| Classical swine fever virus(CSFV) is the causative agent of highly contagious swine disease CSF occurred in many countries over the world including China. Genetic typing based on the 5’NTR(150 nucleotides), E2(190 nt and 1119 nt) and NS5B(409 nt) classified CSFV isolates into 3 genotypes, which were further divided into 11 subgenotypes(1.1-1.4; 2.1-2.3; 3.1-3.4). Tu et al. indicated that CSFV isolates collected from China between 1980 s and 1990 s were divided into sub-genotypes 2.1, 2.2, 2.3 and 1.1 and the first two groups were dominant for CSF outbreaks at that period. Since the genetic diversity present within the sub-genotype 2.1 isolates, it was further segregated into 2.1a and 2.1b. Chen et al. reported that 34 out of 35 CSFV isolates collected from southeastern China in 2004-2007 were grouped into sub-genotype 2.1b and only one isolate collected in 2004 belongs to sub-genotype 2.2, indicating the dominant role of 2.1 isolates in CSF outbreaks in this epidemic area. In 2009, the first and unique 2.1a isolate SXCDK was collected from Shanxi province in northeastern China. More recently, 2.1c and 2.1d isolates collected from Hunan and Guangdong provinces in 2011 were reported to be the agent of many CSF cases.For now, different strategies for disease control including eradication and vaccination were carried out and large scale of CSF outbreaks was not occurred. In this case, many sub-genotype CSFV isolates became history, such as genotype 1 isolates in Europe and genotype 3 isolates in Asia. At the same time, the switch of virus populations from genotype 1 or genotype 3 to genotype 2 was occurred. In Europe, sub-genotype 2.1 has caused the epidemic of outbreaks of many countries in 1990 s and wild boar isolates of sub-genotype 2.2 and 2.3 are currently dominant. In Asia, subgenotype 2.1 isolates became dominant in China and Korea.Recently three CSF-suspected clinical samples from Guangdong province was collected and detected as CSFV positive by RT-PCR. Phylogenetic analysis based on the nucleotide sequence of full-length E2 gene showed that the 3 of CSFV isolate GD176, GD53 and GD19 belongs to sub-subgenotype 2.1b, 2.1c and 2.1d of CSFV. To further understand the replication characteristics of these sub-subgenotype 2.1 isolate, GD176, GD53 and GD19 was isolated and adapted in PK-15 cells, wholegenome sequence of GD176, GD53 and GD19 cell-adapted virus was amplified and sequenced. Adaptation of GD176 in PK-15 cells started at F6 with 102.61 TCID50/mL and has been increased to 108.06 TCID50/ml at F46, Adaptation of GD176 in PK-15 cells started at F6 with 102.61 TCID50/mL and has been increased to 108.06 TCID50/ml at F46, Adaptation of GD176 in PK-15 cells started at F6 with 102.61 TCID50/mL and has been increased to 108.06 TCID50/ml at F46. Growth dynamics of GD176-F46, GD53-F46 and GD19-F46 with a MOI at 0.1 indicated that the peak titer reached at 72 h postinfection(p.i.). To further define the stability of GD176, GD53 and GD19 during cellular adaptation, full-length E2 genes of different cell-adapted viruses of GD176, GD53 and GD19 were sequenced. As a result, E2 gene of F0 was found to be identical with that of F6, F10,F15, F20, F25, F30, F35, F40 and F46, indicating the genetic stability of E2 gene during in vitroadaptation of GD176, GD53 and GD19 in PK-15 cells. In addition, comparison of individual viral protein showed that the most variable protein between GD176, GD53 and GD19 and other 2.1 sub-subgenotype isolates is NS5 A rather than viral envelope protein E2.Overall, highly cell-adapted CSFV sub-subgenotype 2.1b strain GD176, CSFV sub-subgenotype 2.1c strain GD53, CSFV sub-subgenotype 2.1d strain GD19 was obtained in this study and it will be useful for future study on viral virulence, replication and pathogenesis. |
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