In a first life, nearly thirty years ago, I tried to produce the ecthyma virus on cell lines to make a vaccine.

We have never succeeded in correctly infecting the various strains on vero cells which have proved to be insensitive to infection.

The only cell lines we could infect were OA2SK and IDO5 sheep skin cell lines, and primary ovine kidney cells or the IRO4 sheep cell line.

The tropism of the ecthyma virus targeting primarily the skin cells, as well as sheep, it is normal for this type of virus does not infect monkey kidney cells what cell line véro.

Finally, it is very difficult to filter the virus suspension because ecthyma is a large poxvirus so that 99% of the virons can be retained on a pore size filter of 0.8μm. We used to centrifuge strongly viral suspension to pellet the bacteria that may contaminate the sample homogenates, before infecting the cells in the presence of a large dose of antibiotic.

But sometimes the infection did not work because the animal was infected by a pestivirus which is capable of inhibiting the proliferation of the ecthyma virus via the production of IFNα which blocks the synthesis of poxviruses.

Thank you for your useful answer.

I can work with primary ovine kidney cells but did this type of primary cell culture gave the cytopathic effect when it were infected with ecthyma virus and at which passage it gave the Cytopathic effect.

About the pore size which was used in filtration 0.45 μm in my work and according to what you mentioned previously the virus will retain in this pore size but actually I did the infection on Chorioallantoic membrane (CAM) and it gave a positive results through pocks formation. For cell culture infection  did you recommend to prepare the suspension without filtration or to use 0.8 μm instead of 0.45 μm to get more virus in the suspension and what was the speed of centrifugation which you used to prepare the suspension.

Finally, could you please till me the best age of sheep that I will use its kidney in cell culture.

Thank you in advance.

The cytophatic effect is perfectly visible on the renal cells between the third and sixth day postinfection, and this as soon as the second passage on the kidney cells.

The sample used to isolate the poxvirus was made by biopsy at the edges of the lesions present on the lips of the lambs or the udder of the ewes.

For the culture of kidney cells, the kidney is taken from adult animal, disheveled, ground and digested with trypsin.The cell suspension is cultured for six days in MEM medium supplemented with horse serum.

The viral suspension is obtained, after a freeze / thaw cycle at -20 ° C., by grinding with a Poter and centrifugation of the supernatant, 30 minutes at 3500 rpm.The viral suspension is then filtered over 0.8 μm and inoculated on the kidney cell layer after replacement of the medium with 199 medium supplemented with 1% FCS.

Two cycles of infection make it easy to see the characteristic ECP of poxviruses.

But you can follow the infection, from the first pass, by immunofluorescence on cold acetone fixed cells.

Hope this help you.

Thank you very much. These information will help me very much in my project. The last question which I would ask you is about the antibiotics type and concentration because you said previously you used a high concentration of antibiotic to prevent bacterial contamination.

Thank you for your help and useful information.

We used a cocktail of two antibiotics, kanamycin and vancomycin, at twice the usual dose.

Currently we use a mixture of penicillin 10000U / ml and streptomycin 10000 μl / ml, diluted to 1/50.