8 replies to this topic

[ozeir]

Hi everyone

To determine whether the  LDV Virus can be detected at different days after infection(1, 2, 4 and 7 days), I measured expression of orf 7(nucleocapsid proteinof virus ) by real time PCR and I wander how can I interpret the result through these data. this is my perceptive:  in day 1 most abount of virus was detected  and then it’s decreased to its half at 2 day post-infection, for day 4 and 7 it's hardly detectable.

any help would be greatly appreciated

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[Curtis]

your interpretation is correct. Do you check the supernatant too? or you lyse the cells only?

[ozeir]

thanks for your reply, i took the blood form infected mice and then extract the plasma containing virus which would be the supernatent I suppose, mixing with cell line and finally extract the RNA. after cDNA synthesis i did Quantification of cDNA via Real-Time PCR. what can we say about the number of relative expression that I highlightedt?

[Curtis]

Doesn't infect? are you sure your virus particles are active?

[ozeir]

yes i'm sure about the infectivity, this virus reaches its pick 1 day after infection and declines sharply at day 2 and then  decrease progressively till it reaches plateau level later whose level hasnt detected for any reason in this method . what i want to know is: is there anything special about relative expression that i got?

[Curtis]

How do you check activity? I did not ask about infectivity, I'm concerned about activity of the particles. we measure activity by plaque assay before infection

[ozeir]

it was checked by assay of lactate dehydrogenase activity in plasma since The plasma level of lactate dehydrogenase (LDH) activity rises to about ten times by this virus

[Curtis]

yeah, but did you put your virus back to the freezer after the test? we keep our virus in -80 freezer, and it can last for several years. If it's stored in PBS the activity drops dramatically after few months, so we check activity right before infection. Sometimes although we get HA activity, there is no plaque formation. It means the particles are there, but they are not active anymore to cause infection....but if you are sure about your activity then maybe there is another explanation in vivo.

[ozeir]

thanks for your help