Complete genome sequences of three sub-genotype 2.1b isolates of classical swine fever virus in China

A total of eight clinical samples, including the lung, lymphatic nodes, and spleen, were collected from pigs suspected to have CSFV infections during 2014. The animals were from large-scale pig farms in Shandong Province, China. Tissue samples were homogenised using a TissueLyser II (Qiagen, Germany) in Dulbecco’s modified Eagle medium (Gibco, USA). The tissue homogenates or sera were centrifuged at 10,000 × g for 15 min. The supernatant was passed through a 0.22 μm filter and transferred to swine testis (ST) cells which were grown for 36–48 h, and the remaining samples were kept at –70°C until the total RNA was extracted. The cells were subsequently incubated at 37°C with 5% CO₂ for 3-5 days. Viral stocks were harvested from infected cells by three cycles of freezing and thawing and stored at -70°C.

Based on the published known sequence of CSFV Zj0801 (GenBank accession no. FJ529205), six pairs of primers were designed (Table 1). Total RNA was extracted from tissue homogenates or serum samples using a viral RNA extraction kit (Qiagen). Reverse transcription (RT)-PCR was performed using the One Step RT-PCR Kit (Qiagen) according to the manufacturer’s instructions. A total of 8 μL of the RNA template was added to 42 μL of the RT-PCR master mix, including 1 μL each of the primer sets. The reaction conditions were as follows: 95°C for 5 min, 30 cycles of denaturation (95°C for 30 s), annealing at 56°C for 30 s, elongation at 72°C for 2.5 min, and final extension at 72°C for 10 min. Based on the results from the RT-PCR and viral isolation attempts, three samples from three independent pig farms were selected to determine the full-length genomic sequences of CSFV. The three CSFV isolates were designated as SD2014-1, SD2014-2, and SD2014-3.

Table 1

Primers used for amplification of gene fragments of CSFV SD2014-1, SD2014-2, and SD2014-3

RT-PCR products were analysed via 1% agarose gel electrophoresis. The target fragments were excised from the gels for purification using a Gel Extraction Kit (OMEGA, USA) at a later stage. The purified PCR products were cloned into a pMD18-T vector (TaKaRa, Dalian, China). Recombinant clones were sent to Comate Bioscience (Jinlin, China) for sequencing.

The entire genome sequences of SD2014-1, SD2014-2, and SD2014-3 were obtained after sequencing. Phylogenetic trees were constructed with MEGA (version 7) using the neighbour-joining method with 1,000 bootstraps (12). The evolutionary trees of the three CSFV isolates and other known isolates worldwide were determined based on complete genomic sequences or full-length E2 gene sequences (Fig. 1).

Fig. 1

The complete genome sequences and deduced amino acid sequences of SD2014-1, SD2014-2, and SD2014-3 were analysed and their homologies with other eight representatives of CSFV strains were compared using DNAstar (DNAstar Inc., USA) (Tables 2 and 3). To explore the genetic variations of SD2014-1, SD2014-2, and SD2014-3, the nucleotide sequences of the 5′UTR and the 3′UTR and the amino acid sequences of E2 were fully analysed, together with 33 other CSFV isolates from China and other countries.

Table 2

The genome homology of SD2014-1, SD2014-2, and SD2014-3 (%)

Table 3

Detailed comparison of the full-length genomes of SD2014-1, SD2014-2, and SD2014-3 to other representative CSFV isolates (%)

The tissue homogenates or sera of suspected CSFV-infected pigs were transferred to ST cells for virus isolation. After being identified, the three strains, i.e., SD2014-1, SD2014-2, and SD2014-3, were successfully isolated.

The genomic sequences of SD2014-1 (GenBank accession no. MF149061), SD2014-2 (GenBank accession no. MF149062), and SD2014-3 (GenBank accession no. MF149063) were 12,296, 12,333, and 12,296 nucleotides in length, respectively. The three strains shared 97.7%–98.6% homology between each other (Table 2).

Complete nucleotide sequences of the three isolates were further compared with eight other representative CSFV isolates, including Shimen (AF092448), Paderborn (AY072924), HEBZ (GU592790), HNSD-2012 (JX218094), JSZL (KT119352), CSFV39 (AF407339), Alfort/Tuebingen (J04358), and 94.4/IL/94/TWN (AY646427) (Table 3). The three isolates shared 94.9%–95.0% and 94.7%–94.8% homology with sub-genotype 2.1b isolate HEBZ and 2.1d isolate JSZL, respectively; these were the most closely related isolates. In addition, they shared 88.2%–94.2% homology with other genotype 2 isolates, including 2.1a Paderborn, 2.1c HNSD-2012, 2.2 CSFV39, and 2.3 Alfort/Tuebingen. They also shared only 85.5% homology with 1.1 isolate Shimen and 83.4%–83.5% homology with 3.4 isolate 94.4/IL/94/TWN. In sum, these results indicate that all three isolates belong to sub-genotype 2.1b or 2.1d.

To further examine the genomic variation in the two isolates, their genomic characteristics were analysed in detail. Compared with the eight representative isolates, the 5′UTRs of the three isolates were the most conserved regions in the genomes, which exhibited 96.0%–98.1% homology with 2.1 isolates Paderborn, HEBZ, HNSD-2012 or JSZL, 93.0%–95.4% homology with 2.2 isolate CSFV39 or 2.3 isolate Alfort/Tuebingen, and 91.4%–94.1% homology with 1.1 isolate Shimen or 3.4 isolate 94.4/IL/94/TWN. In addition, the amino acid identities of NS3, NS4A, and NS4B among the three isolates and eight other representative isolates were higher than other regions of the genome, and the homologies were 98.1%–99.4%, 95.2%–100%, and 93.7%–100%, respectively. The detailed identities of the three isolates and eight other representative strain isolates are summarised in Table 3.

To understand the genetic relationships among SD2014-1, SD2014-2, SD2014-3, and other CSFV isolates, phylogenetic trees were produced using 36 CSFV entire genome sequences or 205 full-length E2 gene sequences (Fig. 1). CSFV isolates were divided into genotypes 1, 2, and 3. Genotype 2 isolates were further divided into sub-genotypes 2.1, 2.2, and 2.3, and sub-genotype 2.1 isolates could be further subdivided into 2.1a, 2.1b, 2.1c, and 2.1d. The three new isolates, i.e., SD2014-1, SD2014-2, and SD2014-3, belonged to 2.1b.

The 5′UTR and 3′UTR have been documented to be crucial regulatory elements in the CSFV genome (10, 28). Within the CSFV genome, the 5′UTR of CSFV is the most conserved region. Compared with those of other sub-genotype isolates, the 5UTRs of SD2014-2 and SD2014-3 had a nucleotide T deletion at position 44 (T⁴⁴), while the 5′UTR of SD2014-1 had a nucleotide substitution at the very same position (T/C⁴⁴A) (Fig. 2). In addition, different CSFV isolates showed nucleotide A, T, C, or deletions at this position, while some isolates in the same sub-genotype showed different nucleotides at position 44 (Fig. 2). Similarly to most CSFV isolates, the 5′UTRs of SD2014-1 and SD2014-2 had two continuous nucleotide A deletions at positions 357–358, while the 5′UTR of SD2014-3 had only one nucleotide A deletion at these positions (Fig. 2).

Fig. 2

The 3′UTRs of all three new isolates were similar to most of the CSFV isolates. However, the HCLV vaccine strain had 12 continuous nucleotide insertions (TTTTTCTTTTTT) at positions 66–77 compared with the reference Shimen strain (Fig. 3). Similarly, the other two strains, RUCSFPLUM (AY578688) and Rovac (KJ873238), had 13 nucleotide insertions (TATTTATTTAGAT) at these positions (Fig. 3). Among the 14 analysed CSFV isolates, the CSFV-PK15C-NG79-11 (KC503764) and CSFV/IVRI/VB -131 (KM262189) strains had five and three poly T-nucleotide insertions, respectively (Fig. 3). In addition, compared with those of sub-genotype 1.1 isolates, the 3′UTRs of most sub-genotype 2.1 isolates, including the three new isolates, had two discontinuous nucleotide T deletions at positions 173 and 199, respectively (Fig. 3).

Fig. 3

The E2 protein is essential for virus attachment and entry into the target cell and is also an important virulence determinant of CSFV (24). Consequently, the E2 amino acid sequences of the three new isolates and 33 reference isolates were compared and analysed (Fig. 4). There were some unique molecular characteristics for the new isolates, including the amino acid W at position 132 (W¹³²), E²²⁸, I²⁸³, and L³⁴³ of SD2014-1 and L¹² of SD2014-2. In addition, the three new isolates also had some consistent molecular characteristics, including E³¹, Y²¹⁰ and K²⁷⁷. Consistently with most 2.1b and 2.1d isolates, the new isolates had an amino acid substitution at position 182 (L¹⁸² W). In contrast, at position 195, the new isolates were amino acid V, consistent with 1.1 isolates, and all other sub-genotype isolates were amino acid T. Additionally, at position 364, SD2014-1 was V, SD2014-2 was L, SD2014-3 was A, and other sub-genotype isolates were I.

Fig. 4