propagation. After collection of virus after 2 days, viral particles
are subjected to either storage or propagation (up to four
passages). RNA extraction is carried out using the TRIzol
reagent (Invitrogen) followed by cDNA synthesis with the
iScript cDNA Synthesis kit (Bio-Rad) (see Notes 2 and 3).
The 15-nucleotide long insert is amplified using three genespecific forward primers (see Subheading 2.6.2) and Vic-labeled
insertion-specific mini-primer (see Subheading 2.6.2) with the
help of KOD Hot-Start polymerase (Novagen). The PCR reaction is set to the initial denaturation at 95
C for 10 min and at
95
C for 45 s for 30 cycles. Annealing is performed at 52
C
for 30 s, and extension is carried at 72
C for 90 s for 30 cycles;
with a final extension at 72
C for 10 min for 1 cycle. Analysis of
the fluorescent-labeled PCR products is done in duplicate with
a Liz-500 size standard (Applied Biosystems) using a
96-capillary genotyper (3730xl DNA Analyzer, Applied Biosystems). Sequencing data is analyzed using ABI software to
remove nonspecific data and the background noise. To study
in vivo infection scenario, the mutant virus is subjected to
titration and concentration by ultracentrifugation. Retitrated
viral pool is used to inject mice. Lungs are harvested 2 days
postinfection. Homogenized tissues are dissolved in TRIzol for
RNA extraction. PBS or WSN-infected mice are treated as
controls (see Notes 2 and 3).
2. Virus strain generation: Generation of A/H1N1 virus is performed by Influenza A/WSN/1933 by reverse genetics
approach
27 . HEK293T cells are transfected with the eight
plasmids containing the cDNA of A/WSN/33 (Subheading
2.6.2) using TransIT LT-1 (Panvera) following the manufacturer’s protocol. Serial passaging of the virus is performed in
MDCK cells thrice to obtain a final titer of 10
7.4 PFU/ml.
Determination of the MLD 50 of both strains is done in
C57BL/6 mice. Amplification of Influenza virus A/Victoria/
3/75 (seasonal A/H3N2 virus), A/Wisconsin/65/05 (seasonal A/H3N2 virus), and A/Hongkong/68 (seasonal
A/H3N1 virus) is performed in MDCK cells for two to three
passages to a final titer of 10
5.5 PFU/mL, 10
5.4 PFU/mL, and
10
7 PFU/mL, respectively. For the MLD 50 determination,
C57BL/6 and BALB/c mice are used. Influenza virus A/Cambodia/P0322095/05 [42] is propagated in MDCK cells. The
supernatants containing the virion particles are pooled, concentrated by centrifugation, and stored at À80
C. MDCK cells
and BALB/c mice are used for the determination of the
TCID 50 and the MLD 50 of the viruses, respectively, and the
calculation is performed as described previously [43].
3. Virus Titrations: MDCK cells are used to carry out plaque assay
and calculated as PFU/μL of supernatant. Serially diluted viral
348
Dipasree Hajra et al.
are subjected to either storage or propagation (up to four
passages). RNA extraction is carried out using the TRIzol
reagent (Invitrogen) followed by cDNA synthesis with the
iScript cDNA Synthesis kit (Bio-Rad) (see Notes 2 and 3).
The 15-nucleotide long insert is amplified using three genespecific forward primers (see Subheading 2.6.2) and Vic-labeled
insertion-specific mini-primer (see Subheading 2.6.2) with the
help of KOD Hot-Start polymerase (Novagen). The PCR reaction is set to the initial denaturation at 95
C for 10 min and at
95
C for 45 s for 30 cycles. Annealing is performed at 52
C
for 30 s, and extension is carried at 72
C for 90 s for 30 cycles;
with a final extension at 72
C for 10 min for 1 cycle. Analysis of
the fluorescent-labeled PCR products is done in duplicate with
a Liz-500 size standard (Applied Biosystems) using a
96-capillary genotyper (3730xl DNA Analyzer, Applied Biosystems). Sequencing data is analyzed using ABI software to
remove nonspecific data and the background noise. To study
in vivo infection scenario, the mutant virus is subjected to
titration and concentration by ultracentrifugation. Retitrated
viral pool is used to inject mice. Lungs are harvested 2 days
postinfection. Homogenized tissues are dissolved in TRIzol for
RNA extraction. PBS or WSN-infected mice are treated as
controls (see Notes 2 and 3).
2. Virus strain generation: Generation of A/H1N1 virus is performed by Influenza A/WSN/1933 by reverse genetics
approach
27 . HEK293T cells are transfected with the eight
plasmids containing the cDNA of A/WSN/33 (Subheading
2.6.2) using TransIT LT-1 (Panvera) following the manufacturer’s protocol. Serial passaging of the virus is performed in
MDCK cells thrice to obtain a final titer of 10
7.4 PFU/ml.
Determination of the MLD 50 of both strains is done in
C57BL/6 mice. Amplification of Influenza virus A/Victoria/
3/75 (seasonal A/H3N2 virus), A/Wisconsin/65/05 (seasonal A/H3N2 virus), and A/Hongkong/68 (seasonal
A/H3N1 virus) is performed in MDCK cells for two to three
passages to a final titer of 10
5.5 PFU/mL, 10
5.4 PFU/mL, and
10
7 PFU/mL, respectively. For the MLD 50 determination,
C57BL/6 and BALB/c mice are used. Influenza virus A/Cambodia/P0322095/05 [42] is propagated in MDCK cells. The
supernatants containing the virion particles are pooled, concentrated by centrifugation, and stored at À80
C. MDCK cells
and BALB/c mice are used for the determination of the
TCID 50 and the MLD 50 of the viruses, respectively, and the
calculation is performed as described previously [43].
3. Virus Titrations: MDCK cells are used to carry out plaque assay
and calculated as PFU/μL of supernatant. Serially diluted viral
348
Dipasree Hajra et al.