What is adenovirus?
Adenoviruses were first isolated in human adenoids, from which the name is
derived. Adenoviruses are medium-sized (90–100 nm), nonenveloped (naked)
icosahedral viruses composed of a nucleocapsid and linear, non-segmented double
stranded (ds) DNA genome which is about 36kb long. The genes of Adenovirus are
divided into two phases, early and late phases. Genes of early phase are E1, E2,
E3 and E4. Genes of late phases are L1, L2, L3, L4 and L5. The virion use its
unique "spike" or fiber associated with each penton base of the capsid to attach
the host cell via the coxsackie-adenovirus receptor on the surface of the host
cell. There are more than 53 described serotypes in humans. Adenovirus can cause
upper respiratory infections, conjunctivitis, gastroenteritis in human.
Adenovirus type 5 is most extensive studied. Recombinant adenovirus vector
usually delete E1 and E3 genes from genome of wild type adenovirus, making it
replication deficiency so it can be safely used as transgene vector. E1 plus E3
are about 8.0 kb so the maximum insert size of your interesting gene is about
8.0kb, taking it into account that Promoter and Poly A occupy about 1 kb, so the
maximum insert size is about 7 kb. Due to higher transduction efficiency (almost
100%), higher level expression of transgene and broad range of tropism,
adenovirus vector has been widely used as gene therapy vector, vaccine
production, gene knockdown, production of membrane and hard-to-expression
protein and engineering antibody.
Is the recombinant adenovirus safe?
the recombinant vector is replication deficient adenovirus without the E1 and E3
genes. E1 and E3 gene products will be provided by E1 and E3 immortalized human
kidney embryo cell 293.
What are the signs and symptoms of adenovirus infection?
Adenovirus often infects the airways – this can appear as a “cold” (e.g., sore
throat, sneezing, runny nose, cough, headache, chills), croup, or bronchitis.
Illness usually lasts three to five days, but serious infections can last weeks.
Complications can include middle ear infections, pneumonia, or meningitis. Some
types of adenovirus cause other illnesses, such as skin rash, conjunctivitis
(pink-eye,) bladder infections, or bowel infections (e.g., diarrhea).
How is adenovirus infection diagnosed and treated?
Some laboratory tests for adenovirus are available; however, doctors usually
diagnose infection from the person’s symptoms. There is no adenovirus-specific
therapy – in most cases the immune system deals with the infection. Treatment
may include rest, drinking plenty of fluids, over-the-counter medicine (e.g.,
acetaminophen [Tylenol]), a cool-mist humidifier, and sometimes bronchodilators.
See a doctor if fever continues for more than a few days, symptoms worsen,
breathing problems occur, or not enough fluids are being taken. Serious illness
may need hospitalization (e.g., for fluids, oxygen) or other treatment (e.g.,
topical steroids for conjunctivitis) – those with weak immune systems may need
treatment by specialists.
What are the applications for recombinant adenovirus (rAd)?:
Recombinant Adenovirus are used for express gene and shRNA in many mammalian
cells, dividing or non-dividing, in vitro , vaccine studies, gene therapy,
especially good for short term high level expression experiment due to its high
transduction efficiency and broad host range. Medium size amplification is
suitable for tissue culture.
What is the difference between viral particle and Plaque Forming Unit (PFU)?
Viral particles are calculated based on reading of OD260. It represents total
virus (live and dead) in the solution. PFU represents the infectious virus. It
is determined by counting the number of plaques of mono-layer of HEK293 cells
infected by 10-fold serial dilution of virus stock. After virus infect HEK293
cells, it will propagate in the cells, when virus reach certain amount, they are
released from infected HEK293 cell and the infected cell are ruptured, dead and
floated. The released virus infected neighbored cells again, and repeat the
infection and release process, until the plaques are formed and counted. For
most virus preps, the VP/PFU ratio range is 5:1 to 100:1.
What is the cloning capacity of the adenovirus?
The cloning capacity for the transgene, by using (DE1/E3) adenovirus type 5, is
about 7 Kb in length plus 1 kb promoter and Poly A.
How can I find out whether adenovirus will work well in my cell models?
The Adenovirus has a very broad host range; it can infect human and other
mammalian cell lines or primary cells, including replicative as well as non-replicative
cells. There are in fact very few cell lines that cannot be infected. Some
lymphoid cell lines may be more resistant to Adenovirus infection, and so may
need high quantities of viruses to achieve sufficient infection levels.
For your convenience, we offer some marker adenoviruses, such as
Ad-CMV-GFP to allow you to conduct pilot testing in your
systems.
What's the optimal concentration of viruses that I should use for
infection?
The appropriate amount of active/infectious viruses used for infecting cells is
very important for the outcome of your experiments. If not enough virus is used,
it will not give 100% of infection. If too much virus is used, it will cause
cytotoxicity or other undesired effects. You should use the minimal virus
concentration that will give 100% gene delivery. This optimal concentration
differs dramatically between cell types. To determine this optimal concentration
of virus, you could conduct pilot testing in your system by using marker
adenoviruses, such as
Ad-CMV-GFP.
We have tested numerous cell types by exposing cells to virus-containing media
for 6-8 hours or overnight. For most cell types, viral concentrations of 2x105~1x106
PFU (infectious unit)/ml of media gives 100% of infection without visible
toxicity. However, we recommend you test your cell system by using marker
viruses.
There are several viral gene delivery systems, including adenovirus,
retrovirus and lentivirus. Which one should I use for my experiments?
Adenovirus: 100% gene delivery efficiency in most cell types including dividing
and non-dividing or primary cells. There is no integration with the host system.
However, protocols involved in developing recombinant adenovirus are very
complicated.
Retrovirus: gives to <30% efficiency in most cell types, and requires active
cell division. In addition, there is a significant risk of integration into the
host genome, leading to mutation of genes or activation of oncogenes in the host
system, which is a concern for scientists. Unlike adenovirus, recombinant
retrovirus is very easy to develop.
Lentivirus: gives to <30% efficiency, in both dividing and non-dividing cells.
Like retrovirus, there is a significant risk of integration into the host
genome, leading to mutation of genes or activation of oncogenes in the host
system. As with retrovirus, recombinant lentivirus is very easy to develop.
If you are interested in delivering your specific gene or siRNA into all cells,
adenovirus system is the best choice. However, if you are interested in
single-cell level studies, retroviral system, lentiviral system or even direct
transfection of expression plasmid DNA may sometimes serve your need.
What are the advantages and disadvantage of
adenovirus vector and letivirus?
Generally speaking, adenovirus vector has
highest transduction efficiency (100%) than lentivirus(<30%). There are some
exceptions for which letivirus is better than adenovirus such in case of
skeletal muscles and lymphocytes. Adenovirus can reach highest transgene
expression in very short time and gene expression level decline with time, on
the other hand, lentivirus expression is slow and persistent with potential
randomly activation of oncogenes.
I will use adenovirus to do in vitro experiment? do I need the CsCl or
chromatography purification?
Tissue culture dose not require high purification of adenovirus. Medium
amplification is enough. CsCl or chromatography purification is required for in
vivo studies.
Is there a receptor for adenovirus
infection?
Yes, the classical adenovirus receptor is called Coxsackie and
Adenovirus Receptor (CAR). It is believed that CAR determine the tropism of
adenovirus. Recent study showed that there are some molecules such as CD46,
heparan sulfate, or sialic acid which can also mediated adenovirus infection
functioning as other adenovirus receptors.
Does adenovirus infect CAR negative cell?
Yes. CAR is the one of the adenovirus receptors. There are some
molecules which also works as adenovirus receptor s, in addition to as
CD46, heparan sulfate, or sialic acid. One example is
Integrin αvβ5.
Potentially any adhering molecule can function as adenovirus receptor.
Does adenovirus infect any human
cell lines?
Theoretically adenovirus infect almost any human cells, however
transduction efficiency does vary with different cells
, depending on the adenovirus
receptors. It is recommended to do pilot study by using Ad-CMV-GFP (SL100708).
Which cell has the lowest
transduction
efficiency?
Muscle
and lymphocytes are normally CAR deficient and refractory to
transduction.
What is the best choice to
deliver or knockdown gene in nervous system?
Recombinant adenovirus and adeno-associated virus (AAV) have the highest
transduction efficiency (>75% to 97%) for central nervous system and
peripheral nervous
system, for which any non-viral transfection reagent is incomparable.
Adenovirus and AAV can infect different type cells from nervous
system, including neuron, glial cells, retina, neural stem cells,
Y79
retinoblastoma cells,
cochlear hair cell. Adenovirus vector is suitable for quick and short term
expression of transgene and gene knockdown. For stable and long term gene
expression and knockdown, AAV is the choice.
Which viral vector are good for muscle cell and lymphocytes?
Adeno associated virus efficiently transfer genes to nondividing
cells such as muscle cell, for lymphocytes, transduction efficiency is lower
than muscle cell.
Lymphocytes are natural target for lentivirus because it is derived from Human
Immunodeficiency Virus (HIV), making lentivirus less safe than adenovirus and
AAV.
How to store Adenovirus?
Generally speaking. the virus stock should be aliquoted upon receipt and stored
at -80°C. the virus should be stable for up to a year or so. avoiding repeated
thaw and freeze, especially after CsCl or chromatography purification in PBS
solution. Virus stored at -80°C in DMEM supplemented with serum or BSA are
relatively more stable than purified virus. it will remain stable after going
through 2-3 freeze-thaw cycles.
To learn more about adenovirus or have any questions or comments about
adenovirus, please email us at
tech@signagen.com
