CHAPTER 1: INTRODUCTION 1.1 Overview of emerging infectious diseases Despite advances in medical research and treatments during the 20th century, infectious diseases remain the leading causes of death worldwide The elimination of smallpox in 1977 was a great achievement in the fight against infectious diseases However, infectious diseases continue to persist and caused great losses both in the social and economic aspects This is due to the emergence of new infectious diseases, re-emergence of old infectious diseases, and persistence of intractable infectious diseases Emerging infections can be defined as infections that have newly appeared in the population, or have existed but are rapidly increasing in incidence or geographic range (Morse & Schluederberg, 1990) Examples of recent emerging diseases include Acquired Immunodeficiency Syndrome (AIDS) cause by human immunodeficiency virus (HIV), hantavirus pulmonary syndrome, Lyme disease and foodborne infection by O157:H7 Escherichia coli There are several factors that contribute to the emergence of new diseases These include ecologic changes (e.g deforestation), changes in human demographics and behavior (e.g urban migration and intravenous drug use), and increased international air travel Human populations were brought closer and more frequently to the source of the pathogens The outbreak of the new infection, severe acute respiratory syndrome (SARS) may be the result of increased use of exotic animals as food source Close contact with palm civet cats and raccoon dogs in China was identified as a potential route by which the SARS coronavirus (SARS-CoV) was transmitted from animal to human (Guan et al., 2003; Wang et al., 2005) In addition to the identification of new human viruses, “old infectious pathogens” are also re-emerging Microbial adaptation and natural genetic recombination and mutation result in new strains of known pathogens, which are not recognized by the human immune system One example is the emergence of H5N1 influenza A virus strain which has caused human infection since 1997 (Subbarao et al., 1998) The aim of the project was to understand the adaptive immune response against two newly emerged viruses, SARS-CoV and the H5N1 influenza A virus For SARS-CoV, the focus was mainly to investigate the T cell response against the unique accessory 3a protein The study was also extended to T cell response against the structural nucleocapsid (N) protein in SARS recovered individuals The main objective for the H5N1 work was to demonstrate the use of the recombinant baculovirus-expressed hemagglutinin (HA) protein for use as vaccine and as a tool to generate neutralizing antibodies Further characterization of a potent monoclonal antibody (mAb) against the HA protein was also performed 1.2 Severe Acute Respiratory Syndrome (SARS) 1.2.1 Epidemiology of severe acute respiratory syndrome (SARS) SARS first emerged in Guangdong, China in late 2002 (Zhong et al., 2003) By March 2003, the disease spread to Hong Kong, and then to Vietnam, Singapore and Canada (Lee et al., 2003; Poutanen et al., 2003; Tsang et al., 2003) Those infected by SARS were mainly healthcare workers or household members who had cared for patients with severe respiratory illness Contact tracing finally indicated the index case was a healthcare worker from Guangdong province who visited Hong Kong and transmitted the virus to several other guests who, further contributed to global dissemination of the disease (Ksiazek et al., 2003) A novel coronavirus SARS coronavirus (SARS-CoV) was identified as the causative agent (Drosten et al., 2003; Ksiazek et al., 2003; Peiris et al., 2003b) The SARS-CoV went on to infect more than 8000 people in 29 countries across continents with 774 deaths reported by World Health Organization (WHO) (WHO, 2003b) The SARS epidemic was officially controlled by July 2003 with strict isolation of patients The main route of transmission seems to be airborne droplets from infected patients (Booth et al., 2005; Hui & Chan, 2010; Yu et al., 2004b) Blood and fecal-oral transmission has also been suggested (Poon et al., 2003) There are no known vectors for coronaviruses but epidemiological evidence demonstrated that early cases of SARS were linked to exposure to wild game animals in the live wet markets in Guangdong province (Guan et al., 2003) In the nasal and fecal swabs from masked palm civets (Paguma larvata) and raccoon dogs (Nyctereutes procyonoides) in the wet markets, SARS-like viruses which are genetically and antigenically related to the human SARSCoV were detected using reverse transcriptase polymerase chain reaction (RTPCR) and electron microscopy of viral particles from infected cells The SARS-like viruses isolated from the animals were shown to have more than 99% homology with the human SARS-CoV Interestingly, high seroprevalence for the animal SARS-CoV antibodies were found in animal traders working with these live animals in the wet markets, although they did not have a history of SARS-like disease (MMWR, 2003) These observations seem to suggest that the live animal market probably is the site for the animal SARS-CoV to amplify and allow interspecies transfer of the animal virus to the humans However, it is not clear whether these animals are the natural reservoirs of the SARS-CoV in the wild In 2005, reports from several groups identified a virus that was genetically closely related to human SARS-CoV in the Chinese horseshoe bats, suggesting that they may be the natural source of SARS-CoV although attempts to isolate the virus from bats have not been successful (Lau et al., 2005; Li et al., 2005) Bats are known to be the reservoir hosts of several zoonotic viruses, including the Hendra and Nipah paramyxoviruses that have recently emerged in Australia and East Asia (Chua et al., 2000; Murray et al., 1995) Finally, molecular epidemiology showed that at least two strains of SARS-CoV have been found in patients in Hong Kong (Guan et al., 2004a) This suggests that the virus had jumped from animal source to human on two separate occasions This indicates the outbreak of SARS would have been inevitable and the potential of reemergence is also high 1.2.2 Genome Organization Coronaviruses are a diverse group of large, enveloped positivestranded RNA viruses They belong to the order Nilovirales, family Coronaviridae, genus Coronavirus and cause respiratory and enteric diseases in humans and other animals Three serologically distinct groups of coronaviruses have been identified Group I and II contain mammalian viruses, whereas group III contains only avian viruses After its discovery, the SARS-CoV was initially placed in a new group (IV) of coronavirus as the sequence is distinct from those previously reported in animals and humans (Marra et al., 2003; Rota et al., 2003) However, examining sequences within regions of ORF 1a of SARS-CoV showed domains that are unique to the group II coronaviruses, suggesting that SARS-CoV may be more directly related to group II viruses (Snijder et al., 2003) Group II coronaviruses include the bovine coronavirus, human OC43 virus and murine hepatitis coronavirus (MHV) More evidence demonstrated that the 3’UTR of SARSCoV could substitute functionally for that of MHV, but not with 3’ UTR from group I coronavirus (Goebel et al., 2004) Thus some research groups suggested that SARS-CoV belongs within group II coronaviruses, in a subgroup IIb The genome of SARS-CoV is approximately 30 kb, with polyadenylated positive-stranded RNA (Marra et al., 2003; Rota et al., 2003) The genomic organization is typically of a coronavirus, with the characteristic gene order, with the first two open reading frames (1a and 1b) encoding the viral replicase and the downstream mRNAs encoding structural proteins spike (S), envelope (E), membrane (M) and nucleocaspid (N) However, the gene encoding hemagglutinin-esterase found in group II and some group III coronaviruses was not found in SARS-CoV (Rota et al., 2003) The RNA is packaged by the N protein into a helical nucleocapsid, with the S protein forming morphologically characteristic projections on the virion surface 1.2.2.1 Replicase genes The viral replicase genes (ORF 1a and 1b) translate into two polyproteins, pp1a (486 kDa) and pp1ab (790 kDa) (Thiel et al., 2003) Expression of the pp1ab is predicted to involve a ribosomal frameshifting into the -1 frame just upstream of the ORF1a translation termination codon The pp1a and pp1ab polyproteins undergo proteolytic processing by viral cysteine proteinases to yield the functional components of the membrane-bound replicase complex and a group of 16 non-structural proteins (nsp) (Ziebuhr et al., 2000) SARS-CoV uses only two proteinases, PL2pro, a papain-like cysteine proteinase (nsp3) and 3CLpro, a 3C-like proteinase (nsp5) , in contrast to most coronaviruses that use three proteinases (Gao et al., 2003; Rota et al., 2003; Snijder et al., 2003) The replicase complex, which includes an RNAdependent RNA polymerase and RNA helicase mediates both genome replication and transcription of a “nested” set of subgenomic mRNAs The putative functions of the non-structural proteins are summarized in Figure 1.1 The functions have been shown by biochemical assays or predicted based on their functional domains or structural similarities to other proteins while others remain to be further characterized [reviewed by (Cheng et al., 2007)] P Not known 3C-like protease Not known Papain-like protease 2; ADP-ribose 1-phosphatase Putative 2’-O-ribose methyltransferase Uridylate-specific endoribonuclease; Involved in the coronavirus replication cycle 3’→5’-exoribonuclease; supplements the endoribonuclease activity in the replication of the giant RNA genome Helicase (dNTPase and RNA 5’-triphosphatase activities) Deletion attenuates viral growth and RNA synthesis RNA-dependent RNA polymerase Expression promoted degradation of host endogenous mRNAs, which may inhibit host protein synthesis and prevented endogenous IFNβ mRNA accumulation Three-dimensional structure found potential sites for proteinprotein interaction Crystal structure suggests a nucleic acid binding function within a larger RNA binding protein complex for viral gene transcription and replication Three-dimensional crystal structure of a dimer which binds viral RNA and interacts with nsp8 Putative RNA-dependent RNA polymerase; crystal structure of the hexadecameric nsp7-nsp8 possesses a central channel with dimensions and positive electrostatic properties favorable for nucleic acid binding; it is probably another unique RNAdependent RNA polymerase for its large genome Fig 1.1 Summary of the SARS-CoV genome organization and viral protein expression Replicase (ORF 1a and 1b), constituting the first 2/3 of the genome, which translates into two polyproteins, pp1a and pp1ab The putative functions of each of the nsps are shown in the text boxes Open reading frames (ORFs) in the remaining 1/3 of the genome are translated from eight subgenomic mRNAs Four of the ORFs encode the structural proteins, spike (S), membrane (M), and envelope (E) and nucleocapsid (N) Another eight unique ORFs encode accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b), which have no significant sequence homology to viral proteins of other coronaviruses This figure was adapted and modified from Cheng, V C C et al 2007 Clin Microbiol Rev 20(4): 660-694 1.2.2.2 Structural and accessory proteins The subgenomic mRNAs encode the structural proteins, S, E, M and N, and the set of accessory proteins (namely ORF 3a, 3b, 6, 7a, 7b, 8a, 8b and 9b) The surface S protein is involved in the attachment and entry of the host cell The N together with M and E are involved in the assembly of the virion The accessory proteins have no significant homology to viral proteins of other coronaviruses These proteins are dispensable for virus replication in cell culture while some appear to contribute to viral pathogenesis (Narayanan et al., 2008; Tan et al., 2006) The focus of our study is the largest SARS-CoV accessory protein, 3a It is also known as U274 or X1 and this protein will be discussed in detail in section 1.2.2.3 The characteristic, functions, and/or putative roles of the four structural and seven other accessory proteins of the SARS-CoV are outlined in Table 1.1 and Table 1.2 respectively Table 1.1 Summary of the SARS-CoV structural proteins Protein (No of amino acid residues in protein) Spike protein (1,255) Protein characteristic [ref(s)] A type I integral membrane glycoprotein which is N-glycosylated, trimerized in endoplasmic reticulum (ER) (Bosch et al., 2003; de Groot et al., 1987; Rota et al., 2003) It is divided into subdomains of similar size, S1 and S2 with distinct functions S1 domain forms the globular portion of the spike, mediating binding to host cell receptor, angiotensin-converting enzyme (ACE2) (Li et al., 2003) The receptor binding domain (RBD) is localized to amino acids 318 to 510 The S2 ectodomain contains two regions with a 4, hydrophobic (heptad) repeat, HR1 and HR2 and a putative, internal fusion peptide (Bosch et al., 2004; Sainz et al., 2005) Protein’s function(s) or putative function(s) [ref(s)] Biochemical studies have shown that peptides corresponding to the HR1 and HR2 of the SARSCoV S protein can associate into an anti-parallel sixhelix bundle with structural features typical of class I fusion proteins (Ingallinella et al., 2004; Liu et al., 2004; Tripet et al., 2004) This HR1-HR2 structure brings the fusion peptide in close proximity to the transmembrane domain (Bosch et al., 2004), leading to the fusion of the viral and cellular membrane, and consequently the viral entry Effect on cellular response of host [ref(s)] The S protein was known to be responsible for inducing host immune responses and is the primary target for viral neutralizing antibodies (Keng et al., 2005; Zhou et al., 2004) The functional region of S protein from amino acids 324-688 can induce the release of IL-8 in lung cells (Stevens et al., 2006) It induced unfolded protein response in cultured cells as SARS-CoV with accumulation of S protein in the ER, may modulate viral replication (Yamada et al., 2006) Table 1.1 Continued Protein (No of amino acid residues in protein) Envelope protein (76) Protein characteristic [ref(s)] The E protein of SARS-CoV (9-12 kDa) has a short 7-9 amino acid hydrophilic region and a 21-29 amino acid hydrophobic region, followed by a hydrophilic Cterminal region (Shen et al., 2003) Protein’s function(s) or putative function(s) [ref(s)] It was demonstrated that the E protein does form ion channels, which are are more selective for monovalent cations than monovalent anions (Wilson et al., 2004) It was shown to be important for viral assembly as demonstrated by the formation of VLPs (Ho et al., 2004; Nal et al., 2005; Vennema et al., 1996) Membrane protein (221) The M protein contains a long cytoplasmic tail, hydrophobic transmembrane domains, and a short glycosylated Nterminal ectodomain It has been shown to be N-glycosylated at asparagines residue at position (Nal et al., 2005; Voss et al., 2006) Functional analysis showed that the N-terminal region of the M protein, comprising of the transmembrane domains, is sufficient to mediate accumulation of M in the Golgi complex and recruit the S protein to the sites of viral assembly and budding in the ER Golgi-intermediate compartment (Voss et al., 2009) Effect on cellular response of host [ref(s)] It induced apoptosis in transfected Jurkat T cells in the absence of growth factors A novel BH3-like region located in the C-terminal cytosolic domain of SARS-CoV E protein can bind Bcl-xL, whose overexpression can antagonize apoptosis (Durrer et al., 1996) M protein induced apoptosis in HEK293T cells, which could be suppressed by caspase inhibitor (Tsurudome et al., 1992) 10 A J (2003) Identification of severe acute respiratory syndrome in Canada N Engl J Med 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B*570101 Cw*07020101 Cw*06020101 A*2407 A*2407 B*1502 B*5801 Cw*030201 Cw*080101 A*110101 A*2407 B*1502 B*400101 Cw*080101 Cw*030401 10 A*0207 A*300101 B*130201 B*460101 Cw*060201 Cw*010201 11 A*330301 A*020301 B*380201 B*40060101 Cw*080101 Cw*07020101 12 A*0222 A*260101 B*5810 B*400101 Cw*030201 Cw*07020101 13 A*310102 A*02010101 B*1301 B*400101 Cw*030401 Cw*030401 14 A*310102 A*020601 B*400101 B*400101 Cw*030401 Cw*04010101 15 A*330301 A*110101 B*580101 B*550201 Cw*030201 Cw*030301 16 A*2420 A*020601 B*1502 B*1525 Cw*080101 Cw*0403 178 179 ... two newly emerged viruses, SARS- CoV and the H5N1 influenza A virus For SARS- CoV, the focus was mainly to investigate the T cell response against the unique accessory 3a protein The study was also... degradation of viral mRNA The IFN is also important as it enhances the efficiency of the adaptive immunity and activates the NK cells and macrophages In the case of influenza virus infection, the. .. the cytoplasm, a transmembrane domain, the stalk, the head with the catalytic active site (Varghese et al., 19 83) The role of NA in the influenza virus life cycle is still unclear NA can catalyze