Yes, it sounds like the problem is with the titre method from the mouse lung as the animals are clearly getting sick. If you're avoiding freeze thawing after homogenization then the other factor that we've found affects titre a lot is the method of homogenization - we used to use hand-held "mixer" devices (with or without glass beads) and would get low titres, whereas the current Qiagen Tissue Lyser II machine gives us 100-fold higher titres from the same experimental set up. Whether this is because it's faster or simply macerates the tissue better (or both) I don't know.

Bottom line - if the animals are losing substantial amounts of weight, your system is basically working.

Paul

Hi Randy, glad to have your reply.

I anesthetized mice with pentobarbitol in 70 mg/kg (i.p.) and the volumn for inoculum is 50 ul per mice through intranasal injection to nose. 5x10^3 pfu is the virus titer for inoculation. At 48 hrs and 96 hrs post-infection, the virus titer of infected mice lung I detected from plaque assay remained 5x10^3 pfu or less, so the virus did not grow in the lung of mice I guess(at least from the result of what I detected).

I agree with you that something might go wrong in the infection of mice or the virus growth in mice, because when I directly infected 5x10^3 pfu virus in MDCK cells,  the virus titer from plaque assay reached to 10^5 at least. Thanks for your suggestion, I'll see if I can do this experiment to clarify.

Best Regards,

Li-Ming

Hi Randy, I'll discussed with my colleagues and see if we can do these modifications.

Thank you for your kind suggestion.

Best Wishes,

Li-Ming

What strain of virus? Is it one that grows in mice?

A titre of 1e5 PFU/ml from MDCK cells sounds low too, unless it's a pdm2009 strain.

Regarding the lung homogenates, my lab reckon it's much better to plaque immediately after homogenization and that the lysates cannot be frozen without severely decreasing the titre.  Testing your samples in parallel by qPCR might show if this is a problem for you or not - if the genome copy:PFU ratio changes dramatically.

Paul

For most of H1N1 strains in mouse model, 5x10^3 pfu or even less at 48 hpi is too low.  

Sometimes the wrong procedure of plaque assay or poor activity of MDCK cells will lead to an undervalued result of titer, so I suggest that you should reconfirm your cell line and protocol of plaque assay.

However, I think we cannot exclude the possibility  that your virus strain replicate poorly in mammal hosts. So could you tell us which strain you used in your experiments? Did you ever try to use another strain? Had the body weight of infected mice decreased at 3 or more dpi?

Meanwhile,  isoflurane inhaled anesthesia is much more conSavevenient and efficient for intranasal infection of mouse.

Dear Dr. Digard and Dr. Tan:

Thank you for both of your reply and suggestion.

The virus strain I used is A/WSN/33, the strain used both on in vitro testing and in mice. The mice strain I used in BalB/c, which as I know should be a common strain used in influenza mouse model.

Since I rechecked the data that direct virus infection(infection titer: 5x10^3 pfu) to MDCK cells without  mice inoculum is around 10^7 pfu at 48 hpi, but the same experimental procedure done on mice  result in less than 10^4 pfu at 48 hpi. Therefore, I hypothesize that the main problem is our virus poor replication in the mouse model.

The weight of our mouse model  did decrease, there are 3% decrease 48 hpi, 12 % decrease 72 hpi and 17% decrease 96 hpi. And they started to show symptoms like ruffed fur, hunched back since 48 hpi. Dose these weight loss and symptoms serious enough as a flu model for you? Or I just need to elevate the infection titer at the begining? Any kind of suggestion is very welcome.

Many Thanks,

Li-Ming

Hi Li-Ming:

Your data of weight-change was in accordance with similar previous studies which involved with WSN strain on BalB/c model (Jackson., et al. 2008; Wolk., et al. 2008) while your lung viral titer data was significantly lower.    I guess that your virus replicated well on mouse thus the main problems might exist in the preparation of your samples or the procedure of plaque assay. Could you give us more details about the methods of your experiment, for example how did you retrieve the BALF or lung homogenate and the storage condition of you samples?

Best regards

Likai

Jackson, David, et al. "A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity." Proceedings of the National Academy of Sciences 105.11 (2008): 4381-4386.

Wolk, Kendra E., et al. "Influenza A virus inhibits alveolar fluid clearance in BALB/c mice." American journal of respiratory and critical care medicine 178.9 (2008): 969-976.

Yes, it sounds like the problem is with the titre method from the mouse lung as the animals are clearly getting sick. If you're avoiding freeze thawing after homogenization then the other factor that we've found affects titre a lot is the method of homogenization - we used to use hand-held "mixer" devices (with or without glass beads) and would get low titres, whereas the current Qiagen Tissue Lyser II machine gives us 100-fold higher titres from the same experimental set up. Whether this is because it's faster or simply macerates the tissue better (or both) I don't know.

Bottom line - if the animals are losing substantial amounts of weight, your system is basically working.

Paul

Dear Dr. Tan and Dr. Digard:

Glad to see your reply again. About the way to homogenize lung tissue and the storage condition, we did not do extra procedures to retrieve BALF actually (I guess you mean bronchoalveolar lavage fluid?). According to your experiences, will it affect the virus infection efficacy? If it does, what would you usually do to retrieve it?

Second, we did not use hand-held mixer as well, we use Precellys® Minilys Personal Tissue Homogenizer system (Seems to be pretty similar with Qiagen Tissue Lyser II machine to me, only the speed of our system is 5000 rpm) to homogenize our lung tissue. We collected lung tissue from a mice in 1 ml filtered PBS and for Minilys machine to shake twice and 40 seconds each time. There are plastic little beads in every  microtube in the homogenizer system. Before and After the homogenization, all samples remain on ice. After homogenization, we centrifuge 10000 rpm for 3 mins, then collect the supernatant only and separate the samples into several eppendorfs and store in -80 Clausius to avoid freeze and thaw.

Li-Ming

That is strange because  the process you homogenizing tissues  seems correct. However, to exclude the possibility of incomplete homogenization, you can try to use a contusion mortar with  liquid nitrogen or dry ice to homogenize the lungs.

Next time when you perform another plaque assay, I suggest that you could add a virus sample which was titrated accurately before as a positive control.

Dear Dr. Tan and Dr. Albrecht:

Thanks for all your suggestion. I did not place samples on ice in between the homogenize cycle, but sure I can try to do so. And the lung tissue were all completely homogenized. So, it seems that the temperature is the condition that I can modify.

ps: I have heard one of the possible reasons of the low virus load. If the infection virus titer is too high, virus might not be able to proliferate accurately for bad condition of MDCK cells. Does it sounds reasonable for you both? or have you ever heard of this concept before? cause I can not find any references t support this idea.

Many Thanks,

Li-Ming

"ps: I have heard one of the possible reasons of the low virus load. If the infection virus titer is too high, virus might not be able to proliferate accurately for bad condition of MDCK cells."

This might be a garbled description of the DI (defective interfering) phenomenon? If you grow your virus stocks at too high an MOI then the stock will become riddled with DI particles. A stock full of DIs might then struggle in animals when inoculated at a lower input perhaps.  Pubmed "defective interfering + influenza" and you'll find a mass of literature, mostly quite old now.

Paul 

Dear Dr. Digard:

Thank you for pointing out the key words for PubMed searching.

I'll check the literature and see if it plays a role in my issue.

Many Thanks,

Li-Ming