cells. Identity of the viruses is confirmed by sequencing. Viral
titers are determined by standard plaque assay on MDCK cells.
3. Evaluation of Viral Growth kinetics: Multiplicity of infection
(MOI) of 0.001 and 0.01 is chosen for MDCK cells and A549
cells respectively. Cells are maintained in infection medium
supplemented with 1% P/S and tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (1 μg/ml for MDCK cells,
0.5 μg/ml for A549 cells) at 37
C. The supernatant is collected after first, second, and third day of infection. Inoculation
is done with 100 PFU of viruses into allantoic fluid in experiments using embryonated eggs and the allantoic fluid is collected after first, second, and third day of infection. Plaque
assays on MDCK cells are performed to determine the viral
titers.
4. In vivo mouse experiments: Specific pathogen-free female
BALB/c mice aged 4–5 weeks are subjected to intranasal infection. Monitoring of mice body weights are done daily and mice
showing weight loss of more than 25% are euthanized. Lung
tissues are harvested at third and seventh of infection. Harvested tissues are homogenized for titration by the 50% tissue
culture infective dose (TCID 50 ) assay using MDCK cells.
Determination of the measure of the numbers of TCID 50 s
per gram is performed using the Reed and Muench
method [27].
5. Estimation of Immune responses: Twenty-eighth day postinfection, serum is analyzed for the titer of neutralizing antibodies. Heat inactivation of serum is performed at 56
C for
0.5 h. MDCK cells are used for microneutralization assay
with 100 TCID 50 s of viruses [37]. Bronchoalveolar lavage
(BAL) fluid is collected at seventh day postinfection. Profiling
of immune cells isolated from Bronchoalveolar lavage (BAL)
fluid is performed as described previously [38]. Briefly, after
blocking Fc receptor with Fc blocker, cells are subjected to
staining with the following two cocktails of monoclonal antibodies (all from BioLegend) to detect innate and adaptive
immune cells. Cocktail 1 comprises F4/80-phycoerythrin
(PE), I-AE-peridinin chlorophyll protein (PerCP)-Cy5.5,
CD11b-allophycocyanin
(APC)-Cy7,
Gr1-PE-Cy7,
IA8-APC, CD11c-fluorescein isothiocyanate (FITC), and
DAPI (4
0 ,6-diamidino-2-phenylindole). Cocktail 2 consists of
CD3-APC, CD4-APC-Cy7, CD8-PerCP-Cy5.5, Dx5-FITC,
γδT-PE, B220-PE-Cy7, and DAPI. Subsequently, samples are
run through flow cytometry analysis.
The beta interferon mRNA expression is evaluated using
quantitative PCR. An MOI of 5 is chosen for viral infection to
A549 cells and the experiment is conducted in triplicate. Total
RNA extraction is done after 4 h and 8 h of infection followed
346
Dipasree Hajra et al.
titers are determined by standard plaque assay on MDCK cells.
3. Evaluation of Viral Growth kinetics: Multiplicity of infection
(MOI) of 0.001 and 0.01 is chosen for MDCK cells and A549
cells respectively. Cells are maintained in infection medium
supplemented with 1% P/S and tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (1 μg/ml for MDCK cells,
0.5 μg/ml for A549 cells) at 37
C. The supernatant is collected after first, second, and third day of infection. Inoculation
is done with 100 PFU of viruses into allantoic fluid in experiments using embryonated eggs and the allantoic fluid is collected after first, second, and third day of infection. Plaque
assays on MDCK cells are performed to determine the viral
titers.
4. In vivo mouse experiments: Specific pathogen-free female
BALB/c mice aged 4–5 weeks are subjected to intranasal infection. Monitoring of mice body weights are done daily and mice
showing weight loss of more than 25% are euthanized. Lung
tissues are harvested at third and seventh of infection. Harvested tissues are homogenized for titration by the 50% tissue
culture infective dose (TCID 50 ) assay using MDCK cells.
Determination of the measure of the numbers of TCID 50 s
per gram is performed using the Reed and Muench
method [27].
5. Estimation of Immune responses: Twenty-eighth day postinfection, serum is analyzed for the titer of neutralizing antibodies. Heat inactivation of serum is performed at 56
C for
0.5 h. MDCK cells are used for microneutralization assay
with 100 TCID 50 s of viruses [37]. Bronchoalveolar lavage
(BAL) fluid is collected at seventh day postinfection. Profiling
of immune cells isolated from Bronchoalveolar lavage (BAL)
fluid is performed as described previously [38]. Briefly, after
blocking Fc receptor with Fc blocker, cells are subjected to
staining with the following two cocktails of monoclonal antibodies (all from BioLegend) to detect innate and adaptive
immune cells. Cocktail 1 comprises F4/80-phycoerythrin
(PE), I-AE-peridinin chlorophyll protein (PerCP)-Cy5.5,
CD11b-allophycocyanin
(APC)-Cy7,
Gr1-PE-Cy7,
IA8-APC, CD11c-fluorescein isothiocyanate (FITC), and
DAPI (4
0 ,6-diamidino-2-phenylindole). Cocktail 2 consists of
CD3-APC, CD4-APC-Cy7, CD8-PerCP-Cy5.5, Dx5-FITC,
γδT-PE, B220-PE-Cy7, and DAPI. Subsequently, samples are
run through flow cytometry analysis.
The beta interferon mRNA expression is evaluated using
quantitative PCR. An MOI of 5 is chosen for viral infection to
A549 cells and the experiment is conducted in triplicate. Total
RNA extraction is done after 4 h and 8 h of infection followed
346
Dipasree Hajra et al.