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FAQs for adenovirus

Q1. Are the recombinant adenovirus vectors safe to handle? 
Q2. What is the requirement for handling recombinant adenovirus vectors?  
Q3. How are virus titers determined?  
Q4. What are the differences between viral particle (VP), plaque formation unit (PFU) and infectious unit (IFU)? Which one of these better reflects the amount of active virus used?


 Q1. Are the recombinant adenovirus vectors safe to handle?
Recombinant adenovirus vectors are currently derived from the defective adenovirus serotype 5 that are deleted in the E1 and E3 region, which will not replicate in cells other than Human Embryonic Kidney (HEK) 293 cells.

The probability of producing replication competent adenovirus (RCA), although low, increases with each successive amplification. RCA is produced when adenoviral DNA recombines with E1-containing genomic DNA in HEK 293 cells. It is suggested to use early amplification stocks when needed to produce additional quantities of adenovirus.

Q2. What is the requirement for handling recombinant adenovirus vectors? 

The recombinant adenoviruses we made are replication deficient due to deletions in the E1 and E3 regions. they will not replicate in cells other than complementing cells (293 cells). According to references issued by the NIH Office of Biosafety, recombinant human adenovirus has been classified in biosafety level II for agents considered of ordinary potential harm, and you need BL-2 level facility to work with it. It should be noted that cell culture facilities in most institutes are certified as BL-2 level.

Wild type, replication competent adenoviruses provoke cold symptoms and strong immune responses in healthy individuals and generally do not cause serious illness. For more information on biosafety levels, please refer to the following CDC publication: Biosafety in Microbiological and Biomedical Laboratories,5th Edition, Dec. 2009; this publication is also available at http:oba.od.nih.gov and http://www.cdc.gov/biosafety/publications/bmbl5/.

 Q3. How are virus titers determined?
There are 3 commonly used protocols for determining adenovirus titer:
(1) OD260 Assay, (2) Plaque Formation Assay, and (3) End-point Dilution Assay.
OD260 assay measures the concentration of viral DNA and protein. It does not distinguish between intact, infectious viruses and damaged, non-infectious viruses. It is a physical assay measuring the concentration of total viruses, live and dead. Based on OD260, the concentration of viral particles (VP) could be obtained. To measure the OD260, the virus stock has to be purified first.

Q4.What are the differences between viral particles (VPs), plaque formation unit (PFU) and infectious unit (IFU)? Which one of these better reflects the amount of active virus used?
Viral particles (VPs) represent the total number of viral particles (infectious and non-infectious combined). The ratio of infectious/non-infectious varies because of varations in virus preparations.

PFU (plaque formation unit) represents the number of infectious viruses. IFU (infectious unit) is equivalent to PFU. For most virus preparations, the VP/PFU ratio is 20:1 to 50:1.

Using VP (viral particles) as the unit will result in significant variations in the amount of actual live viruses used, and using IFU or PFU as the viral unit will give more consistent outcomes. We provide empirical formula to estimate PFU if the virus purified our kit ( for more details, please read the manual of Adeno-Purification Kits).

  Q5. What are RCAs (Replication competent adenoviruses) and how can I avoid them? 
One concern when working with Adenoviral vectors is the possible occurrence of replication competent adenoviruses (RCAs) in a population of replication deficient adenoviruses (Ad). RCAs can emerge as a result of a double crossover event between the homologous overlapping sequences present in the recombinant Ad and the 293 genome (Lochmüller, 1994). This event results in the loss of the transgene and its replacement by the E1 region (Zhu, 1999) thus rendering the Ad replication competent without the need of a complementing cell line. Lochmüller et al. demonstrated that Ad stocks contained an increasing amount of RCAs after increasing number of passages in 293 cells.

Q6. What are the conditions recommended for the storage of recombinant Adenovirus preparations?
The viruses should be stored at -80°C especially after purification from culture media. The virus will be stable for 1-2 years. Storage at -20°C for purified virus is not recommended. Virus in cultured media (DMEM supplemented with serum) is stable in room temperature in short time (1-2 days) or at 4 °C for up to 1 week. For longer storage, keep it at -20 °C or -80 °C.
Q7. For in vivo use (animal modles), is the cesium chlordie purification required?

Ideally Yes, but not absolutely necessary: CsCl purification is most effficient in order to 1) remove defective particles, 2) remove condition media with its contaminants from the viral preparation, 3) concentrate the virus to a level suitable for injection and 4) resuspend the virus in a buffer suitable for injection. However, adenovirs prepared by our kit (Adeno-Purification Kits) showed efficient transduction without any cytotoxicity or immune response. 

Q8.  What type of cell is used to amplify recombinant Adenoviruses in your systems?
The cells used for the amplification of recombinant Adenoviruses is Human Embryonic Kidney 293 cell line (HEK-293 cell). The HEK293 cell contains the full E1 region of the Adenovirus type 5, nucleotides 1 to 4355, making HEK293 cell suitable for the generation and growth of helper-independent recombinant Adenoviruses.

Q9.  Will the adenovirus work with my specific cells or tissues?

The Adenovirus has a very broad host range; it can infect human and other mammalian cell lines or primary cells, and replicative as well as non-replicative cells.

In general, the viral infectious efficiency (e.g. 100% infection) varies considerably from one cell type or tissue to another. To know how many viral dose will be efficient in certain experiment, we strongly recommend performing an infectivity test with a control reporter virus (i.e. GFP or LacZ) when starting a new project with recombinant Adenoviruses.

Q10. What determines the tropism of Adenovirus in infecting cells?

Cell lines with a high number of Adenovirus receptors (CAR, coxsackievirus-adenovirus receptor, and Integrins), like Cos-7 and HeLa, typically show significant adenovirus infection, while cell lines with low numbers of adenovirus receptors (e.g. NIH 3T3 and U-937) show little and need higher doses (MOI) of viruses to infect almost all the cells. We provide AdenoBooster Solution (cat# AD1012) for these cells to facilitate infection.

Q11. What MOI (multiplicity of infection) should I use in my cells?

 A MOI of 10 is suitable to infect 293 cells. As a general guideline, the transfer of a reporter gene to 100% of cells can generally be achieved with a MOI of 10-100 for most cell lines. More detailed guideline is provided in the user manual.

Q12. If I have viral expression of GFP, when could I start seeing some fluorescence after infection? how quickly does adenovirus express proteins?

Infected cells should start expressing a detectable level of GFP after 8-20 hours infection. This is also true for other proteins expression, however, it will vary because stability and expression of proteins and mRNAs could vary.

Q13. How much media should I use during infection?

For your reference, we recommend the following amount virus-containing media for infection:
10-cm plate:  8-10 ml per plate
6-well plate:  1 ml per well
12-well plate:  0.5 ml per well
24-well plate:  0.2 ml per well

It roughly reflects the surface area of each well or plate.



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