Adenovirus FAQs

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.

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