by DNase treatment and reverse transcription (see Notes 2 and
3). Expression levels of cDNAs of beta interferon [39] and
β-actin [40] is determined by real-time qPCR. For beta interferon, template is denatured initially at 95
C for 20 s, followed
by denaturation for 3 s at 95
C and annealing for 30 s at 60
C
for 40 cycles. In case of β-actin, initial denaturation of 20 s at
95
C is performed, followed by denaturation of 3 s at 95
C
and annealing for 30 s at 64
C for 40 cycles. A melting curve
analysis of the amplicons is done from 60
C to 95
C at the
rate of 0.1
C/s to determine the specificity of the assay.
Normalization of beta interferon mRNA expression is done
with respect to the level of β-actin expression.
6. Vaccine efficacy evaluation: 12 female BALB/c mice are subjected to intranasal infection. Mock vaccination is performed
with phosphate-buffered saline (PBS) in another group of
12 mice. Infected and mock treated mice are challenged with
MA-WT virus, PR8, HK68-MA20C [36], or PBS on 28th day
postvaccination. Loss in body weight is monitored daily for a
period of 14 days. Lungs are harvested from each group for
viral titration and immunohistochemistry analysis after third
and seventh day of viral challenge. Harvested lung tissues are
homogenized for titration by the TCID 50 assay using MDCK
cells. Fixation of tissues is done in 10% formalin and processed
further as described previously [41] for immunohistochemical
studies. Staining of the tissues is done with monoclonal antibody against the NP (clone HB65; ATCC) of influenza A virus.
7. Protein expression Studies Using Western blotting: Infection
to A549 and DF1 cells is done at an MOI of 5. Whole-cell
lysates are harvested at indicated time points postinfection.
Protein samples are run in 12% SDS-polyacrylamide gels. Western blotting is performed using the following primary antibodies: PB2, PB1, PA, HA, NP, NA, M1, NS1, and β-actin
(as previously mentioned in Subheading 2.5.7). β-Actin is
used as loading controls. Detection of primary antibody is
performed using corresponding IRDye-conjugated secondary
antibodies.
3.6 Transposon
Mediated Mutagenesis
[16]
1. Creation of M Gene Segment Mutant Plasmid Library: A
stretch of 15-nucleotide long sequence 5
0 -NNNNNTGCGG
CCGCA-3
0 is inserted at random region by Mu-transposonmediated mutagenesis (MGS kit, Finnzymes) [16] (see Notes
4–6). The M gene mutant is electroporated into E. coli DH10B
at 2.0 kV, 200 Ω, 25 μF (ElectroMax DH10B, Invitrogen).
Transfection of HEK293T cells is done with the M gene
mutant plasmid and seven other wild type plasmids for generation of virion particles. The supernatant is harvested at third
day posttransfection and transferred to MDCK cells for
Live Vaccine Attenuation
347
3). Expression levels of cDNAs of beta interferon [39] and
β-actin [40] is determined by real-time qPCR. For beta interferon, template is denatured initially at 95
C for 20 s, followed
by denaturation for 3 s at 95
C and annealing for 30 s at 60
C
for 40 cycles. In case of β-actin, initial denaturation of 20 s at
95
C is performed, followed by denaturation of 3 s at 95
C
and annealing for 30 s at 64
C for 40 cycles. A melting curve
analysis of the amplicons is done from 60
C to 95
C at the
rate of 0.1
C/s to determine the specificity of the assay.
Normalization of beta interferon mRNA expression is done
with respect to the level of β-actin expression.
6. Vaccine efficacy evaluation: 12 female BALB/c mice are subjected to intranasal infection. Mock vaccination is performed
with phosphate-buffered saline (PBS) in another group of
12 mice. Infected and mock treated mice are challenged with
MA-WT virus, PR8, HK68-MA20C [36], or PBS on 28th day
postvaccination. Loss in body weight is monitored daily for a
period of 14 days. Lungs are harvested from each group for
viral titration and immunohistochemistry analysis after third
and seventh day of viral challenge. Harvested lung tissues are
homogenized for titration by the TCID 50 assay using MDCK
cells. Fixation of tissues is done in 10% formalin and processed
further as described previously [41] for immunohistochemical
studies. Staining of the tissues is done with monoclonal antibody against the NP (clone HB65; ATCC) of influenza A virus.
7. Protein expression Studies Using Western blotting: Infection
to A549 and DF1 cells is done at an MOI of 5. Whole-cell
lysates are harvested at indicated time points postinfection.
Protein samples are run in 12% SDS-polyacrylamide gels. Western blotting is performed using the following primary antibodies: PB2, PB1, PA, HA, NP, NA, M1, NS1, and β-actin
(as previously mentioned in Subheading 2.5.7). β-Actin is
used as loading controls. Detection of primary antibody is
performed using corresponding IRDye-conjugated secondary
antibodies.
3.6 Transposon
Mediated Mutagenesis
[16]
1. Creation of M Gene Segment Mutant Plasmid Library: A
stretch of 15-nucleotide long sequence 5
0 -NNNNNTGCGG
CCGCA-3
0 is inserted at random region by Mu-transposonmediated mutagenesis (MGS kit, Finnzymes) [16] (see Notes
4–6). The M gene mutant is electroporated into E. coli DH10B
at 2.0 kV, 200 Ω, 25 μF (ElectroMax DH10B, Invitrogen).
Transfection of HEK293T cells is done with the M gene
mutant plasmid and seven other wild type plasmids for generation of virion particles. The supernatant is harvested at third
day posttransfection and transferred to MDCK cells for
Live Vaccine Attenuation
347