1. ADENOVIRUSES

2. ADENOVIRUS- Classification
- DNA viruses first isolated from adenoidal tissue in 1953
- approximately 100 serotypes have been recognized, at least 47 of which infect humans.
Subdivided into 6 subgroups based on hemagglutination (A-F)
Human pathogens belong to 49 serotypes
Common serotypes:- 1-8, 11, 21, 35, 37, 40
Enteric Adenoviruses belong to subgroup F
Molecular biology research : splicing……
Gene therapy : cystic fibrosis………..

3. Table 52-1. Illnesses Associated with Adenoviruses
Disease
Patient Population
Respiratory Diseases
Febrile, undifferentiated upper respiratory tract infection
Infants, young children
Pharyngoconjunctival fever
Children, adults
Acute respiratory disease
Military recruits (serotype 4, 7)
Pertussis-like syndrome
Pneumonia
Infants, young children; military recruits; immunocompromised patients
Other Diseases
Acute hemorrhagic cystitis
Children; bone marrow transplant recipients
Epidemic keratoconjunctivitis
Any age; renal transplant recipients
Gastroenteritis
Hepatitis
Liver transplant recipients; other immunocompromised patients
Meningoencephalitis
Children; immunocompromised patients

4. ADENOVIRUS - Structure
Non-enveloped DNA virus
Icosadeltahedrons (20면체)
70-90 nm in size
Linear ds DNA genome (36kb) with a terminal protein (molecular mass, 55 kDa)

5. -12 pentons : have a penton base and a fiber.
- capsid comprises 240 capsomeres, which consist of hexons and pentons.
-12 pentons : have a penton base and a fiber.
fiber : viral attachment proteins
can act as a hemagglutinin.
penton base and fiber are : toxic to cells, carry type-specific antigens.
BOX Unique Features of Adenovirus
Naked icosadeltahedral capsid has fibers (viral attachment proteins) at vertices.
Linear double-stranded genome has 5' terminal proteins.
Synthesis of viral DNA polymerase activates switch from early to late genes.
Virus encodes proteins to promote messenger RNA and DNA synthesis, including its own DNA polymerase.
Human adenoviruses are grouped A through F by DNA homologies and by serotype (more than 42 types).
Serotype is mainly a result of differences in the penton base and fiber protein, which determine the nature of tissue tropism and disease.
Virus causes lytic, persistent, and latent infections in humans, and some strains can immortalize certain animal cells.

6. Simplified genome map of adenovirus type 2
Transcription : both strand
Early transcription – early protein : E1A, E1B, E2A, E2B, E3, E4
Late transcription –late protein
11 polypeptide : 9 – structural protein (capsid)
2 – core (DNA-binding protein)

7. Gene Number M.W (kDa2) Function E1A*
Activates viral gene transcription
Binds cellular growth suppressor: p105RB promotes transformation
Deregulates cell growth
Inhibits activation of interferon response elements
E1B
Binds cellular growth suppressor: p53 promotes transformation
Blocks apoptosis E2
Activates some promoters
Terminal protein on DNA
DNA polymerase E3
Prevents tumor necrosis factor-α (TFN-α) inflammation E4
Limits viral cytopathologic effect
VA RNAs
Inhibit interferon response
Capsid II
120
Contains family antigen and some serotyping antigens
III 85
Penton base protein
Toxic to tissue culture cells IV 62
Fiber
Responsible for attachment and hemagglutination; contains some serotyping antigens VI 24
Hexon-associated proteins
VIII 13
Penton-associated proteins IX 12
IIIa 66
Core V 48
Core protein 1: DNA-binding protein
VII 18
Core protein 2: DNA-binding protein

8. Entry and replication
Fiber protein determines target cell specificity and attachment
Viral DNA enters host cell nucleus
Virus replicates in nucleus
Fiber receptor : Ig superfamily
Coxsackie B viruses 도 이용
- Coxsackie adenovirus receptor
- MHC I 도 이용
Penton base interact with av integrin
receptor-mediated endocytosis
(clathrin-coated vesicles)
Capsid delivers the DNA genome
to the nucleus
Early transcription
Replication
Late gene transcription
: capsid proteins in cytoplasm 
nucleus  viral assembly

9. Pathogenesis and Immunity
Lytic (용해감염) : mucoepithelial cells (점막상피세포)
Latent (잠복감염) : lymphoid and adenoid cells
Transforming (형질전환) : hamster, not human
BOX Disease Mechanisms of Adenoviruses
Virus is spread by aerosol, close contact, or fecal-oral means to establish pharyngeal infection. Fingers spread virus to eyes.
Virus infects mucoepithelial cells in the respiratory tract, gastrointestinal tract, and conjunctiva or cornea, causing cell damage directly.
Disease is determined by the tissue tropism of the specific group or serotype of the virus strain.
Virus persists in lymphoid tissue (e.g., tonsils, adenoids, Peyer's patches).
Antibody is important for prophylaxis and resolution.
viral fiber proteins determine the target cell specificity.
toxic activity of the penton base protein can result in inhibition of cellular mRNA transport and protein synthesis, cell rounding, and tissue damage.

10. ADENOVIRAL INCLUSION BODIES
Histologic appearance of adenovirus-infected cells. Inefficient assembly of virions yields dark basophilic nuclear inclusion bodies containing DNA, proteins, and capsids
The histologic hallmark of adenovirus infection is a dense, central intranuclear inclusion within an infected epithelial cell that consists of viral DNA and protein (Figure 52-3).
These inclusions may resemble those seen in cells infected with cytomegalovirus, but adenovirus does not cause cellular enlargement (cytomegaly).
Mononuclear cell infiltrates and epithelial cell necrosis are seen at the site of infection.

11. Viremia : immunocompromised patients
latent and persist in lymphoid and other tissue, such as adenoids, tonsils, and Peyer's patches
- Antibody : important for resolving lytic adenovirus infections and protects the person from reinfection with the same serotype
- Cell-mediated immunity is important in limiting virus outgrowth, as borne out by the fact that immunosuppressed people suffer more serious and recurrent disease.
host defenses evasion
(1) encode small virus-associated RNAs (VA RNA) : prevent the activation of the interferon-induced protein kinase R mediated inhibition of viral protein synthesis.
(2) viral E3 and E1A proteins block apoptosis induced by cellular responses to the virus or by T cell or cytokine (e.g., TNF-α) actions.
(3) adenoviruses can inhibit CD8(+) cytotoxic T-cell action by preventing proper expression of MHC I molecules and therefore antigen presentation.

12. Epidemiology Disease/Viral Factors
BOX Epidemiology of Adenoviruses
Disease/Viral Factors
Capsid virus is resistant to inactivation by gastrointestinal tract and drying.
Disease symptoms may resemble those of other respiratory virus infections.
Virus may cause asymptomatic shedding.
Transmission
Direct contact via respiratory droplets and fecal matter, on hands, on fomites (e.g., towels, contaminated medical instruments), close contact, and inadequately chlorinated swimming pools.
Who Is at Risk?
Children younger than 14 years of age.
People in crowded areas (e.g., daycare centers, military training camps, swimming clubs).
Geography/Season
Virus is found worldwide.
There is no seasonal incidence.
Modes of Control
Live vaccine for serotypes 4 and 7 is available for military use.

13. Clinical syndromes
Adenoviruses primarily infect children and less commonly infect adults.
Disease from reactivated virus occurs in immunocompromised children and adults.
BOX Clinical Summaries
Pharyngoconjunctival fever (인두결막염) : A 7-year-old student develops sudden onset of red eyes, sore throat, and a fever of 38.9°C (102°F). Several children in the local elementary school have similar symptoms.
Gastroenteritis: An infant has diarrhea and is vomiting. Adenovirus serotype 41 was identified by polymerase chain reaction analysis of stool for epidemiologic reasons.

14. ACUTE FEBRILE PHARYNGITIS (급성 열성 인두염)
AND PHARYNGOCONJUNCTIVAL FEVER (인두 결막염)
pharyngitis, which is often accompanied by conjunctivitis (pinkeye) and pharyngoconjunctival fever.
young children
mild, flulike symptoms (including nasal congestion, cough, coryza, malaise, fever, chills, myalgia, and headache) that may last 3 to 5 days.
- Pharyngoconjunctival fever occurs more often in outbreaks involving older children.
ACUTE RESPIRATORY DISEASE (급성 호흡기 질환)
fever, cough, pharyngitis, and cervical adenitis.
adenovirus serotypes 4 and 7.
military recruits stimulated the development and use of a vaccine for these serotypes.
OTHER RESPIRATORY TRACT DISEASES
coldlike symptoms, laryngitis, croup, and bronchiolitis.
pertussis-like illness, viral pneumonia.
CONJUNCTIVITIS AND EPIDEMIC KERATOCONJUNCTIVITIS (유행성 결막염)
follicular conjunctivitis : (Figure 52-6).
Swimming pool conjunctivitis
Epidemic keratoconjunctivitis may be an occupational hazard
for industrial workers.
GASTROENTERITIS AND DIARRHEA
- major cause of acute viral gastroenteritis;
- Adenovirus serotypes 40 to 42 have been grouped as enteric adenoviruses (group F)

15. Laboratory Diagnosis Prevention Gene therapy Good handwashing
- Immunoassays, including fluorescent antibody and enzyme-linked immunosorbent assays
- genome assays, PCR to detect, type, -must be used for enteric adenovirus serotypes 40 to 42, which do not grow readily in available cell cultures.
Prevention
Good handwashing
Contact precautions
Chlorination of water
Disinfection or sterilization of ophthalmologic equipment
Use of single dose vials
Oral vaccine- restricted use
Gene therapy
Used as VECTORS to transfer desired genetic material into cells
Viral genome is relatively easily manipulated in vitro
Efficient expression of inserted DNA in recipient cell

16. Gene Therapy The transfer of selected genes into a host
with the hope of ameliorating or curing a disease state

17. Sustained drug delivery
Human many diseases
absence or inappropriate presence of a protein
Isolate and produce these natural proteins
genetic engineering and recombinant technology
Protein delivery
Sustained drug delivery
Gene therapy
Ultimate method of protein delivery
Body’s cells
Small factories
produce a therapeutic protein
for a specific disease over a prolonged period

20. Gene Transfer Technology
Viral Vectors for Gene Transfer
Retroviral Vectors
HIV
MMLV
Adenoviral Vector
Adeno-Associatedviral Vector
Herpes Simplex Viral Vector
Nonviral Techniques
Naked DNA
Liposome
Molecular conjugates
Antisense Technology
Non-catalytic antisense
Catalytic antisense molecules
Ribozymes: hammerhead or hairpin

21. Retrovirus Vectors Adenovirus vectors Herpes simplex virus
Advantages Disadvantages
High transduction efficiency Requires dividing cells for infectivity
Insert size up to 8kB Low titers( )
Integrates into host genome resulting Integration is random
in sustained expression of vector
Extremely well studied system In vivo delivery remains poor.
Vector proteins not expressed in host Effective only when infecting helper
cell lines
Adenovirus vectors
Advantages Disadvantages
High transduction efficiency Expression is transient
Insert size up to 8kb (viral DNA does not integrate)
Viral proteins can be expressed in
High viral titer ( ) host following vector administration
Infects both replicating and In vivo delivery hampered by host
differentiated cells immune response
Herpes simplex virus
Advantages Disadvantages
Large insert size System currently under development
Could provide long-term CNS Current vectors provide transient expression
gene expression
High titer Low transduction efficiency

22. AAV : Useful for a vector for gene therapy
-Lack of association with disease
-The ability to latently infect a high fraction of
exposed cells
-A minimal number of viral antigens to induce a host
immune response
-The possible ability to latently infect
non-dividing cells
-The possible advantage of site-specific integration
-The ability to latently infect a broad range of
human cell types

23. Absence of viral genes that may be responsible for causing an
Delivery System Requirements
A practical gene delivery system must meet five demanding requirements.  It must be:
Efficient
Capable of achieving the duration of expression of protein
from the gene required by the applicable medical situation
Flexible with respect to the tissues it can deliver to
Able to handle a wide range of therapeutic genes
Able to demonstrate a dose-response relationship
Efficient delivery of genes to both dividing and non-dividing target cells
Absence of viral genes that may be responsible for causing an
undesirable immune response
In vivo administration to patients
High levels of gene expression
Excellent stability allowing AAV vectors to be manufactured, stored and handled like more traditional pharmaceutical products.

24. Structure of wild type and vector AAV genome

25. Structure of AAV Virus Genomic DNA
AAV Vector Delivery System
                                                 
AAV Virus Particles
                                            
Structure of AAV Virus Genomic DNA
                                             
Structure of AAV Vector DNA

26. Adenovirus-dependent and Adenovirus-free production of rAAV

27. Gene Therapy