Isolation and Characterization of Encephalomyocarditis Virus from Dogs in China

Encephalomyocarditis virus (EMCV) is as a potential zoonotic agent with a wide host range. Here, we describe an EMC virus isolate, identified as EMCV C15, which was successfully obtained from the serum of dogs from animal hospitals. Virus production in cell culture was confirmed by EMCV-specific real-time RT-PCR, indirect immunofluorescence assays and electron microscopy. In addition, the open reading frame sequence (ORF) of the EMCV C15 virus was determined. From sequence comparison and phylogenetic analysis among 24 reference EMCV strains, it appears that the EMCV C15 strain is closely genetically related to strain BEL2887A/91 (>99.0% nucleotide identity). In artificially challenged dogs, the heart and brain were important targets of EMCV C15. This study provides genetic and pathogenic characterization of the EMCV C15 strain isolated in Beijing and calls for sustained surveillance of EMCV infection in China to support better prevention and control of the disease.


Real time RT-PCR for EMCV detection.
Real-time RT-PCR assay utilized the SYBR Premix Ex TaqTM kit (Takara, Dalian, China) in a total volume of 25 μL. The assay was performed following the manufacturer's instructions. The EMCV-specific primer set described by Wang et al. 16 was used as follows: forward primer (position 1116-1140) 5′-GACGCTTGAAGACGTTGTCTTCTTA-3′; reverse primer (position 1302-1326) 5′-CCCTACCTCACGGAATGGGGCAAAG-3′. The primers were designed based on a 3D sequence and have been confirmed to be highly specific for EMCV. Real-time RT-PCR was conducted on an ABI H7900 Fast instrument (Life Technologies, Carlsbad, CA) and the results were analyzed with the included system software.
Clinical Samples. A total of 69 serum specimens were collected from dogs at pet hospitals in Beijing in 2015. The dogs presented high fever symptoms (69/69), diarrhea (45/69) and dyspnea (32/69). Colloidal gold strip detection was used to detect canine parvovirus (CPV) and canine distemper virus (CDV). The detection results showed that 44 and 36 of 69 dogs were infected with CPV and CDV, respectively, while 22 of 69 dogs were co-infected with CPV and CDV. The results of real-time RT-PCR showed that 4 of 69 dogs were co-infected with CPV and EMCV.
Virus isolation, propagation, and titration. The EMCV-positive serum samples were incubated with 1.0 IU/mL penicillin/streptomycin for 10 min at 37 °C. Then, serum samples were filtered through a 0.22-μm filter and used as an inoculum for EMC virus isolation. Isolation of EMCV C15 was attempted using BHK21 cells as previously described with some modifications 17 . Confluent BHK21 cells in 6-well plates were washed twice with postinoculation medium and inoculated with 300 μL of sample and 100 μL of postinoculation medium. After 45 min, another 1.0 mL of DMEM supplemented with 10% FBS and 1.0 IU/mL of penicillin and streptomycin was added to each well in the 6-well plate. Inoculated cell (passages 0 [P 0]) were incubated at 37 °C with 5% CO 2 . When a 70% cytopathic effect (CPE) developed, the plates were subjected once to freezing and thawing. The mixtures were centrifuged at 3,000× g for 15 min at 4 °C. The supernatants were harvested for further propagation or saved at −80 °C. If no CPE was observed three days post-inoculation, the plates were frozen and thawed once, after which the supernatants were inoculated on new BHK21 cells for a second passage. Inoculated cells at each passage were also tested using a real-time RT-PCR assay. If the CPE tests and real-time RT-PCR results were negative after four passages, the virus isolation result was considered negative. Virus titration was performed in 96-well plates with 10-fold serial dilutions performed in triplicate per dilution. Virus titers were determined according to the Reed and Muench method and expressed as the 50% tissue culture infective dose (TCID 50 )/100 μL. The virus isolated and characterized in this study was designated EMCV C15.
Electron Microscopy (EM). Samples were prepared for negative staining examination by electron microscopy (EM) following previously described procedures with some modifications 18 . BHK21 cells infected with the EMCV-C15 P3 virus isolate were frozen and thawed 48 h post-infection, after which they were centrifuged at 3,000× g for 15 min. A total of 5.0 mL of supernatant was prepared in this section. The virus was pelleted from the supernatant by ultracentrifugation at 159,000× g for 1.5 h at 4 °C and resuspended in PBS. The resulting pellet was resuspended in 500 μL of 0.01 M PBS (pH 7.2 to 7.4), which was nebulized onto coated EM grids. The grids were stained with 1% phosphotungstic acid (pH 7.0) and observed with a transmission electron microscope. instructions. An equal amount of each sample was loaded onto a 12% SDS-PAGE gel, followed by protein transfer to a PVDF membrane (Millipore). Each membrane was blocked for 1 h with blocking buffer (5% skimmed milk and 0.1% Tween-20 in PBS) and incubated with anti-VP1 primary antibody (1:1,000 dilution) 19 or with mouse anti-GAPDH antibodies (Cell Signaling Technology, Inc.) to detect endogenous GAPDH, which served as a protein-loading control. Each membrane was washed three times with washing buffer (0.1% Tween-20 in PBS) and incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution) for 1 h, after which the membranes were washed and exposed to the detection agent 3,3′-N-diaminobenzidine tertrahydrochloride (DAB, CWBIO, China). Protein sizes were determined by comparison with prestained protein ladders (Thermo Scientific, U.S.A.).

Animal experiments and detection of viral load in dogs.
Eight 25-to-30-day-old dogs were obtained from a commercial breeding herd that was free of CPV, CDV, CPIV, CHV, ICHV and EMCV. The dogs were observed for two weeks to ensure that none were symptomatic and were then randomly assigned to two groups: group A (n = 5) and group B (n = 3). Each dog in group A was treated with 1.0 mL (10 5 TCID 50 ) of EMCV-C15, whereas each dog in group B was treated with 1.0 mL of DMEM. At 35 DPI, the animals in group A and group B were euthanized and the viral load in several organs (heart, liver, spleen, lung, kidney and brain) was determined by real-time RT-PCR performed as described above. Each sample was tested three times.
Sanger sequencing and phylogenetic analysis. EMCV-specific primers (Table 1) were designed based on the ORF of the BJC3 strain (DQ464062). DNA fragments corresponding to the complete ORFs of EMCV C15 strain were amplified and the amplicons were sequenced commercially (HuaDa, Beijing). Sequence assembly was carried out using the SeqMan program included with the DNASTAR software package (Madison, WI). The complete nucleotide sequence of the EMCV-C15 ORF was deposited in GenBank under accession number KU664327. Phylogenetic analysis was performed using the nucleotide sequence of the EMCV-C15 virus from this study, as well as the sequences of 24 reference strains for which full genome sequences were available in GenBank. A phylogenetic tree was constructed based on the ORF sequences of EMCV-C15 using the

Results
Virus isolation and characterization. An EMCV isolate designated EMCV-C15 was successfully obtained from a mixture of four serum samples from pet hospitals in Beijing (collected in March 2015) which had shown positive real-time RT-PCR results. The EMCV-C15 particles in infected BHK21 cells were examined by EM 48 h post-infection. As shown in Fig. 1, negatively stained samples contained a small virus with a diameter of 27-30 nm 18 . Virus growth was confirmed by IFA using an anti-EMCV VP1 specific monoclonal antibody 19 . The EMCV VP1 protein stained by the monoclonal antibody was distributed in the cytoplasm but not in the nucleus ( Fig. 2A). Negative control cells are shown in Fig. 2B. Western blot analysis demonstrated that capsid protein VP1 expression was similar in BHK-21 cells infected with EMCV-C15 or EMCV HB10, which was used as a positive control (Fig. 3). Taken together, these results show that EMCV-C15 and EMCV HB10 are indistinguishable with regard to viral replication and spreading in BHK-21 cells and that the monoclonal antibody recognizes an epitope common to both strains.

EMCV-C15 infection in artificially challenged mice. Mice injected with EMCV-C15 showed clinical
symptoms including anorexia, paralysis and sudden death, and displayed high mortality. At 15 DPI, the mice that were still alive were bled and subjected to pathological examination. The presence of EMCV-C15 was confirmed by real-time RT-PCR. The LD 50 of EMCV-C15 in mice was 10 2.98 TCID 50 (Table 2).

Quantity and distribution of EMCV-C15 in artificially challenged dogs. The five dogs in group A,
which had been inoculated with EMCV-C15, presented with pulmonary edema (Fig. 4A), actuated pericardial effusion, myocarditis, hind limb paralysis and encephalitis (pictures not shown). However, the dogs in group B displayed no clinical signs (Fig. 4B). The presence of EMCV-C15 in the organs of each group was detected by real-time PCR. In group A, EMCV was detected in the heart, liver, spleen, lung, kidney and brain, which had average viral loads of approximately 4.37 × 10 5 , 3.52 × 10 4 , 3.64 × 10 4 , 9.72 × 10 3 , 9.27 × 10 3 , and 2.68 × 10 5 EMCV genomes, respectively (Fig. 5).  otide sequences were performed to examine the degree of sequence similarity between EMCV C15 isolate and 24 EMCV reference strains retrieved from GenBank nucleotide database (Table 3). The complete 6,879-nt EMCV C15 ORF GenBank accession number (KU664327) encoding 2,292 amino acids with no insertions or deletions was examined. Multiple sequence alignment based on the ORF sequence of EMCV-C15 and other EMCV reference strains was performed using the Clustal X program 17 . The results showed the newly isolated virus (EMCV C15) shared the highest sequence similarity (99.0-99.75%) with GX0601, BJC3, NJ08, GS01, HB10 and BEL-2887A/91. However, it shared lower similarity (80-83.52%) with PV2, EMCV-B, EMCV-D, and the D variant. Phylogenetic analyses of the ORF sequence of the EMCV C15 in this study demonstrated that all EMCV strains could be divided into two mian groups (groups 1 and 2). Group 1 was subdivided into group 1a and b (Fig. 6). Except EMCV-R and EMCV C15 strains, the rest of strains were isloated form pigs in group 1a. The EMCV-C15 strain was clearly most closely related to the EMCV strain BEL2887A/91 which was detected in 1991 in Belgium. Group 2 comprises five EMCV isolates from sus scrofa.  Table 2. Outcome of infection with EMCV C15 in mice. a Data were analyzed using SPSS 17.0 software. Oneway ANOVA followed by Duncan's multiple range test was used to compare the parameters among the different groups.

Discussion
EMCV has often been described as a potential zoonotic agent with a wide range of hosts. Reports have clearly shown that humans are susceptible to EMCV infection; indeed, such infection is likely to be fairly common, but most human cases are probably asymptomatic and/or unrecognized 21 . Although human exposure to EMCV is quite common and associated with low morbidity, the virus is a potential threat to public health security. Therefore, it is essential that researchers focus on EMCV infection in humans and dogs. Infection of dogs with EMCV was reported by Schwab et al., who described a high proportion of dogs and cats with non-suppurative meningoencephalitis, for which immunohistochemical examination with antibodies against 18 different infectious agents failed to reveal a cause. However, the significance of positive immunoreactions obtained with antibodies against proteins produced by West Nile virus (WNV) and EMCV requires further investigation 22 .
In this study, 4 of 69 dog serum specimens tested positive for EMCV using real-time RT-PCR. EMCV-C15 was isolated from a mixture of four serum samples that were co-infected with CPV and EMCV. The EMCV-C15 isolate was serially propagated via cell culture methods and characterized. By examining CPE development, IFA staining, EM, western blot analysis, infectious virus titers and complete ORF sequences, we clearly demonstrated that the C15 isolate is phenotypically and genetically stable in cell culture.
Experiments in mice showed that the EMCV isolate was highly virulent and caused hind limb paralysis. In addition, we revealed that the LD 50 of EMCV-C15 in mice was 10 2.98 TCID 50 . To enhance our understanding of EMCV loads in various organs in dogs, real-time RT-PCR was used to quantify the presence of the EMCV genome in various tissues from artificially challenged dogs. The heart and brain are the most important targets for the isolated EMCV C15 in challenged dogs. Inflammation of the heart and brain were obvious in dogs infected with EMCV-C15 and might have been important in the development of acute myocarditis and pulmonary edema.
Phylogenetic analysis showed that the C15 strain is closely genetically related to strain BEL2887A/91, which circulated in Belgium in 2002. The hosts of BEL2887A/91 are pigs or piglets; however, the mechanism by which EMCV was introduced to dogs remains unknown. The history of the spread of EMCV among species and geographical areas merits further investigation.
In this study, the EMCV isolate EMCV-C15 was obtained and characterized. To our knowledge, this is the first report describing the isolation and characterization of EMCV from dogs. All experimental data indicate that the dog is a host for EMCV. Therefore, people living in areas with dogs, especially owners of pet dogs, should be aware of this potential health threat.