High attenuation of GPV-CZM-142 through adaptation in GEF
In order to generate an attenuated GPV vaccine candidate, a highly pathogenic GPV strain GPV-CZM was serially passaged in GEF cells. A total of 142 generations of GPV-CZM were generated. To evaluate the pathogenicity of these adapted GPV-CZM in GEF cells, the ELD50 of wild type GPV-CZM, the 70th generation GPV-CZM-70 and the 142th generation GPV-CZM-142 was tested in goose embryos. The mortality of the goose embryos infected with the wild type GPV-CZM was 100%, 100%, 80%, 60% and 40% respectively at the infection dose of 1:10, 1: 102, 1: 103, 1: 104 and 1: 105 dilutions, whereas that of the goose embryos infected with GPV-CZM-70 was 20% , 0%, 0%, 0% and 0% respectively. Notably, GPV-CZM-142 was not lethal to goose embryos at all these infection doses tested. Based on the viral TCID50 titer of these GPV strains, one ELD50 of GPD-CZM, GPV-CZM-70 and GPV-CZM-142 was 1.46 TCID50 ,105 TCID50 and > 105 TCID50 respectively as shown in Table2. This data clearly demonstrate that the GPV-CZM-70 and GPV-CZM-142 strains are highly attenuated in comparison with the wild type GPV-CZM.
Genome amplification and sequencing
To assay the mutation profiles for the GEF adapted GPV-CZM, the genomes of the GPV-CZM, GPV-CZM-70 and GPV-CZM-142 strains were amplified as six fragments using primers listed in Table1. As shown in Fig1, the six fragments with corresponsing sizes could be efficiently amplified. These PCR products were then cloned into pGEM®-T Easy vector and the recombinant plasmids were sequenced. After sequence alignment and joint, the whole genome size of GPV-CZM, GPV-CZM-70 and GPV-CZM-142 was 5106bp, 5120bp and 5128bp, respectively. Genome sequence analysis revealed that the a novel insertion in the ITR region was found in the adapted GPV-CZM-70 and GPV-CZM-142 respectively when compared with the wild type GPV-CZM. Further assay showed that GPV-CZM-142 carried 101mutations for nucleotide in comparison with GPV-CZM. Among these mutations, 54, 16 and 31 sites were located in the non-coding region, the NS gene and the VP genes respectively. Genome phelogenic tree analysis showed that GPV-CZM, GPV-CZM-70 and GPV-CZM-142 strains were clustered into the same branch as strains GDaGPV, SYG61v, GPV-98E and GPV-98D15 as described in Fig2. Among them, GPV-98D15 is an attenuated GPV through passaging in duck embryo.
Insertion and mutation 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 444bp, 451bp and 455bp, 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, an insertion with 7bp and 11bp length was identified in the ITR of GPV-CZM-70 and GPV-CZM-142 respectively in comparison with that of the wild type GPV-CZM as shown in Fig3. Notably, such insertions were occurred at the same position in the GPV-CZM-70 and GPV-CZM-142, which were not found in other GPV strains analyzed, indicating the host adaptation of these insertions and their potential roles in the attenuation.
Mutation sites in NS and VP1 of GPV-CZM-142
Different from the ITR region, no deletion and insertion was found in the non-structural protein NS and the structural protein VP1 of GPV-CZM-70 and GPV-CZM-142 in comparison with GPV-CZM. However, the host adapted mutations were found in both proteins of the two strains as described in Table 4 and 5. Compared with GPV-CZM, GPV-CZM-70 had a total of18 mutations for amino acid. 2 of 18 mutations were located at NS protein (Table 4) whereas other sites were located in VP1 protein (Table 5). GPV-CZM-142 carried33mutations for amino acid in comparison with GPV-CZM. Among them, 8were located in NS protein (Table 4) and 25 were located in VP1 protein (Table 5). Notably, of these mutations, 7 and 16 mutations in NS and VP1 respectively were common in GPV-CZM-70 and GPV-CZM-142 compared with the wild type GPV-CZM, highlighting the host adaptation of these sites. In addition, 7 and 6 mutations respectively in NS and VP1 of GPV-CZM-142 were also found in GPV strain SYG61v, an attenuated vaccine strain, indicating the roles of these mutations in the viral attenuation.