Medically Important Viruses and Prions PDF

Medically important viruses and prions Maria Huachen Zhu ([email protected]) School of Public Health Learning Objectives • Define the terms “virus” & “prion” • Explain how viruses & prions differ from other organisms • Classification of the viruses • Describe the structure and life cycle of a typical...

Medically important viruses and prions Maria Huachen Zhu ([email protected]) School of Public Health Learning Objectives • Define the terms “virus” & “prion” • Explain how viruses & prions differ from other organisms • Classification of the viruses • Describe the structure and life cycle of a typical virus & a prion • List some medically important viruses & prions • Discuss reasons for studying viruses & prions Pathogens for Infectious Diseases Infectious diseases: 1) diseases caused by microbes; and 2) that spread non-cellular Virus Protozoan prokaryotic Prion Bacterium Helminth Fungus eukaryotic Distribution of 1407 human pathogen species Group % of total (Arthropods) excluded Helminths 20 Fungi 22–23 Protozoa 4–5 Bacteria 38–41 Viruses and prions 14–15 Viruses infect all living things Virus definition A virus is a small, infectious, obligate intracellular parasite, capable of replicating itself in a host cell. Its genome is composed of either DNA or RNA, enclosed in a protein coat. Electron Microscopy Herpes virus (Varicella) - A Parapoxvirus (Orf virus) - B Adenovirus Icosahedral form Rotavirus Wheel like FEI Tecnai TEM Roingeard P. 2008 Biology of the Cell 100:491 About virus n n Viruses are parasites; they depend on cells for molecular building blocks, machinery and energy. Virus particles are small; dimensions usually range from approx. 20 – 200 nm*, should be observed by Electron Microscopy. * Ribosomes 20 nm * Most viruses: 20-200nm * Giant viruses: • • • Mimivirus (2003) 400nm Megavirus (2011) 440nm Pandoravirus (2013) 1000nm * E.coli 3000nm * Red blood cell 8000nm * Human 1.5-2.0m Genome size kb (= kbp) = kilo base pairs = 1,000bp Mb (= Mbp) = mega base pairs = 1,000,000 bp Gb (=Gbp)= giga base pairs = 1,000,000,000 bp Organism Genome size No. of genes* Human 3 Gb ~ 21,000 Yeast (S. cerevisiae) 12.1 Mb ~ 6,300 E. coli 4.6 Mb ~ 4,000 Pandoraviruses 1.9 - 2.5 Mb (dsDNA) ~1,500 - 2,500 Herpes viruses 120 - 230 kb (dsDNA) 60 - 120 Coronaviruses 27 - 32 kb (+ssRNA) 14 -16 Influenza virus 14 kb (-ssRNA) 14 Hepatitis B virus 3 kb (dsDNA-RT) 4 Hepatitis D virus ~1.7 kb (-ssRNA) 1 (with 2 isoforms) *Protein coding genes Hepatitis delta virus (HDV): the smallest "virus" known to infect animals • a subviral satellite depending on HBV • closed circular ssRNA of 1679nt • 200 molecules of HDAg Nucleocapsid • 2 isoforms of HDAg (L, S) • envelope from host • Envelope proteins from HBV Outer membrane HBV envelop proteins Lipid envelope from host 36 nm Group: Group V ((−)ssRNA) Genus: Deltavirus Coronavirus: the largest RNA viral genome Group: Order: Family: Group IV ((+)ssRNA) Nidovirales Coronaviridae SARS-CoV-2: viral genome encoding >29 proteins Structure & Schematic Diagram a typical virus a typical bacteria prokaryotic a typical animal cell eukaryotic non-cellular Structure of a virus n Nucleic acid (genome) - either DNA or RNA - segmented or in one piece - inside the capsid (often with virus proteins) n Capsid (protein shell, matrix) - made of protein subunit called capsomeres/protomeres - accounts for the main structure and major mass of virus n Influenza A virus Envelope (with or without) - lipid bilayer lying outside of the capsid - providing antigenic structures of lipid, protein and carbohydrate molecules n Viral Spikes (surface proteins) - protruding from the envelope or matrix, usually made of glycoprotein - have enzymatic and/or adsorption and/or hemagglutinating activity - highly antigenic, targeted by the immune response Classifying viruses n By morphology - Helical - Icosahedral - Complex morphologies n By envelop - enveloped - non-enveloped (naked) n By genome - dsDNA, ssDNA, dsRNA, ssRNA - linear, circular n By mechanism of mRNA production q Baltimore classification Classifying viruses (by morphology) • Helical-structured viruses Paramyxoviridae Measles Mumps Orthomyxoviridae Influenza Coronaviridae SARS MERS Common colds COVID-19 Cann 2005 Principles of Molecular Virology, Elsevier Academic Press Coronavirus Classifying viruses (by morphology) • Icosahedral-structured viruses 5nm Carter and Saunders 2007 Virology: Principles and Applications, Wiley Icosahedral viruses The most optimal way of forming a closed shell using identical protein sub-units. A virus must be very economical in the number of genes it has. This shape lets the virus do a lot with a little. Goddard et al. (2005) Structure, 13, 473 Classifying viruses (by morphology) • Complex morphologies Poxviridae Smallpox Rhabdoviridae Rabies Flint et al 2009 Principles of Virology, ASM Press; Cann 2005 Principles of Molecular Virology, Elsevier Academic Press Classifying viruses (by envelop) • enveloped virus Cut away of envelope Capsid - blue circle, made of capsomers (eg M1 protein) contains the genome (RNA or DNA) and virus proteins. Envelope - lipid bilayer (orange-pink) covers capsid holds the antigenic glycoproteins (HA and NA here) virus proteins may be between envelope and capsid. Flint et al 2009 Principles of Virology, ASM Press; Baker et al 1999 Microbiol Mol Biol Rev 63:862 Classifying viruses (by envelop) • non-enveloped virus VP2 VP1/3 VP4/6/7 capsomers, making up the icosahedral capsid virus proteins with dsRNA in the capsid outer shell with VP4 as a “spike” protein Baker et al 1999 Microbiol Mol Biol Rev 63:862 Classifying viruses (by envelop) Enveloped virus: Has lipid membrane, which will hold surface proteins, outside the capsid Naked virus: does not have an envelope. Enveloped viruses take a lipid layer from the infected cell and usually “bud” from it. Naked viruses usually destroy or lyse the infected cell to escape Flint et al 2009 Principles of Virology, ASM Press Classifying viruses (by genome) A virus genome is composed of one of: Linear, circular Single, double stranded DNA, RNA Classifying viruses (by genome & transcription) Baltimore classification (based on the mechanism of mRNA production) Class Type Virus I (ds): Adenovirus Herpesvirus Poxvirus II (ss): Parvovirus III (ds): Reovirus (eg rotavirus) IV(+ss): Picornavirus (eg EV71) Astrovirus Coronavirus (eg SARS-CoV) V(-ss): Orthomyxovirus (eg flu) Paramyxovirus (eg Nipah) Rhabdovirus (eg rabies) Deltavirus (eg HDV) VI(RT): Retroviruses (eg HIV) VII(RT): Hepadnaviruses (eg HBV) I, VII II III IV, V, VI Virus ‘life’ cycle Enter a host cell virus surface protein contacts a cell receptor for attachment (adsorption) followed by penetration The type of receptor a virus can attach to will define its host and tissue tropism (key & lock) Baker et al 1999 Microbiol Mol Biol Rev 63:862 Virus ‘life’ cycle Extract viral genome in the cytoplasm – uncoating cellular proteolytic enzymes digest the viral capsid releasing the virus’ nucleic acid Protein synthesis – transcription & translation structural and functional proteins are made Baker et al 1999 Microbiol Mol Biol Rev 63:862 Virus ‘life’ cycle Replication of the virus’ genome – copying the nucleic acid (DNA or RNA) Maturation of virus particle: (assembly of) synthesized nucleic acid, proteins, capsid, envelopes Release of particles from the host cell - lysis of cell or virus “buds” from cell. Baker et al 1999 Microbiol Mol Biol Rev 63:862 Virus ‘life’ cycle Uncoating Replication budding Poliovirus (+ssRNA) lysis Flint et al 2009 Principles of Virology, ASM Press Baker et al 1999 Microbiol Mol Biol Rev 63:862 AETTGAE Routes of Transmission By respiratory droplets and aerosols （air-borne） Through the faecal – oral route (food/water) By the (faecal-)genito-urinary tract (sex) Vertically (mother to foetus/child) By mixing blood & body fluids By vectors (eg. Insects/arthropods) Associations with clinical medicine n Viruses can only be grown in living cells / animals n Viruses are host and tissue specific (by using specific receptors on host cells) n Antiviral therapy is more difficult than antibiotic therapy for bacteria n Vaccination is the best medical way to prevent infection and disease caused by viruses Important viral infections • Gastroenteritis: rotavirus, norovirus, adenovirus, astrovirus… • Common cold: associated with over 200 different viral types (eg. rhinovirus and other enteroviruses, coronavirus, influenza viruses, adenoviruses, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, etc) Important viral infections Influenza: influenza virus - causes seasonal epidemics (A,B,C) - can cause pandemics (influenza A) • Hepatitis: hepatitis virus (A, B, C, others) infectious, can cause liver failure / cancer • • AIDS: Human Immunodeficiency Virus • • SARS/MERS: SARS/MERS coronavirus COVID-19: SARS-CoV-2 Zika/Dengue/Yellow fever/Japanese encephalitis: Vector (mosquito) borne viruses (Flaviviruses) • Oncogenic viruses (oncoviruses) • Liver Cancer - Hepatitis B/C Virus (HBV, HCV) • Cervical cancer - Human Papilloma Virus (HPV) • Nasopharyngeal Carcinoma / Burkett’s Lymphoma Epstein-Barr virus (EBV/HHV-4) • Kaposi's sarcoma - Human Herpes Virus 8 (HHV-8) • Adult T-cell leukemia/lymphoma (ATL or ATLL) - Human Tlymphotropic virus type 1 (HTLV-1) * All act through interference with the host cell’s normal regulatory processes and activation of the viral or cellular oncogenes. 09:47:45 36 Diagnosing a viral infection • Virus detection antigen detection nucleic acid detection shell viral cultures culture inclusion bodies direct electron microscopy (hours) (hours-days) (days) (weeks) (days) (days) • Serology (acute infection) IgM rising antibody titres (days) (weeks) • Serology (postinfection/serostatus) IgG (hours) Specimens for virus culture n n n Send to the lab within hours of collection. Otherwise the viruses will ‘die’. If delayed, keep cool (at 4°C; do not freeze). Put viruses in transport medium (with antibiotics) Transport medium Effect of a virus on a cell culture Normal cells Infected cells showing CPE “cytopathic effect” Virus culture in embryonated eggs Detecting virus genes by PCR Polymerase Chain Reaction Requires species-specific “primers” Use “polymerase” to amplify (and Reverse Transcriptase [RT] for RNA viruses) Visualize by hybridization with complimentary DNA probes; gel electrophoresis; “Real-time” PCR. Real-time (quantitative) RT-PCR to identify and distinguish the recent H7N9 viruses from other H7 viruses. Wong et al 2013, ClinChem 59:1062 Strategy used by the viruses n Use different receptors and transmission routes n Have multiple hosts and can cross species barriers n n n n Some may lie latent or integrate within the host cell chromosome, others replicate at a slow rate, persisting as a source of infection in symptomless carriers, waiting for the host defense to be lowered use the cellular machinery for replication, making it difficult for an antiviral agent to target the virus without affecting the host cell RNA viruses have high mutation rates and can evolve rapidly, escaping the antiviral agents, vaccines or immune response, and causing different clinical illness spectrum Reassortment & recombination can also help to generate novel variants Virus evolution c Mutation Prion coined in 1982 by Stanley B. Prusiner; a portmanteau derived from protein & infection n A prion is an infectious agent composed entirely of protein material, called PrP (prion protein) n is short for “proteinaceous infectious particles of modified forms of a normal cellular protein” (host-derived molecules) n Causes degenerative changes in the brain, with large vacuoles in the CNS & motor disturbances (neurodegenerative disorders): transmissible spongiform encephalopathies (TSEs) Characteristics of prions – small size (< 100 nm à filterable) – proteinaceous infectious particles of modified forms of a normal cellular protein – lack of a nucleic acid genome – extreme resistance to heat, disinfectants & irradiation – susceptible to high concentrations of phenol, periodate, sodium hydroxide, sodium hypochlorite – slow replication, transmissible within or across species • usually long incubation period (up to 35 years) & appear late in life • variant CJD (vCJD) can produce symptoms much more rapidly – cannot be cultured in vitro – do not elicit immune or inflammatory responses Prions are host-derived molecules q PrPSc (prion protein scrapie) n a 30–35 kDa glycoprotein derived from PrPc n associated with intracellular fibrils in diseased tissue n can be found in the lymphoreticular system (tonsils, spleen & neurological tissues) n q may be carried in the blood by lymphocytes PrPc n a naturally occurring cellular prion protein n expressed predominantly on surface of nerve cells n coded by a single copy gene (on chromosome 20 in humans), with unknown function Prions are host-derived molecules q PrPSc & PrPc highly similar sequence differ in structure & protease resistance q PrPc Soluble, linear enzyme susceptible q PrPSc Insoluble globular enzyme resistant Soluble linear α-helices proteins (PrPc) à modified (misfolded) & accumulate as insoluble ß-sheet rich amyloid fibrils (PrPSc) Normal cell, linear proteins at cell membrane as a free globular glycoprotein (misfolded) conformational changes from alpha helices to beta-pleated sheets (more stable, resistant to proteolysis) s Diseases caused by prions n n n have different strains spectrum of disease onset & severity were determined by combinations of host & prion variation causing human & animal diseases q q q q scrapie in sheep Creutzfeldt–Jakob diseases (CJD), Gerstmann-Sträussler-Scheinker disease, Kuru & fatal familial insomnia in humans Sporadic CJD à most common prion disease in humans àaffecting ~ 1.5 per million people bovine spongiform encephalopathy (BSE) in cattle (mad cow disease) à variant CJD (vCJD, 1990s) in humans (the only human prion disease from animals) n 1st reported in the UK in 1996 by the National CJD Surveillance Unit (by oral route?) n By Jul 2010, 220 people developed vCJD in 11 countries (173 in UK), underestimate? n Has much shorter incubation period, different pathological phenotype Prions can cross species boundaries n same species transmission à more effective n Spread of scrapie agents between species: nearly all have been transmitted to laboratory rodents and primates q Transfer: scrapie infected sheep à cattle (BSE) à humans (vCJD) n Most infectious agents have mutations at a.a. residue 129 Infection & Transmission n Endogenously acquired by mutation (can be inherited) n Transmission & spread requires exposure to the infective agent by: q eating contaminated food material kuru à eating the brains of dead humans in funeral rites vCJD à eating beef products from BSE infected cattle q use of contaminated medical products (blood, hormone extracts, transplants) q introduction of prions from contaminated surgical instruments q Possibly mother–fetus transmission Yet none of the infants born to mothers with kuru developed disease * prions survive digestion à taken up across the intestinal mucosa à carried in lymphoid cells à transferred into neural tissues & enter the CNS Prion diseases are difficult to diagnose p cannot be cultured p no immune response àcan't be diagnosed easily in early stages Clinical appearances àprobable occurrence? Confirmed histologically post mortem q Tonsillar tissue n n q good source of PrPSc in clinical cases tested for prions by immunoblotting or immunohistochemistry Tissue homogenates n tested for abnormal prion protein by enzyme immunoassays Prevention & treatment of prion diseases n Prion diseases à incurable n no effective treatment nor vaccine q chemotherapeutic strategies à reduce, stop or destabilize PrPSc formation n q polyanionic & tricyclic compounds (rodent) immunomodulation & mucosal immunization n potential therapeutic & preventative approaches since alimentary tract is likely to be the main route of transmission Prions – Key Facts n host-derived glycoproteins lack a nucleic acid genome extremely resistant to disinfection procedures n Unusual infectious agents n n q n Transmission q q n causing spongiform encephalopathies & motor disturbances usually by ingestion of contaminated tissues can occur via medical procedures Diseases include q q q q Kuru Creutzfeldt–Jakob disease (CJD) variant CJD (vCJD) bovine spongiform encephalopathy (BSE, mad cow disease) human-pathogen conflict (prions) n Resistant to disinfectant n Minimal immune response n No nucleic acid and no metabolic systems ànot targeted by antimicrobial drugs n Arise by mutation then hijack protein-folding control àresistant to enzymes n Hard to detect n Interspecies transmissible n Meat-based food àintestine àlymphoid àCNS n Host genetic predisposition Learning Outcomes By the end of this lecture you should be able to •Define the terms “virus” & “prion” •Explain how viruses & prions differ from other organisms •Classification of the viruses •Describe the structure and life cycle of a typical virus & a prion •List some medically important viruses & prions •Discuss reasons for studying viruses & prions References • Carter J & Saunders V. Virology: Principles and Applications. Wiley, 2007 (or 2012 2nd). • Goering R, et al. Mims' Medical Microbiology (5th Edition). Elsevier, 2013.