The evidence of porcine hemagglutinating encephalomyelitis virus induced nonsuppurative encephalitis as the cause of death in piglets

Sample collection and testing

On March 2014, there was an acute outbreak of suspected porcine hemagglutinating encephalomyelitis in suckling pigs on a farm with a total of 502 sows in Changchun, Jilin province, China. At the time of the outbreak, these pigs had not been immunized with any PHEV vaccines; the total proportion of deaths in piglets that had not been weaned was 46.7% (140 dead piglets). The collected samples were tested for PHEV using real-time reverse transcription-polymerase chain reaction (RT-PCR) targeting the HE gene, as well as for other viruses that cause similar clinical symptoms among swine, including porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and pseudorabies virus (PRV). All experiments on piglets research were performed in accordance with Animal Welfare Ethical Committee of Jilin University guidelines and regulations (permission number 2012-CVM-12). The involved RT-PCR primers were designed based on the most conserved segment of their genomes (Table 1), and subsequently validated by BLAST (http://www.ncbi.nlm.nih.gov/BLAST) with sequences from GenBank. The original samples were diluted 10-fold with phosphate-buffered saline (PBS) and were centrifuged at 3,000× g at 4 °C for 10 min. The supernatant was filtered through a 0.22-µm syringe filter, and was used as inoculums for BALB/c mice or for virus isolation in Neuro-2a cell culture.

Histopathologic examination

Postmortem examinations were performed and samples submitted for histopathologic examination, including tissues from the brain, heart, spleen, liver, kidneys, and lungs. Paraffin-embedded sections of brain that had characteristic microscopic lesions were examined by hematoxylin-eosin staining. Selected paraffin sections for PHEV antigen detection by immunohistochemistry (IHC) tests were treated with normal goat serum for 1 h and anti-HEV 67N monoclonal antibody (Chen et al., 2012) (diluted 1:100) overnight; then, the staining procedure was performed according to the kit instructions.

Inoculation of BALB/c mice with PHEV strains

Thirty 3-week-old male BALB/c mice were randomly and equally divided into three groups. The mice in group 1 were inoculated with the original filtered brain tissue by the intranasal route, the mice in group 2 were inoculated with HEV 67N (GenBank: AY048917) in the same manner, and the mice in the third group formed a negative control group. The permission to work with laboratory animals was obtained from the Animal Welfare Ethical Committee of the College of Veterinary Medicine, Jilin University, China (permission number 2012-CVM-12). All of the mice experiments were carried out at Bio-Safety Level 2 (BSL-2) facilities at the Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University. Clinical signs were monitored, and immunofluorescence assay (IFA) was performed. The PHEV monoclonal antibody (diluted 1:500) was used as the primary antibody, and a 1:200 dilution of affinity purified fluorescein-labeled goat anti-mouse IgG was used as the second antibody. Cell staining was examined using a fluorescence microscope.

Virus isolation and propagation

The Neuro-2a cell line was used to isolate PHEV from the original field and from mouse-passaged PHEV samples. Cultured cells were propagated in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT, USA) and 1% antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Briefly, a monolayer of cells was washed twice with 2% DMEM, and then was inoculated with the filtered samples. After adsorption for 1 h at 37 °C in 5% CO₂, the cells were washed 3 times, and 2% DMEM was added. The cell cultures were examined daily for cytopathic effect (CPE). When more than 80% CPE was evident in the inoculated cell monolayers, the cells and supernatants were harvested together and used as seed stocks for the next passage. After serial passage, the cell cultures were clarified by centrifugation at 3,000× g for 30 min at 4 °C and then were further ultracentrifuged at 20,000× g for 2 h at 4 °C using an ultracentrifuge. The pellet was resuspended and submitted for virological investigation using electron microscopy (EM). The virus isolate was designated PHEV-CC14.

Virus titration and purification by plaque assay

The 100% confluent Neuro-2a cells in 6-well plates were used for plaque assays of PHEV-CC14 propagation and purification. Briefly, the wells were inoculated with 10-fold serially diluted virus (0.2 mL/well), followed by adsorption for 1 h at 37 °C in 5% CO₂; then, the wells were washed 3 times, and 2 mL of the agarose/MEM mixture (1:1) were added. After the plaques were counted and confirmed, uniform and clear plaques were chosen to inoculate 6-well plates directly. When CPE was observed, the positive clones were harvested, and the viral titers were determined. When the Neuro-2a cells were confluent in 96-well plates, 100 mL of 10-fold dilutions of the purified virus were absorbed for 1 h. Viral CPE was monitored for 5 to 7 days, and virus titers were determined by 50% tissue culture infectious dose (TCID₅₀).

PHEV-CC14 structural gene sequencing and phylogenetic analysis

All five of the main structural protein genes, hemagglutinin-esterase (HE), spike (S), small membrane (E), membrane (M) and nucleocapsid (N), of PHEV in the original specimen, in the BALB/c mice infected with passaged PHEV-CC14, and in cell culture were amplified, cloned and sequenced. All of the primers were designed according the sequence of the HEV 67N genome. Viral RNA was extracted from the brain tissue suspensions of symptomatic piglets using a commercial kit (QIAGEN, Hilden, Germany), and the RNA was quantified using a spectrophotometer (BIO-RAD, USA). The RNA was converted to cDNA by an oligo (dT)-priming strategy, and the genes were amplified using PrimeSTAR MAX DNA Polymerase (TaKaRa, Kyoto, Japan). The purified PCR products were cloned into the pMD18-T vector (TaKaRa, Kyoto, Japan) and were introduced into E. coli DH5a by transformation. The recombinant plasmids were extracted and verified by PCR and then were sequenced at Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (China).

The sequence data were assembled and analyzed using DNASTAR and NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The percentage similarities of the nucleotides and amino acids were analyzed using DNAMAN and DNASTAR software. The structural gene sequences and other coronavirus strains sequence were subjected to phylogenetic analysis using the neighbor-joining method in MEGA software, version 6.06.

Statistical analysis

Statistical analysis was performed with either Student’s t-test or one-way ANOVA with a Bonferroni post hoc test with software provided by GraphPad Prism version 5. Data were presented as means ± S.E.M. P values of <0.05 were considered statistically significant.

Pathological examination and pathogen detection

Clinical signs of these suspected infected suckling piglets were consisted of vomiting, diarrhea, wasting, dullness, screaming, anorexia, trembling, and ataxia (Fig. 1A). Pathological examination showed that the main changes in the piglets were congestion, edema and hemorrhage in brain tissue (Fig. 1B). None significant histopathological changes were found in other substantive organs. A total of 54 homogenized tissue suspensions of the brain, spinal cord, lungs, kidneys, spleen and intestinal contents from nine suspected piglets were tested for PHEV by RT-PCR. Of these tested samples, eight of nine brain samples from young nursing pigs on the farms were PHEV positive, as well as eight of nine spinal cord samples and four of nine intestinal content samples (Table 2). Of the 20 PHEV-positive samples, all were negative for PEDV, TGEV, PDCoV, and PRV.

Histopathologic examination of the PHEV-infected piglets

Postmortem examinations were performed on seven infected piglets for pathologic evaluation. Samples submitted for histopathologic examination included brains from PHEV- infected piglets and antigen-negative piglets. Microscopic examination of brain samples showed characteristics of non-suppurative encephalitis. A large number of glial cells were aggregated to glial nodules in the infected brains (Figs. 2A and 2B). Neurons in the cerebral cortex were degenerated and necrotic, and neuronophagia was widespread (Figs. 2C and 2D). Selected paraffin sections of brain samples that had characteristic microscopic lesions were examined for PHEV antigen by IHC tests with an anti-PHEV monoclonal antibody. In the brains, antigen-positivity in the cytoplasm of nerve cells was distributed widely in the cortical neurons (Fig. 2E). Brain samples from the healthy pig were normal (Fig. 2F).

Pathogenicity of HEV 67N and PHEV-CC14 in BALB/c mice

Mice in two infected groups were inoculated with HEV 67N and PHEV-CC14, respectively, and were monitored daily for clinical signs of disease. Mice in the HEV 67N-infected group showed typical neurological damage, with symptoms of depression, arched waists, standing and vellicating front claws at three days post-inoculation (dpi). The same symptoms occurred in the PHEV-CC14-infected group (Figs. 3A and 3B), but the emergence time was slightly delayed (Fig. 3C, P < 0.05). All of the infected mice died within a week, and the mice in the control group survived normally. Paraffin-embedded sections of the infected mouse brain samples were positive for PHEV in the cytoplasm of nerve cells by IFA using a mouse anti-PHEV monoclonal antibody. In the brain, antigen-positive neurons were distributed widely in the cerebral cortex and hippocampus (Fig. 4). In the cerebellum, viral-specific antigen was detected in the Purkinje cells (Fig. 4) but in only a few granular cells.

Isolation and purification of PHEV-CC14 strain

The Neuro-2a cell monolayer was inoculated with original field and mouse-passaged PHEV-positive samples. At 3 dpi, the inoculated cell monolayer showed visible CPE, in the form of gathering pyknosis and rounded cells that rapidly detached from the monolayer on 4 dpi (Fig. 5A), the mock-inoculated Neuro-2a cells showing normal cells (Fig. 5B). The virus was further serially passed in Neuro-2a cells for a total of 18 passages. Virus growth was confirmed by IFA using the antiserum PHEV, and the antigens were mostly located in the cytoplasm (Fig. 5C). To confirm PHEV replication, viral RNA was extracted from the culture supernatants and was tested by RT-PCR. The presence of PHEV particles in the infected cells was also examined by EM. The EM results showed multiple virus particles approximately 110 to 130 nm in diameter with typical coronavirus morphology (Fig. 5D). Thus, the PHEV strain was successfully isolated and was designated as PHEV-CC14.

Plaque assay was used to plaque isolates and to purify PHEV on Neuro-2a cells, and large clear plaques were evident under an agar overlay medium on the cells. The cloned virus PHEV-CC14 was tested by RT-PCR and was further serially passaged to 20 passages on Neuro-2a cells (5.2 log₁₀ PFU/mL). During the serial passages, significant increases in viral RNA titers were observed following each cell passage. The infectious titers of PHEV-CC14 were determined by TCID₅₀ and were calculated according to the Reed-Muench method. As shown in Fig. 6, there were no significant differences in replication or proliferation between the PHEV-CC14 and HEV-67N strains in the Neuro-2a cells, but the RNA virus titers in the PHEV-CC14-infected cells (10^6.03TCID₅₀∕mL) were slightly higher than those in the HEV 67N-infected cells (10^5.43TCID₅₀∕mL) after 72 h post-inoculation.

Sequence and phylogenetic analysis

To examine whether genetic changes occurred in the PHEV-CC14 strain (GenBank: KU127229) compared with other PHEVs available in GenBank, the major structure genes were amplified by specific primers (Table 3) and sequenced. A total of 8,123 nucleotides were determined for strain PHEV-CC14, covering five complete structure genes—HE, S, E, M and N—and the locations of the organization of the targeted genes were sketched in a conceptual map (Fig. 7A). Therewith, the corresponding nucleotides and deduced amino acid sequences of the PHEV-CC14 strain were compared with the homologous sequences of PHEVs. The results showed that the PHEV-CC14 strain shared 95%–99.2% nt identities with the other PHEV strains available in GenBank. The structural genes of the PHEV-CC14 strain had the greatest nucleotide sequence similarity (99.2%) to the HEV-JT06 strain (GenBank: ED919227.1), and it shared 99% with HEV 67N (GenBank: AY078417.1). Compared with the HEV 67N strain, there were four nucleotide sense mutations at positions 12 and 114 in the HE gene, 381 in the S gene, 146 in the M gene. These nucleotide changes all induced corresponding amino acid (aa) changes (S12G and T114I in the HE protein; R381H in the S protein; A146T in the M protein). However, residues in the E and N genes of PHEV-CC14 strains were highly conserved in identity with other PHEV reference strains in the GenBank database.

A phylogenetic tree was constructed using the five genes (HE, S, E, M, and N) of PHEV-CC14 with some other PHEV strains obtained from GenBank Database, as well as several members of the coronaviruses (Fig. 7B). Phylogenetic analysis of the five genes clearly showed that the PHEV-CC14 strain clustered into a subclass with a HEV-JT06 strain from China isolated in 2006, and a similar finding showed that the PHEV stains in China were highly homologous with a North American strain (AY078417). Additionally, the homology of the deduced amino acid sequences between the PHEV-CC14 strain and HCoV-OC43 (GenBank: KF530085) was as high as 91%.