ADENOVIRUSES

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ADENOVIRUSES
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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..

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Table 52-1. Illnesses Associated with
Adenoviruses
Disease Patient Population
Respiratory Diseases 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 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
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ADENOVIRUS - Structure

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

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• - 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.
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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)

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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-a (TFN-a) inflammation
E4     Limits viral cytopathologic effect
VA RNAs     Inhibit interferon response
Capsid Capsid Capsid 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 Core Core Core
  V 48 Core protein 1 DNA-binding protein
  VII 18 Core protein 2 DNA-binding protein

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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 ? ??
• - Coxsackie adenovirus receptor
• - MHC I ? ??
• 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

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Pathogenesis and Immunity

• Lytic (????) mucoepithelial cells (??????)
• Latent (????) lymphoid and adenoid cells
• Transforming (????) 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.

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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.
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• 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-a) actions.
• (3) adenoviruses can inhibit CD8() cytotoxic
  T-cell action by preventing proper expression of
  MHC I molecules and therefore antigen
  presentation.

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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.
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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 (?????) A 7-year-old student develops sudden onset of red eyes, sore throat, and a fever of 38.9C (102F). 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.
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ACUTE FEBRILE PHARYNGITIS (?? ?? ???) AND
PHARYNGOCONJUNCTIVAL FEVER (?? ???)

• 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 (?? ??? ??)

• 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
(??? ???)

• 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)
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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.
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

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Gene Therapy The transfer of selected genes
into a host with the hope of ameliorating or
curing a disease state
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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 Bodys cells Small factories produce a
therapeutic protein for a specific disease over
a prolonged period
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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
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Retrovirus Vectors
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
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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
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• 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.

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Structure of wild type and vector AAV genome
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AAV Vector Delivery System
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Adenovirus-dependent and Adenovirus-free
production of rAAV
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Gene Therapy