| Classical swine fever virus(CSFV),a single stranded positive-sense RNA virus belonging to Pestivirus genus within the Flaviviridae family,is the causative agent of CSF,which is a highly contagious disease that caused severe impact and significant economic losses to pig industry.The obligate immunization with attenuated vaccine C strain is an important strategy to prevent and control CSF in China.At the same time,surveillance of CSFV through molecular epidemiology is also essential for CSF prevention and control.To date,phylogenetic analysis based on full-length E2 gene sequences of CSFV is more reliable and statistically significant,thus which has been widely used for CSFV molecular epidemiology.In addition,genetic diversity was presented in the CSFV field isolates from China,but the biological characteristics of these strains,such as replication,virulence and pathogenicity,are still to be understood.Furthermore,antigenic differentiation of attenuated vaccine C strain from CSFV field isolates remained to be resolved.The basis to perform all studies mentioned above is to obtain cell adapted strains in vitro.In order to investigate the epidemic situation,geographical distribution and genetic evolution characteristics of CSFV in China,254 suspected CSF clinical samples collected between 2014 and 2017 from 21 provinces and autonomous regions of China were detected for CSFV by RT-PCR,and the positive samples together with 95 CSFV isolates collected between 1990 and 1999 were used to further amplify the full-length E2 gene fragments.Phylogenetic relationship and genetic variation of CSFV field isolates from China were analyzed together with the reference strains from different countries.Results showed that 98 of the 254 clinical samples were CSFV positive,and the positive rate was 38.6%.Sequence comparison of full-length E2 gene showed that 120 CSFV field isolates collected between 1990 and 2017 shared 85.7%?100%and 89.5%?100%sequence identity at the nucleotide and amino acid level respectively,the sequence identity of which with C strain were 81.1%?84.9%and 86.9%?90.9%at nucleotide and amino acid level respectively.Phylogenetic analysis based on the full-length E2 gene sequences showed that CSFV isolates collected between 1990 and 1999 in this study belong to CSFV subgenotypes 2.1a,2.1b,2.1c,2.1h,2.2 and 2.3,and isolates collected between 2014 and 2017 were clustered to 2.1b,2.1c,2.1h and 2.1j subgenotypes,of which subgenotype 2.1b was dominant.These epidemiological data will provide new insights into establish reasonable strategy for CSF prevention and control as well as vaccination.In order to obtain the highly cell-adapted strains in vitro,CSFV positive tissue samples were inoculated into PK-15 cell lines and adapted with "cell passage" followed by "virus passage".As a result,76 CSFV strains were successfully isolated from 95 CSFV positive tissue samples and gradually adapted through continuous passages.The infectious titers of 50 cell-adapted CSFV strains were above 106.5 TCID5O/mL and the hightest tone reached 108.3 TCID50/mL.The establishment of viral stocks of CSFV cell-adapted isolates laid the foundation for further analysis of the biological characteristics of different subgenotype virus strains.To explore the factor associated with "genotype shift" of CSFV field isolates in China,CSFV IRES translation activity and cross-neutralization of serum produced with C strain for field isolates were conducted.Results showed that the IRES activity of different virus strains belonging to the same subgenotypes or different subgenotypes was varied,but no significant and expected results were obtained.Cross-neutralization assay showed that the neutralization titers of porcine anti-C strain sera against CSFV subgenotype 2.1 isolates were lower than those against strains belonging to subgenotypes 2.2 and 2.3.The above results will be useful for in-depth analysis of genetic evolution mechanism of the CSFV field strains under vaccination environment. |
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