Epidemiological and genetic characteristics of swine pseudorabies virus in mainland China between 2012 and 2017

Samples collection and virus isolation

A total of 16,256 samples including tissue from lungs, lymph nodes, brains, serums, stillbirths, kidneys, and spleens were collected from pigs of different ages with signs suspected of PRV infection in farms (no. of sows ≥100) in 27 Provinces in mainland China, excluding Ningxia and Tibet, between January, 2012 and December, 2016 (Fig.  1). The number of samples collected in 2012, 2013, 2014, 2015, 2016 and 2017 was 1,284, 1,846, 2,523, 2,424, 2,640 and 5,539, respectively. After collection, tissues were minced, immersed with Dulbecco’s modified Eagle medium (DMEM), and homogenized using a QIAGEN TissueLyser II (QIAGEN Germany). Sample homogenates were then frozen at −80 °C and thawed for three times. Following centrifugation at 5,000 rpm for 5 min, the supernatants were harvested for DNA and/or virus isolation.

Template DNA for PCR detecting PRV was isolated using a Universe Genomic DNA Kit (CWBIO, Beijing, China) following the manufacturer’s instructions. For virus isolation, homogenate supernatants were filtered using a 0.22 µm membrane, and inoculated into PK-15 cells (Purchased from ATCC, Manassas, VA, USA). Cells were then incubated in a 37 °C incubator supplemented with 5% CO2. Cell culture with obvious CPE was used for further plaque purification assays. Briefly, PK-15 cells were plated into a 6-well plate, and a series of 10-fold dilutions (from 10⁻¹ to 10⁻⁶) of virus was inoculated. The plate was incubated at 37 °C under an atmosphere containing 5% CO₂ for 2 h and was shaken one time every 15 min. After incubation, the virus was discarded and the cells were washed using DMEM for two times. Finally, DMEM medium containing 1% low melting agarose was added into each of the wells, and the plate was incubated at 4 °C until the medium solidified. The plate was then moved into a 37 °C cell incubator for plaque formation. Plaques with suitable size were selected and inoculated into 500 µL DMEM, frozen and thawed for three times, and then diluted 2-fold in DMEM for the second round of plaque purification assay. After that, plaque fluid was inoculated into PK-15 cells and cultured in flask.

PRV detection

Polymerase chain reaction (PCR) assays were designed to detect the presence of PRV gE gene from the clinical samples using the DNA isolated as template as well as the primers listed in Table 1. The gE gene was used as the target gene because the gE-deleted pseudorabies virus (PRV) vaccine has been used in China extensively, and the detection of the gene could be applied for determining wild-type virus infection. As shown in Table 1, primers gE1-F and gE1-R were designed for the detection of gE. PCR reactions were performed in a 25 µL volume mixture containing 12.5 µL 2 ×Taq Master mix (TAKARA, Shiga, Japan), 8.5 µL nucleotide-free water, each of the forward and reverse primers 1 µL, 1 µL DMSO, and 1 µL template DNA. Thermocycler conditions used for PCR were 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C, 30 s for gE, and extension at 72 °C for 1 min, with a final extension at 72 °C for 10 min before storage at 4 °C. The PCR product was visualized using 1% agarose gel electrophoresis under ultraviolet light.

Sequencing and phylogenetic analysis

PCR assays were also designed for analysing the gB, gC, and gE genes of the PRVs currently circulating in China. The PCR volumes were the same as that used for PRV detection from the samples. Cycling conditions were 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 10 min before storage at 4 °C. The PCR product was visualized using 1% agarose gel electrophoresis under ultraviolet light.

After amplification, PCR products were purified using a TIANgel Midi Purification Kit (Tiangen Biotechnique Inc., Beijing, China) and cloned into a pMD19-T vector (TAKARA, Shiga, Japan). Plasmids carrying either gB, gC, or gE were extracted and sent to Genscript (Nanjing, China) for DNA sequencing. The nucleotide sequences of gB, gC, and/or gE were edited and aligned (using BioEdit software) and then translated into aminoacids from each gene and then compared using DNAStar. Phylogenetic trees were generated through MEGA X, using neighbor-joining algorithm with 1,000 bootstrapping. Sequences of PRV strains listed in Table 2 retrieved from NCBI were used as references.

PCR detection of PRV in mainland China

Of the 16,256 samples, 1,345 samples were positive for the detection of PRV-gE, yielding an average positive rate of 8.27%. The positive rates of PRV detection from 2012 to 2017 were 11.92% (153/1284), 12.19% (225/1846), 6.70% (169/2523), 11.10% (269/2424), 5.57% (147/2640), and 6.90% (382/5539), respectively. Monthly, higher positivity rates of PRV were detected in January, February, March, April, June, October, November and December; and winter (December, January and February), spring (March, April, and May) and autumn (September, October and November) were the seasons with the high positivity rate of PRV detection during 2012 and 2017 (Figs. 2A and 2B).

Mainland China is divided into seven parts including Northeastern China, Northern China, Eastern China, Central China, Southern China, Northwestern China, and Southwestern China (Fig. 1). Among these graphic regions, the positive rates of PRV detection in Eastern China and Central China between 2012 and 2017 were higher than 10.00%, while the positive rates in other parts of China between 2012 and 2017 ranged from approximately 7.00% and 10.00% (Fig. 1). In addition, the positivity rate of PRV detection in different graphic regions between different years displayed diversity. For instance, the positive rates of PRV detection in Northern China in 2012, 2013 and 2015 were higher than 15.00%, but the positivity rates in 2014 and 2016 were lower than 5.00%; the positive rate of PRV detection in Southwestern China in 2012 was 26.15%, while it was only 1.80% in 2013, and 6.90% in 2014, 5.97% in 2015, but zero in 2016; in Northwestern China, the positivity rate of PRV detection in 2012 and 2013 were higher than 20.00%, and approximately 10.40% in 2014 and 2015, but zero in 2016 (Fig. 3).

PRV isolation

To understand the genetic characteristics of PRVs currently circulating in China, a total of 25 PRV strains isolated herein were used for further analysis in this study (Table 3). Most of those strains were isolated from lungs and their TCID₅₀/0.1 mL values ranged from 10^6.72 to 10^7.96.

Phylogenetic characteristics

Phylogenetic analysis using gB, gC, and/or gE sequence showed that the PRV isolates from China were located on a phylogenetic branch, which was distinct from the those isolates from other countries of the world (Figs. 4A, 4B, 4C). According to previous study (Ye et al., 2015), those isolates from China belonged to genotype II, while those isolates from the other parts of the world including Bartha, Becker, Kaplan, and NIA3 were genotype I strains. Interestingly, one isolate from China in 2016, which we designed HuB, should be determined as a genotype II strain (Fig. 4B) according to Ye’s study, in which PRVs were phylogenetically divided into two major genotypes, genotype I and genotype II, based on the analysis of gC gene (Ye et al., 2015). However, this isolate had a closer relationship to the genotype I strains when using gB to perform the phylogenetic analysis (Fig. 4A).

Analysis of gB, gC and gE

The maximal amino acid sequence divergence for gB, gC, and gE proteins of the 25 PRV isolates were 5.2, 2.7, and 2.6% within the isolates, and were 8.4, 9.2, and 5.5% compared to those isolates from the other countries, respectively. The maximal amino acid sequence divergence for the three proteins of the 25 isolates were 4.8, 9.9, and 2.8% compared to those strains prevalent in China before 2012, and were 4.8, 2.7, and 2.8% after 2012.

Alignment of amino acid sequences of gB found the isolates from China mainly had three types of mutations within the protein compared those strains from the other countries. The isolates from China harbored an-amino acid insertion at site 94 (G), a substitution of ten amino acids at sites 53 (A→T), 55 (P→T), 70 (T→A), 81 (N→D), 82 (D→G), 83 (V→F), 87 (A→E), 93 (E→D), 96 (F→V) and 102 (E→D), and a deletion of three-amino acids at sites 75–77 (S, P and G) compared to Bartha-Hungary, Kaplan-Hungary and NIA-3-Japan. In addition, there were also some different substitutions at different sites within the gB protein of the 25 isolates compared to Bartha-Hungary. For example, HuBHZ-China-2016 harbored a substitution of one amino acid at sites 11 (P→A) and 12 (R→G), while strains JSZL-China-2016, HuB-China-2016, and SDRZ-China-2016 had an amino acid substitution at sites 12 (R→H), 67 (A→V), and 228 (K→E), respectively (Fig. 5).

For the gC protein, the isolates from China had an insertion of seven amino acids at sites 63-69 (A, A, A, S, T, P and A) within the protein compared to the genotype I strains; while strain LXB6-China-2009 harbored an insertion of six amino acids at sites 64-69 (A, A, S, T, P and A). In particular, those isolates from China after 2012 contained a substitution of twenty-three amino acids substitutions at sites 14 (P→L), 16 (A→T), 52 (P→S), 55 (A→E), 57 (A→V), 59 (P→G), 60 (E →T), 76 (A→V), 87 (P→Q), 90 (N→G), 102 (A→S), 130 (F→V), 163 (S→P), 186 (T→A), 188 (V→A), 190 (E→V), 191 (D →V), 243 (S→H), 431 (L→M), 449 (A→T), 457 (S→T), 461 (V →T) and 467 (G→A) compared to the genotype I strains (Fig. 6). In addition, some other substitutions were also found during the analysis: HuN-China-2016, SDRZ-China-2016, HuB-China-2016, HuN-China-2016, HuBAL-China-2016, and SDRZ-China-2016 harbored amino acid substitutions at sites 106 (K→T), 107 (R→C), 210/227 (A→T), 235 (A→V), 300 (L→P) and 386 (W →R), respectively.

The sequence alignments of the gE protein found also found some amino acid mutations within the protein of the genotype II strains compared to the genotype I isolates. Compared with Kaplan-Hungary and NIA3-Japan, the 25 isolates contained an insertion of one amino acid (D) at site 48 (Fig. 7). Compared with Min-A-China-2002, the 25 isolates contained a deletion of one amino acid (D) at site 493. In particular, HeNJYG-China-2016, HuBWX-China-2016, HeNXY-China-2016 and GDHDYC-China-2016 deleted an amino acid (D) at site 491, while HuN-China-2016 and SDRZ-China-2016 had a deletion of one amino acid at sites 489 (Y) and 495 (D) compared to Min-A-China-2002, respectively; strain HuB-China-2016 deleted four amino acids (DLNG) at sites 61-64 compared to Min-A-China-2002. In addition to amino acid deletion, sequence alignments also identified amino acid substitutions within the gE protein of the strains isolated herein. For example, strains HeNJYG-China-2016, HuBHZ-China-2016, HuBLLP-China-2016, HuN-China-2016, HuN-China-2016, and SX-China-2016 contained one amino acid substitution at sites 336 (D→G), 2 (R→G), 473 (T→M), 49 (L→R), 573 (A→T), and 526 (D→G) compared to the genotype I strains, respectively. Particularly, one strain, SDRZ-China-2016, contained four substitutions at sites 407 (V→L), 487 (E→D), 499 (E→D) and 535 (E→D) compared to the genotype I strains.