Pathogenicity and Whole Genome Sequence Analysis of a Pseudorabies Virus Strain FJ-2012 Isolated from Fujian, Southern China

The outbreaks of pseudorabies have been frequently reported in Bartha-K61-vaccinated farms in China since 2011. To study the pathogenicity and evolution of the circulating pseudorabies viruses in Fujian Province, mainland China, we isolated and sequenced the whole genome of a wild-type pseudorabies virus strain named “FJ-2012.” We then conducted a few downstream bioinformatics analyses including phylogenetic analysis and pathogenic analysis and used the virus to infect 6 pseudorabies virus-free piglets. FJ-2012-infected piglets developed symptoms like high body temperature and central nervous system disorders and had high mortality rate. In addition, we identified typical micropathological changes such as multiple gross lesions in infected piglets through pathological analysis and conclude that the FJ-2012 genome is significantly different from known pseudorabies viruses, in which insertions, deletions, and substitutions are observed in multiple immune and virulence genes. In summary, this study shed lights on the molecular basis of the prevalence and pathology of the pseudorabies virus strain FJ-2012. The genome of FJ-2012 could be used as a reference to study the evolution of pseudorabies viruses, which is critical to the vaccine development of new emerging pseudorabies viruses.


Introduction
Pseudorabies virus (PRV), also called Aujeszky's disease virus or Suid herpesvirus 1, is the causative agent of pseudorabies (PR), which infects a wide variety of animals from mollusks to mammals and damages world economy. PRV is a member of the subfamily Alphaherpesvirinae in the family Herpesviridae belonging to the genus Varicellovirus [1].
ough the virus was rst described in cattle by Aujeszky in 1902, pigs are the natural reservoir for PRV [2,3]. e clinical symptoms of PR in pigs are characterized by central nervous system (CNS) disorders in piglets, abortion in pregnant swine, and respiring signs in older pigs [4]. e PRV genome encompasses a unique long segment (UL) and a unique short region (US) anked by the internal and terminal repeat sequences (IRS and TRS, resp.), encoding more than 70 proteins. e virulence of PRVs and the immunology mutual protection between them are determined by multiple genes, and thus the genome-wide analysis is necessary to de ne all the characteristics of the viruses [5,6].
Pseudorabies had been well controlled in China due to the wide usage of gE-deleted vaccines and the serum distinguish test [7]. However, in late 2011, outbreaks of PR were reported in Bartha-K61-vaccinated farms, and the disease rapidly spread to 11 provinces from northern to eastern China including Heilongjiang, Jilin, Liaoning, Tianjin, Jiangsu, Zhejiang, and Fujian. A few studies showed that the current PR outbreaks on farms were caused by PRV variants, and the PR vaccine could not provide e ective protection against the prevalence of PRV strains in China [8][9][10]. However, the complete genomes of the variants and their molecular characteristics are unclear, which is an obstacle in producing e ective vaccines. As a result, the outbreaks have caused a great economic loss to swine-feeding industry in China.
In this study, we thoroughly assessed a pseudorabies virus named "PRV FJ-2012," which was isolated from a Bartha-K61-vaccinated pig farm in Fujian Province during a PR outbreak. e outbreak has the following characteristics: (1) the mortality of infections can be as high as 100% and (2) plenty of pregnant sows aborted. We have characterized the pathogenicity in pigs and analyzed the complete genome of the PRV FJ-2012 in order to characterize its molecular properties and virulence.

Materials and Methods
2.1. Virus, Cells, and Genomic Viral DNA Preparation. PRV FJ-2012 was isolated from pig brain samples collected from a Bartha-K61-vaccinated farm with a PR outbreak in Fujian Province, southern China, in 2012. e virus was determined to be a pseudorabies viral strain by the PCR analysis and the sequence analysis of its partial gE gene. Virus was propagated on PK-15 cells, cultured in Dulbecco's modi ed Eagle's medium (DMEM, Hyclone, USA) containing 1% fetal bovine serum (FBS, Gibco, USA), 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO 2 . Cells were harvested when the cytopathic e ect (CPE) of PK-15 cells that were inoculated by PRV FJ-2012 strain reached 80%. After freezethaw for three times, the cell debris was removed by centrifugation at 5000 ×g for 30 min at 4°C. en, the supernatant involving PRV was centrifuged by a Beckman ultracentrifuge (LE-80K) at 30,000 ×g for 2 h at 4°C; the supernatant was discarded, and the pellets were then resuspended in 2 mL PBS (0.01 mol/L, PH7.2). Discontinuous mass fraction sucrose gradients (30%, 35%, 40%, and 45%) that were formulated with PBS were further puri ed at 26,000 ×g for 2 h at 4°C. And then, the virus band (between 35% and 40%) was drawn to a centrifuge tube, and the sucrose was removed by centrifugation at 30,000 ×g for 1 h at 4°C. e puri ed virus particle was obtained and used to prepare genomic viral DNA using QIAamp DNA Mini Kit (QIAGEN, Germany) according to the manufacturer's instructions.

Experimental PRV Inoculation of Pigs.
Ten healthy, 28day-old Duroc × Landrace × Yorkshire (DLY) hybrid pigs were collected from PRV-free swine farm and con rmed to be serologically negative for PRV antibodies with a gB ELISA kit (IDEXX, USA). e pigs were randomly allocated to two groups, namely, the challenge and control group, and each group was housed in separate pens. Four pigs in Group 1 (the challenge group) were each inoculated intranasally (i.n.) with 1 mL 10 6 TCID50 FJ-2012 strain, and the other two pigs in Group 1 were i.n. with DMEM with the same dose as the cohabit infective test. e remaining four pigs were served as uninfected control. Clinical signs and rectal temperatures of pigs were recorded daily throughout the study. At 14 dpi, all surviving pigs were euthanized.

Tissue Sampling and Histological Analysis.
After macroscopic examination, the tissue samples required for histological examination were obtained from the brains, kidneys, lungs, tonsils, livers, and lymph nodes (super cial inguinal). ese samples were xed in 10% formalin, processed routinely, and embedded in para n. Each para n sample was sectioned to 4-5 μm and stained with HE. e sections were viewed with a Motic BA210 microscope.

Genomic Sequencing, Assembly, Annotation, and Analysis.
e genome of PRV FJ-2012 strain was sequenced with a Paci c Biosciences RS II sequencer (Paci c Biosciences, Menlo Park, CA, USA), using a single-molecule long-read sequencing technology with a 10K SMRTbell template library. We are fully aware that the long reads from the Paci c Biosciences RS II sequencer have some disadvantages like high error rate compared to short reads. However, we still prefer long reads since they are better at identifying gene isoforms and assembly, which are critical to this study. We then removed host reads by comparing the reads against the pigs (Sus scrofa) using BLAST. e remaining reads were de novo assembled; the obtained contigs were assembled with Celera software (https://sourceforge.net/projects/wgsassembler/ les/wgs-assembler/wgs-8.3/), and the sca olds were constructed by comparing the contigs with reference PRV genomes (GenBank Accession # NC_006151) using the NCBI BLAST program. Finally, a Perl program was used to check gaps between the sca olds, and the alignments were extremely high coverage with no gap. e open reading frames (ORFs) of FJ-2012 genome were searched by ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/), and genes were predicted and analyzed by GeneMarkS [11]. e ORF annotations of the FJ-2012 strain were created by BLAST homology-based transfer as previously described in [5,12,13]. e alignments were performed using the mVISTA genomic analysis tool with a LAGAN global alignment [14]. e phylogenetic trees were constructed using the neighborjoining algorithm with 1000 bootstrap repetitions using the Kimura 2-parameter substitution model in MEGA 5.0 [15][16][17].
e distribution of polymorphic sites in eight PRV genomes was inferred using the software Base-By-Base [18].

Pathogenicity of the PRV Strain FJ-2012 in Pigs.
After intranasal infection of the 28-day-old PRV-free piglets, all pigs in the challenge group developed high fever beginning at 2 dpi with temperatures 41.9°C-42.5°C. e clinical signs were consistent with typical pseudorabies syndrome, from listlessness, anorexia, to high fever and then displayed respiratory symptoms such as cough, sneeze, and central nervous system (CNS) symptoms. Finally, all pigs in the challenge group exhibited opisthotonus and were dead in 8-14 dpi. e pigs in the cohabitation infection group also showed high fever with temperatures 41.9°C-42.5°C, but the time of onset was 4 days later and the clinical signs were milder than those in the challenge group. e pigs were also dead in the end (Table 1). In contrast, pigs in the control group remained healthy without any abnormal symptom.

Gross Lesions.
e pigs in the FJ-2012-infected group showed multiple gross lesions. Macroscopic encephalic hemorrhages or encephalemia was observed in all pigs (4/4).
ree of those pigs (3/4) showed pinpoint hemorrhages in the kidney. Small white foci were seen in the liver of four pigs (4/4). Di use reddened foci and edema of the lungs were observed in all pigs (4/4). e dark-red hemorrhage and congestion were noted in the lymph node of four pigs (4/4). Tonsil anabrosis was observed in three pigs (3/4). Two pigs in the cohabitation infection group showed milder lesions (such as slight hemorrhages in the brain and lung). No pig in the control group displayed gross lesions.

Histopathological Analysis.
To further study the pathology of these organs, samples from brains, kidneys, livers, lungs, lymph nodes, and tonsils of pigs were stained with hematoxylin and eosin (H&E). On one hand, the histopathological examination showed multiple lesions in several organs of PRV-infected pigs. For example, nonsuppurative ganglioneuritis, characterized by gliosis, hemorrhage ( Figure  1(a)), pronounced perivascular in ammatory in ltrates, and nerve cell necrosis were observed in the brain (Figure 1(b)). e lungs showed severe hemorrhage, congestion, and edema, and bronchiolar cavities were lled with cellular serous exudates that were red-stained (Figure 1(c)). Multiple small focal necrosis were discovered in the liver (Figure 1(d)) and the kidney with lymphocytic in ltration and congestion ( Figure 1(e)). e striking changes in the lymph nodes include brownish-stained hemosiderosis and lymph follicle swelling (Figure 1(f)). e strati ed squamous epithelium of tonsil appeared modi ed, necrosed and exfoliative (Figure 1(g)). On the other hand, the histopathological results of these organs from the control group had no signi cantly pathological changes (Figures 1(h)-1(n)).

Complete Genomic Characterization of the PRV FJ-2012
Strain. We performed the whole genome sequencing of FJ-2012 and performed a few downstream analyses on the sequencing data (Figure 2). e total length of its genome is 144,873 bp with a high G + C content of 73.5%. e overall genomic composition is the same as that of a few previously studied strains (Bartha, Kaplan, and Becker), consisting of a unique long (UL) region (position at 1-102,119), a unique short (US) region (118,893-128,096), internal repeat sequences (IRs, 102,120-118,892), and terminal repeat sequences (TRs, 128,097-144,873), which are located at the ank of the US region (Figure 1(a)). 70 open reading frames (ORFs) were identi ed, which relate to 70 genes that encode 68 di erent proteins (Figure 1(a)) since there are the two putative genes (US1 and IE180). It is of note that US3 and US3.5 were treated as di erent genes due to their distinct functions [19]. e unique long regions containing 59 genes, most of which are involved in DNA replicative mechanisms and virus particle assembly and mature, are transcribed in both directions. Unique short regions containing 7 genes transcribed backward are likely a ecting pathogenesis and host range functions. IRS and TRS containing US1 and IE180 genes, which function as an accessory regulatory protein and interact with the IE protein to enhance gene transactivation, are transcribed in reverse directions [20].

Comparison and Phylogenetic Analysis among PRV
Strains. We compared the genomes of FJ-2012 with those of other 7 PRV strains including Kaplan, Becker, Bartha, JS-2012, HeN1, TJ, and ZJ01. e four strains JS-2012, HeN1, TJ, and ZJ01 were isolated from China ( Table 2). As can be seen, FJ-2012 exhibited 94.2% to 98.32% nucleotide identity with other strains from China and 89.8% to 91.9% nucleotide identity with strains outside China. Speci cally, FJ-2012 is highly homologous to TJ strains (98.32%) and shared only 89.8% identity with the strain Bartha, which is deemed as an excellent vaccine strain to control pseudorabies.
In addition, most genetic variations among the PRV strains are located in noncoding regions, internal repeat sequence regions, and terminal repeat sequence regions ( Figure 3 and see Table S1 available online at https://doi. org/10.1155/2017/9073172). ere are also variations in a few coding regions such as US1, UL36, gN, gB, and gC. ere are overall 11,893, 12,621 and 14,956 genomic changes, including 3759, 3724, and 3789 single-nucleotide substitutions between FJ-2012 and three non-China-origin viruses Kaplan, Becker, and Bartha, respectively (Table 2). e genomic changes between FJ-2012 and non-China-origin viruses are higher than those between FJ-2012 and China-origin viruses. Speci cally, there are 4649, 4628, 2445, and 8557 genomic changes, including 716, 560, 206, and 1025 single-nucleotide substitutions between FJ-2012 and four China-origin viruses JS-2012, HeN1, TJ, and ZJ01, respectively.
We then performed the phylogenetic analysis of the 8 viruses based on their full-length genome sequences, which indicates that the PRV strains can be separated into 2 major groups corresponding to their geographic locations, namely, group genotype I consisting of strains in China and group genotype II consisting of European and American strains ( Figure 4). FJ-2012 is phylogenetically most close to TJ strain.

Distribution of Polymorphic Sites and Protein Coding
Variations of PRVs. We used Base-by-Base software to infer  To further study the FJ-2012 genome, we compared its protein-coding regions with those of other viruses using local BLAST in BioEdit. e viruses in the group genotype I share 100% sequence identity with FJ-2012 for 16 ORFs and 94.7-99.9% identity for 53 ORFs. It is of note that HeN1 only shares 22.1% sequence identity with other strains on the protein IE180 due to a frame shift by upstream substitution. As for European and American strains (genotype II), FJ-2-12 shares 90.8-99.9% sequence identity in 69 ORFs (Table 2). e sequence alignment showed that FJ-2012 displayed extensive variations with previously isolated PRV strains including Kaplan, Becker, and the vaccine strain Bartha in most viral proteins, such as glycoproteins, for example, gN (UL49.5), gC (UL44), gL (UL1), gE (US8), and gB (UL27), tegument proteins, for example, UL36, UL46, UL16, and UL13, and nonstructural proteins, for example, IE180 and UL52 ( Figure 5).

Discussion
Previous studies suggest that pseudorabies virus (PRV) can cause serious disease to piglets. e mortality rate is up to 100% for infected two-week old piglets with neurologic symptoms, while that for weaned piglets, the mortality rate is about 50% [19]. In this study, we tested the pathogenicity of FJ-2012 using six 28-day-old PRV-free piglets, four of which were in the challenge group and the other two were in the cohabit group. e piglets in the challenge group received intranasal challenge with 1 mL 10 6.0 50% tissue culture infecting dose, and those in the cobabit group were inoculated intranasally with DMEM with the same dose. Unfortunately, all 4 piglets in the challenge group died on day 8, 10, 12, and 14 post infection (DPI), respectively, and one piglet in the cohabit group died on 12 DPI (Table 1). e clinical symptoms of the piglets in the challenge group were consistent with those of typical pseudorabies syndromes including listlessness, anorexia, and high fever and respiratory symptoms such as cough, sneeze, and central nervous system symptoms. In postmortem and histopathological examinations of dead piglets, multiple lesion sites were observed in several organs (Figure 1). e ndings suggest that FJ-2012 strain can cause severe pathological changes, which are more serious than previously observed [19].
In general, the virulence of virus and the immune failure are potentially associated with gene mutations. Previous surveys have showed that gene variants of isolated viruses contributed to the epidemics of PRV [8][9][10]21]. erefore, it is important and necessary to further analyze the genetic variations of the current PR for disease control and surveillance. Here, we have sequenced the whole genome of the PRV strain FJ-2012 by a Paci c Biosciences RS II sequencer, and the resulting sequences were assembled, predicted, and annotated of genes. e resulting PRV FJ-2012 genome sequence is 144,873 bp in length with a high G + C content of 73.5% and contains 70 ORFs. e analysis of genome variations indicated that the isolate strain showed high variations with other abovementioned strains, including substitutions, insertions, and/or deletions that occurred in most proteins, revealing that the PRV strain in southern China is quite di erent from previous ones.
Additionally, the main antigen of gB, gC, and gD genes which induces the neutralization antibody is important for protecting PRV infection, and the genes of gE, gI, and TK are related to virus virulence [22,23]. Our study showed that FJ-2012 has 96.9-97.4%, 93.1-93.3%, and 97.5-98.0% sequence identity with the 7 compared strains on gB, gC, and gD, respectively (see Table S1), while the sequence identities on gE, gI, and TK are 95.8-96%, 94.2-94.5%, and 99.4-99.7%, respectively. Moreover, the US1 protein, which functions as an accessory regulatory protein and  interacts with the IE protein to enhance gene transactivation [20], and the UL36 protein, which is thought to function both in early infection and in later stages of viral maturation [24], showed the highest variability between genotype I and II PRV strains. e envelope of PRV virion, glycoprotein N protein, is encoded by UL49.5 gene, a small O-glycosylated protein that forms a disul de-linked complex with gM and functions in viral immune evasion [25,26]. In veterinary varicelloviruses (PRV, EHV-1, and BHV-1), the UL49.5 gene product is an inhibitor of TAP, the transporter associated with processing antigens into peptides for presentation by major histocompatibility complex (MHC) class I molecules at the cell surface [26]. In this study, gN only shares 87.9% identity with the vaccine strain Bartha and shares high homology with other Chinese PRV strains (Table 2, Figure 3). e great variations between vaccine strains and novel Chinese strains might be a potential source of the novel isolate PRV strains to evade the immune response.
Finally, the apparent genetic relationships among PRVs and the su cient genomic variants in the eld isolate strain FJ-2012 are identi ed to distinguish it from 2 major genotypes

Conclusions
Since late 2011, pseudorabies outbreaks have been reported and spread in many farms in China. However, all novel isolated PRV genome reports were isolated in northern China, and there is no systematic research in southern China. In this study, we observed that the PRV FJ-2012 isolated from Fujian Province, southern China, is highly pathogenic and has extensive variation in genomic sequence with the reference PRV. is study contributes to the study of epidemiology and genetic evolution on PRV and lays a scienti c foundation for the development of new PR vaccine.