adenovirus and gene therapy Flashcards
- general structure of the adenovirus virion
naked, icosahedral, 90 nm in diameter, with capsids composed of 252 capsomers. Linear dsDNA. Since they contain no lipid, are resistant to lipid solvents such as bile salts (can be isolated from feces). Stable to heat.
adenovirus replication
Attaches to cell via fiber protein > internalized via receptor mediated andocytosis > Early gene products promote cell’s transition into S-phase and activate viral genes for viral DNA replication > viral DNA replication involves attachment of viral terminal protein to first base of 5’ end of each DNA strand to serve as primer for virally encoded DNA polymerase > cessation of host DNA replication after 8-10 hrs and protein synthesis 6-10 hrs after that.
Adenovirus hexons
The hexonsare components of the viral capsid and contain family-reactive determinants that cross-react with a similar Ag in all mastadenoviruses (the family that includes human adenoviruses). Hexons also possess a type-specific reactive site that is the prevalent antigen exposed when hexons are assembled in virions
Adenovirus pentons
contain minor antigens of the virions and a family-reactive soluble Ag found in infected cells.
Which cells does adenovirus infect
Epithelium-like and embryonic kidney are best. T and B cells are productively infected, but yield low levels of virus
adenovirus transmission
Person-to-person spread in respiratory and ocular secretions is the most common mode. Among children, fecal-oral transmission is important. Dissemination in swimming pools has been implicated in epidemics of pharyngoconjunctival fever and conjunctivitis. Excreted in feces (even in respiratory/ ocular infections) for days or even years. Serotypes 40 and 41 are the only types clearly transmitted by fecal-oral route.
types of diseases caused by adenovirus
subclinical, pharyngitis, tonsillitis, and nasopharyngitis are most common. acute respiratory illness, pneumonia, conjunctivitis, pharyngoconjunctival fever, diarrhea in children, ARD, ocular infections, disseminated infections, CNS infections, epidemic keratoconjunctivitis, fatal pneumonia in military recruits (rare),
Which adenovirus serotype is associated with acute respiratory disease
Serotype 14 recently in the US. Serotypes 4 and 7 are most common. Plus 11, 14 and 21
Which adenovirus serotype is associated with obesity
serotype 36- Induces Commitment Differentiation and Lipid Accumulation in Human Adipose-derived Stem Cells.
Adenovirus pathogenesis
Able to replicate in all areas of the respiratory tract, producing acute upper and lower respiratory tract infections. Induce latent infections in tonsils, adenoids, and other lymphoid tissues that may be reactivated. Persistent infections occur despite adequate immune function, leading to asymptomatic chronic shedding of viable viral particles. Can disseminate to CNS, liver, skin, myocardium and kidneys
Adenovirus diagnosis
Recovery from the eye, genital tract, lung, or brain is diagnostic; from the throat of a patient with respiratory disease, suggestive; and from feces, ambiguous, because adenoviruses are shed in large numbers and for long periods of time in the feces. Serology and ELISA and cultures in human epithelial cells
Adenovirus treatment
ribavirin and cidofovir have been used to treat systemic infections in immune-suppressed transplant patients
Adenovirus prevention
Since many healthy persons are carriers, isolation of sick people has little effect. Live virus is used to vaccinate US protect military recruits with serotypes 3, 4, 7 and 21.
List the viruses used for gene therapy
retroviruses, adenoviruses, and adeno-associated virus (AAV)
retrovirus vectors- length of time the transduced gene is expressed
Long term due to DNA integration into host DNA
Which retroviral genes are retained in the retroviral vector
Only the long terminal repeats, the sequence that encodes the signal to package the genomic RNA within the virion, and sequences required for initiation of reverse transcription of the viral RNA need be retained in the virus vector.
retrovirus vectors host range/ stabilization
Stabilization of the virion and broadening of its host range occurs through the use of pseudotyping with the vesicular stomatitis G protein as the envelope glycoprotein
Which cells cant retroviruses infect
retroviruses cant transduce non-mitotic cells b/c th cDNA that is synthesized within the cytoplasm after infection is unable to enter the nucleus prior to cell division. This problem is being addressed through the use of lentivirus (e.g., HIV) based vectors. These vectors take advantage of the ability of lentivirus proteins to direct the viral cDNA to the nucleus in certain cells that are not dividing. However, many non-mitotic cells still cannot be transduced.
retrovirus vectors advantages and disadvantages
advantages: 8kb of foreign DNA can be used, stable expression of the transduced gene because of proviral integration in host DNA. Disadvantages: relatively small amounts of virus obtained during growth, virions are not very stable, low efficiency of transduction of certain non-mitotic cells
Structure of adeno-associated virus
AAV is a parvovirus with a naked capsid and a single-stranded DNA genome of approximately 5 kb.
Advantages of adeno-associated virus as a vector
high titers can be obtained, the virions are very stable, approximately 5 kb of foreign DNA can be introduced, AAV has not been shown to cause clinical symptoms
disadvantages of adeno-associated virus as a vector
the viral chromosome integrates, although inefficiently, into host DNA, a potentially mutagenic event. second strand DNA synthesis, gene expression, and integration are typically very slow in the absence of helper virus. the small size of the genome means that large genes cannot be introduced into the vector. in non-mitotic cells, transduction may be inefficient in the absence of adenovirus helper function
Advantages of adenovirus vectors
very high titers can be obtained, virions are stable, the viral chromosome does not normally integrate, non-mitotic cells are efficiently transduced, -36 kb of foreign DNA can be inserted, depending on the vector used, adenoviruses can be made to selectively replicate in, and kill, tumor cells
Disadvantages of adenovirus vectors
elicit a strong inflammatory response and mst people have Abs against adenoviruses, making repeated use a problem. Requirement that most of adenovirus genes are retained in vector. expression of the transduced gene will be transient in mitotic cells. death of patient in gene therapy trial
What kind of vector has shown success in treatment of SCID
retrovirus vector
