this is typical given the stress freezing and then thawing has on the virion. Adding FBS to your frozen stock should increase the stability of the frozen virus but the titer will still decline over time.

Viruses also have optimum temperature requirements for their incubation and replication. comparing freshly prepared viral stock and the one from the stock, it is obvious that at -80oC, the virus is frozen (clumped virions) and all the biological reactions are halted, therefore no replication. And also the cells of the host organism would be considered dead as no biological function can take place, which is another prerequisite for virus replication and cell to cell movement. Under these conditions, the virulence of the virus stored at -80oC is dramatically reduced.

Another method of storing viruses for long term is: after purification of the virus, mix with Glycerol at 50/50 vol. This reducesthe clamping, and the virulence is preserved, as the glycerol do not freeze. For use you will have to centrifuge the glycerol out.

All the best!

Long stock virus storage may affect the both virus titer and integrity, and by consequent  virus virulence.

A virus is a complex microscopic structure. There are three major types: filament (like Ebola), icosohedral (like cold viruses), and enveloped, like influenza and HIV. Of the set, the most sensitive to freeze damage are the enveloped viruses for the same reason cells are sensitive. The envelope is a cell membrane from the host and it isn't very strong.

Water expands when it freezes. It doesn't expand by much, but it's a very strong force. There's an old demo of a cast iron ball filled with distilled water. The ball was completely filled, with no air pocket. A tight fitting screw was used as a stopper. The ball was placed in dry ice inside an explosion containment chaimber. The ball exploded with enough force to embed fragments of iron into the steel of the door.

So that's a major factor. When ice forms, it pushes all viruses apart somewhat. When they thaw, the question is, will they go back together well enough or not to function. Even the tougher icosahedral, or filament viruses won't all be ok after thawing.

When you add glycerin, that acts as a glassifying agent. It prevents water from forming crystals, so it doesn't expand.

Another factor is local desiccation. As water freezes, it forms crystals, and those crystals force salts and non-water elements out. You can experience this in sea-ice in the arctic. Fresh ice you can't use for water because as the sea-ice forms, it creates tiny pockets of concentrated brine. But as the brine drains down through the ice, it becomes fresh water. The same process happens inside cells and viruses that get frozen, but at a tiny scale.

Those dry pockets with high salt concentrations damage proteins and DNA. Molecules can also be compressed and sheared into pieces by the ice crystals. One technique for dealing with that is to add sugars. Even sucrose will help in high concentration. It helps protect DNA and protein molecules during freezing.

Most of the damage is done by either the freezing process or by thawing. The more times you cycle it, the more damage you will do. There is some slower degradation that happens at very low temperature, but it's pretty slow. It also depends on your storage temp. Storing in liquid nitrogen or at -80 C will do better than storage at -20.

Thank you all (Dr. Brent, Ogada, Nouhoum and Brian)for your answers and suggestions especially to Dr. Brian Hanley for his clear, deep and detailed concept.

Decrease in viral titer means that the virulence goes down and also its infectivity capacity

Thanks Prof. (Dr) Luis Rodrigo for ur answer.

An interesting question and some very good and clear answers. Thank you all for giving me more mechanistic insight.

I have a question related to this.

Intro:

There are several definitions for virus titer and each comes with their own unit (e.g. genomic titer; GC/mL and infectious units: IU/mL). The later being the most stringent and best option.

I know that stored viruses (-80C or -20C) in time wil decline due to damage.

What I could imagine is that while the IU/mL goes down in time, the GC/mL will remain the same or decline with a much lower rate.

Question:

Does anyone knows by experience or a publication if this is the case?

Brent Stanfield:

I have experienced the difference between storing retroviruses in the -80C in OptiMEM versus FBS. After 1 year, the FBS stored virus had a much higher IU/mL compared to the OptiMEM stored. Although colleagues use the OptiMEM method because it eases concentrating the virus (and contains some kind of 'FBS'), I immediately returned back to FBS (and longer waiting while using a column-based centrifugation concentrating method). Good to read from an independent source that FBS does matter!

There is a parallelism with the viral load

I am currently working with rotaviruses. I want to use a particular strain as a control for FFA experiment. I used to keep multiple small aliquots of control virus.Still I have experienced difference in FFA titers . Can anyone suggest the best method to store the virus ?

This means a decrease of the concentration of de viral particles by one unit of serum (cubic ml)