Adenovirus Packaging Service

Adenovirus is a linear, double strand DNA virus, the length of its genome is 36 Kb. The Adenovirus genome is flanked by inverted terminal repeats(ITRs), which are required for viral DNA replication, and near the left ITR is a packaging signal (Ψ), which is required for the encapsidation of the Adenovirus genome. The Adenovirus genome is comprised of two main regions: early (E) and late (L) gene. Early genes (E1 to E4) are necessary for viral replication; whereas, late genes(L1 to L5) are required for viral assembly. See the transcription map of human Adenovirus serotype 5 below.

Base on the genome structure and function of human adenovirus serotype 5, recombinant adenovirus vector is generated by removing both E1 and E3 regions. Although the E1 gene is essential for adenovirus packaging, it can be provided in trans in HEK293 packaging cells, that are E1-expressing cells, whereas, the E3 region counteracts host defense mechanism, it is not essential for in vitro viral growth. Rcombinant adenovirus without E1 and E3 genes is able to accommodate larger recombinant genes (up to 7.5 Kb).

Adenovirus infection starts with the recognition and binding of the distal knob domain of the viral fiber to the adenovirus receptor (coxsackie and adenovirus receptor, CAR). Following CAR binding, the penton base engages integrins αvβ3 and αvβ5 to initiate endocytosis and viral entry. This process is facilitated by the RGD motif in the penton base. The process of Adenovirus infection is shown below:

1. The adenovirus has a very broad host range. It can infect a wide range of cells including dividing and non-dividing mammalian cells. Some lymphoid cell lines are excepted, because they are resistant to adenoviral infection.

2. Adenovirus has high infection efficiency. It is able to deliver genes with 100% efficiency under optimal infection conditions.

3. Adenovirus can easily obtain very high titer, and concentrated to 10^12-13VP/ml(10^10-11pfu/ml).

4. Adenovirus is easily amplified in HEK293 cells compared with other viruses such as lentivirus and adeno-associated virus which are need to be repackaged.

5. Adenovirus genome rarely integrate into the host chromosome and does not inactivate genes or activate oncogenes.

6. Adenovirus is relatively stable. It can be stored at fridge for weeks, -20◦C freezers for months and -80◦C freezers for years.

The efficiency of Ad5 infection depends on CAR, and av integrins(αvβ3 & αvβ5) density. However, some immune cells express scarcely CAR and av integrins, such as hemopoietic stem cells. Therefore, numerous studies focused on the development of a new adenovirus serotype. Finally, serotype 35 emerged as the variant with the highest tropism for hemopoietic stem cells. Therefore, a chimeric vector (Ad5/F35) was developed by scientists, which contained the short shafted Ad35 fiber incorporated into an Ad5 capsid. The structure and difference of Ad5 & Ad5/F35 are given in the below.

1. Hello, the cells I infected are trophoblast cells. 100,000 cells were inoculated in a six-well plate firstly, then virus (MOI=40) was added after 16 hours of attachment. 30 hours later, the cells became full. I would like to ask: After virus infection, the cell production of the experimental group is similar to that of the control group. Is it normal?

If the state of cells infected with adenovirus is not significantly different from that of the uninfected group, it means that the virus has no obvious toxic effect on cells. Therefore, it may not be that the number of viruses is insufficient. Non-adenovirus packaging cells will not have any obvious characteristics after being infected by adenovirus. If you add the virus to the culture dish after diluting the virus with the medium in advance when adding the virus, then the high MOI value (causing the cells to float) is relatively high for the target cells. Therefore, don’t worry, you can first check whether there is protein expression.

2. Is it necessary to replace the fresh culture medium after adenovirus infects cells?

Whether it is necessary to replace the fresh culture medium depends on the amount of virus added. When the amount of virus is large, it will be toxic to the cells: if there is a change in the cell state during the microscopic examination, the fresh medium needs to be replaced in about 4-8 hours; If there is no obvious change in the cell state, there is no need to replace the fresh culture medium after the virus infects the cell. Or, if you are worried that the virus will be toxic after acting on the cells for a long time, you can also replace the fresh culture medium after 12 hours.

3. Why the cells are all floating after 4-6 hours of adenovirus infection?

Three main reasons can explain the phenomenon above:

1) Cell status: The cells plated before infection must be healthy cells;

Improper operation: When adding virus, if the local virus concentration is too high, it will cause cell death. Therefore, we recommend mixing the virus and medium before adding.

If the 2 points above are both correct, then the amount of virus added is too large.

4. What is the basis for the amount of adenovirus virus when infection the cells?

Selecting the optimal virus amount is the key for getting the best experimental results.
Insufficient amount can’t reach 100% infection efficiency; too high amount will cause toxicity to cells. So how to determine it? Multiplicity of infection (MOI) plays a decisive role. MOI is the ratio of virus to infection cell number. The number of adenovirus receptors on cell surface is different in different cell lines, which determines that the MOI of different cell lines will be different. Generally, for susceptible cell lines, the MOI range used is 10-100. However, for some hard-to-infect cell lines, MOI may need to be as high as 1,000. For most cell lines, the optimal MOI range is very narrow. We recommend you to read the relative literature, or to test the best MOI by infecting the target cells with the adenovirus of the reporter gene before the formal experiment.

Calculation formula for optimal virus dosage
The amount of virus pfu=optimal MOI *cell number/ virus titer
For example, if the optimal MOI of target cell is MOI=10, 106 cells will be infected, then 107pfu virus are needed. If the virus titer is 1×1010 pfu/mL, then 1ul will be used for the experiment.

5. How long can the expression of adenovirus be detected at the gene level and protein level after infecting cells? Is there a difference in the detection time between secreted proteins and non-secreted protein?

The target gene carried by adenovirus expresses quickly. If you want to detect gene expression at the gene level, then perform the test between 12-24 hours of virus-infected cells; if you want to detect the gene expression at protein level, then perform the test between 48-72 hours of virus-infected cells.

For secreted protein and non-secreted protein, the detection time is the same. If you worried about protein secretion, you can do western blot together with the medium.

6. The adenovirus seed is contaminated by bacteria, there is no extra virus seed, and there is no constructed vector, what should I do?

The diameter of adenovirus is 90-100nm, and the diameter of bacteria is much larger, so it can be filtered with a 22um filter. If your virus load is very low and you are worried that you will lose too much after filtering and cannot proceed with the subsequent amplification work, you can directly amplify the contaminated adenovirus firstly. Of course, adenovirus contaminated with bacteria are obtained after amplification. But the amount of virus is increased, then use a 22um filter for filtration.

7. How to identify the target gene that adenovirus delivered?

To identify as follow:

①Design primers for genes, which can also be used as amplification and sequencing;
②Virus is the template which is treated with proteinase K. If it is an unpurified virus, it needs to pass through a purification column to remove the medium components.
③Perform PCR with the processed virus as a template;
④Sequencing the PCR products with PCR primers.