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 serotypesCommon serotypes:- 1-8, 11, 21, 35, 37, 40Enteric Adenoviruses belong to subgroup FMolecular biology research : splicing……Gene therapy : cystic fibrosis………..
3 Table 52-1. Illnesses Associated with Adenoviruses DiseasePatient PopulationRespiratory DiseasesFebrile, undifferentiated upper respiratory tract infectionInfants, young childrenPharyngoconjunctival feverChildren, adultsAcute respiratory diseaseMilitary recruits (serotype 4, 7)Pertussis-like syndromePneumoniaInfants, young children; military recruits; immunocompromised patientsOther DiseasesAcute hemorrhagic cystitisChildren; bone marrow transplant recipientsEpidemic keratoconjunctivitisAny age; renal transplant recipientsGastroenteritisHepatitisLiver transplant recipients; other immunocompromised patientsMeningoencephalitisChildren; immunocompromised patients
4 ADENOVIRUS - Structure Non-enveloped DNA virusIcosadeltahedrons (20면체)70-90 nm in sizeLinear 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 proteinscan act as a hemagglutinin.penton base and fiber are : toxic to cells, carry type-specific antigens.BOX Unique Features of AdenovirusNaked 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 strandEarly transcription – early protein : E1A, E1B, E2A, E2B, E3, E4Late transcription –late protein11 polypeptide : 9 – structural protein (capsid)2 – core (DNA-binding protein)
7 Gene Number M.W (kDa2) Function E1A* Activates viral gene transcriptionBinds cellular growth suppressor: p105RB promotes transformationDeregulates cell growthInhibits activation of interferon response elementsE1BBinds cellular growth suppressor: p53 promotes transformationBlocks apoptosisE2Activates some promotersTerminal protein on DNADNA polymeraseE3Prevents tumor necrosis factor-α (TFN-α) inflammationE4Limits viral cytopathologic effectVA RNAsInhibit interferon responseCapsidII120Contains family antigen and some serotyping antigensIII85Penton base proteinToxic to tissue culture cellsIV62FiberResponsible for attachment and hemagglutination; contains some serotyping antigensVI24Hexon-associated proteinsVIII13Penton-associated proteinsIX12IIIa66CoreV48Core protein 1: DNA-binding proteinVII18Core protein 2: DNA-binding protein
8 Entry and replicationFiber protein determines target cell specificity and attachmentViral DNA enters host cell nucleusVirus replicates in nucleusFiber receptor : Ig superfamilyCoxsackie B viruses 도 이용- Coxsackie adenovirus receptor- MHC I 도 이용Penton base interact with av integrinreceptor-mediated endocytosis(clathrin-coated vesicles)Capsid delivers the DNA genometo the nucleusEarly transcriptionReplicationLate gene transcription: capsid proteins in cytoplasm nucleus viral assembly
9 Pathogenesis and Immunity Lytic (용해감염) : mucoepithelial cells (점막상피세포)Latent (잠복감염) : lymphoid and adenoid cellsTransforming (형질전환) : hamster, not humanBOX Disease Mechanisms of AdenovirusesVirus 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 capsidsThe 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 AdenovirusesDisease/Viral FactorsCapsid 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.TransmissionDirect 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/SeasonVirus is found worldwide.There is no seasonal incidence.Modes of ControlLive vaccine for serotypes 4 and 7 is available for military use.
13 Clinical syndromesAdenoviruses primarily infect children and less commonly infect adults.Disease from reactivated virus occurs in immunocompromised children and adults.BOX Clinical SummariesPharyngoconjunctival 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 childrenmild, 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 DISEASEScoldlike symptoms, laryngitis, croup, and bronchiolitis.pertussis-like illness, viral pneumonia.CONJUNCTIVITIS AND EPIDEMIC KERATOCONJUNCTIVITIS (유행성 결막염)follicular conjunctivitis : (Figure 52-6).Swimming pool conjunctivitisEpidemic keratoconjunctivitis may be an occupational hazardfor 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.PreventionGood handwashingContact precautionsChlorination of waterDisinfection or sterilization of ophthalmologic equipmentUse of single dose vialsOral vaccine- restricted useGene therapyUsed as VECTORS to transfer desired genetic material into cellsViral genome is relatively easily manipulated in vitroEfficient 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 diseasesabsence or inappropriate presence of a proteinIsolate and produce these natural proteinsgenetic engineering and recombinant technologyProtein deliverySustained drug deliveryGene therapyUltimate method of protein deliveryBody’s cellsSmall factoriesproduce a therapeutic proteinfor a specific disease over a prolonged period
20 Gene Transfer Technology Viral Vectors for Gene TransferRetroviral VectorsHIVMMLVAdenoviral VectorAdeno-Associatedviral VectorHerpes Simplex Viral VectorNonviral TechniquesNaked DNALiposomeMolecular conjugatesAntisense TechnologyNon-catalytic antisenseCatalytic antisense moleculesRibozymes: hammerhead or hairpin
21 Retrovirus Vectors Adenovirus vectors Herpes simplex virus Advantages DisadvantagesHigh transduction efficiency Requires dividing cells for infectivityInsert size up to 8kB Low titers( )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 helpercell linesAdenovirus vectorsAdvantages DisadvantagesHigh transduction efficiency Expression is transientInsert size up to 8kb (viral DNA does not integrate)Viral proteins can be expressed inHigh viral titer ( ) host following vector administrationInfects both replicating and In vivo delivery hampered by hostdifferentiated cells immune responseHerpes simplex virusAdvantages DisadvantagesLarge insert size System currently under developmentCould provide long-term CNS Current vectors provide transient expressiongene expressionHigh 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 ofexposed cells-A minimal number of viral antigens to induce a hostimmune response-The possible ability to latently infectnon-dividing cells-The possible advantage of site-specific integration-The ability to latently infect a broad range ofhuman cell types
23 Absence of viral genes that may be responsible for causing an Delivery System RequirementsA practical gene delivery system must meet five demanding requirements. It must be:EfficientCapable of achieving the duration of expression of proteinfrom the gene required by the applicable medical situationFlexible with respect to the tissues it can deliver toAble to handle a wide range of therapeutic genesAble to demonstrate a dose-response relationshipEfficient delivery of genes to both dividing and non-dividing target cellsAbsence of viral genes that may be responsible for causing anundesirable immune responseIn vivo administration to patientsHigh levels of gene expressionExcellent stability allowing AAV vectors to be manufactured, stored and handled like more traditional pharmaceutical products.