Amplification of E2 gene was performed using AccuPrime Taq DNA Polymerase High Fidelity according to the manufacturer’s instruction (Invitrogen). The obtained cDNA was served as template to amplify the full-length E2 gene using forward primer CSFV-E2-F: 5′-GGYRAATATGTGTGTGTWAGACC-3′ and reverse primer: CSFV-E2-R: 5′-TGGTCTTRACTGGRTTGTTRGTC-3′. Briefly, total RNA of the infected kidney tissue was extracted using Trizol reagent (Ambion, Carlsbad, CA) according to the manufacturer’s instruction and subsequently reverse transcribed with Super Script III first strand cDNA synthesis kit (Invitrogen, Carlsbad, CA) using random primer. Detection of wild-type CSFV and other virusesĬSFV in the specimen was detected by amplification of full-length E2 gene with RT-PCR. The kidney specimen was collected from one dead piglet and sent to our laboratory for diagnosis. All these piglets in this farm were imported from the neighboring Guangxi province. Furthermore, the complete genome sequence of this CSFV isolate was determined and analyzed.Īn outbreak of suspected CSF in a pig farm in Guangdong province was reported in 2011, in which 42 of 50 piglets around 30-day old showed clinical signs resembling CSF, and 8 piglets died. In the present study, a sub-subgenotype 2.1c CSFV was isolated from the kidney of a CSFV-infected piglet and adapted to cell culture with high titer following 46 times of continuous passages in PK-15 cells. Most recently, sub-subgenotype 2.1d was identified from outbreaks in Shandong, Jilin, Heilongjiang and Jiangsu Provinces, indicating the presence of genetically divergent subgenotype 2.1 viruses in China. Recent reports showed that a novel CSFV sub-subgenotype, 2.1c, was characterized from CSF outbreaks in Hunan and Guangdong provinces in 2011. reported that 34 out of 35 CSFV isolates collected from Southeast China between 20 were sub-subgenotype 2.1b. Further analysis showed that subgenotype 2.1 isolates could be segregated into sub-subgenotypes 2.1a and 2.1b. In China, subgenotypes 2.1, 2.2, 2.3, 1.1, and 3.4 were reported with the first two subgenotypes causing the most CSF outbreaks in the country. It was reported that CSFV circulating in Europe belong to genotype 2 and isolates from South America belong to genotype 1. CSFV isolates can be classified into 3 genotypes and 11 subgenotypes (1.1–1.4 2.1–2.3 3.1–3.4) based on the 5′UTR (150 nucleotides), E2 (1 nt), and NS5B (409 nt). CSF virus is a member of the genus Pestivirus within the family Flaviviridae, which is an enveloped virus harboring a single-strand positive-sense RNA genome. As an OIE notifiable disease, CSF usually causes significant economic losses in pig industries. Taken together, a field CSFV strain GD53/2011 was isolated, fully sequenced, and adapted to high growth titer in PK-15 cells, which might be suitable for future studies on CSFV infection, replication, and vaccine development.Ĭlassical swine fever (CSF) is a highly contagious swine disease with high morbidity and mortality, featuring symptoms of hemorrhagic fever and immunosuppression. Full-length protein sequence comparison of GD53/2011 with other 2.1 sub-subgenotype isolates showed that Core and NS5A, rather than E2, are more genetically variable. Furthermore, E2 gene sequences at passages 6, 15, 25, 35, and 46 was found identical to that in the original tissue, indicating that the E2 gene was stable during CSF virus adaptation in PK-15 cells. Sequence comparison revealed that the E rns gene at passages 6, 15, and 25 of GD53/2011 was identical to that in the original tissue, but one amino acid substitution (S476R) was detected at passages 35 and 46. After adaptation in PK-15 cells, the titer of GD53/2011 was significantly increased from 10 3.39 TCID 50/ml at passage 6 (F6) to 10 8.50 TCID 50/ml at passage 46 (F46) with the peak titer obtained at 48 h post-inoculation. To further understand the replication characteristics, GD53/2011 was subsequently adapted in PK-15 cells, and its full-length genome was sequenced. Phylogenetic analysis based on the full-length E2 gene sequence revealed that this isolate belongs to CSFV sub-subgenotype 2.1c. This study reports the isolation and characterization of a field CSF virus named GD53/2011 from pig kidney tissue collected during a CSF outbreak in Guangdong province, China. Classical swine fever (CSF) still causes substantial economic losses in the pig industry in China.
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