adenovirus and gene therapy

Question 1

1. general structure of the adenovirus virion

Answer 1

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.

Question 2

adenovirus replication

Answer 2

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.

Question 3

Adenovirus hexons

Answer 3

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

Question 4

Adenovirus pentons

Answer 4

contain minor antigens of the virions and a family-reactive soluble Ag found in infected cells.

Question 5

Which cells does adenovirus infect

Answer 5

Epithelium-like and embryonic kidney are best. T and B cells are productively infected, but yield low levels of virus

Question 6

adenovirus transmission

Answer 6

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.

Question 7

types of diseases caused by adenovirus

Answer 7

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),

Question 8

Which adenovirus serotype is associated with acute respiratory disease

Answer 8

Serotype 14 recently in the US. Serotypes 4 and 7 are most common. Plus 11, 14 and 21

Question 9

Which adenovirus serotype is associated with obesity

Answer 9

serotype 36- Induces Commitment Differentiation and Lipid Accumulation in Human Adipose-derived Stem Cells.

Question 10

Adenovirus pathogenesis

Answer 10

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

Question 11

Adenovirus diagnosis

Answer 11

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

Question 12

Adenovirus treatment

Answer 12

ribavirin and cidofovir have been used to treat systemic infections in immune-suppressed transplant patients

Question 13

Adenovirus prevention

Answer 13

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.

Question 14

List the viruses used for gene therapy

Answer 14

retroviruses, adenoviruses, and adeno-associated virus (AAV)

Question 15

retrovirus vectors- length of time the transduced gene is expressed

Answer 15

Long term due to DNA integration into host DNA

Question 16

Which retroviral genes are retained in the retroviral vector

Answer 16

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.

Question 17

retrovirus vectors host range/ stabilization

Answer 17

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

Question 18

Which cells cant retroviruses infect

Answer 18

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.

Question 19

retrovirus vectors advantages and disadvantages

Answer 19

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

Question 20

Structure of adeno-associated virus

Answer 20

AAV is a parvovirus with a naked capsid and a single-stranded DNA genome of approximately 5 kb.

Question 21

Advantages of adeno-associated virus as a vector

Answer 21

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

Question 22

disadvantages of adeno-associated virus as a vector

Answer 22

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

Question 23

Advantages of adenovirus vectors

Answer 23

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

Question 24

Disadvantages of adenovirus vectors

Answer 24

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

Question 25

What kind of vector has shown success in treatment of SCID

Answer 25

retrovirus vector