Highly attenuated GPV-CZM-142 generated through adaptation to GEF
To generate an attenuated GPV vaccine candidate, a highly pathogenic GPV strain GPV-CZM was continuously passaged in GEFs underwent a total of 142 generations. To evaluate the pathogenicity of the adapted GPV-CZM, the ELD50 of wild type GPV-CZM, the 70th generation of GPV-CZM-70 and the 142th generation of GPV-CZM-142 was tested in goose embryos. The mortalities of the goose embryos infected with the wild type GPV-CZM were 100%, 100%, 80%, 60% and 40% at the infection dose of 1:10, 1: 102, 1: 103, 1: 104 and 1: 105 dilutions, respectively. In contrast, those of the goose embryos infected with GPV-CZM-70 were 20%, 0%, 0%, 0% and 0%, respectively. Notably, GPV-CZM-142 was not lethal to goose embryos at these doses. Based on the viral TCID50 titers of these GPV strains, The ELD50 of GPD-CZM, GPV-CZM-70 and GPV-CZM-142 were 1.46 TCID50, 105 TCID50 and > 105 TCID50, respectively (Table 2). These data clearly demonstrate that the GPV-CZM-70 and GPV-CZM-142 strains are highly attenuated compared with the wild type GPV-CZM.
Sketch on the genome sequences of different GPV strains
To profile sequence mutations in the genome of the GEF adapted GPV-CZM strains, the genomes of the GPV-CZM, GPV-CZM-70 and GPV-CZM-142 strains were amplified using primers listed in Table 1. As shown in Fig. 1, six fragments with the expected sizes were efficiently amplified. These amplicons were cloned into pGEM®-T Easy vector, followed by sequencing the recombinant plasmids. The whole genome sizes of GPV-CZM, GPV-CZM-70 and GPV-CZM-142 were 5106 bp, 5120 bp and 5128 bp, respectively. The sequence of GPV-CZM, GPV-CZM-70 and GPV-CZM-142 had been submitted to the GenBank (the accession number for GPV-CZM, GPV-CZM-70 and GPV-CZM-142 was MT939902, MT939903 and MT939904, respectively). Alignment analysis of the genomes of the adapted GPV-CZM-70 and GPV-CZM-142 against the wild type GPV-CZM identified a novel insertion in the ITR region. Moreover, 101 single nucleotide mutations in the genome of GPV-CZM-142 compared with the genome of GPV-CZM. Among these mutations, 54, 16 and 31 sites were located in the non-coding region, the NS gene and the VP gene, respectively. Genome phylogenic analysis indicated that GPV-CZM, GPV-CZM-70 and GPV-CZM-142 strains were clustered into the same branch as the strains GDaGPV, SYG61v, GPV-98E and GPV-98D15 (Fig. 2). It is noteworthy that GPV-98D15 is an attenuated GPV through passaging in duck embryo.
Mutations identified in ITR of GPV-CZM-142
Since ITR plays vital roles in gene expression and viral replication, ITR of the adapted GPV-CZM-70 and GPV-CZM-142 were further analyzed. The size of ITR of GPV-CZM, CZM-70 and CZM-142 was 444 bp, 451 bp and 455 bp, respectively. The homology of these ITRs from GPV-CZM, GPV-CZM-70 and GPV-CZM-142 was 95.5-97.1%. Except for several site mutations, the insertions with 7 bp and 11 bp lengths were identified in the ITRs of GPV-CZM-70 and GPV-CZM-142, respectively, compared with that of the wild type GPV-CZM (Fig. 3). Notably, such insertions were occurred at the same position in the genomes of GPV-CZM-70 and GPV-CZM-142 but not in the other GPV strains analyzed, suggesting the insertions have a potential role in the attenuation of the virus.
Mutations identified in NS and VP1 of GPV-CZM-142
Different from the ITR region, no deletion and insertion were identified in the non-structural protein NS and the structural protein VP1 of GPV-CZM-70 and GPV-CZM-142 compared with the wild type strain of GPV-CZM. However, there were some single nucleotide mutations identified in the protein-encoding genes of the two strains (Table 4 and 5). Compared with GPV-CZM, GPV-CZM-70 had a total of 18 mutations for amino acid with 2 mutations located in NS gene (Table 4) and the other mutations located in VP1 gene (Table 5). In contrast, GPV-CZM-142 carried 33 mutations for amino acid with 8 mutations located in NS gene (Table 4) and the other mutations located in VP1 gene (Table 5). Notably, of these mutations, 7 and 16 mutations in NS and VP1 were common to GPV-CZM-70 and GPV-CZM-142, respectively. In addition, 7 and 6 mutations in NS and VP1 of GPV-CZM-142 were also found in GPV strain SYG61v, an attenuated vaccine strain. These findings suggest that these mutations are involved in host adaptation of the virus.
Table 3. The information for 17 GPV reference strains used in this study
Strain
|
Genome/bp
|
Source
|
Login ID
|
GPV-98E
|
5106
|
Heilongjiang province, China
|
KT598506
|
GPV-98D15
|
5114
|
Heilongjiang province, China
|
KT598505
|
SYG61v
|
5102
|
Jiangsu province, China
|
KC996729
|
DY16
|
5046
|
Jiangsu province, China
|
MH209633
|
LH
|
5047
|
Jiangsu province, China
|
KM272560
|
SH
|
5106
|
Shanghai province, China
|
JF333590
|
SHFX1201
|
5050
|
Shanghai province, China
|
KC478066
|
Y
|
5106
|
Anhui province, China
|
KC178571
|
Yan2
|
5106
|
Anhui province, China
|
KR136258
|
GDaGPV
|
5106
|
Fujian province, China
|
HQ891825
|
G7
|
5106
|
Fujian province, China
|
KR029617
|
FJ
|
5049
|
Fujian province, China
|
KY511292
|
FJ01
|
5104
|
Fujian province, China
|
KT232256
|
MDE
|
5106
|
Fujian province, China
|
MF438102
|
RC16
|
5046
|
Sichuan province, China
|
KY475562
|
82-0321
|
5050
|
Taiwan province, China
|
EU583390
|
B
|
5106
|
Hungary
|
U25749
|
Table 4. Mutations in NS protein of GPV-CZM-70 and GPV-CZM-142
Position
|
GPV-CZM
|
CZM-70
|
CZM-142
|
SYG61v
|
72
|
I
|
V
|
V
|
I
|
149
|
Q
|
Q
|
L
|
Q
|
199
|
E
|
E
|
D
|
E
|
203
|
A
|
A
|
S
|
A
|
501
|
L
|
L
|
P
|
P
|
507
|
R
|
R
|
P
|
P
|
508
|
K
|
E
|
E
|
E
|
573
|
E
|
E
|
A
|
E
|
579
|
T
|
T
|
T
|
E
|
Table 5. Mutations in VP1 protein of GPV-CZM-70 and GPV-CZM-142
Position
|
GPV-CZM
|
CZM-70
|
CZM-142
|
SYG61v
|
3
|
T
|
N
|
N
|
T
|
5
|
L
|
V
|
V
|
L
|
89
|
Q
|
Q
|
R
|
Q
|
110
|
D
|
D
|
N
|
D
|
142
|
D
|
N
|
N
|
D
|
146
|
T
|
K
|
K
|
T
|
193
|
T
|
P
|
P
|
P
|
201
|
K
|
K
|
K
|
E
|
217
|
V
|
L
|
L
|
V
|
302
|
I
|
I
|
L
|
I
|
360
|
P
|
A
|
A
|
P
|
381
|
N
|
N
|
K
|
N
|
449
|
S
|
R
|
R
|
S
|
503
|
F
|
V
|
V
|
V
|
516
|
L
|
P
|
P
|
P
|
532
|
K
|
Q
|
Q
|
Q
|
534
|
I
|
M
|
M
|
I
|
537
|
I
|
L
|
L
|
L
|
544
|
S
|
P
|
P
|
S
|
545
|
G
|
G
|
S
|
G
|
546
|
S
|
S
|
T
|
S
|
547
|
T
|
T
|
S
|
T
|
549
|
A
|
A
|
T
|
A
|
555
|
R
|
M
|
M
|
M
|
593
|
D
|
E
|
E
|
D
|
643
|
V
|
V
|
I
|
V
|