ADENOVIRUSES

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………..

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

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)

-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 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 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

Pathogenesis and Immunity 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. 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.

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.

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 Disease/Viral Factors 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.

Clinical syndromes 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.

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 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

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

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

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 Adenovirus vectors Herpes simplex virus Advantages Disadvantages High transduction efficiency Requires dividing cells for infectivity Insert size up to 8kB Low titers(106-107) 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 (1010-1013) 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

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

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.

Structure of wild type and vector AAV genome

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

Adenovirus-dependent and Adenovirus-free production of rAAV

Gene Therapy