Introduction

Classical swine fever (CSF) is a highly contagious swine disease with high morbidity and mortality, featuring symptoms of hemorrhagic fever and immunosuppression [1]. As an OIE notifiable disease, CSF usually causes significant economic losses in pig industries. 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 [2]. 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 (190 and 1119 nt), and NS5B (409 nt) [3, 4]. It was reported that CSFV circulating in Europe belong to genotype 2 and isolates from South America belong to genotype 1 [3–7]. 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 [8, 9]. Further analysis showed that subgenotype 2.1 isolates could be segregated into sub-subgenotypes 2.1a and 2.1b [9]. Chen et al. [10] reported that 34 out of 35 CSFV isolates collected from Southeast China between 2004 and 2007 were sub-subgenotype 2.1b. Recent reports showed that a novel CSFV sub-subgenotype, 2.1c, was characterized from CSF outbreaks in Hunan and Guangdong provinces in 2011 [11, 12]. Most recently, sub-subgenotype 2.1d was identified from outbreaks in Shandong, Jilin, Heilongjiang and Jiangsu Provinces [13], indicating the presence of genetically divergent subgenotype 2.1 viruses in China.

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. Furthermore, the complete genome sequence of this CSFV isolate was determined and analyzed.

Materials and methods

Clinical sample

An 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. All these piglets in this farm were imported from the neighboring Guangxi province. The kidney specimen was collected from one dead piglet and sent to our laboratory for diagnosis.

Detection of wild-type CSFV and other viruses

CSFV in the specimen was detected by amplification of full-length E2 gene with RT-PCR. 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. 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′. Amplification of E2 gene was performed using AccuPrime Taq DNA Polymerase High Fidelity according to the manufacturer’s instruction (Invitrogen). The PCR products were analyzed by 1 % agarose gel electrophoresis and the positive amplicon was then sequenced using ABI 3730 sequencer (Kumei Biotechnology Company, Changchun, China). To obtain consensus sequences of full-length E2 gene, three independent sequencing of PCR amplicons were performed.

The CSF-suspected piglet may have also been co-infected with other viruses [14], thus RT-PCR or PCR was performed to detect porcine reproductive and respiratory syndrome virus (PRRSV), bovine viral diarrhea virus (BVDV), porcine circovirus type 2 (PCV-2), pseudorabies virus (PRV), and porcine parvovirus (PPV) in the clinical sample. The cDNA obtained above was used as the template for PCR detection of PRRSV and BVDV.

For PCR detection of PCV-2, PRV, and PPV, 200 µl of the kidney tissue suspension was subjected to DNA extraction with QIAGEN DNA extraction kit (QIAGEN, Germany), and the extracted DNA was used as template for PCR. Briefly, for each virus type, 50 µl of PCR reaction mixture was prepared with 5 µl of 10 × Ex-Taq Buffer (Mg2+ Plus), 1 µM of forward primer, 1 µM of reverse primer, 4 µl of dNTPs (2.5 mM each), 2 µl of cDNA or DNA template, 1 µl of Ex-Taq (5 U/µl), and 36 µl of ddH2O. Specific primers for detection of the above viruses were listed in Table 1. The above PCR reagents were purchased from TaKaRa (TaKaRa, Dalian, China). The following PCR programs were performed: 3 min denaturation at 95 °C, 35 cycles of 45 s at 95 °C, 45 s at 55 °C, 45 s at 72 °C, and 10 min at 72 °C for final elongation. The resulting PCR products were analyzed by 1 % agarose gel electrophoresis.

Table 1 Primer sets used for PCR detection of BVDV, PRRSV, PCV-2, PRV, and PPV
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Isolation and adaptation of field CSFV in PK-15 cells

PK-15 cells were cultured in MEM (Sigma, St. Louis, MO) containing 10 % fetal bovine serum (FBS), 100 U/ml of penicillin G, 100 µg/ml of sodium streptomycin sulfate, and 2 mM l-glutamine (Invitrogen) at 37 °C. For in vitro adaptation of CSFV field isolate in PK-15 cells, virus-infected cells were continuously passaged for the first 18 generations (cell passage) until most of the cells were positive, and the obtained cellular viruses were then inoculated onto naïve PK-15 cells (virus passage) to yield virus with high infectivity titer.

For “cell passage,” the supernatant of 10 % tissue homogenate of CSFV-positive kidney was inoculated onto PK-15 cells in T25 flasks with 60–80 % confluency. After incubation at 37 °C, the virus-infected PK-15 cells were subcultured into new flasks after trypsin-EDTA treatment at a 96-h interval. Meanwhile part of the cell suspension was seeded into a 96-well culture plate in parallel to monitor the virus growth in each cell passage by indirect fluorescent antibody test (IFA) described below after 72 h incubation. When >90 % cells in the plate were shown to be infected, CSFV culture in the T25 flasks at that passage was harvested as cell-adapted isolate by freezing and thawing three times and then served as the inoculum for “virus passage.”

For “virus passage,” CSFV from “cell passage” was used to inoculate PK-15 cells. At 96-h post-inoculation, the virus culture was collected by freezing and thawing three times and used for the next round of virus passage until highly adapted virus was obtained.

To characterize the growth dynamic, cell-adapted isolate was inoculated on PK-15 cells in 24-well plate with a MOI of 0.1 and then harvested at different time point p.i. by freezing and thawing three times. Meanwhile, extraneous viruses in the CSF virus stocks of GD53/2011-F46 (passage #46) were also examined using RT-PCR or PCR methods as described above. In addition, mycoplasma in the virus stocks of GD53/2011-F46 were detected using LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich, St. Louis, USA) according to the manufacturer’s instruction.

Virus titration and IFA test

To determine the TCID50 of the virus culture collected at different passages or time points, the cell culture supernatants were serially diluted from 10−1 to 10−8 and 100 µL of the diluted virus was inoculated onto PK-15 cells in each well of 96-well culture plates with six wells for each dilution. After incubation at 37 °C for 72 h, the infected wells were examined by IFA. Briefly, wells were washed four times with PBS and then fixed with 80 % cold acetone in PBS at −20 °C for 1 h. The fixed cells were incubated with specific E2 mAb WH303 [15] and followed by incubation with FITC-conjugated goat anti-mouse polyclonal antibody (Sigma, Saint Louis, MO). The incubation with antibody was performed in a humid box at 37 °C for 1 h. Stained cells were observed under Axioskop-40 fluorescent microscope (Zeiss, Goettingen, Germany). Virus titers were calculated by the Kärber method [16] and expressed as TCID50/ml.

Virus genome sequencing and phylogenetic analysis

The complete genome sequence of cell-adapted isolate after 46 passages (F46) was obtained by RT-PCR described above using seven primer pairs covering whole genome (Table 2). The amplicons were sequenced as described above and then assembled into a complete genome sequence. To further analyze the genetic stability of the virus during cell passage, its full-length Erns and E2 genes at different passages were amplified and sequenced as described above.

Table 2 Primer sets used for amplification of complete CSFV genome
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Alignment of nucleotide/amino acid sequences of individual protein gene or complete genome sequence were conducted using CLC sequence viewer 7.6.1 (QIAGEN, Germany). Phylogenetic analysis were performed using Molecular Evolutionary Genetics Analysis software MEGA 6.06 (Center for Evolutionary Functional Genomics, Tempe, AZ). Neighbor-joining method including Bootstrap value of 1000 repetitions was used for construction of phylogenetic tree.

Results

Detection of wild-type CSFV and other swine viruses

Full-length E2 gene was successfully amplified by RT-PCR from the CSF-suspected sample and the field CSFV was named GD53/2011. After three independent sequencing of the positive PCR products, nucleotide sequence of full-length E2 gene of GD53/2011 was obtained. The phylogenetic analysis in Fig. 1 showed that GD53/2011 was classified into the recently identified sub-subgenotype 2.1c [11, 12] and shared 94.8–99.0 % nucleotide sequence and 96.7–98.7 % amino acid similarity with other 2.1c isolates obtained from pigs in Guangdong and its neighboring Guangxi and Hunan provinces.

Fig. 1
figure 1

CSFV GD53/2011 belongs to subgenotype 2.1. Full-length E2 sequence-based Neighbor-Joining tree indicates the phylogenetic relationship of GD53/2011 with those of other CSFV subgenotypes identified in China. The black triangles indicate subgenotype 2.1 isolates with whole-genome sequences, which were used for further nucleotide and amino acid sequence alignments with the newly isolated GD53/2011 (also marked with an asterisk)

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In addition, this CSFV-positive kidney tissue also tested positive for PRRSV and PCV-2, but negative for BVDV, PRV, PPV, and mycoplasma, using RT-PCR and PCR methods described above (data not shown).

Adaptation of GD53/2011 in PK-15 cells

In order to generate a highly cell-adapted CSFV strain, the CSFV-positive kidney tissue homogenate was inoculated onto PK-15 cells and subjected to 46 passages in PK-15 cells (18 times of cell passage, followed by 28 times of virus passage), resulting in the cell-adapted strain GD53/2011-F46. Result of titration showed that the TCID50 of GD53/2011 significantly increased from 103.39 TCID50/ml at passage 6 (F6) to 108.50 TCID50/ml at F46 (Fig. 2a), which was comparable to that of CSFV strain Alfort 187 in subgenotype 1.1, one of the viruses with the highest infectivity titer [17].

Fig. 2
figure 2

Adaption and growth characteristics of GD53/2011 in PK-15 cells. a the titers of GD53/2011 were significantly increased over the time of passaging in the cells; b the titers of cell-adapted GD53/2011-F46 reached its peak at 48 h p.i. All data are shown as mean ± SEM from three independent experiments

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Furthermore, the growth characteristics of GD53/2011-F46 were analyzed by titration of the virus cultures collected at 12, 24, 36, 48, 72, and 96 h. As depicted in Fig. 2b, more than tenfold increase of the virus titer of GD53/2011 was detected for every 12 h interval from 12 to 48 h p.i., the peak titer for replication of GD53/2011 reached 108.40 TCID50/ml at 48 h p.i. These results confirmed the efficient replication of GD53/2011 in PK-15 cells observed earlier. In addition, detection of extraneous viruses in cell-adapted GD53/2011-F46 showed that PRRSV and PCV-2 were not present in the virus stocks, indicating that a pure and cell-adapted CSFV GD53/2011 was obtained.

To test whether the genetic variation occurred during passage, full-length genes of viral glycoproteins Erns and E2 from original kidney tissue and different passages of virus culture were compared. Results showed that the Erns gene at passages 6, 15, and 25 was identical to that in the original tissue specimen, but one amino acid substitution (S476R) was identified at passages 35 and 46. However, the E2 gene sequence of GD53/2011-F0 was identical to that of GD53/2011 passages 6, 15, 25, 35, and 46, showing the genetic stability of viral E2 gene of GD53/2011 during the cellular adaptation.

Comparison of GD53/2011 whole-genome sequence with those of other CSFVs

To identify the genetic variations, full-length genome sequence of GD53/2011-F46 was compared with other whole-genome CSFV sequences available from GenBank. The length of GD53/2011 whole genome is 12,296 nt and contains a 372-nt 5′UTR, a 11697-nt ORF, and a 227-nt 3′UTR. GD53/2011 shared 98.8 % nt and 99.2 % AA sequence similarity with CSFV sub-subgenotype 2.1c isolate HNLY2011 [12]. Comparison of full-length genome sequences or polyproteins between GD53/2011 and other 12 isolates of subgenotype 2.1a and 2.1b, which were also used for construction of the phylogenetic tree (Fig. 1), indicates that the genetic distance between GD53/2011 and 2.1a and 2.1b isolates is 6.1–7.5 and 6.9–7.7 % at the genomic level, respectively, and 2.6–3.4 and 3.0–3.7 % at the polyprotein level.

Sequence analysis of viral polyproteins showed that several important functional areas were completely conserved in the GD53/2011 sequence as well as other 2.1c CSFV isolates. These functional areas include the RNase sites of Erns, the cysteines in E2, protease, and the epitope recognized by E2-specific monoclonal antibody WH303 (Fig. 3). Comparison based on the viral polyproteins further revealed that GD53/2011 and another 2.1c isolate HNLY2011 harbor 29 sub-subgenotype-specific amino acid substitutions when compared with other groups (2.1a, 2.1b, 2.2, 2.3, 1.1, and 3.4) of CSFV isolates at the genomic level. Interestingly, four of the sub-subgenotype-specific amino acid substitutions were found in the Core, and nine were located in the NS5A proteins, respectively (Table 3; Fig. 3).

Fig. 3
figure 3figure 3figure 3

Amino acid alignment of Core/Erns, E2 and NS5A proteins of GD53/2011 and 22 reference CSFV isolates. a alignment of full-length Core/Erns protein; b alignment of full-length E2 protein; c alignment of full-length NS5A protein. Unique substitutions in sub-subgenotype 2.1c including GD53/2011 as well as RNase sites of Erns, cysteines (c), and WH303 epitope are indicated by boxes

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Table 3 Amino acid substitutions of protein sequences between GD53/2011 and other subgenotypes used for phylogenetic analysis in the study
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Furthermore, the amino acid identity of individual protein encoding regions of GD53/2011 and other 13 CSFV subgenotype 2.1 isolates used for construction of the phylogenetic tree was analyzed. Among all 12 viral proteins, the more variable regions were Core (90.9–93.9 %) and NS5A (93.4–95.6 %), indicating that Core and NS5A were relatively less conserved compared to other viral proteins.

Discussion

CSFV continues to cause significant economic losses in the pig industry in many countries. Although mandatory vaccination with attenuated vaccine C strain was carried out twice a year, sporadic CSF outbreaks still occur in China. In the present study, outbreak strain GD53/2011 was classified into CSFV sub-subgenotype 2.1c together with isolates from two other neighboring provinces (Guangxi and Hunan) in south of China. Because piglets in the GD53/2011 CSFV-positive farm in Guangdong Province were transported from the neighboring Guangxi Province, strict quarantine measures should be considered to prevent the spread of CSFV through trading. To cultivate a cell-adapted strain for further studies, highly cell-adapted GD53/2011 was obtained through cell passage followed by virus passage. Previous experiment to isolate the field CSFV by blind passage using freezing and thawing virus culture produced low-titer virus (data not shown), therefore we started the isolation and adaption by cell passage and then virus passage. The result showed that this procedure worked effectively and yield high viral titers after several dozens of passages.

Notably, PRRSV and PCV-2 in the original kidney tissue of GD53/2011 were eliminated during cellular adaption and not detected in the GD53/2011-F46, and this was probably because PK-15 cells are not susceptible to PRRSV and the replication efficiency of PCV-2 in PK-15 cells is much lower than of CSFV. In addition, mycoplasma in the viral stock of GD53/2011-F46 was also detected as negative. Therefore, the cell-adapted GD53/2011 can be used for future virulence evaluation and vaccine development.

CSFV glycoproteins Erns and E2, as the key factors for virus entry and infection [18, 19], are genetically variable. However, we found that E2 of GD53/2011 was genetically stable during in vitro adaptation in PK-15 cells, and Erns had only one amino acid substitution (S476R) at the late stage of virus adaption. It has been reported that envelope protein Erns harboring Ser-to-Arg change is critical for the interaction of CSFV with membrane-associated heparin sulfate (HS), and acquisition of Arg was sufficient to alter the HS-independent virus to a virus that uses HS as an Erns receptor [18]. Therefore, the Ser-to-Arg change at Erns may contribute to the enhanced replication rate of GD53/2011-F46 in PK-15 cells. Meanwhile, it has been shown that the Ser-to-Arg mutation has no effect on the virulence of CSFV [19], thus the GD53/2011-F46 isolate can be used for further virulence studies.

To further analyze the genetic diversity of individual protein genes of CSFV isolates, the whole genome of GD53/2011-F46 was sequenced and compared with 12 other CSFV subgenotype 2.1 isolates. Surprisingly, comparison between GD53/2011 and isolates belonging to 2.1a and 2.1b revealed that there are more sub-subgenotype 2.1c group mutations present in Core and NS5A than in the E2 protein, which was recognized previously as the most variable protein [20]. Among the four group-specific substitutions in Core (Table 3; Fig. 3a), two of them V256I and L261M were present in the area of signal peptide of Erns (248–267 AA), which is the determinant of glycosylation and extracellular secretion of Erns [21]. It has been demonstrated that CSFV Erns can induce lymphocyte apoptosis and abundant Erns protein was detected in the serum of infected pigs and cell culture supernatant [22]. The amount of Erns secretion may be related to the pathogenesis of CSFV [23]. Thus, it is important to determine whether the substitutions in the signal peptide area of Erns have any impact on the production and secretion of Erns of GD53/2011 in future studies.

CSFV NS5A is a viral phosphoprotein and can induce autophagy of CSFV-infected cells to promote virus replication [24] and is also involved in the translation of CSFV polyprotein [25]. In the present study, 7 of 8 group substitutions were found in both N-terminus (n = 3) and C-terminus of NS5A (n = 4) (Fig. 3c). It remains to be examined whether these mutations can alter the function of NS5A and then influence the replication of GD53/2011 and other viral phenotypes.

Taken together, we present here isolation and in vitro adaptation of CSFV sub-subgenotype 2.1c isolate GD53/2011 in PK-15 cells. Whole-genome analysis revealed that individual protein encoding regions exhibit distinct genetic variations between GD53/2011 and other CSFV field isolates, and Core and NS5A were found to be more variable than glycoprotein E2 within CSFV subgenotype 2.1.