From personal experiences, I think that 3~4 dpi is too long that virus might degrade. I usually choose supernatant in 36~48 hpi for HA test.

Why don't you try to propagate your virus in chicken embryo? That is more efficient.

Sometimes some viruses grow poorly in MDCK.

Again, all of above are my personal experiences.

Good luck!

It is true that virus won't degrade completely, nonetheless, the HA test is a very insensitive assay which can hardly react with low concentration of virus, say, lower than 2log10 PFU/mL (The exact figures depend on your virus and experimental condition).

Meanwhile it should be mentioned that the copies of flu RNA can not represent the real titer of live virus since sometimes some viruses degrade and sometimes excessive RNA or inactive virions are produced by cells.

To another possibility, I had heard about a theory that sometimes the TPCK trypsin you added in your medium will gradually degrade thus the HA0 of progeny virions won't be splited into HA1 and HA2, thus the virions are inactive. So adding some more TPCK trypsin into your supernatant 1 hour before the HA test might be helpful. However, I failed to find out any reference to support this assumption while never tried it by myself,  but I think you can have a tentative trial since it is not laborious.

I advice you to culture the virus in 6-well plate with MDCK cell and the infected virus dilution is 10-3 or 10-4.Culturing time is about 48-72h.If you want to concentrate your virus, the speed must above 25,000rpm.Hope it could help you. Best.

It Is advice to see CPE every day when you find 70-80 CELL DEATH go for HA titeration and preserve that virus even after more CPE you get more HA titer but may be that virus are not live, AND also not good for further experiment

We routinely set up infuenza A and B in culture and grow them to high titers (> 1:16).  Some influenza viruses do not produce high HA titers.  There are many factors for harvesting and running the HA assay.  Without a more complete protocol on how you set up the cultures and HA assay, but in my experience it may take 2 to 3 passages to get higher titers in routine culturing when everything works correctly.  Freshly washed RBC's (Guinea Pig or Turkey) are also critical.

I think before performing the test hemagglutination , it should be ensured that the virus grows well on cells. sera neutralization could confirm if there is an actual virus activity on cells

I'm amazed you got so much viral genome in the supernatant but not surprised at the lack of HA titre. In our hands the Cal7 virus does not grow in normal MDCKs because of their low 2,6 sialic acid levels, but instead needs to be grown in MDCK-SIATs. Even then, it's a poor-grower, best done at 35°C instead of 37°C because it's temperature sensitive. Using this method, we can get measurable HA titres, albeit low compared to better yielding strains. I don't have cell data to hand, but from eggs we'll get 2e3 HAU for Cal7 or Eng195 (a UK prototype strain) whereas PR8 will give 2e10. We use chicken blood, having tested turkey but not found any benefit.  Remember that the qPCR assay is going to be orders of magnitude (10E3 or 10E4) more sensitive that the HA assay too. Growth time depends on the input MOI of course - 2 to 3 days for a multicycle infection is generally about right.

So - get some SIATs, drop the temperature, stick to a consistent low MOI infection protocol and do a timecourse to work out what's best, and (perhaps) change virus. I'm told that more recent pdm2009 isolates grow better in the lab, despite not having an significant antigenic changes.

cheers

paul

At what MOI do you infect you MDCKs? You might want to lower that and try out different MOI (0.1, 1, 10) so that you reduce the amount of interfering particles. Low MOI will often result in higher titers.

Furthermore RT-PCR identifies viral copies, not infectious particles which you will measure with titration on MDCK cells. Finally, as mentioned before, some viruses don't produce high HA titers, check the literature for your isolate if anyone states something about that and try to add a positive control virus that will certainly have a HA titer.

If possible you might want to try to grow the virus in SPF eggs... that makes it way easier. and just checking when the embryo dies as to harvest the allantoic fluid.

Thanks for your answers guys

Unfortunately I have my research lab in the diagnostic department of the hospital so we don't have access to eggs. We chose California/07pdm09 because we have 2300 sera with a tonne of clinical data collected over the pdmo9 so I` restricted to this strain.

I`ve set up several T75 flasks. I don't know the MOI of the virus, I`m trying to skip the MOI determination step to save time. The virus was sent to me by a college and measured 62HAU. I`m inoculating 1ul, 10ul and 100ul into separate flasks and testing supernatant 8, 24, 48 and 72 hpi.

I don't know what strain of MDCK I`m using, but I`ve never seen specific strains mentioned in the literayre and don't have access to other types of MDCK

My media is DMEM containing 0.2% BSA (final conc), gentamycin, amphotericin and vancomycin and TPCK trypsin (1ug/ml). It has been in the fridge for 2 weeks so should I add more trypsin and BSA ?

My blood arrives on Wednesday but 8 hpi will be on the day before, will the supernatant be OK at +4 overnight?

I`m going to spin the debris out at 8000rpm for 10 minutes - is this important for the assay?

Is there anything else I need to do to the supernatant before I test it with the HA, will anything else in the media interfere with the assay

Cheers gang

Mark - PMID: 12857911 or 16081961 are a couple of the first examples of engineering MDCKs to better support highly 2.6-dependent flu strains. You should be able to get some from several of the London area labs. Mill Hill, Colindale etc. Ordinary MDCKs are notorious for changing phenotype upon prolonged passage anyway (e.g. 6911762), so if you don't know what your cells are or (I'm guessing) their passage history, you're on shaky ground even for a less finicky virus than Cal7. And yes, bin the elderly media and make fresh. Don't simply add more trypsin/BSA.  The base medium is fine for quite some while, but only make up what you need with the trypsin & BSA. Virus supernatant will be absolutely fine at 4°C for a day or so (freeze thaws do more damage than short cold storage periods) and a low speed spin to remove cells and junk is fine.

Good luck!

Paul

Cheers PauL

They have been passages 67 times

Add fresh TPCK-trypsin to the culture. Should work between 1-2ug/ml.  Usually see good replication between 2-3days.  Best to harvest before CPE is too high.  you can e-mail me directly at [email protected] and I can send you some SOPs and maybe help you troubleshoot if you still have issues.