2 ms·

I'm specifically referring to Koch's Postulates here, and as far as I'm aware the viruses you're referring to weren't isolated. I've always seen them being used

I'm specifically referring to Koch's Postulates here, and as far as I'm aware the viruses you're referring to weren't isolated. I've always seen them being used either in models or in some other host cells from cows, monkeys, etc. though I've never worked directly in the field so please point me to a study if you've seen one truly isolate a virus.

anonymouskimmer 3y ago
Here's a simple isolation protocol for a fish virus. The isolated virus is imaged in figure 3. https://www.researchgate.net/publication/50409110_Cardiomyopathy_Syndrome_of_Atlantic_Salmon_Salmo_salar_L_Is_Caused_by_a_Double-Stranded_RNA_Virus_of_the_Totiviridae_Family/download https://www.researchgate.net/publication/50409110_Cardiomyop... > Virus propagation in cell culture. Heart tissue from freshly dead Atlantic salmon (Salmo salar L.) was collected from a clinical outbreak of CMS (the diagnosis was confirmed by histopathological examination). The heart tissue was homogenized, followed by centrifugation at 4,000 g and 4°C for 20 min to remove cellular debris, before being processed through a 0.22-m filter. The homogenate was subsequently inoculated onto GF-1 cell cultures (5). The GF-1 cell line, derived from the fin tissue of orange-spotted grouper, Epinephelus coioides, was grown in plug seal cap culture vessels at 15°C after inoculation in Leibowitz 15 supplemented with 1% L-glutamine (2 mM final concentration), 0.1% gentamicin sulfate (50 g/ml final concentration) (all from Sigma Aldrich), and 10% fetal bovine serum (Invitrogen). Development of cytopathic effect (CPE) was monitored at 6, 14, and 21 days postinoculation (p.i.). At 21 days p.i., the supernatant was harvested and passaged further in cell culture. Notice that since this wasn't a stringent purification they used a subsequent passage to further dilute out anything carrying over. As stated in the other comment it would also be possible to use something such as tangential flow filtration or size-exclusion techniques in order to get an even purer first sample.
anonymouskimmer 3y ago
> the viruses you're referring to weren't isolated They're made de novo (adenovirus and baculovirus derivatives, and yes, I've done this for baculovirus), which is better than isolation as even with isolation it's difficult to prove a pure sample. Even Koch couldn't prove an isolated sample back in the day. Not to our standards, at least. Throw a GFP protein in the modified adenovirus or baculovirus genome and you can literally see the exact specific virus that you made move from cell to cell while the cells start dying.

anonymouskimmer 3y ago

Here's a simple isolation protocol for a fish virus. The isolated virus is imaged in figure 3. https://www.researchgate.net/publication/50409110_Cardiomyopathy_Syndrome_of_Atlantic_Salmon_Salmo_salar_L_Is_Caused_by_a_Double-Stranded_RNA_Virus_of_the_Totiviridae_Family/download https://www.researchgate.net/publication/50409110_Cardiomyop... > Virus propagation in cell culture. Heart tissue from freshly dead Atlantic salmon (Salmo salar L.) was collected from a clinical outbreak of CMS (the diagnosis was confirmed by histopathological examination). The heart tissue was homogenized, followed by centrifugation at 4,000 g and 4°C for 20 min to remove cellular debris, before being processed through a 0.22-m filter. The homogenate was subsequently inoculated onto GF-1 cell cultures (5). The GF-1 cell line, derived from the fin tissue of orange-spotted grouper, Epinephelus coioides, was grown in plug seal cap culture vessels at 15°C after inoculation in Leibowitz 15 supplemented with 1% L-glutamine (2 mM final concentration), 0.1% gentamicin sulfate (50 g/ml final concentration) (all from Sigma Aldrich), and 10% fetal bovine serum (Invitrogen). Development of cytopathic effect (CPE) was monitored at 6, 14, and 21 days postinoculation (p.i.). At 21 days p.i., the supernatant was harvested and passaged further in cell culture. Notice that since this wasn't a stringent purification they used a subsequent passage to further dilute out anything carrying over. As stated in the other comment it would also be possible to use something such as tangential flow filtration or size-exclusion techniques in order to get an even purer first sample.

anonymouskimmer 3y ago

> the viruses you're referring to weren't isolated They're made de novo (adenovirus and baculovirus derivatives, and yes, I've done this for baculovirus), which is better than isolation as even with isolation it's difficult to prove a pure sample. Even Koch couldn't prove an isolated sample back in the day. Not to our standards, at least. Throw a GFP protein in the modified adenovirus or baculovirus genome and you can literally see the exact specific virus that you made move from cell to cell while the cells start dying.