3.3 Viral Attenuation
by Sequential
Passaging [13]
1. Vaccine Preparation: Varicella (Oka strain) virus is serially
grown in human embryonic lung (HEL) cells for eleven times
and then in GPE cells. After a twelfth passage in GPE cells, the
virus is subsequently passaged in human diploid cells (WI-38)
cells (Flow Laboratories). GPE cells are obtained by trypsinization of skin and muscle tissues from 3- to 4-week-old guinea
pig embryos. Passage of virus is carried out by transfer of
infected cells. Cell-free varicella virus is obtained essentially by
the method of Caunt and Taylor-Robinson and Brunell
[28, 29]. The tissue-culture fluid is removed, and infected
cells are washed with phosphate-buffered saline solution
(PBS) and collected by edetic acid treatment. Harvested cells
are suspended in 3 ml of Eagle’s minimum essential medium
and lysed by sonication by a 20KC sonifier for 30–40 s. The
disrupted cells are centrifuged for 15 min at 4000 r.p.m. and
the supernatant is collected. The collected supernatant is used
as an, experimental vaccine after addition of a suitable stabilizer
like 5% sugar. The vaccines are prepared from the virus at the
sixth or seventh passage in GPE cells and second passage in
WI-38 cells. They are examined for the presence of bacteria and
mycoplasmas. Absence of viruses other than varicella virus is
confirmed by electron microscopy.
3.4 MicroRNA-Based
Viral Attenuation
1. Eggs and Cell culture: Embryonated eggs are incubated at
37.5
C for up to 9 days. MDCK cells are cultured in MEM
supplemented with 10% FBS, 50 μg/ml gentamicin, and 1 mM
sodium pyruvate. HEK293T, MEF, MEF DicerÀ/À, DF1,
and A549 cells are cultured in DMEM supplemented with
10% FBS, 1% penicillin, and 1 μg/ml streptomycin.
2. Artificial microRNA design and expression: The miR-93 cassette with a scrambled control sequence, the miR-93 locus, and
amiR-93NP are synthesized by GenScript and cloned into the
microRNA-expressing plasmid pLL3.7 [30]. For transfection,
8 Â 10
5 293T cells are seeded per well into 6-well plates. The
following day, the cells are transfected with 1 μg plasmid
pcDNA-NP with 1 μg pLL3.7, pLL3.7-ctl, pLL3.7-miR-93,
or pLL3.7-amir-93NP. After 24 h of transfection, cells are
harvested and lysed. Expression of NP and amiR-93NP are
detected by western blot and northern blot, respectively.
3. Virus design, rescue, and titration: Modified NS gene segments
with miR-93 locus and amir-93NP cassette insertions are
synthesized by GenScript. Reorganization of the NS gene segment is done as described [31]. Viruses are rescued using a
plasmid-based rescue system [32]. The viruses designed were
wild-type PR8 (PR8-wt), PR8-control (PR8-ctl), PR8-miR93, and PR8-amiR-93NP. Viral stocks are titrated in chicken
eggs and expressed as EID50. Briefly, tenfold serial dilutions of
Live Vaccine Attenuation
343
by Sequential
Passaging [13]
1. Vaccine Preparation: Varicella (Oka strain) virus is serially
grown in human embryonic lung (HEL) cells for eleven times
and then in GPE cells. After a twelfth passage in GPE cells, the
virus is subsequently passaged in human diploid cells (WI-38)
cells (Flow Laboratories). GPE cells are obtained by trypsinization of skin and muscle tissues from 3- to 4-week-old guinea
pig embryos. Passage of virus is carried out by transfer of
infected cells. Cell-free varicella virus is obtained essentially by
the method of Caunt and Taylor-Robinson and Brunell
[28, 29]. The tissue-culture fluid is removed, and infected
cells are washed with phosphate-buffered saline solution
(PBS) and collected by edetic acid treatment. Harvested cells
are suspended in 3 ml of Eagle’s minimum essential medium
and lysed by sonication by a 20KC sonifier for 30–40 s. The
disrupted cells are centrifuged for 15 min at 4000 r.p.m. and
the supernatant is collected. The collected supernatant is used
as an, experimental vaccine after addition of a suitable stabilizer
like 5% sugar. The vaccines are prepared from the virus at the
sixth or seventh passage in GPE cells and second passage in
WI-38 cells. They are examined for the presence of bacteria and
mycoplasmas. Absence of viruses other than varicella virus is
confirmed by electron microscopy.
3.4 MicroRNA-Based
Viral Attenuation
1. Eggs and Cell culture: Embryonated eggs are incubated at
37.5
C for up to 9 days. MDCK cells are cultured in MEM
supplemented with 10% FBS, 50 μg/ml gentamicin, and 1 mM
sodium pyruvate. HEK293T, MEF, MEF DicerÀ/À, DF1,
and A549 cells are cultured in DMEM supplemented with
10% FBS, 1% penicillin, and 1 μg/ml streptomycin.
2. Artificial microRNA design and expression: The miR-93 cassette with a scrambled control sequence, the miR-93 locus, and
amiR-93NP are synthesized by GenScript and cloned into the
microRNA-expressing plasmid pLL3.7 [30]. For transfection,
8 Â 10
5 293T cells are seeded per well into 6-well plates. The
following day, the cells are transfected with 1 μg plasmid
pcDNA-NP with 1 μg pLL3.7, pLL3.7-ctl, pLL3.7-miR-93,
or pLL3.7-amir-93NP. After 24 h of transfection, cells are
harvested and lysed. Expression of NP and amiR-93NP are
detected by western blot and northern blot, respectively.
3. Virus design, rescue, and titration: Modified NS gene segments
with miR-93 locus and amir-93NP cassette insertions are
synthesized by GenScript. Reorganization of the NS gene segment is done as described [31]. Viruses are rescued using a
plasmid-based rescue system [32]. The viruses designed were
wild-type PR8 (PR8-wt), PR8-control (PR8-ctl), PR8-miR93, and PR8-amiR-93NP. Viral stocks are titrated in chicken
eggs and expressed as EID50. Briefly, tenfold serial dilutions of
Live Vaccine Attenuation
343