Differentiation of virulent Shimen and vaccine C strains of classical swine fever virus by duplex reverse-transcription polymerase chain reaction

ABSTRACT It is difficult to differentiate pigs that have been injected attenuated vaccines from those that are infected by a virulent virus. In this study, a duplex reverse transcription polymerase chain reaction (RT-PCR) was developed for simultaneous detection and identification of the pathogenic strain (Shimen-strain) and attenuated vaccine strain (C-strain) of classical swine fever virus (CSFV). A fragment of 438 bp or 265 bp was amplified from the genomic RNA of the Shimen-strain or the C-strain, respectively. Both types of viruses were simultaneously identified from the mixed samples of Shimen-strain and C-strain, while no PCR bands were obtained from the templates of some other porcine original viruses with the designed primers. The lowest limit of detection was 0.0616 pg for the C-strain RNA and 0.0844 pg for the Shimen-strain RNA. The developed RT-PCR was adopted to test CSFV from 65 collected samples. Compared with quantitative real-time PCR, the results indicated that the developed RT-PCR can not only be used to distinguish the virulent and attenuated CSFV, but also possesses high sensitivity for detecting CSFV. This is a convenient method to detect and differentiate pigs infected with virulent CSFV from the pigs immunized with the C-strain.


Introduction
Classical swine fever virus (CSFV) can spread via vertical and horizontal transmission directly, and via pork products and contaminated fomites indirectly [1]. Thus, strict control measures for CSFV should be applied to the suspicious herds that may have been infected [2]. It is effective to control classical swine fever (CSF) with attenuated vaccines [3] and some vaccines against CSF have been developed [4]. The attenuated lapinized vaccine strains are widely used around the world [5]. The hog cholera lapinized virus (HCLV strain) has been maintained in many countries for its good efficacy and safety, and was named 'Chinese vaccine strain (C-strain)' [6].
The standard CSFV reference virulent strain in China named Shimen was isolated in 1945 and the HCLV strain was developed in 1954, derived by serial passages of a virulent virus through rabbits [6]. A phylogenetic tree shows that Shimen and HCLV cluster into the same subgroup [7].It is difficult to differentiate the animals infected with the wild-type virus from HCLV vaccinated pigs by serology [8,9]. Therefore, it is of particular importance to develop a sensitive and specific approach for distinguishing the virulent CSFV from the vaccine virus.
Many studies in this area are based on the different nucleotide sequences of the viruses. There is a notable insertion of 12-13 continuous T-rich nucleotide sequence in the 3'-nontranslated region (3'-NTR) of the attenuated CSFV genome [10,11]. However, this is not an optimal location for primer design for the 3'-NTR is difficult to reverse into cDNA. The real-time polymerase chain reaction (PCR) assay is faster and more sensitive than conventional reverse transcription PCR (RT-PCR) [12][13][14][15][16][17]. However, the expensive instruments and reagents make it difficult to apply widely [18]. Here, we compared the genomic sequences of epidemic CSFV and attenuated vaccine strains to identify different sequences between virulent strains and the C-strain. Two pairs of specific primers were designed for developing a duplex RT-PCR, in the hope that the simple duplex RT-PCR can become a convenient approach in identifying the CSFV-infected pigs within nonvaccinated and/or vaccinated swine herds.

Viruses and cells
Virulent Shimen strain (F114) was obtained from China Institute of Veterinary Drug Control (Beijing). A virulent CONTACT Kang-kang Guo guokk2007@nwsuaf.edu.cn 1 These authors contributed equally to this work. CSFV isolate from pathological tissue was maintained in our lab. C-strain (HCLV) was purchased from China Animal Husbandry Group (Beijing). PRRSV (porcine reproductive and respiratory syndrome virus), PCV2 (porcine circovirus type 2), PPV (porcine parvovirus), PRV (porcine pseudorabies virus) and TGEV (transmissible gastroenteritis virus of swine) were obtained from the Laboratory of Veterinary Prevention(Yangling, Shaanxi). Swine umbilical vascular endothelial cells (SUVECs), porcine kidney epithelial (PK-15)cells and swine testicular (ST) cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM; GIBCO, Carlsbad, UK) containing 10% fetal bovine serum (FBS; GIBCO, UK) and 50 mg/mL heparin (Sigma-Aldrich, Merch KGaA, Darmstadt, Germany) at 37 C with 5% CO 2 . SUVEC is an immortalized porcine cell line established and maintained at our lab. PK-15 and ST cells were gifts from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

Samples
A total of 65 samples were collected, including 5 lymph nodes samples from non-CSF pigs, 5 spleens and whole blood samples from CSFV-infected experimental pigs which manifested clinical and pathological symptoms of CSF, 10 whole-blood samples from HCLV immunized pigs 7-21 d post-immunization, 42 whole-blood samples from clinically healthy pigs from three farms in Shaanxi Province and three cell cultures of PK-15, ST, SUVEC [19], which were simultaneously infected with Shimen and the C-strain.
Care of laboratory animals and animal experimentation were performed in accordance with the animal ethics guidelines and approved protocols. All animal experiments were approved by the Animal Ethics Committee of Northwest A&F University.

RNA extraction and cDNA synthesis
RNA was extracted from infected cell cultures, blood (100 mL) or tissue (100 mg) using TRIzol® reagent (Life Technologies, Shanghai, China),according to the manufacturer's instructions. The total RNA was measured with NanoDrop2000 Spectrophotometers (Thermo Fisher, Shanghai, China),according to the instructions. Reverse transcription (RT) was performed using PrimeScript TM 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China) with a final volume of 10 mL.

Duplex PCR for distinguishing shimen and C-strain of CSFV
A duplex PCR for distinguishing the Shimen strain from the C-strain was performed with a total volume of 20 mL, including 2 £ PCR Mix 10 mL (Cwbiotech, Beijing, China), cDNA reversed from the co-infected cells 2 mL, each of four primers (10 mmol/L) S-F/S-R and C-F/C-R 0.5 mL and 6 mL of ddH 2 O. The amplification conditions were as follows: 95 C for 5 min, 35 cycles at 95 C for 30 s, 54 C for 30 s, 72 C for 30 s and a final extension at 72 C for 10 min (Bio-Rad S1000, USA). Meanwhile, single PCR was also performed with corresponding cDNA and specific primers. PCR products (10 mL) were visualized by electrophoresis in a 2% (w/v) agarose gel.
The PCR products were purified and sequenced (TaKaRa, Dalian, China) for alignment.

Specificity of the duplex RT-PCR
The duplex RT-PCR was evaluated for its specificity by testing the Shimen strain, the C-strain and several porcine original viruses including PRV, PCV2, PRRSV, PPV and TGEV. RNA or DNA was extracted (by using TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0) from infected cell cultures.

Sensitivity test of the duplex RT-PCR
Serial 10-fold dilutions of C-strain or Shimen genomic RNA (10 ¡1 -10 ¡6 ) extracted from infected ST cells were subjected to amplification by duplex RT-PCR.

Repeatability of the duplex RT-PCR
Reproducibility tests of the duplex RT-PCR were operated in triplicate by testing three different titers of cell cultures infected with the Shimen strain and the C-strain.

Detection of CSFV by RT-PCR
The RNA of the Shimen strain and the C-strain was detected by using single RT-PCR with designed primers. Fragments of 265 bp or 438 bp were amplified ( Figure 1A and Figure 1B). A strong fluorescent signal was also detected in Shimen or C-strain infected cells with real-time RT-PCR ( Figure 2).
The sequence alignment of the PCR products showed that the PCR product of the Shimen strain was 98.4% homologous to AF092448, and that of the C-strain was 99.6% homologous to AF091507.

Specificity of duplex RT-PCR
After optimization, the duplex RT-PCR reaction system was determined to be 20 mL, with the primers concentration being 0.5 mmol/L and the annealing temperature, 54 C. Two bands of 438 and 265 bp were detected simultaneously from the mixed RNA of the Shimen strain and the C-strain ( Figure 3). As expected, the PCR bands from a virulent CSFV isolate were similar to that of the  Shimen strain, and the amplification pattern from a commercial attenuated vaccine was similar to that of the Cstrain (Figure 4). No PCR bands were obtained from TGEV, PRV, PCV2, PRRSV and PPV-infected cells ( Figure 5).
Considering the comparable serology presentation of the swine infected with virulent and attenuated CSFV [20][21][22], a rapid, sensitive and specific method for distinguishing virulent CSFV from vaccine virus is justified to be of key importance [23].In this respect, the diagnostic method described here would be significant and useful.

Sensitivity of the duplex RT-PCR
Serial 10-fold dilutions of C-strain, Shimen and C+Shimen genomic RNA (10 ¡1 -10 ¡6 ) were subjected to the duplex RT-PCR. The minimum detectable quantity was 0.0616 pg for the C-strain RNA, 0.0844 pg for Shimen RNA ( Figure 6A,B), and a similar result was observed for C+Shimen RNA. Three independent repetitions showed consistent results.

Applicability of the duplex RT-PCR
The 65 collected samples were detected by the developed duplex RT-PCR and the results showed that 8 samples were tested to be Shimen-positive from CSFV infected pigs and cell cultures; 55 samples were tested to be HCLV-positive (1 infected pig, 51 non-infected pigs and 3 cell cultures). The obtained results were consistent with those determined by real-time RT-PCR developed in previous studies for CSFV detection ( Table 1).
The diagnostic sensitivity estimates for this duplex RT-PCR resulted in 100% sensitivity for detection of virulent CSFV and 96.4% for the vaccine strain. Additionally,    100% specificity for both of the viruses ensured the reliability of the test.
Since there is a biannual compulsory vaccination in China (spring and autumn), and the vaccine virus is detectable two weeks following vaccination [24,25], this result could be expected. HCLV and Shimen strains were simultaneously detected in 1 pig (1/5), implying that it may have been infected with the Shimen strain before or after the vaccination.
In summary, the two pairs of primers can effectively distinguish the nucleotide difference between Shimen and HCLV strains by RT-PCR. This method is sensitive enough to detect a mild CSFV infection with a superb specificity.

Conclusions
The developed duplex RT-PCR was demonstrated to be a useful method for rapid detection and differentiation of pathogenic CSFV and vaccine strains. The protocol presented here is a rapid, sensitive and specific method to differentiate pigs infected with virulent CSFV from immunized pigs.  -CSF pig  Lymph nodes  5  0  3  0  3  Infected pigs  Spleen/ blood  5  5  1  5  1  Immunized pigs  Blood  10  0  9  0  10  Clinical healthy pigs  Blood  42  0  37  0  38  Co-infected cells  Cells  3  3  3  3  3  Total  65  8  53  8  55