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Adenoviridae Molecular Virology HISTORY first isolated in 1953 (Rowe, et al. (1953), Proc. Soc. Exp. Biol. Med. 84:570–573) from tonsils and adenoids of children passage normal cell culture cytopathic effect virions similar viral agents were isolated from febrile military personnel with respiratory illnesses adenoid degeneration (AD), adenoid-pharyngeal conjunctival (APC), and acute respiratory disease (ARD) agents 1956 ADENOVIRUS TAXONOMY GROUP FAMILY GENUS Group I dsDNA Adenoviridae (Greek, adenos, “gland”) Mastadenovirus (Greek, mastos, “breast”) Aviadenovirus (Latin, avis, “bird”) Atadenovirus (English, adenine and thymine) Siadenovirus (English, sialidase) Ichtadenovirus (Greek, ichthys, “fish”) TYPE SPECIES Sturgeon Adv A Frog Adv Fowl Adv A Ovine Adv D Human Adv C CLASSIFICATION SCHEMES FOR HUMAN ADENOVIRUS (MASTADENOVIRUS H) Subgroup (species) A B C a Hemagglutination groups Serotypes IV (little or no agglutination) I (complete agglutination of monkey RBCs) III (partial agglutination of rat RBCs) 12, 18, 31 D II (complete agglutination of rat RBCs) E F III III Percent of GC in DNA 48-49 3, 7, 11, 14, 16, 21, 34, 35, 50 50-52 1, 2, 5, 6 57-59 8-10, 13, 15, 17, 19, 20, 22-30, 36-39, 42-49, 51 4a 40, 41 57-61 57-59 The genome sequence of Ad4, the one member of subgroup E, is related to Ads of subgroup B Knipe and Howley. Field’s Virology. 5th ed. GENOME linear dsDNA; 30-36 Kb inverted terminal repeat (ITR) sequences at 3’-end; 30-200 bp linear DNA forms a loop due to terminal protein (covalently linked to each 5’ end) and terminal base pairing all genes are transcribed by host RNA pol II except VA gene which is transcribed by RNA pol III mRNAs are polycistronic and are differentiated by alternative splicing and use of different poly(A) sites STRUCTURE non-enveloped, icosahedral capsid; 90-120 nm 20 equilateral triangular faces, 12 vertices each face has 12 identical hexons and each vertex has 1 penton; each penton is attached to a fiber protein 12 x 20 = 240 hexons; also 12 pentons total of 252 capsomers CAPSID Protein II (hexon monomer) - structural - neutralizing Abs directed against the ε epitope - type specific Ag sites CAPSID Protein II Protein III (penton base) - penetration CAPSID Protein II Protein III Protein IIIa - penetration CAPSID Protein II Protein III Protein IIIa Protein IV (fiber protein) - receptor attachment - hemagglutination - type-specific and some species-specific Ag sites CAPSID Protein II Protein III Protein IIIa Protein IV Protein VI - hexon minor polypeptide - stability/particle assembly CAPSID Protein II Protein III Protein IIIa Protein IV Protein VI Protein VIII - hexon minor polypeptide - stability/particle assembly CAPSID Protein II Protein III Protein IIIa Protein IV Protein VI Protein VIII Protein IX - hexon minor polypeptide - stability/particle assembly CAPSID Protein II Protein III Protein IIIa Protein IV Protein VI Protein VIII Protein IX - hexon minor polypeptide - stability/particle assembly CORE Protein V - links DNA to penton base - histone-like, packaging CAPSID Protein II Protein III Protein IIIa Protein IV Protein VI Protein VIII Protein IX - hexon minor polypeptide - stability/particle assembly CORE Protein V - links DNA to penton base - histone-like, packaging Protein VII - histone-like, packaging CAPSID Protein II Protein III Protein IIIa Protein IV Protein VI Protein VIII Protein IX - hexon minor polypeptide - stability/particle assembly CORE Protein V - links DNA to penton base - histone-like, packaging Protein VII Protein X (µ) - packaging CAPSID Protein II Protein III Protein IIIa Protein IV Protein VI Protein VIII Protein IX - hexon minor polypeptide - stability/particle assembly CORE Protein V - links DNA to penton base - histone-like, packaging Protein VII Protein X (µ) 55kD Terminal protein - genomic replication CAPSID Protein II Protein III Protein IIIa Protein IV Protein VI Protein VIII Protein IX - hexon minor polypeptide - stability/particle assembly CORE Protein V - links DNA to penton base - histone-like, packaging Protein VII Protein X 55kD Terminal protein dsDNA ATTACHMENT & ENTRY CD46 for Human Adv subgroup B Disassembly of proteins IIIa, IV, III, VIII Intracellular reducing environment activates viral protease and cleavage of protein VI that links the viral core to capsid EARLY TRANSCRIPTION & TRANSLATION <8 hr GENOME REPLICATION LATE TRANSCRIPTION & TRANSLATION >12 hr ASSEMBLY & RELEASE 100K protein facilitates folding and assembly of hexon trimers Protein VI stabilizes capsid and facilitates hexon importation IVa2, L1 52/55K, L4 22kD promote viral DNA packaging Cleavage of precursors of VI, VII, VIII, X by viral protease Viral escape and spread of progeny virus by: 1) L3 protease cleavage of cellular cytokeratin 2) ADP kills cells 3) free fiber trimers released from infected cells interfere CAR oligomerization at tight junctions Immune Evasion 1. Inhibition of IFN functions - VA-RNA and E1A – binds to PKR 2. Inhibition of TNF and Fas-mediated apoptosis - E1B19K, E314.7, E310.4K/14.5K 3. Downregulation of surface class I MHC - E3gp19, E1A – retention of MHC I in ER E3 proteins and their functions Knipe and Howley. Field’s Virology. 5th ed. E4 proteins and their functions E4 orf6/7 orf6 orf4 orf3 Modulates E2F orf2 ? Interacts with E1B 55K facilitating RNA metabolism Binds to DNA PK Interacts with E1B 55K Relocates nuclear pods orf1 Facilitates transformation Inhibits E1A activation of E2F Binds to DNA PK Journal of General Virology (2000), 81, 2573–2604 Journal of General Virology (2000), 81, 2573–2604 Oncogenic potential of human adenoviruses Subgroup (species) A B C D E F Tumor in animals High, within 4 months Moderate, within 4 to 8 months Low or none Low or none (mammary tumors, within 3 to 5 months) Low or none Unknown Transformation in tissue culture + + + + + Knipe and Howley. 2007. Field’s Virology. 5th ed. CLINICAL FEATURES OF DISEASE meningoencephalitis • ENTRY OCULAR INFECTIONS conjunctivitis keratoconjunctivitis myocarditis SPREAD RESPIRATORY INFECTIONS pharyngitis pertussus pneumonia acute respiratory disease • • GASTROINTESTINAL INFECTIONS gastroenteritis hepatitis hemorrhagic cystitis • Adenovirus diseases, associated serotypes, hosts and clinical specimens for diagnosis Zuckerman, et al. 2009. Principles and Practice of Clinical Virology. 6th ed. Diseases of Domestic Animals Associated with Adenoviruses NUMBER OF ANIMAL SPECIES SEROTYPES DISEASE Dogs 2 Infectious canine hepatitis (Canine Adv 1) Infectious canine tracheobronchitis (Canine Adv 2) Horses 2 Usually asymptomatic or mild respiratory disease. Bronchopneumonia and generalized diseases in foals with primary severe combined immunodeficiency disease Cattle 10 Usually asymptomatic or mild respiratory disease Swine 4 Usually asymptomatic or mild respiratory disease Sheep 6 Usually asymptomatic or mild respiratory disease Goats 2 Usually asymptomatic or mild respiratory disease Deer 1 Pulmonary edema, hemorrhage, vasculitis Rabbits 1 Diarrhea Chickens 12 Egg drop syndrome, inclusion body hepatitis Turkeys and 3 Hemorrhagic enteritis (turkey); marble spleen disease (pheasant); egg pheasants drop syndrome in both Quail 1 Bronchitis Duck 2 Rarely, duck hepatitis Geese 3 Isolated from liver, intestines Murphy, et al. Veterinary Virology. 3rd ed. DIAGNOSIS 1. DIRECT METHODS a. Viral isolation – human epithelial cell lines b. Histopathology – enlarged nuclei with basophilic inclusions c. Direct antigen detection i. IFA ii. EIA iii. Immunochromatography iv. IHC d. Direct particle detection i. EM – acute gastroenteritis e. Direct genome detection i. PCR, real-time PCR 2. INDIRECT METHODS a. Serology – IgM/IgG USE OF ADENOVIRUSES AS: I. GENE THERAPY VECTORS - gene to correct genetic defect II. CANCER THERAPY VECTORS - gene induces cell death III. VACCINE VECTORS - gene is antigen http://en.wikipedia.org/wiki/Image:Gene_therapy.jpg Types of Adenovirus Vectors 1) Replication-defective vectors - one or more viral genes deleted 2) Replication-competent vectors Replication-defective vectors FIRST GENERATION VECTORS -E1 deleted SECOND GENERATION VECTOS -E1, E2, E4 deleted - helper-dependent vector Knipe and Howley. Field’s Virology. 5th ed. Replication-competent vectors Knipe and Howley. Field’s Virology. 5th ed. ADENOVIRUSES AS VECTORS FOR VACCINATION AND GENE THERAPHY Advantages can be grown to produce stable, high titer stocks; can infect broad range of tissues including nondividing cells; rarely integrate into the host chromosome Disadvantages duration of the expression of the transgene will be limited chances of the vector to bind to non-target cells size of foreign gene is limited Journal of General Virology (2000), 81, 2573–2604 Thanks for listening