Isolation and identification of mutant isolate of classical swine fever virus from Guangxi and genetic characteristic analysis of E2 gene

【Objective】 This study clarified the molecular epidemiological and etiological characteristics of the Guangxi isolates of classic swine fever virus(CSFV), to provide reference for the scientific use of the CSF vaccine and effective prevention and control of the CSFV epidemic in Guangxi.【Method】 From December 2019 to October 2020, tissues(hearts, livers, spleens, lungs, kidneys, lymph nodes) of aborted fetuses, stillbirths, mummified fetuses, and weak piglets suspected of CSFV infection were collected from Nanning, Long'an, Liuzhou, Baise and Beihai in Guangxi for CSFV testing. The virus was isolated through PK-15 cells, and identified by RT-PCR and indirect immunofluorescence tests(IFA). The E2 gene of the CSFV isolates was cloned. The nucleotide sequence and deduced amino acid sequence were derived via DNASTAR to conduct homology comparison and analysis and the genetic evolutionary tree was constructed with MEGA 6.0. At the same time, with the Shimen standard strain as a control, the growth curve, virus mRNA, and E2 protein levels of the isolates after infecting PK-15 cells were tested separately.【Result】 A strain of CSFV was isolated from 104 suspected CSFV-infected samples and named GXNN2019-13. There was no cytopathy after inoculation with PK-15 cells. The similarity of E2 gene nucleotide sequence between the GXNN2019-13 strain and the standard Shimen strain(strong toxic) as well as the HCLV strain(weak toxic) in China was low, which was 84.6% and 83.5% respectively. The similarity of E2 gene nucleotide sequence between the GXNN2019-13 strain and the Japanese CSF0745 strain, the German CSF0496 and the Alfort Tuebingen strains was 93.9%, 92.2%, and 91.5% respectively. The similarity of E2 amino acid sequence between the GXNN2019-13 strain and Shimen as well as HCLV strain was 89.3% and 88.7% respectively. The similarity between the GXNN2019-13 strain and the Japanese CSF0745 strain, the German CSF0496 and Alfort Tuebingen strains was 95.5%, 94.6%, and 93.8% respectively. Taking the HCLV strain as a reference for multiple sequence comparison, it was found that the E2 protein in the GXNN 2019-13 strain had a higher degree of variation than that ofthe Shimen standard strain. Infected with PK-15 cells, the virus replication ability of the GXNN2019-13 strain was weaker than that of the Shimen standard strain, and the viral titer and mRNA levels at various time points in the growth curve were lower than that of the Shimen standard strain.【Conclusion】 The GXNN2019-13 strain obtained from suspected CSFV-infected material is an independent small branch of subtype 2.3. It is far from related to the previous Guangxi isolates, which may be a new mutated strain with weaker replication ability than that of the Shimen standard strain (strong toxic).