1. Adenoviridae Molecular Virology

2. HISTORY  first isolated in 1953 (Rowe, et al. (1953), Proc. Soc. Exp. Biol. Med. 84:570–573) from tonsils and adenoids of children  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 normal cell culture passage cytopathic effectvirions

3. TAXONOMY GROUPGroup I dsDNA FAMILYAdenoviridae (Greek, adenos, “gland”) GENUSMastadenovirus (Greek, mastos, “breast”) Aviadenovirus (Latin, avis, “bird”) Atadenovirus (English, adenine and thymine) Siadenovirus (English, sialidase) Ichtadenovirus (Greek, ichthys, “fish”)

4. TYPE SPECIES Ovine Adv D Human Adv C Fowl Adv A Frog Adv Sturgeon Adv A

5. CLASSIFICATION SCHEMES FOR HUMAN ADENOVIRUS (MASTADENOVIRUS H) Knipe and Howley. Field’s Virology. 5 th ed.

6. 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

8. 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

9. CAPSID Protein II (hexon monomer) - structural - neutralizing Abs directed against the ε epitope - type specific Ag sites

10. CAPSID Protein II Protein III (penton base) - penetration

11. CAPSID Protein II Protein III Protein IIIa - penetration

12. CAPSID Protein II Protein III Protein IIIa Protein IV (fiber protein) - receptor attachment - hemagglutination - type-specific and some species-specific Ag sites

13. CAPSID Protein II Protein III Protein IIIa Protein IV Protein VI - hexon minor polypeptide - stability/particle assembly

14. CAPSID Protein II Protein III Protein IIIa Protein IV Protein VI Protein VIII - hexon minor polypeptide - stability/particle assembly

15. CAPSID Protein II Protein III Protein IIIa Protein IV Protein VI Protein VIII Protein IX - hexon minor polypeptide - stability/particle assembly

16. 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

17. 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

18. 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

19. 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

20. 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

21. ATTACHMENT & ENTRY CD46 for Human Adv subgroup B

30. 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

33. EARLY TRANSCRIPTION & TRANSLATION <8 hr

49. GENOME REPLICATION

58. LATE TRANSCRIPTION & TRANSLATION >12 hr

63. ASSEMBLY & RELEASE 100K protein facilitates folding and assembly of hexon trimers Protein VI stabilizes capsid and facilitates hexon importation

65. IVa2, L1 52/55K, L4 22kD promote viral DNA packaging Cleavage of precursors of VI, VII, VIII, X by viral protease

67. 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

68. 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

69. E3 proteins and their functions Knipe and Howley. Field’s Virology. 5 th ed.

70. orf6/7orf6orf4orf3orf2orf1 E4 Modulates E2F Interacts with E1B 55K facilitating RNA metabolism Binds to DNA PK Inhibits E1A activation of E2F Binds to DNA PK Interacts with E1B 55K Relocates nuclear pods ? Facilitates transformation E4 proteins and their functions Journal of General Virology (2000), 81, 2573–2604

72. Oncogenic potential of human adenoviruses Knipe and Howley. 2007. Field’s Virology. 5 th ed.

73. CLINICAL FEATURES OF DISEASE ENTRY SPREAD RESPIRATORY INFECTIONS pharyngitis pertussus pneumonia acute respiratory disease OCULAR INFECTIONS conjunctivitis keratoconjunctivitis GASTROINTESTINAL INFECTIONS gastroenteritis hepatitis meningoencephalitis myocarditis hemorrhagic cystitis

74. Adenovirus diseases, associated serotypes, hosts and clinical specimens for diagnosis Zuckerman, et al. 2009. Principles and Practice of Clinical Virology. 6 th ed.

75. Diseases of Domestic Animals Associated with Adenoviruses Murphy, et al. Veterinary Virology. 3 rd ed.

76. 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

77. I. GENE THERAPY VECTORS - gene to correct genetic defect II. CANCER THERAPY VECTORS - gene induces cell death III. VACCINE VECTORS - gene is antigen USE OF ADENOVIRUSES AS: http://en.wikipedia.org/wiki/Image:Gene_therapy.jpg

78. Types of Adenovirus Vectors 1)Replication-defective vectors - one or more viral genes deleted 2)Replication-competent vectors

79. Replication-defective vectors FIRST GENERATION VECTORS -E1 deleted SECOND GENERATION VECTOS -E1, E2, E4 deleted -helper-dependent vector Knipe and Howley. Field’s Virology. 5 th ed.

80. Replication-competent vectors Knipe and Howley. Field’s Virology. 5 th ed.

81. 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

82. Journal of General Virology (2000), 81, 2573–2604

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