Mouse as Model System to Study Host-Pathogen Interactions in Influenza A Infections

Figure 1 Pipetting scheme for performing the hemagglutination assay.



4. Discard the 50 µl from the last column.

5. Add 50 µl RBC suspension to all wells.

6. Mix the plate carefully and seal it with a cover plate.

7. Incubate for 30 to 45 min at room temperature.


Interpretation of results


No agglutination results in an accumulation of cells at the bottom, forming a solid button-shaped structure. Agglutination in the form of a clumped suspension indicates the presence of virus particles in the respective well. The highest dilution with agglutination activity is considered as the endpoint representing one hemagglutination unit (HAU). The number of HAU/50 µl is the reciprocal of the highest dilution with agglutination: e.g., if the first five wells (1:32) reveal complete hemagglutination, then there are 32 HAU/50 µl in the starting volume.


TITRATION OF INFECTIOUS VIRUS BY FOCUS-FORMING UNIT ASSAY


Several different methods exist to quantify the amount of infectious virus in a sample, e.g., egg infectious dose (EID50; Matsuoka et al., 2009b) or tissue culture infectious dose (TCID50; Cottey et al., 2001; Matsuoka et al., 2009b). Here, we describe the focus-forming assay as a way to determine the titer of infectious virus particles (FFU, focus-forming units). In our hands, this is the most reliable and informative assay. The assay determines exact titers for infectious viral particles (e.g., in harvested allantoic fluid or tissue homogenates). Quantification is done by serial dilutions, usually performed in triplicate or quadruplicate. The basic principle is the same as for the plaque-formation assay, where you estimate foci of cells that were infected and killed by the virus (and which thus become evident as plaques in the cell culture lawn). Therefore, in some protocols, plaque-forming assays (PFU) are used to determine the number of infectious viral particles. The FFU assay combines the PFU assay with the use of virus-specific antibodies. The advantage of the FFU assay is that one does not have to wait 3 to 5 days until plaques have formed; the results can already be obtained on the second day after infection. The protocol described here uses an enzymatic staining protocol to visualize viral proteins in infected cells. Alternatively, fluorescent labeling may be used, whereby the number of cell foci is quantified by fluorescence microscopy.


Another fast and sensitive method for detection of virus is quantitative PCR. But in contrast to the above described methods, this method cannot differentiate between intact, functionally active virus and incomplete, noninfectious particles, since all nucleic acids are amplified. Several commercial kits for this method are available from different companies.


NOTE: Work under aseptic conditions with sterile material and equipment.


Materials



MDCK II cells (Madin Darby Canine Kidney), (ATCC)


MDCK II cell culture medium (see recipe)


Virus (e.g., harvested from infected embryonated eggs, Basic Protocol 1, or lung homogenates, Support Protocol 3)


Infection medium (see recipe)


Overlay solution (see recipe)


Fixative: 4% (v/v) formalin in PBS


Quencher solution (see recipe)


Blocking buffer (see recipe)


Washing buffer: 0.5% (v/v) Tween 20 in phosphate-buffered saline (PBS; no Ca2+ or Mg2+; Invitrogen)


Primary antibody (polyclonal, against influenza A virions, H1N1; Virostat, http://www.virostat-inc.com/)


Secondary antibody (anti-goat-IgG-HRP; KPL, http://www.kpl.com/)


Substrate: TrueBlue peroxidase substrate (KPL, http://www.kpl.com/)



96-well plates, flat bottom


Infrared heat lamp


Day 1

1. Seed MDCK II cells in MDCK II cell culture medium at a concentration of 6 × 104/100 µl/well in 96-well flat-bottom plates.

2. Incubate cells for 24 hr at 37°C with 5% CO2.

Cells have to be 100% confluent.

3. Thaw virus in a water bath at 37°C.

4. Perform serial ten-fold dilutions of the virus in another sterile 96-well plate (use a multichannel pipet). For example, add 225 µl of infection medium to all wells needed. Transfer 25 µl of virus-containing sample to the first column and mix well. Prepare serial dilutions by transferring 25 µl to the next column and so on. Discard remaining volume of last column.

Final volume in all wells is 225 µl.

Important note for lung homogenates: Add 50 µl of infection medium to the first column and 225 µl to all other wells. Transfer 200 µl of lung sample suspension (see Support Protocol 3) to the first column and mix thoroughly. Prepare serial dilutions by transferring 25 µl to the respective next column. Discard volume of last column. Final volume in all wells is 225 µl.

5. Remove cell culture medium from MDCK II cells by dumping and wash twice with 100 µl/well infection medium. After washing, remove residual medium by dumping.

6. Transfer 25 µl/well of the virus-containing dilutions to the MDCK II cells growing in the wells of the plate prepared in step 1. Use a multichannel pipet if available, and pipet the number of replicates required for your experiment (generally three to five replicates).

7. Incubate for 1 hr at 37°C with 5% CO2, rocking gently every 20 min.

8. Pipet 100 µl of overlay solution directly to the wells and incubate for 24 hr at 37°C with 5% CO2.

Day 2

9. Aspirate the overlay and subsequently wash twice with 100 µl PBS/well. Remove all residual fluid by dumping.

10. Add 100 µl formalin-containing fixative to each well and incubate for 10 min at room temperature.

11. Remove supernatant and wash as described in step 9.

12. Pipet 100 µl of quencher solution into all wells and incubate again for 10 min at room temperature.

13. Dump supernatant and wash once with 100 ml washing buffer per well.

14. Remove washing buffer, add 50 µl of blocking buffer per well, and incubate for 30 min at 37°C with 5% CO2.

15. Prepare 1:1.000 dilutions of primary and secondary antibody in blocking buffer.

16. After dumping residual blocking buffer, add 50 µl of appropriately diluted primary antibody and incubate 1 hr at room temperature.

17. Wash cells three times with washing buffer and add 50 µl of appropriately diluted secondary antibody to each well. Incubate for 1 hr at room temperature in the dark.

18. Before adding 50 µl of TrueBlue substrate, wash six times with washing buffer.

19. Wait until blue spots from infected cells appear (∼10 min).

20. Count number of blue foci.

Viral titers can be calculated as focus-forming units (FFU) per ml (of allantoic fluid, lung homogenate, etc.). For calculation of viral titers, select and count wells containing about 3 to 30 plaques. The titer (FFU/ml) is the number of foci in the selected well multiplied by the dilution factor and multiplied by 1000, and divided by the starting volume (here 25 µl).

INTRANASAL INFECTION WITH INFLUENZA A VIRUS


Mice are anesthetized with a mixture of ketamine/xylazine before intranasal infection with influenza A virus. When using a general anesthetic, the infection will take place in the entire respiratory tract. This protocol generates the most reproducible results because it reduces the variance caused by individual differences due to defense and sneezing of the animal (see Commentary, Anticipated Results, for further discussion).


NOTE: Work under aseptic conditions with sterile material and equipment.


Materials



Ketamine (100 mg/ml, e.g., Bela-Pharm GmbH & Co., http://www.bela-pharm.com/)


Xylazine (20 mg/ml; cp-pharma, http://www.cp-pharma.de/)


0.9% (w/v) NaCl (Merck)


Influenza A virus (e.g., PR8; see Strategic Planning, Different variants of standard laboratory virus strains) stock solution in PBS


Phosphate-buffered saline (PBS; Invitrogen)


8- to 12-week-old mice (see Strategic Planning, Choice of mouse strain and age of mice)


Eye ointment (Bepanthen, Bayer)


1-ml syringe and 26-G, 0.45 × 12 mm needle



1. Prepare a solution of 10% (v/v) 100 mg/ml ketamine and 5% (v/v) 20 mg/ml xylazine in 0.9% (w/v) NaCl. Dilute the virus stock solution on ice to the required concentration of 2 × 102 to 2 × 105 FFU/20 µl with sterile PBS. Hold and restrain mouse.

2. Anesthetize the mouse with 200 µl of the ketamine/xylazine solution per 20 g body weight by intraperitoneal injection using a 26-G needle and 1-ml syringe. Place a small drop of ointment into each eye to prevent exsiccation of eyes.

Monitor respiration, relaxation, and loss of reflexes, e.g., by pinching the toe with a forceps to monitor the stage of anesthesia.

3. Hold the unconscious mouse with the nose pointed up and the head overextended; the diluted virus can now be administered to the nostrils using 20 µl with the required concentration of virus. Pipet 10 µl slowly into each nostril and wait until the mouse has aspirated it drop by drop.

It might be necessary to let the mouse recover for a few minutes before you can administer the second half of the virus solution.

Monitor the animal for a while with respect to respiratory problems that might occur due to the inhaled liquid. In our hands, volumes larger than 20 µl cause strong respiratory distress that may be fatal, and smaller volumes increase experimental variance.

4. Make the animals warm and allow them to recover by placing them in the cage on their backs next to each other and using an infrared light.

Lying on the side favors a unilateral infection of the lung.

WEIGHT LOSS AND SURVIVAL


The course of an influenza virus infection and associated pathology can easily be monitored by recording body weight loss. The kinetics of body weight loss may be unique to the virus variant, the infection dose, or the mouse strain used. Recording of weight loss serves as a useful parameter to confirm successful infection, but also supports the decision-making process as to whether an experiment has to be terminated because of ethical reasons. In addition, survival will be recorded in a typical experiment as a basic parameter. At the end of the experiment, blood may be collected from surviving animals and the presence of influenza-specific antibodies (seroconversion) may be used to verify successful infection.


Materials



Animal balance (with box/container/cage to place the mouse in)


Software for statistical calculations (e.g., GraphPad Prism 5; http://www.graphpad.com/)



Additional reagents and equipment for infection of mice (Basic Protocol 4) and for necroscopy and tissue preparation (Basic Protocol 6)



1. Infect mice with virus of interest and include one control group (e.g., PBS alone), as described in Basic Protocol 4).

2. Weigh mice every day at the same time of day. Check the progress of infection (and for dead animals if present), and record the weight of the animals.

3. Depending on your local regulations for animal experimentation, check for termination criteria: e.g., body weight loss of more than 30%.

4. Sacrifice mice depending on the specific experimental settings, perform necropsy, and harvest tissue samples (see Basic Protocol 6).

Data evaluation


Survival data are usually represented as Kaplan-Meier curves. The survival function is shown as a series of declining horizontal steps that represent the percent of surviving animals in a group. The logrank test is generally used to test for significant differences between two groups. Weight curves start with 100% (initial body weight as reference) and display the percentage of weight loss compared to day 0 (the day of infection). Variation within groups is best presented as standard error of the mean. The Mann-Whitney test represents a suitable nonparametric test for the pairwise comparison of groups, e.g., between different days post infection or between treatment groups at a given day post infection; the Kruskal-Wallis test can be used as a nonparametric test to find significant differences between more than two groups. For further details, see Anticipated Results.


NECROPSY AND PREPARATION OF TISSUE


For more detailed analysis, mice are sacrificed and analyzed at certain days after infection. Here, we describe some of the most commonly employed methods that will be important in studying the course of an influenza infection in mice, focusing especially on pathological processes in the lung. In addition, a thorough post-mortem examination (Antal et al., 2011) may also lead to important new findings and should be considered. Besides the lung, one might be interested in investigating other parts of the respiratory system (trachea, nasal turbinates), or in determining viral load in other organs resulting from systemic spreading of the virus. Appropriate protocols can be found in Matsuoka et al. (2009b).


Depending on the focus of interest, different modifications of the protocols have to be considered. If one is interested in the lung only and wants to avoid signals from the circulating blood, the mice may be bled via the retroorbital plexus before preparing the lung. Larger amounts of blood may be obtained via heart puncture (also described in Donovan and Brown, 2006). However, some bleeding into the chest space cannot be circumvented. To prepare lungs for determination of viral load, see Support Protocol 3.


To analyze infiltrating immune cells in the lung, protocols may be used that employ enzymatic digestion (including DNase and collagenase). In this way, a cell suspension is obtained that may e.g., be analyzed by flow cytometry (see steps below for Homogenization of lung using collagenase D and DNase I). It has to be noted that procedures using enzymes may directly affect surface markers, or gene expression may be strongly influenced by this procedure. Furthermore, damage to cells from digestive enzymes will release more enzymes and DNA, leading to clotting of cells, which may interfere with flow cytometry. To avoid these problems, we use mechanical homogenization, which may be followed by a density centrifugation to enrich the cells of interest (see steps below for Homogenization of lung).


Alternatively, the blood ressels of the lung may be flushed by introducing a draining syringe into the left ventricle of the heart (see steps below for Flushing blood from lung vascular system). This procedure is not recommended for histological studies. In this case, it is preferable to prepare the lung in toto and immediately fix it in formalin (see Basic Protocol 8).


To isolate RNA from infected lungs, it is important to inactivate endogenous RNase activity as fast as possible (see Basic Protocol 10). It should be pointed out that the lung is composed of several compartments that are relevant to the host immune response (Tschernig and Pabst, 2009). The formation of bronchus-associated lymphoid tissue (BALT)—intraepithelial lymphocytes infiltrating into interstitial lung tissue, intravascular leukocyte pool, and the periarterial space—can only be examined with histological techniques. However, draining lymph nodes and bronchoalveolar space (by bronchoalveolar lavage, BAL; see Basic Protocol 7) are easily accessible.


Materials



Infected mouse (see Basic Protocol 4)


70% ethanol


Phosphate-buffered saline (Invitrogen)


Optional: Lympholyte M (Cedarlane Laboratories)


Digestion medium (see recipe)


2× PBS: dissolve two PBS tablets (Invitrogen) in 500 ml distilled H2O



Surgical instruments including scissors and forceps


Winged infusion set (Multifly, Sarstedt)


100-µm cell strainer (Becton Dickinson)


Refrigerated centrifuge


50-ml tube to accommodate cell strainer


30-µm filter (CellTrics; Partec, http://www.partec.com; optional)



1. Euthanize mouse. Place the euthanized mouse on its back (see Control for infectivity of viral inoculates under Critical Parameters in the Commentary for more details).

For specific recommendations and guidelines regarding method of euthanasia, please check with your local authorities. We use CO2 asphyxiation. Alternatively, mice can be killed by bleeding the animals to death via the retro-orbital plexus after anesthesia with isoflurane. The latter avoids bleeding into the body cavity during preparation of organs.

2. Wet the fur with 70% ethanol.

3. Perform a midline incision from the belly up to the chin.

Optionally, take heart blood by inserting a needle with 1-ml syringe between the first and second ribs and aspirating.

4. Open the chest by cutting diaphragm and ribs.

5. Take out the lung, and, if needed, the mediastinal lymph node for further analyses.

Most studies prepare only the lung, but for influenza infection also the trachea may be investigated.

Flushing blood from lung vascular system


6. Perform steps 1 to 3, above, but without blood extraction from the heart.

7. Open the abdomen and cut the V. cava inferior.

8. Place a “butterfly” needle (from a winged infusion set) into the left ventricle of the heart and flush with 5 ml PBS until the lung tissue is turning pale.

Homogenization of lung

9. Place the cell strainer in an appropriate 50-ml tube and wet the mesh of the filter with PBS.

10. Prepare the lung as described above and place it in the cell strainer.

11. Cut the lung into small pieces with scissors.

12. Pass the lung pieces through the strainer by using the plunger of a syringe as a pestle.

13. Flush the strainer with PBS (10 to 15 ml in total).

Optionally, perform a density centrifugation using e.g., Lympholyte M (underlay your lung suspension with 15 ml Lympholyte M solution and centrifuge at room temperature for 20 min at 1000 × g, then extract the interphase and wash twice with PBS).

For some subsequent analyses, an additional filtration through a 30-µm mesh might be necessary, e.g., for FACS analysis and cell sorting.

Homogenization of lung using collagenase D and DNase I


14. Prepare lung as above. Cut it into small pieces and transfer it to a 50-ml tube.

15. Add 10 ml of digestion medium and incubate for 1.5 hr at 37°C.

16. Pass the lung pieces through a 100-µm strainer placed on a 50-ml tube with the plunger of the syringe used as a pestle.

17. Centrifuge the suspension for 10 min at 300 × g, 4°C.

18. Discard supernatant and lyse erythrocytes by adding 10 ml distilled water and incubating at room temperature for 30 sec.

19. Stop lysis by adding 10 ml of 2× PBS, and resuspend well.

20. Centrifuge again as in step 17, then wash twice with PBS using the same centrifugation conditions.

For some subsequent analyses, an additional filtration through a 30-µm mesh might be necessary, e.g., for FACS analysis and cell sorting.

BRONCHOALVEOLAR LAVAGE (BAL)


Samples from the lung lumen can be obtained by bronchoalveolar lavage (BAL). The BAL sample can be analyzed for cellular or soluble factors (e.g., cytokines, chemokines). Many commercially available kits, mainly ELISA-based, exist that may be used to analyze BAL for the presence of many chemokines/cytokines simultaneously (e.g., RayBio Cytokine Array, Luminex-based assays, cytometric bead arrays from BD). It should be noted that the analysis of BAL does not necessarily reflect the host response in other compartments of the lung.


Materials



Infected mouse (Basic Protocol 4)


70% ethanol


Phosphate-buffered saline (PBS; Invitrogen)



Surgical instruments including scissors and forceps


Intravenous indwelling cannula (18-G, Braun)


1-ml syringe



1. Euthanize mouse. Place the euthanized mouse on its back (see Monitoring survival in Anticipated Results for more details).

When collecting BAL, contamination with blood should be avoided! Bleeding the mouse to death via the retro-orbital plexus after anesthesia will be the best alternative. Sacrificing mice by cervical dislocation is not recommended.

2. Wet the fur with 70% ethanol to minimize contamination with hairs.

3. Perform a circular incision of the skin around the trunk of the mouse. Skin the mouse by pulling the fur cranially until the cervical area is easily accessible.

4. Open the abdomen and cut the vena cava inferior to bleed the mouse.

5. Remove the three subcutaneous salivary glands.

6. Expose the trachea by removing the overlaying neck muscles.

7. Introduce an 18-G intravenous indwelling cannula into the trachea and pull back the needle when it is placed correctly.

8. Inject approximately 1 ml PBS into the lung via the indwelling cannula.

An increase in lung volume indicates the correct application.

9. Massage the lung carefully while pulling the piston of the syringe.

10. Process BAL for further studies.

HISTOPATHOLOGICAL ANALYSIS OF THE INFECTED LUNG


Histology is the study of the microscopic anatomy of cells and tissues by examining thin (in the µm range) tissue sections using a light microscope. In diagnostic pathology and biomedical research, diagnosis of various disease processes also requires microscopic examination of the affected tissue. In the case of an inflammatory process of the lung (pneumonia), the microscopic features of the lesion (e.g., distribution of lesions, anatomical structures involved, type and degree of infiltrating immune cells) can be highly informative for a particular causative agent (e.g., influenza virus). Moreover, it can be evaluated if unexpected secondary processes occur in the examined organ, which may influence the study (e.g., a lung tumor, secondary infection). Finally, the host response and associated pathology may vary considerably depending on host or pathogen genetics. These differences are not necessarily evident as differences in weight loss, survival, or virus titer.


For sectioning of the tissue, specimens have to be embedded in paraffin wax. Tissue samples taken during necropsy are fixed in 4% formalin, which is an aqueous solution of formaldehyde (CH2O), for 24 to 72 hr. After fixation, samples are gradually transferred from the aqueous formalin to the paraffin wax through a series of alcoholic solutions of increasing concentration.


Generally, sections of 0.5 µm are prepared with a microtome and can be further processed. The tissue structures and cells are visualized by specific histochemical stains. The type of staining depends on the research interest. Specific antibodies are used to detect specific proteins, including viral proteins to identify infected cells. For a first analysis, we recommend that cross-sections be performed along the cranio-caudal axis and that about 5 to 10 consecutive sections from 3 to 4 levels be collected. This will provide a good rough coverage of the lung, which can then be followed up in detail later.


Hematoxylin and eosin (HE) staining is the most commonly used method in histology. Hematoxylin is an alkaline blue/violet dye which binds to acidic substances in tissue sections. The cell nucleus contains large amounts of DNA (deoxyribonucleic acid), and thus stains dark blue in H&E. Eosin is an acidic red dye which binds to alkaline substances in tissue sections, e.g., the cytoplasm, resulting in a pale to bright pink color. Fixation of tissue, embedding in paraffin, preparation of sections, and H&E staining represent routine methods that are established in all histology laboratories. Therefore, a detailed protocol is not provided.


Here, we describe a protocol for immunohistological staining of influenza A virus particles. This method labels infected cells, and thereby makes it possible to estimate spread of the virus in the lung, to determine the cell types that are infected, and to associate pathological changes and host response with infected areas in the lung.


For some applications, alternative methods are needed—for example, if the antibody is not detecting formalin-fixed epitopes or if fluorescence microscopy is required. In this case, the lung tissue can be embedded in special medium, frozen in liquid nitrogen, and later be processed in a cryomicrotome.


A broad variety of potential applications, like multicolor staining, laser microdissection, and confocal microscopy might also be used for more detailed studies in influenza research, but are not described here.


IMMUNOHISTOLOGICAL STAINING OF INFLUENZA A VIRIONS


Immunohistology is used to visualize proteins or in tissue sections, e.g., viral proteins, cell membrane components, or cytoskeletal proteins.


Antibodies that specifically recognize epitopes from the protein of interest (here structural proteins of H1N1 influenza virus) can be used to perform qualitative and quantitative analyses.


During the immunohistological procedure, the tissue is exposed to antibodies that will bind to the respective antigens (e.g., viral proteins) in the tissue section. In the second step, these primary antibodies are visualized by exposing the tissue to secondary antibodies directed against species-specific structures of the primary antibody. The secondary antibodies are usually coupled to biotin, which is visualized by binding a streptavidin-coupled enzyme (HRP, horseradish peroxidase). The enzyme converts a chromogenic substrate to an insoluble colored product to visualize the presence of the targeted protein in the tissue section. Finally, cellular structures are slightly stained with hematoxylin to relate the signals to tissue structures.


Materials



Xylol (J.T. Baker)


100%, 90%, and 70% ethanol


1× TBS (TBS tablets, Merck)


Paraffin sections of influenza-infected tissue on slides (Basic Protocol 8)


Citrate buffer, pH 6.0 (see recipe)


3% H2O2


Primary antibody (polyclonal, against influenza A virions, H1N1; Virostat, http://www.virostat-inc.com/)


Antibody diluent (Zytomed Systems)


Secondary antibody (biotin-labeled rabbit α-goat IgG H+L, KPL, http://www.kpl.com/)


Streptavidin-HRP (Zytomed Systems)


Diaminobenzidine (DAB) substrate buffer (Zytomed Systems)


DAB chromogen (Zytomed Systems)


Hematoxylin (Merck)


Mounting solution (Shandon Consul-Mount Histology Formulation, Thermo Scientific)

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Mar 17, 2017 | Posted by in MICROBIOLOGY | Comments Off on Mouse as Model System to Study Host-Pathogen Interactions in Influenza A Infections

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