting reduced access but in addition the foci remain small in size relative to the areas of necrosis produced by CVB3 (RK) indicating that EM1 replication and spread in heart tissue is also restricted. 96 4. Characterisation of EM1 and CVB3(RK) In Vitro After characterizing the pathogenesis of (EM1) in vivo, a number of experiments were characterised to compare its characteristics in vitro with the wild type CVB3 (RK) strain. 1. Temperature Sensitivity Assay The ability of both strains to grow at 36°, 39°, and 40°C was determined by a plaque assay on CVB3(RK) and EM1 stocks carried out at the 3 different temperatures. The plaque assay was done as described previously except that after adsorption at 36°C, the samples were incubated for 48 hours at the appropriate temperature. The plaques were then counted and are presented in figure 6.4.1. According to the result, the titre of both strains at 39°C was essentially the same as at 36°C. In contrast at 40°C the titre of EM1 was reduced by 4 logs while that of the CVB3(RK) strain reduced only about 10-fold indicating that the EM1 is more sensitive to elevated temperatures than CVB3(RK). Since EM1 is an antibody escape mutant, there is a high possibility that its structural gene region is modified and this change leads to a less stable capsid structure at higher temperatures. 97 Figure 6.4.1 Plaque assay on the same s t o c k o f EM1 and CVB3(RK) s t ra ins were ca r r i ed ou t at 36*C, 39 C and 40°C t o de termine tempera tu re sens i t i v i t y in V e r o ce l ls . P laques w e r e s ta ined at 48 hours pos t - in fec t ion . As can be seen, the t i t re of CVB3(RK) is app rox ima te l y the s a m e at 36°C and 39°C and Is reduced about ten f o l d at 40°C. EWI1 t i t re is a lso s tab le at 39°C bu t it is reduced by 4 logs a t 4 0 ° C . 2. CVB3 decay curve To examine further the relative stabilities of CVB3(RK) and EM1, an experiment was done to compare the decay rate of the two strains. Simply, a sample of each virus was incubated at 37°C incubator and a small aliquot was removed from each sample on days 1 to 6 post infection and frozen at -70°C. The infectivity present in each of these aliqouts for each strain up to 6 days post infection was measured by plaque assay. The result of the decay curve is presented in figure 6.4.2.. According to this figure, at 37°C after 72 hours incubation, the reduction in titre of CVB3(RK) is about 5-fold while that of EM1 is about 15-fold. Similarly, by the 6th day CVB3(RK) titre was reduced by 50-fold as opposed to a thousand-fold reduction for EM1. This indicates that EM1 is less stable at 37°C and also that CVB3(RK) retains remarkable levels of infectivity on prolonged incubation at this temperature (see figure 6.4.2). 99 Figure 6.4.2. T o de te rmine the s tab i l i ty of CVB3(RK) and ENI1 the decay rate of the t w o s t ra ins at 37°C w a s e x a m i n e d A s a m p l e of each v i r u s w a s incubated at 3 7 ° C and a sma l l a l iquot w a s r e m o v e d on days 1 t o 6. Plaque t i t ra t ion w a s car r ied ou t on the s a m p l e s . The resu l ts s h o w that by day 6 the t i t re of EWI1 w a s reduced by a 1000 f o l d as o p p o s e to a 50 f o l d reduc t ion fo r CVB3(RK) . This ind icates that EM1 is less s tab le than CVB3(RK) at 37°C. 100 3. Viral growth curves in Vero, J774A.1, EL-4, and Wehi 231 cells The ability of Vero cells and three lymphoreticular cell-lines to support the replication of the CVB3(RK) and EM1 stains was examined in growth curve experiments. The different lymphoreticular cell-lines were used in order to determine their potential ability to play a role in dissemination of CVB3 to its target organs. The cells were infected with 20 pfu/cell of each strain of the virus and washed thoroughly 5-6 times after 1 hour of absorption at 37°C. At 2,4,6,8,10,12 and 24 hours post infection, supernatant medium containing virus was collected, the cells were washed up to 5 times and were scraped into non-supplemented DMEM and stored at -70°C. Each of these experiments was done in triplicate, and the amount of virus in both intracellular and supernatant fractions was determined by plaque titration in order to determine the intracellular and extracellular CVB3 titres at each time point. Vero cells: The titres of each strain detected in the supernatant medium or cell lysate of Vero cells is presented in figures 6.4.3 and 6.4.4 respectively. As can be seen in fig 6.4.3, release of progeny virus commences around 4 hours post-infection for both strains. In addition, the EM1 strain is slightly growth restricted relative to the CVB3(RK) strain in Vero cells (p= 0.1). The peak of virus release for both strains is at 8 hours post-infection. Figure 6.4.3. Growth Curves of EM1 and CVB3(RK) strains in Vero cells Virus in Supernatant Medium 1,000 800 600 P F U / m l x IO 3 400 200 0 2 4 6 8 10 12 24 Hours Post infection CVB3(RK) "*• CVB3(EM1) The titre of virus released from Vero cells over 24 hours was measured for each strain. The result suggests that EM1 is slightly growth restricted relative to CVB3(RK). The peak of virus release is at 8 hours post-infection with both strains. 102 In figure 6.4.4, the intracellular titre of both CVB3 strains can be seen to be higher than the titre of the virus found in the extracellular medium. Again the peak of infection is at 8 hours post-infection with each strain and therefore the reduction in viral replication and release is due to massive cytopathology in the cultures when infected at high multiplicity. Figure 6.4.4. Growth Curves ofEM1 and CVB3(RK) in Vero cells Intracellular virus titres 2.000 6 8 10 12 Hours Post Infection — CVB3(RK) CVB3(EM1) Intracel lu lar v i r u s t i t res in cel ls harves ted at 2,4,6,8,10,12 and 25 hours pos t - in fec t ion were m e a s u r e d Plaque t i t ra t ion w a s ca r r ied ou t t o de te rm ine the in t racel lu lar v i ra l t i t res . The resu l ts sugges t that the in t racel lu lar t i t res of bo th s t ra ins of CVB3 are h igher than the t i t res of the v i rus f o u n d in the ext race l lu lar m e d i u m . ENI1 is s l igh t ly g r o w t h res t r i c ted re lat ive t o w t and the peak of v i r us p r o d u c t i o n is at 8 hours pos t - in fec t ion w i t h bo th s t ra ins . 103 J774A.1 cells: The results of the viral growth curves in the macrophage cell line, J774A.1, are presented in figures 6.4.5 and 6.4.6 for supernatant and intracellular virus. According to these results, both strains of the virus grow to a lower titre in the J774A. 1 in comparison to the Vero cells. Again the escape mutant EM1 appears to be slightly growth restricted relative to the CVB3(RK) strain (p= 0.1). The peak of virus release was at 8hrs post-infection giving yields of 104-105 pfu/ml , 10-100 fold less than the 108-107 pfu/ml obtained in Vero cells. In addition, intracellular titres are higher than titres of released virus. The drop in virus titre at 24 hours post-infection in J774A.1 cells, is not due to cell death (as with Vero cells), but to clearance of virus by the macrophages which do not show signs of cytopathology. 104 Figure 6.4.5. Supernatant titres of virus released from J774A.1 cells 812,4,6,8,10,12 and 24 hours post-nfection. The result suggests that EM1 is slightly growth restricted relative to the wt*. The peak of virus release is at 8 hours post-infection giving yields of 104-10s pfu/ml. Figure 6.4.6. 105 Growth Curves of EM1 and CVB3(RK) in J774A. 1 cells Intracellular virus titres 50 Hours Post Infection ' CVB3(RK) * CVB3(EM1) In t racel lu lar v i rus t i t res in in fected J774A.1 ce l ls were d e t e r m i n e d at 2,4,6,8,10 and 24 hours pos t - in fec t ion for CVB3(RK) and EM1 s t ra ins . The resu l ts s h o w tha t J774A.1 cel l s u p p o r t a low level of rep l icat ion relat ive to Vero cel ls of b o t h CVB3(RK) and E M 1 . The peak t i t re is at 8 h o u r s pos t - in fec t ion . 106 Wehi 231 cells: The result of the viral growth curves in the B-cell line, Wehi 231, is presented in figures 6.4.7 and 6.4.8 for supernatant virus and intracellular virus respectively. The results show that the CVB3(RK) strain grows to approximately the same titre as in J774A.1 cells. In contrast titres of EM1 are approximately 10 fold lower than the CVB3(RK) strain in Wehi cells. The peak of infection, however, is still at 8 hours post-infection with either strain of the virus. Figure 6.4.7 Growth Curves ofEM1 and CVB3(RK) in Wehi 231 cells Virus in Supernatant Medium 100 — PFU/ml x 103 4 6 8 10 12 24 Hours Post Infection "*~ CVB3(RK) " CVB3(EM1) Growth of CVB3(RK) and EM1 in the B-cell line Wehi 231 was examined over 24 hours. The CVB3(RK) strain was shown to replicate to high levels in this cell line while the EM1 strain showed essentially no rise in supernatant virus titre. 107 Similar results were found with the intracellular titres of each strain indicating that the repliacation of E M 1 in B-cells is significantly restricted. Figure 6.4.8. Growth Curves of EM1 and CVB3(RK) strains in Wehi 231 cells Intracellular Virus titres 140 PFU/ml x 103 2 4 6 8 10 12 24 Hours Post Infection CVB3(RK) -~ CVB3(EM1) G r o w t h of CVB3(RK) and EM1 in the B-cell l ine W e h i 231 w a s m e a s u r e d over 24 hours post-in fec t ion . A low level o f rep l i ca t ion of CVB3(RK) w a s de tec ted peak ing at 8 hours but essent ia l ly no rep l i ca t ion of E M I w a s o b s e r v e d . 108 EL-4 cells Viral growth curves in T-cell line, EL-4, showed that neither strain of the virus was capable of infecting EL-4 cells. This observation agrees with the findings of other researchers (Huber et al.) 1990 who reported no CVB growth in murine T-cells. Data is presented in figures 6.4.9 and 6.4.10 for the supernatant medium and the intracellular virus respectively. Figure 6.4.9 Growth Curve experiment in EL-4 cells Supernatant Medium PFU/mlx 102 8 10 Hours Post Infection G r o w t h of C V B 3 ( R K ) and EM1 s t ra ins in the mur ine T-cell l ine, EL-4, w a s de te rm ined ove r 24 hours . The resu l ts sugges t that nei ther s ta in of the v i r u s w a s capab le of in fec t ing EL-4 T-cell l ine. 109 Figure 6.4.10. Growth Curves of CVB3(RK) and EM1 in EL-4 cells Intracellular PFU/mlx 102 t -4 6 8 10 12 24 Hours Post Infection Intracel lu lar v i r us t i t res o f CVB3(RK) and EW11 were m e a s u r e d over 24 hours in mur ine EL-4 cel ls (T-cell l ine). No g r o w t h of e i ther s t ra ins were detec ted. 110 5. Sequence Analysis of the two CVB3 variants: The final series of experiments was to compare the sequence of two regions (NTR & P1) of the genome of EM1 relative to the CVB3(RK) strain. Since the escape mutant was selected using a neutralizing monoclonal antibody, we predicted that a mutation had occurred in the proteins of the P1 structural region of the genome which contain the neutralizing epitopes for antibody binding. Using this rationale, the structural region of both strains were sequenced and compared using Reverse Transcriptase PCR (RT/PCR) sequencing. In addition, research by others has identified two mutations associated with lack of cardiovirulence in CVB, and so these regions were also compared. More specifically, Tracy et al. (1994) identified a U/C mutation at position 234 of the non translated region (NTR) of a natural non-cardiovirulent isolate of CVB3 (CVB3J and a cardiovirulent CVB3m strain 1 2 3 . In addition, Huber et al. (1991) have isolated an Ab-escape mutant of CVB3 (H310A1) that was less myocarditic and contained a single mutation in nucleotide 1442 of the VP2 protein resulting in an asparagine to asparatate change in the mutant. Since our EM1 mutant is also less cardiovirulent than its parental strain, the NTR region in addition to P1 was sequenced for both strains. The sequencing was carried out at the Biotechnology Labs at UBC using FS Taq and ABI's AmpliTaq cycler. The sequencing was done in both directions, or at least twice in one direction using different primers and the results are shown in figures 6.5.1- 6.5.4. 111 NTR region The results show that both the CVB3(RK) and EM1 strains contain a uracil at position 234 of the NTR region. Thus the mutation identified by Tracy et al. (1994) as the difference between their myocarditic and amyocarditic strains was not observed with EM1. There is however a C to U mutation at position 448 of the NTR region of EM1 which is in stem loop F at the start of the IRES region in the predicted secondary structure shown in fig 6.5.1 of the introduction. P1 region VP4: No mutations were observed in the VP4 protein which is the most highly conserved of the structural proteins. Also since VP4 is hidden inside the icosohedral capsid and is therefore not available on the surface for binding of neutralising antibody, the method used for isolation of EM1 would not be expected to select for mutations in this protein. VP2: Two point mutations were observed in VP2. The first mutation was at position 1329 from the 5' end of the genome that results in a silent mutation and is therefore unimportant. The second mutation occurred at position 1421 resulting in an amino acid change from lysine (K) to arginine (R). As these are both basic amino acids, this mutation is considered to be conservative (see figure 6.5.2). 112 VP3: Three point mutations were detected in the VP3 capsid protein. At position 2225, a serine to threonine amino acid change is detected (conservative). A silent mutation was observed at position 2286, and finally, a valine to leucine amino acid mutation was observed at position 2332 which again is considered a conservative mutation. Therefore, none of the mutations in the VP3 protein of the EM1 capsid are likely to be integral to changing the properties of the mutant (see figure 6.5.3). VP1: The VP1 protein of the P1 structural region, contained four point mutations and a deletion mutation. The first point mutation was observed at position (2554) resulting in a glutamic acid to glutamine (E to Q) amino acid change. The second point mutation was observed at position 2718 and results in a silent mutation. The third and fourth point mutation are conserved mutations resulting from valine to alanine (2723) and serine to threonine (2986) amino acid changes respectively. The addition-deletion mutation was observed at position 2800 where an ACG TTT was mutated to CAC GTT which changed the encoded amino acids from threonine-phenyl alanine (T-F) to histidine-valine (H-V) (see figure 6.5.4). 11 Figure 6.5.1 NTR region NTR 1 UUAAAACAGC CUGUGGGUUG AUCCCACCCA CAGGCCCAUU GGGCGCUAGC ACUCUGGUAU 61 CACGGUACCU UUGUGCGCCO GUUUUAUACC CCCUCCCCCA ACUGUAACUU AGAAGUAACA 121 CACACCGAUC AACAGUCAGC GOGGCACACC AGCCACGUUU OGAOCAAGCA CUUCUGUUAC 181 CCCGGACUGA GUAUCAAUAG ACUGCUCACG CGGUUGAAGG AGAAAGCGUU CGUUAUCCGG 241 CCAACUACUU CGAAAAACCU AGUAACACCG UGGAAGUUGC AGAGOGUUUC GCUCAGCACU 301 ACCCCAGUGa AGAUCAGGUC GAUGAGUCAC CGCAUUCCCC ACGGGCGACC GUGGCGGUGG 361 CUGCGUUGGC GGCCUGCCCA UGGGGAAACC CAOGGGACGC UCUAAUACAG ACAUGGUGCG 421 AAGAGUCUAU UGAGCUAGUU GGOAGUCCUC CGGCCCCOGA AUGCGGCUAA OCCOAACOGC U 481 GGAGCACACA CCCUCAAGCC AGAGGGCAGU GUGUCGUAAC GGGCAACUUO GCAGCGGAAC 541 CGACUACUUU GGGOGUCCGU GUUUCAUUUU AUUCCUAUAC UGGCUGCUUA UGGUGACAAU 601 OGAGAGAUOG UUACCAUAUA GCOAUUGGAO UGGCCAUCCG GUGACUAAUA GAGCUAUUAU 661 AUAaCCCOUU GUUGGGUUUA OACCACUUAG CUUGAAAGAG GUUAAAACAU UACAAOUCAO 721 UGUUAAGUUG AAOACAGCAA A The NTR region of the EM1 is compared to that of the CVB3(RK) using RT/PCR sequencing (see method section for details). The first line corresponds to the CVB3(RK) strain and the dotted line shows the EM1 strain. Note that the mutations In EM1 are Indicated by bolded letters. 114 V P 2 capsid Protein VP 2 950 UCCCCCACAG UAGAGGAGUG CGGAUACAGU GACAGGGCGA GAUCAAUCAC AUUAGGUAAC 1011 UCCACCAUAA CGACUCAGGA AUGCGCCAAC GUGGUGGUGG GCUAUGGAGU AUGGCCAAAU 1071 UAUCUAAAGG AUAGUGAGGC AACAGCAGAG GACCAACCGA CCCAACCAGA CGUUGCCACA 1131 UGUAGGUUCU AUACCCUUGA CUCUGUGCAA UGGCAGAAAA CCUCACCAGG AUGGUGGUGG 1191 AAGCUGCCCG AUGCUUUGUC GAACUOAGGA CUGUUOGGGC AGAACAUGCA GUACCACUAC 1251 UUAGGCCGAA CUGGGUAUAC CGUACAUGUG CAGUGCAAUG CAUCUAAGUU CCACCAAGGA 1311 UGCUUGCUAG UAGUGUGUGU ACCGGAAGCU GAGATGGGUU GCGCAACGCU AGACAACACC 1371 CCAUCCAGUG CAGAAUUGCO GGGGGGCGAU ACGGCAAAGG AGUUUGCGGA CAAACCGGUC 1431 GCAUCCGGGU CCAACAAGUU GGUACAGAGG GUGGUGUAUA AOGCAGGCAU GGGGGUGGGU 1491 GOUGGAAACC UCACCAUUUU CCCCCACCAA UGGAUCAACC UACGCACCAA UAAUAGUGCU 1551 ACAAUUGUGA UGCCAUACAC CAACAGOGUA CCUAUGGAUA ACAUGUUUAG GCAUAACAAC 1611 GUCACCCUAA UGGUUAUCCC AUUUGUACCG CUAGAUUACU GCCCUGGGUC CACCACGUAC 1671 GOCCCAAUUA CGGUCACGAU AGCCCCAAUG OGUGCCGAGU ACAACGGGUU ACGUUUAGCA 1731 GGGCACCAG Figure 6.5.2 The VP2 region of EM1 is compared to the CVB3(RK) strain using RT/PCR sequencing (see method section for detail). The mutations In the EM1 (dotted line) are indicated by bold letters. 115 Figure 6.5.3 VP3 Capsid Protein VP 3 1740 GGCUUACCAA CCAUGAAUAC UCCGGGGAGC UGUCAAUUUC UGACAUCAGA CGACUUCCAA 1800 UCACCAUCCG CCAUGCCGCA AUAUGACGUC ACACCAGAGA UGAGGAUACC UGGUGAGGOG I860 AAAAACUUGA UGGAAAUAGC UGAGGUUGAC UCAGUUGOCC CAGCJCCAAAA UGUUGGAGAG 1920 AAGGUCAACU CUAUGGAAGC AUACCAGAUA CCUGUGAGAU CCAACGAAGG AUCUGGAACG 1980 CAAGUAUUCG GCUOUCCACU GCAACCAGGG UACUCGAGUG UUUUUAGUCG GACGCUCCUA 2040 GGAGAGAUCU UGAACUAUUA UACACAUUGG UCAGGCAGCA UAAAGCUUAC GUUUAUGUUC 2100 UGUGGUUCGG CCAUGGCUAC UGGAAAAUUC CUUUUGGCAU A C U C A C C A C C AGGUGCUGGA 2160 GCUCCUACAA AAAGGGUUGA UGCCAUGCUU GGUACUCAUG UAAUUUGGGA CGUGGGGCUA 2220 CAAUCAAGUU GCGUGCOGUG UAUACCCUGG AUAAGCCAAA C A C A C U A C C G GUUUGUUGCU 2280 UCAGAUGAGU AUACCGCAGG GGGUUUUAUU ACGUGCUGGU AUCAAACAAA CAUAGUGGUC 2340 CCAGCGGAUG CCCAAAGCOC CUGUUACAUC AUGUGUUUCG UGUCAGCAUG CAADGACUUC 2400 UCUGUCAGGC UAOUGAAGGA CACUCCrjUUC AUUUCGCAGC AAAACOOUUU CCAG The VP3 region of EM1 is compared to the CVB3(RK) strain using RT/PCR sequencing (see method section). The mutations in EM1 (dotted line) are shown in bold letters. Figure 6.5.4 VP1 Capsid Protein V P l 2455 GGCCCAGUGG AAGACGCGAU AACAGCCGCU ADAGGGAGAG UUGCGGAUAC CGUGGGUACA 2515 GGGCCAACCA ACUCAGAAGC UAUACCAGCA CUCACUGCUG CUGAGACGGG UCACACGUCA 2575 CAAGUAGUGC CGGGUGACAC UAUGCAGACA CGCCACGUUA AGAACUACCA UUCAAGGUCC 2635 GAGUCAACCA U AG AG AAC UU CCUAUGUAGG UCAGCAUGCG UGUACUUUAC GGAGUAUAAA 2695 AACUCAGGUG CCAAGCGGUA UGCUGAAUGG GUAUUAACAC CACGACAAGC AGCACAACUU 2755 AGGAGAAAGC UAGAAUUCUU UACCUACGUC CGGUUCGACC UGGAGCTG ACGUUUGUCAUA 2815 ACAAGUACUC AACAGCCCUC AACCACACAG AACCAAGAUG CACAGAUCCU A A C A C A C C A A 2875 AUUAUGUAUG UACCACCAGG UGGACCUGUA CCAGAUAAAG UUGAUUCAUA CGUGUGGCAA 2935 ACAUCUACGA AUCCCAGUGU GUUUUGGACC GAGGGAAACG CCCCGCCGCG CAUGUCCAUA 2995 CCGUUUUUGA GCAUUGGCAA CGCCUAUUCA AAUUUCUAUG ACGGACJGGUC UGAAUUUUCC 3055 AGGAACGGAG UUUACGGCAU CAACACGCUA AACAACAUGG GCACGCUAUA UGCAAGACAU 3115 GUCAACGCUG GAAGCACGGG UCCAAUAAAA AGCACCAUUA GAAUCUACUU CAAACCGAAG 3175 CAUGUCAAAG CGUGGAUACC UAGACCACCO AGACUCUGCC AAU AC GAG A A GGCAAAGAAC 3235 GTGAACUUCC AACCCAGCGG AGUOACCACU AC UAGGC AAA GCAUCACUAC AAUGACAAAU 3295 ACGGGCGCAU UU The VP1 sequence of EM1 was compared to the CVB3(RK) strain by RT/PCR sequencing (see method section). The mutations in the EM1 strain (dotted line) are indicated in bold letters. 117 Sequence analysis of virus isolated from hearts of EM 1-infected animals In order to confirm that the virus present in the heart of EM1 infected animals was identical to EM1, virus from heart tissue was also subjected to sequence analysis. As most of the mutations in EM1 were found in VP1, this region was amplified by RT/PCR for sequence analysis. All the mutations found in EM1 were also present in EM1/heart indicating that the genotype of EM1 remained stable in vivo. o Location of the mutations in EM1 in the crystallographic structure of CVB3 proteins The 3D structure of CVB3 has been determined by X-ray crystallography (Muckelbauer. In order to locate the mutations identified in EM1 in the capsid structure, a stereographic image of CVB3 was produced from the coordinates available in the protein data bank for VP1 alone and VP1, 2, 3, 4 capsomers with the help of Dr. M. Murphy. The structures obtained using the program "O" are shown in figures 6.5.5 and 6.5.6 and the altered amino acids are numbered. These mutation in VP1 region of EM1 may be responsible for the altered properties of EM1, particularly its temperature-sensitivity and reduced stability, and possibly also its reduced virulence in vivo. However, conclusive evidence will only be obtained by using site-directed mutagenesis of this region of the CVB3 genome, and assessing the phenotype of the mutagenised virus. 118 Figure 6.5.5. Structure of VP1 Capsid Protein The a b o v e p ic tu re is a s t e r e o g r a p h i c image of CVB3 p r o d u c e d f r o m c o o r d i n a t e s ava i lab le in t h e p ro te in d a t a b a n k fo r VP1 c a p s i d p ro te in o f CVB3. The s t r u c t u r e w a s o b t a i n e d u s i n g p r o g r a m "O" a n d t h e a l te red a m i n o a c i d s are n u m b e r e d . 119 Figure 6.5.6. Structure of CVB3 capsomers The above p ic tu re Is a s te reograph lc Image of CVB3 caps id p ro te ins p r o d u c e d f r o m c o o r d i n a t e s avai lab le in t h e pro te in da ta bank. Th is s t ruc tu re was ob ta ined u s i n g p r o g r a m "O" and t h e a l tered a m i n o ac ids are n u m b e r e d . 120 7.Conclusions: A mutant EM1 has been isolated from CVB3(RK) stock preparation by reaction with a neutralising MAb which displays a different phenotype in vitro and an altered pathogenesis in A/J mice. Using equivalent input multiplicity of infection (MOIs), the yield of EM1 in Vero cells is generally reduced 2-3 fold relative to CVB3(RK), suggesting that it possesses a mutation making it slightly growth-restricted. Similar reductions in viral yield were found in J774A.1 cells while a much greater reduction in titre was found in the B-cell line Wehi 231. Thus there appears to be a fundamental alteration in EM1 which affects growth, and in addition to this, tissue-specific effects which determine the increased restriction in B-cells and the total restriction in T-cells. Reduction in viral yield in specific tissue may be predicted to be due to variations in host factors required for viral translation and RNA replication, or even to difference in the intracellular "milieu" in terms of pH or salt concentration which could affect those processes. On the other hand, the mild growth restriction found in Vero cells may be due to the mutation detected in the E loop of the NTR region which forms part of the IRES. Slight disruption of this loop could affect the binding of host translation factors to the NTR or the attachment of the ribosome itself. The general reduction in Vero cells could also be explained by the presence of mutations in P2 and P3 non-structural regions of the genome which were not examined in the present study. For example a mutation in the viral RNA polymerase encoded in the P3 region could affect the degree of RNA replication and therefore virus yield. This possibility should 121 be examined in future studies. EM1 was also found to be temperature-sensitive at 40°C and more susceptible to decay on prolonged incubation at 37°C. These are likely manifestations of alterations to the structural proteins rendering the capsid structure less stable. Several mutations were identified in the P1 structural gene region including a major alteration in VP1 involving two consecutive amino acid changes, threonine to histidine at position 117, and phenylalanine to valine at position 118. A charge modification at amino acid 35 (glutamic acid to glutamine) might also affect structure, possibly destabilizing the capsid. These alterations in VP1 most likely also explain the loss of reactivity of EM1 with the neutralising MAb. Which specific mutations are involved in each phenotypic change will require the use of site-directed mutagenesis to introduce each mutation separately and/or together into CVB3 (RK) and examining the phenotype of the mutant produced. The differences in pathogenesis in vivo are more difficult to define at the molecular level. The slight reduction in viral titres found in pancreas and liver may reflect the growth inhibition found in Vero cells in vitro but other factors could also play a role. For example, a slight fever in infected animals could retard EM1 replication, or this virus may induce an altered cytokine profile affecting both innate and acquired immune responsiveness. This has been shown by Huber and co-workers (1994) with their amyocarditic H310A1 strain. Moreover, even a 2-fold reduction in viral titre may have consequences for disease manifestations which have no obvious counterparts in cultures in vitro. Thus in infected animals, the disease manifestations depend only 122 partly on the viral genotype/ phenotype but also on a wide array of host factors. While the use of inbred strains of mice, of determined age, reduces the variability in response seen, there is always a spectrum of susceptibility to any infection which requires the use of multiple animals for each sample point to determine mean titres of virus in specific tissues and associated histopathology. Despite these limitations there was a clear significant difference in the titres of EM1 and CVB3 (RK) found in the spleen and heart, specifically a 50-fold reduction in infection virus in spleen and a 100-fold reduction in heart. In the spleen according to our results and the detailed studies of Anderson et al. (1996), CVB3 genome is found in the germinal centres of lymphoid follicles which contain primarily B-cells surrounded by the macrophages of the marginal zone. The fact that B-cells were relatively non-permissive to EM1 in vitro may explain the 50-fold reduction of its titre in spleen. Most probably EM1 replicates less and is cleared much faster and more efficiently from the spleen than CVB3(RK) accounting for the low viral load. In the heart the 100-fold difference between EM1 and CVB3(RK) titres may be due either to the inability of EM1 to penetrate the heart through the endothelial layers and/or to replicate in myocytes following access. The serum titre of EM1 was 2-3 fold lower than that of CVB3(RK) at day 3 post-infection which was previously shown to be the peak of viraemia (Anderson et al. 1996). This could partly account for the reduced titres of EM1 in the heart. However, the 2-3 fold reduction in virus available to infect the heart can not entirely explain the 100-fold difference in the viral load in heart tissue. 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