ADENOVIRUSESADENOVIRUSES

ADENOVIRUS- ClassificationADENOVIRUS- 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………..

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 Infants, young children

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 Infants, young children

Hepatitis Liver transplant recipients; other immunocompromised patients

Meningoencephalitis Children; immunocompromised patients

Table 52-1. Illnesses Associated with Adenoviruses

ADENOVIRUS - StructureADENOVIRUS - Structure

• Non-enveloped DNA virus• Icosadeltahedrons (20 면체 )• 70-90 nm in size • Linear ds DNA genome (36kb) with a

terminal protein (molecular mass, 55 kDa)

- 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 52-1. 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.

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)

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

Entry and replicationEntry 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 v 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

Pathogenesis and ImmunityPathogenesis and Immunity

• 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.

-Lytic ( 용해감염 ) : mucoepithelial cells ( 점막상피세포 ) -Latent ( 잠복감염 ) : lymphoid and adenoid cells-Transforming ( 형질전환 ) : hamster, not human

BOX 53-2. 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.

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.

ADENOVIRAL INCLUSION BODIESADENOVIRAL INCLUSION BODIES

Histologic appearance of adenovirus-infected cells. Inefficient assembly of virions yields dark basophilic nuclear inclusion bodies containing DNA, proteins, and capsids

• 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.

Epidemiology

BOX 53-3. 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.

Adenoviruses primarily infect children and less commonly infect adults. Disease from reactivated virus occurs in immunocompromised children and adults.

BOX 52-4. 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.

Clinical syndromes

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)

Laboratory Diagnosis - 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.

PreventionPrevention• Good handwashing• Contact precautions• Chlorination of water• Disinfection or sterilization of

ophthalmologic equipment• Use of single dose vials• Oral vaccine- restricted use

• 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

Gene therapy

Gene Therapy

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

Human many diseases absence or inappropriate presence of a protein

Isolate and produce these natural proteins genetic engineering and recombinant technology

Protein deliverySustained drug delivery

Gene therapyUltimate method of protein delivery

Body’s cells Small factories

produce a therapeutic protein for a specific disease over a prolonged period

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

Retrovirus Vectors Advantages DisadvantagesHigh transduction efficiency Requires dividing cells for infectivityInsert size up to 8kB Low titers(106-107)Integrates into host genome resulting Integration is randomin sustained expression of vectorExtremely well studied system In vivo delivery remains poor.Vector proteins not expressed in host Effective only when infecting helper cell lines

Adenovirus vectors

Advantages DisadvantagesHigh transduction efficiency Expression is transientInsert size up to 8kb (viral DNA does not integrate) Viral proteins can be expressed in High viral titer (1010-1013) host following vector administrationInfects both replicating and In vivo delivery hampered by hostdifferentiated cells immune response

Herpes simplex virus

Advantages DisadvantagesLarge insert size System currently under developmentCould provide long-term CNS Current vectors provide transient expressiongene expressionHigh titer Low transduction efficiency

-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

AAV : Useful for a vector for gene therapy

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.

Structure of wild type and vector AAV genome

                                                 

AAV Virus Particles

                                            

Structure of AAV Virus Genomic DNA

                                             

Structure of AAV Vector DNA

AAV Vector Delivery System

Adenovirus-dependent and Adenovirus-free production of rAAV

Gene Therapy