Numerous myths exist about the production and properties of recombinant viruses and vectors.
Some of the most common myths are addressed below:
Some researchers believe all viruses infect all cells. In fact, recombinant viruses can have very different species and cell type specificities (“tropisms”) with respect to their infection capabilities. Therefore, it is important to select a virus that will infect your cells of interest. One way to determine if a virus infects your cells is to do a literature search or ask other researchers working in your field which virus is best suited to a particular application. If you are not able to determine if a given virus will infect your cells of interest in this way, then you may have to test the infectivity of your cells. The virus core can provide a wide variety of stock viruses for this purpose.
Virus production is the same for every vector
Typically there may be a 10-fold range of virus productivities between various vectors. In the virus core, the cells, helper plasmids, and media that are used for virus production are pretested for performance and are largely the same from one virus production to another. Customer vectors are the main material that varies from one virus production to another and, therefore, when virus production fails, it is often a problem with the vector. Examples of common vector problems are:
Genes expressed by vectors in virus producing cells that make the cells unhealthy may inhibit virus production because virus production depends on a wide variety of cellular proteins. Typically, the best productivities are obtained from vectors that encode “innocuous” genes, genes driven by promoters that are weak in virus-producing cells or other cases where gene expression is low or off. Productivities may be lower when virus producing cells express genes that affect cell physiology or genes that directly interfere with processes needed for virus production.
All viruses have nucleic acid sequence-dependent steps in their production, and, therefore, different vector sequences can result in different levels of virus production.
Inappropriate signals in viral genomes
Some sequences can inhibit virus production because they serve as inappropriate signals and were inadvertently inserted during vector construction. Viral vectors are a hybrid between viral genomes and protein expression vectors. Therefore, some of the rules that might apply to construction of a protein expression vector may not apply to the construction of a viral vector.
Viruses are inert materials that can be treated without care and still infect cells
All “enveloped” viruses (those with lipid membranes such as lentiviruses, retroviruses, herpes, rabies, etc.) are very fragile and can be easily inactivated, just like cells. In addition, many are very pH sensitive. Non-enveloped viruses (AAV, adenovirus, etc.) are more stable but can still be inactivated by certain treatments. Therefore, it is important to understand the physical properties of the viruses you work with. The virus core can produce high titer virus but if they are not handled properly after delivery they may not work as well as expected. See Care and Handling of Viruses for more information.
Transfection efficiency predicts virus production
While efficient transfection is a prerequisite for high level virus production, it does not guarantee it. This is because the sequences required for gene expression and virus production are completely independent in most cases. In vectors that express a fluorescent reporter such as GFP, transfection efficiency can be judged by the number of cells expressing GFP. However the promoter-GFP sequences are not required for virus production.
Genome packaging limits can be ignored
Genome packaging limits should never be exceeded. All recombinant viruses are derived from wild type viruses. All wild type viruses have limits on the size of genome that they can package. Therefore, all recombinant viruses can typically efficiently package a genome that is less than that of the wild type virus it was derived from. (The exception to this would be for certain filamentous capsids that can have varying sizes.) The consequences of exceeding genome packaging limits vary from virus to virus but are never good, and should not be exceeded.
The maximum size a recombinant viral genome should be is as follows:
Replication competent ALV derived retroviruses: ~9400 bases
MLV derived retroviruses: ~8300 bases
EIAV derived lentiviruses: ~8000 bases
HIV-1 derived lentiviruses: ~9200 bases
AAV: ~4700 bases
The viral genome size limit correlates to insert size or plasmid size
The proper way to determine viral genome size is to determine the size of the region within a viral vector that encodes the viral genome, the sequence of which varies from virus to virus. As such, the genome size is not the insert size. This is because different viral vectors may already have inserts in them that reduce the size of other inserts that can be added. Conversely, if one is replacing one large gene (or promoter) with a smaller one then that can increase the amount of space that is available before the genome size limits are exceeded. In addition, the genome size is not the plasmid size. This is because only part of the entire plasmid encodes the viral genome.
Viruses last forever when frozen at -80oC
This may be true for AAVs but it is not true for any virus that has a lipid membrane. For example, lentiviruses can lose ~10-fold in titer every 6 months at -80oC. This limited shelf life may mean such viruses should be used within 2-3 months after production unless their concentration is well above what is needed. In addition, it may be better to order smaller lots of virus repeatedly rather than one large lot.
Low “apparent” titer means virus production was poor
Any viral concentration (titer) that is determined by using surrogate markers (fluorescent reporters, drug resistant genes, etc.) is termed an “apparent” titer because it may not be an accurate measure of the real (so-called physical) titer of the virus. Assays, such as those that are Q-PCR based, which involve direct detection of viral genomes are often more accurate.
More volume equals more virus
Some customers have thought a larger volume of virus literally means they have “more” virus to do more experiments. However, what really matters is the total virus produced and its concentration. Because of the very low final volumes we can achieve (20 microliters) some customers have thought they “are not getting much virus” compared to what is produced by other virus cores when in fact they are getting the same total amount of virus, but it may be 10-fold or more concentrated. If a virus concentration is higher than necessary for an experiment, then it should be diluted.
AAV is an adenovirus
AAV is an acronym for adeno-associated virus. The name has historical origins. Wild type AAVs cannot replicate and always require “helper viruses” that supply missing functions to replicate. The first AAV that was identified was a “contaminant” of an adenovirus stock and was named adeno-associated virus because its presence was always associated with an adenovirus.
There is only one type of AAV (or other recombinant virus)
Recombinant viruses are derived from viruses found in nature. Viruses in nature recombine and mutate such that numerous variants exist that often have different properties. As such it can be critical for certain experiments to be sure the right strain, type, or serotype of a virus is used.
Controls aren’t needed because viruses themselves have no effects on cells
Viral infection has numerous effects on cell physiology at all stages of virus infection. Viruses have a biology and that biology interacts with and depends on cellular processes, both during production of a virus and infection by a virus. Even recombinant viruses that lack a genome can alter cellular gene expression. Therefore, one should always do controls using viruses with or without the gene that is thought to be causing a certain effect.
Cells in culture cannot be infected with more than one virus
For the most part this is false. It is possible to infect cells simultaneously with viruses that bind to the same receptor as long as the virus amounts were saturating. It is also possible to infect one cell with two different viruses in vivo. However, in vivo, the issue of immune response can come into play. Some closely related AAVs can exhibit cross-neutralization (injection of AAV-1 almost always results in an immune response to AAV-6.). Different individual animals of the same species as well as different species can respond differently to different viruses. Furthermore, some animals have pre-existing immunity to viruses or may have other factors in tissue or blood that result in virus neutralization. As such, it is a good idea to prescreen sera from animals to be used in experiments to be sure it does not contain any activities that may neutralize the virus. Such neutralization activity is not too common in rodents but can occur.