viruses are prepared in PBS. Each egg is inoculated with a
100 μl dilution. Virus from allantoic fluid is tested by hemagglutination (HA) assay, and the titer is calculated according to
the Reed and Muench method [27].
4. Mammalian cell infection: Cells are seeded in different culture
vessels 1 day prior to infection. For the infection, cells are
washed with Dulbecco’s phosphate-buffered saline (DPBS)
supplemented with Ca
2+
/Mg
2+ and infected with influenza
virus at specific MOIs diluted in fresh medium without
serum. After 1-h incubation, cells are washed with DPBS
again supplemented with Ca
2+
/Mg
2+ before adding culture
medium supplemented with 0.3% BSA. Cells are harvested
according to assay-dependent requirements. For infection in
MDCK cells, culture medium is supplemented with tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (Sigma).
5. Northern blot analysis: RNAs are isolated from different cell
lines using the miRNeasy Mini Kit (Qiagen) and stored at
À80
C.Probes for U6 (5
0 -CACGAATTTGCGTGTCA
TCCTT-3
0 ), miR-93 (5
0 -CTACCTGCACGAACAGCACTTT
G-3
0 ), and amiR-93NP (5
0 -GAGGCTTCTTTATTCTAGG-3
0 )
are used (see Note 2). Northern blot experiments are performed using the Highly Sensitive miRNA Northern Blot
Assay Kit as per the manufacturer’s protocol (Signosis). Membranes are developed with chemiluminescent HRP substrate (T
akara Bio). Images are acquired using the Image Quant
LAS400 (GE Healthcare).
6. Western blot analysis: Lysed MDCK cell samples are loaded
and separated on 10% SDS-PAGE. Using a semidry transblot
apparatus (Bio-Rad), proteins are transferred onto a nitrocellulose membrane. Blocking of the membrane is performed in
PBS with 1% Tween (PBST) and 5% nonfat milk for 1 h.
Primary antibody probing is done with an anti-NP monoclonal
antibody (Abcam) at 4
C overnight. After washing with PBST,
the membrane is incubated with alkaline phosphatase–conjugated goat anti-mouse IgG antibody (Cell Signaling) at room
temperature for 1 h. After PBST washing, the membrane is
developed with chemiluminescent HRP substrate before imaging. Finally, expression of NP protein is normalized with
β-actin (actin).
7. Virulence test in vivo: Mice (6- to 8-week-old) are divided
randomly into groups of four mice. For determination of the
MLD50, virus is serially diluted in DPBS, and 50 μl is intranasally inoculated into mice anesthetized by injection with ketamine and xylazine. The MLD50 is calculated according to the
method of Reed and Muench [27]. After infection, mice are
monitored daily for clinical symptoms, weight loss, and death.
344
Dipasree Hajra et al.
100 μl dilution. Virus from allantoic fluid is tested by hemagglutination (HA) assay, and the titer is calculated according to
the Reed and Muench method [27].
4. Mammalian cell infection: Cells are seeded in different culture
vessels 1 day prior to infection. For the infection, cells are
washed with Dulbecco’s phosphate-buffered saline (DPBS)
supplemented with Ca
2+
/Mg
2+ and infected with influenza
virus at specific MOIs diluted in fresh medium without
serum. After 1-h incubation, cells are washed with DPBS
again supplemented with Ca
2+
/Mg
2+ before adding culture
medium supplemented with 0.3% BSA. Cells are harvested
according to assay-dependent requirements. For infection in
MDCK cells, culture medium is supplemented with tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (Sigma).
5. Northern blot analysis: RNAs are isolated from different cell
lines using the miRNeasy Mini Kit (Qiagen) and stored at
À80
C.Probes for U6 (5
0 -CACGAATTTGCGTGTCA
TCCTT-3
0 ), miR-93 (5
0 -CTACCTGCACGAACAGCACTTT
G-3
0 ), and amiR-93NP (5
0 -GAGGCTTCTTTATTCTAGG-3
0 )
are used (see Note 2). Northern blot experiments are performed using the Highly Sensitive miRNA Northern Blot
Assay Kit as per the manufacturer’s protocol (Signosis). Membranes are developed with chemiluminescent HRP substrate (T
akara Bio). Images are acquired using the Image Quant
LAS400 (GE Healthcare).
6. Western blot analysis: Lysed MDCK cell samples are loaded
and separated on 10% SDS-PAGE. Using a semidry transblot
apparatus (Bio-Rad), proteins are transferred onto a nitrocellulose membrane. Blocking of the membrane is performed in
PBS with 1% Tween (PBST) and 5% nonfat milk for 1 h.
Primary antibody probing is done with an anti-NP monoclonal
antibody (Abcam) at 4
C overnight. After washing with PBST,
the membrane is incubated with alkaline phosphatase–conjugated goat anti-mouse IgG antibody (Cell Signaling) at room
temperature for 1 h. After PBST washing, the membrane is
developed with chemiluminescent HRP substrate before imaging. Finally, expression of NP protein is normalized with
β-actin (actin).
7. Virulence test in vivo: Mice (6- to 8-week-old) are divided
randomly into groups of four mice. For determination of the
MLD50, virus is serially diluted in DPBS, and 50 μl is intranasally inoculated into mice anesthetized by injection with ketamine and xylazine. The MLD50 is calculated according to the
method of Reed and Muench [27]. After infection, mice are
monitored daily for clinical symptoms, weight loss, and death.
344
Dipasree Hajra et al.