Hi,
I am trying to rescue VSV virus in BHK-21 cells using In Vitro Transcription system and co-transfection of N, P, L & G helper plasmids and FL VSV genome RNA's by Lipofectamine 3000.
Questions I have are:
1. Can you confirm virus rescue by a positive RT-PCR for negative sense VSV-L genome ?
2. At the maximum, how many blind passages you do before you decide virus rescue is a failure if you don't see CPE in 1 or 2 blind passaging after rescue transfection?
Thanks in advance!
VSV virus belongs to the Family Rhabdovirade which is a negative sense RNA virus of glucprotein cause different diseases in domestc animals it,s importance due to misdiagnosis as FMD.while very important main disease for horse. Transmission as contagious one (direct& indirect methods)and by insect bite. Virus can be propsgated in every cell culture and prduce CPE and visiable plaque.
Ref.https:// stacks.cdc.gov>cdc.
Continue about RT_ PCR used but coast while tissue culture easily propagated in tissue without need of blind passage. Virus also repliccate in mosquitoes.
Salameh Barhoom thank you for the answers. I was not referring to the cultivation of VSV from clinical isolates. Instead, VSV virus rescue from full length DNA supplied along with the support plasmids which passes through the invitro transcription step to produce positive sense RNA. I was wondering, as I am supplying positive sense RNA, does the presence of negative sense viral RNA by RT-PCR indicates viral production ? How does the conversion of positive to negative sense RNA happens in the absence of replication competent viruses ?
I am so surprized and appreciated for you to supply me with such really new scientfic method which lead conversition of negative to positive sense RNA.Thanks
Are you in need of kits to isolate viral RNA/DNA? My non-profit has free kits available from Zymo. Please see my post if you need these kits.
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