8. Humoral immune response and protective immunity: Mice (6to 8-week-old) are randomly divided into groups and intranasally inoculated with 50 μl of diluted influenza virus. Mouse
blood is collected on days 15 and 29, and serum is isolated for
analysis by microneutralization assay and ELISA for anti-HA
responses. For testing the IgG antibody concentration in
mouse serum, plates are coated with HA of the PR8 virus,
and specific IgG, IgG1, and IgG2a are measured in the sera
of immunized mice. After the last bleeding, mice are
challenged with 100Â LD50 mouse adapted PR8 (H1N1),
A/California/04/2009(H1N1) (CA09), or 104 PFU
A/Hong HK/1/68 (H3N2) (HK68) influenza virus. The
challenged mice are monitored for clinical symptoms and
survival.
3.5 Exploiting Codon
Usage Bias for Viral
Attenuation
1. Development of a human influenza virus having avian influenza
virus-like codon bias sequences [15]: Codon usage study is
performed using the Influenza A/Brisbane/59/2007
(H1N1) (BR59) strain. Number of mutations required to be
introduced to alter the codon bias from human to avian like
influenza virus are selected by comparing the codon usage
frequency of wild-type (WT) BR59 with that of the frequency
of avian influenza virus. Introduction of such mutations into
the viral genome led to the emergence of the required mutant
with the avian virus–like codon usage. The minimum free
energy of the mutated gene sequences and their nucleotide
usage frequencies are more identical to that of the avian influenza virus [34]. Synthesis of the RNA segments of the wild
type and mutated virus are then performed using GenScript.
2. Cells and Viral Maintenance: MEM and DMEM are used to
maintain MDCK, 293T, A549 cells and DF1 cells, respectively.
Supplementation of both MEM and DMEM is done with 1%
P/S and 10% fetal bovine serum FBS. All cells are incubated at
37
C humidified incubator with 5% CO 2 . WT BR59 virus,
8 segment mutated virus (8-mut), and 1–4 segment mutated
virus are generated by reverse genetics techniques [32]. BR59
wild-type or mutant genes and HA and NA (previously stated
in Subheading 2.5.2) containing recombinant viruses are also
rescued. Mouse-adapted WT (MA-WT) BR59 is rescued by
introduction of three site directed mutations that are known
to enhance virus pathogenicity in mice (T89I, N125T, and
D221G in the HA gene) [35]. Reverse genetics approach is
used to rescue PR8 [32]. Heterosubtypic virus challenge is
performed with a mouse-adapted A/HK/1/68 clone,
MA20C (HK68-MA20C; H3N2) [36]. Amplification of all
the viruses are performed in embryonated eggs or MDCK
Live Vaccine Attenuation
345
blood is collected on days 15 and 29, and serum is isolated for
analysis by microneutralization assay and ELISA for anti-HA
responses. For testing the IgG antibody concentration in
mouse serum, plates are coated with HA of the PR8 virus,
and specific IgG, IgG1, and IgG2a are measured in the sera
of immunized mice. After the last bleeding, mice are
challenged with 100Â LD50 mouse adapted PR8 (H1N1),
A/California/04/2009(H1N1) (CA09), or 104 PFU
A/Hong HK/1/68 (H3N2) (HK68) influenza virus. The
challenged mice are monitored for clinical symptoms and
survival.
3.5 Exploiting Codon
Usage Bias for Viral
Attenuation
1. Development of a human influenza virus having avian influenza
virus-like codon bias sequences [15]: Codon usage study is
performed using the Influenza A/Brisbane/59/2007
(H1N1) (BR59) strain. Number of mutations required to be
introduced to alter the codon bias from human to avian like
influenza virus are selected by comparing the codon usage
frequency of wild-type (WT) BR59 with that of the frequency
of avian influenza virus. Introduction of such mutations into
the viral genome led to the emergence of the required mutant
with the avian virus–like codon usage. The minimum free
energy of the mutated gene sequences and their nucleotide
usage frequencies are more identical to that of the avian influenza virus [34]. Synthesis of the RNA segments of the wild
type and mutated virus are then performed using GenScript.
2. Cells and Viral Maintenance: MEM and DMEM are used to
maintain MDCK, 293T, A549 cells and DF1 cells, respectively.
Supplementation of both MEM and DMEM is done with 1%
P/S and 10% fetal bovine serum FBS. All cells are incubated at
37
C humidified incubator with 5% CO 2 . WT BR59 virus,
8 segment mutated virus (8-mut), and 1–4 segment mutated
virus are generated by reverse genetics techniques [32]. BR59
wild-type or mutant genes and HA and NA (previously stated
in Subheading 2.5.2) containing recombinant viruses are also
rescued. Mouse-adapted WT (MA-WT) BR59 is rescued by
introduction of three site directed mutations that are known
to enhance virus pathogenicity in mice (T89I, N125T, and
D221G in the HA gene) [35]. Reverse genetics approach is
used to rescue PR8 [32]. Heterosubtypic virus challenge is
performed with a mouse-adapted A/HK/1/68 clone,
MA20C (HK68-MA20C; H3N2) [36]. Amplification of all
the viruses are performed in embryonated eggs or MDCK
Live Vaccine Attenuation
345