Combine the flow cytometry results with an image analysis software, you´ll have a lot of fun calibrating the desired intensities but it is possible and well accepted. You can add to your results if you measure your virus entry against a competitor for the cell receptor, as a second control and another against your virus. Alternatively you may use real time pcr to measure how much entry was inhibited against your controls. Classical virology is also an option, plaque reduction assays are really cool for these questions and provide very accurate results, which are treatable and less subjectibe than the also powerful statistical TCID aproach.

Do you have different time points when you stop the experiment?

How many controls are available to you?

What sort of material are you preserving for later on?

Boy do I love virology...

Best of luck and I hope it was useful in any way.

Daniel

Hi there are many ways to do this. One is to have GFP or luc fused to a psuedovirus that enters the cell but do not replicate. That way all u have to do is to measure the readout and convert them into percentage inhibition. A easier way to find a drug to block post entry replication of the virus. Good luck!

Combine the flow cytometry results with an image analysis software, you´ll have a lot of fun calibrating the desired intensities but it is possible and well accepted. You can add to your results if you measure your virus entry against a competitor for the cell receptor, as a second control and another against your virus. Alternatively you may use real time pcr to measure how much entry was inhibited against your controls. Classical virology is also an option, plaque reduction assays are really cool for these questions and provide very accurate results, which are treatable and less subjectibe than the also powerful statistical TCID aproach.

Do you have different time points when you stop the experiment?

How many controls are available to you?

What sort of material are you preserving for later on?

Boy do I love virology...

Best of luck and I hope it was useful in any way.

Daniel

Hi Lisandro

I'm sorry but I have the virologist opinion..that is more complicated, but is better in a standard way. The virus tritation is the best method for quantify infectious particles, you can count fluorescent cells but never you don't have the real data if you can not count PFUs or at least Fluorescent Foci. Working with a one-step growth curve, the entry step of viral infection is the first one on this dynamic. Lisandro if you like you can take our seminar in the next weeks (usually friday in mornings) about these important principles....please give us a touch by mean of skipe.

The answer of Daniel is very nice...he is trying to tell you about the One-Step Growth Curve, do you have this one? Take a look in the Flint's Virology, or the classical Luria textbook...he was the Father of our science. If you like I can share you a dropbox folder with the whole anciente book.

sincerely,

Juan Carlos Gallego-Gómez. B.Sc., Ph.D.

Professor of Microbiology and Parasitology Department

Medicine Faculty

Viral Vector Core and Gene Therapy

Neuroscience's Group of Antioquia

Sede de Investigación Universitaria, SIU.

Universidad de Antioquia

Medellín, COLOMBIA

ZIP 05001000

http://neurociencias.udea.edu.co/es/research/neurobiology

email: [email protected]

the best way to do it is calculate CP.

Dear Lisandro,

Personnally speaking I would suggest firstly to use imaging (high resolution confocal or TEM) to document the way your virus enters cells. The best method to show that would be the 3D Focused Ion Beam/ Scanning Electron Microscopy (FIB/ SEM) (see an excellent paper published by the Ari Helenius's lab in Zurich). Of note, even interesting, the proposed methods so far are very late readouts since they all propose to quantify viral progeny or events related to the viral genes expression. Just to remind some basic knowledges, viral entry steps encompass binding, endocytosis (or direct fusion at the plasma membrane) and capsid shuttling to the nucleus in the case of enveloped viruses (after fusion in endosomes). Given that, if you are unable to use heavy technologies to answer your question one can assume that a good approximation of an inhibitory effect can be obtained by calculating infectious rates with or w/o drugs as followed : (nbr infected cells/ total cells)x100 (this percentage is given by the percentage of GFP+ cells if you use cytometry). You can then index the obtained values on the infectious rate w/o drug (100%). Doing this you have to keep in mind that it does not rule out the possibility to get adverse effects with your drugs that may bias your results. So you will have also to provide robust control experiments to demonstrate that the drug you have used only or mainly act on the entry steps and not later.

I hope this will help you.

All the best,

Franck.

@Lulan Wang:

@Daniel Neto:

@my dear friend Juantxo Gallegomez:

@Priyanka K:

@Franck Halary

Thank you for your great advices. All of you have made some elightening comments. To Juantxo: I hope can take the seminar.

Best.

Dear Franck

in my experience on virology I've never seen anyone starting by electron microscopy to study antivirals.

I think that Lisandro needs quantitative reed out.

I suggest Lisandro to follow the advice of Professor Gallego-Gómez ,

know that for each virus there is a method for assaying antivirals

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1186929/

you do not say which virus you are studying

all the best

paola

Dear Paola,

In my small experience as an immuno-virologist it's never too late to use TEM or related technologies to control the effect of the drug you test. I simply said TEM is complementary to a readout by cytometry for instance (i.e., GFP+ infected cells). The virus I am interested in is the HCMV.

All the best,

Franck

I agree never is too late

may be my collegues microscopist are too lazy because they ask me so many experiments before to do some greeds for me, next time I will send my samples to you Franck

Lisandro did not say us his virus

ciao ciao

Dear Dr. Di Bonito, I work with Respiratory Syncytial Virus.

Best.

Hi Everybody!

It's important for our scientific community to remember and recognize the history, profile and behavior of our human associations.

There is a lot of information about the scientific practices, customs and the way of observing reality. I mean about the literature of SSK (Sociology of Scientific Knowledge), is the field which are studying these traditions, in this sense...we can wait many sessions, during weeks or even years, and it is possible that never we could get any agreement...because there is no epistemic reason for these "scientific beliefs", these social constructs are working in a subtle way, only when one had been in a laboratory and finished the training, a new woman/man had emerged...it is similar to initiation rituals for primitive and tribal groups.

I can explain many times that the PFUs (plaque forming unit), are the method for tritation viruses, but usually the immunologists are trying to tritation by mean of cytometer or different method. I can try to give more data about the relationship between genomes, physical viral particles and virions with assembly process, trying to give additional data related to the pros and cons...

Our colleague Di Bonito has reason about the tritation and the use of electron microscopy...I have used one of these during four years, and the growth curves and virus titer, it was the requirements for preserving, fixation and embedding in epoxic reagents.

Lisandro if you have some reporter gene or antibody for labeling your viruses, you can test with the shorter method for tritation: seeding cells and infect with viruses and several days after this, fixation with methanol and immunolabeling the fluorescent foci or immunostaining by mean of DAB and enzymatic protocol.

Good luck Lisandro, Paola and Franck

Best wishes

juantxo gallegomez

Thanks you for these comments but we are talking

About "early steps of the viral entry". So methods to assess the viral progeny

Do not make sens here.

All the best,

Franck

Of course we are studying early times of infection with DENV (Dengue Virus), now my PhD Student is working in the PI3K/Akt and RhoGTPases Signalling Pathways. She is looking the infection from 1 min, 5 mins.....until 2 hours postinfection, and the using larger intervals she ulteriorly take supernatants for tritation .

But if we were very strict with the early events in the entry of virus into the host cells, Lisandro will need a engineered virus with GFP, and cell line with fluorescent compartment involved in the entry (clatrin, lipid rafts, plasma membrane, early endosomes, others), in a sort of dynamic research like Live Cell Imaging. Attached you will fin two papers of this topic.

The point at the final is that Lisandro only needs by now, how many infectious particles are blocking the entry into the host cell by mean of a drug? is it right Lisandro?. The conventional protocols is in the antiviral research: treatment of cell cultures before the infection with the drug, is correlated with entry, adhesion and uncoating virus into the cell. Treatment during the infeciton (put together drug with virus inoculum), is evaluating the virucidal effect. And if the drug is putted after the infection, it is supossed that the drug are blocking ulterior steps of the viral cycle: maturation, exit and spreading cell-to-cell.

I know the TEM technology, I know all the procedures for embedding the samples, contrasting, cutting ultrafine sections in the ultramicrotome...but if I were Lisandro, first I will take the dynamics studies using growth methods (Frank did you see the curves that I have drawn the other day?....one of this is postulating when a virus has difficulty to entry step). And if I will have some data after this I prefer confocal microscopy with live cell imaging....is the best way. The TEM is a very nice tool but the main use is in the morphogenesis and assembly process.

Lisandro if you like I can send you the paper of my first PhD student, who have tested the antiviral effect of statins in DENV infections.

Sincerely,

Juan Carlos Gallego-Gómez. B.Sc., Ph.D.

Professor of Microbiology and Parasitology Department

Medicine Faculty

Viral Vector Core and Gene Therapy

Neuroscience's Group of Antioquia

Sede de Investigación Universitaria, SIU.

Universidad de Antioquia

Medellín, COLOMBIA

ZIP 05001000

Dear Lisandro

Best procedure to study binding and internalization is to use radiolabelled virus protocols. Since you have standardized the infection and the use of the inhibitor and your virus have a reporter (GFP), I suggest you to use flow cytometry to both quantified the number of positive cells as well as the level of fluoresce in the infected cells instead because it will produce good enough data. If number of cells is better than intensity (I don't know if your blocking agent is 100% effective), then just a simple rule of 3 will be enough.

good luck

Daniel Neto :· I am agree with you.