samples (in dilution buffer previously mentioned in Subheading 2.6.4) are added to MDCK cells and are incubated for 1 h
at 37
C. It is then supplemented with growth medium (Subheading 2.6.4). Staining solution (Subheading 2.6.4) is used to
stain infected cells after second day for visualization of the
plaques. End-point titration is performed in MDCK cells to
measure virus titer. Inoculation in MDCK cells is performed
with tenfold serial dilutions of the virus. One hour postinoculation PBS wash is given once and cells are allowed to grow for
2 days in DMEM to visualize cell viability. Luminescence assay
or plaque assay is used to determine the viral titre. In vitro
growth analysis of individual mutants is performed using Gaussia luciferase (gLuc) reporter system responsive to influenza
virus. Briefly, the gLuc coding region is incorporated between a
human RNA polymerase I promoter and a murine RNA polymerase I terminator in the reverse-sense orientation [16]. The
gLuc coding sequence is flanked by the UTRs from the PA
segment of influenza virus A/WSN/33 strain to enable influenza
virus
infection
dependent
gLuc
expression
[16]. HEK293Ts are transfected with the gLuc reporter for a
day. Then the supernatants comprising the mutant or WT
influenza viruses are added. gLuc gets successfully released
into the supernatant upon active infection. gLuc can be quantified with Renilla luciferase substrate (Promega).
4. Mouse Immunization and Challenge: Groups of female
C57BL/6 and BALB/c mice are subjected to intranasal or
intratracheal inoculation with either PBS or W7-791. Each
group consisted of five to six mice. Prior to intratracheal injection, anesthetization of the mice is performed intraperitoneally
with a ketamine–xylazine mixture. Subsequently, the trachea is
exposed surgically and a solution of 30 μL is injected directly
with a sterile 27G needle [44]. All mice are challenged after
four weeks of immunization intranasally or intratracheally with
the following influenza strain: A/WSN/1933 (H1N1) at
4 MLD 50 , A/Puerto Rico/8/1934 (H1N1) at 4 MLD 50 ,
A/Cambodia/P0322095/05 (HPAI-H5N1) at 2 MLD 50 , or
A/Victoria/3/75 (H3N2) at 2 MLD 50 . Daily monitoring of
the mice is performed to detect symptoms of illness. Weight
loss of 30% or more is reported and the mice are euthanized.
Female C57BL/6 mice are randomly divided into two groups
of vaccinated or unvaccinated mice for the adoptive-transfer
experiment. Unvaccinated groups are mock immunized. On
the other hand, the vaccinated group is immunized with a
single dose of W7-791 at 10
6 PFU/mouse. Cells are harvested
from one set of mice from each group after 4 weeks of vaccination for the transfer experiment. The other set served as a
vaccinated, nontransferred, control. Isolation of the total
Live Vaccine Attenuation
349
at 37
C. It is then supplemented with growth medium (Subheading 2.6.4). Staining solution (Subheading 2.6.4) is used to
stain infected cells after second day for visualization of the
plaques. End-point titration is performed in MDCK cells to
measure virus titer. Inoculation in MDCK cells is performed
with tenfold serial dilutions of the virus. One hour postinoculation PBS wash is given once and cells are allowed to grow for
2 days in DMEM to visualize cell viability. Luminescence assay
or plaque assay is used to determine the viral titre. In vitro
growth analysis of individual mutants is performed using Gaussia luciferase (gLuc) reporter system responsive to influenza
virus. Briefly, the gLuc coding region is incorporated between a
human RNA polymerase I promoter and a murine RNA polymerase I terminator in the reverse-sense orientation [16]. The
gLuc coding sequence is flanked by the UTRs from the PA
segment of influenza virus A/WSN/33 strain to enable influenza
virus
infection
dependent
gLuc
expression
[16]. HEK293Ts are transfected with the gLuc reporter for a
day. Then the supernatants comprising the mutant or WT
influenza viruses are added. gLuc gets successfully released
into the supernatant upon active infection. gLuc can be quantified with Renilla luciferase substrate (Promega).
4. Mouse Immunization and Challenge: Groups of female
C57BL/6 and BALB/c mice are subjected to intranasal or
intratracheal inoculation with either PBS or W7-791. Each
group consisted of five to six mice. Prior to intratracheal injection, anesthetization of the mice is performed intraperitoneally
with a ketamine–xylazine mixture. Subsequently, the trachea is
exposed surgically and a solution of 30 μL is injected directly
with a sterile 27G needle [44]. All mice are challenged after
four weeks of immunization intranasally or intratracheally with
the following influenza strain: A/WSN/1933 (H1N1) at
4 MLD 50 , A/Puerto Rico/8/1934 (H1N1) at 4 MLD 50 ,
A/Cambodia/P0322095/05 (HPAI-H5N1) at 2 MLD 50 , or
A/Victoria/3/75 (H3N2) at 2 MLD 50 . Daily monitoring of
the mice is performed to detect symptoms of illness. Weight
loss of 30% or more is reported and the mice are euthanized.
Female C57BL/6 mice are randomly divided into two groups
of vaccinated or unvaccinated mice for the adoptive-transfer
experiment. Unvaccinated groups are mock immunized. On
the other hand, the vaccinated group is immunized with a
single dose of W7-791 at 10
6 PFU/mouse. Cells are harvested
from one set of mice from each group after 4 weeks of vaccination for the transfer experiment. The other set served as a
vaccinated, nontransferred, control. Isolation of the total
Live Vaccine Attenuation
349