I am currently doing some virus infectious clones work.
I have tried to in-vitro transcribe the coxsackievirus A16 (picornavirus) infectious clone to make rna, and then transfect them into HEK 293 cells
next day, I have saw the cells showed kind of CPE and then I directly harvest the supernatant. I re-infect again the rhabdomyosarcoma cells (which is susceptible to infection of this virus) with harvested supernatant which i think contains viruses. however, it doesnt show any CPE till third day.
Is that any problem in the virus recovery, or is the harvested virus titer is too low.
Can anyone explain this for me and give some useful advise or suggestion?
First of all, mentioning which virus you are working with will help people to give specific advice. Now from my experience of transfecting genomic RNA (flaviviruses) into cells, 24h might be too early to harvest infectious virus (again, depends on the virus in question). The "CPE" you thought you saw could very well be cell death due to stress from transfection. Another question is whether the virus has a 5'cap and whether you have capped the RNA. You could collect supernatant different days after transfection, do a titration to see when you reach maximum titre and then optimize your harvest according to that.
I am doing coxsackievirus A16 virus. this virus is without cap.
The cell line i used is HEK 293, which is not permissive for virus infection and multiplication. That's why i just harvest it after about 24 hr, not sure if i can incubate it for longer period.
the other option would be to co-culture the two cells together and transfect in vitro derived RNA. Also in case of coxsackie virus infection people wait more than 3 days to observe CPE. The other option would be to perform immunoflourescence assay in infected cells with viral specific antibody. The Vero cells show good CPE, you may want to try it. goo luck!!
you meant co-culture two different cells together just in one flask? it works?yea, may be if things doesnt go better, i can try with other cells, perhaps Vero
yes, both the cells together in the same flask. This is often done for influenza recovery where 293T are mixed with MDCK cells. The 293 are used for better transfection while MDCK for infectivity.
I have worked with Tick-borne encephalitis virus (a flavivirus). I used in-vitro transcribed capped RNA and Lipofectamin 2000 to transfect co-Culture of Vero B4 and HEK 293 (1:1). Vero B4 are really good for RNA replication whereas HEK293 cells offer better tranfection efficiency. I transfected the RNA in 24-well format, changed the media after 24h to remove dead cells & transfected RNA. As I did not see any CPE after 72h so split the cells in 6 well format. It took about 7 days to get sufficient virus in the supernatant.
Good luck!!
Naveed Asghar,
Thanks for kind sharing.
You meant that after 72h, you didnt get CPE, so you just harvest the supernatant from infected cells and re-infect in 6 wells plate? is that what you meant?
@ Antonio
No, after 72h I detached the cells by pipetting and moved them (with same medium) from 24-well plate to 6-well plate and added addition 1.5ml media/well.
So about 7 days post transfection, I took supernatant from the cells, now in 6-well plate, and infected a T25 flask having about 70-80 confluent VeroB4 cells. Viral stock was made after observing CPE in T25 flask.
you can initially check the concentration of virus in the supernatant and then go for second time of infection. see that MOI of first harvest could able to give the infection in the experimental rhabdomyosarcoma cells.
all the best
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