Isolation, molecular and phylogenetic analysis of porcine encephalomyocarditis virus strain HLJ in China

Encephalomyocarditis virus (EMCV) is a positive single-stranded small RNA virus without envelope, which can infect a variety of mammals. Swine are the most susceptible animals, which can cause acute myocarditis and respiratory failure in piglets and reproductive failure in pregnant sows. Diseases caused by EMCV have a wide range of effects on the global swine industry. Samples were prepared for negative staining examination by electron microscopy (EM). BHK-21 cells were infected with the third generation virus of HLJ isolate, frozen and thawed 48 h later, and centrifuged at 3,000 × g for 10 min. The supernatant was harvested and the virus was granulated from the supernatant by ultracentrifugation at 159,000 × g for 1.5 h at 4 °C. The resulting pellet was resuspended in 500 µl of 0.01 M PBS (pH 7.2 to 7.4), which was atomized onto coated EM grids. The grids were stained with 1% phosphotungstic acid (pH 7.0) and observed with a transmission electron microscope. After 12 h post-infection, the infected BHK-21 cells were xed with 4% formaldehyde for 30 min at room temperature. The cells were treated with glycine for 6 min and permeated by 0.1%Triton for 10 min. The cells were incubated with EMCV-specic rabbit polyclonal antibody (1:200 dilution) for 1 h and incubated with uorescein isothiocyanate (FITC) labeled goat anti-rabbit IgG (1:100 dilution; Sigma-Aldrich) for 1 h at 37 °C, then re-stained the nucleus with DAPI (Beyotime, China). It was observed under an inverted uorescence microscope. EMCV-specic rabbit polyclonal antibody was prepared in our laboratory.


Background
Encephalomyocarditis virus (EMCV) is an animal pathogen, which is characterized by encephalitis, myocarditis or sudden death [1,2]. EMCV was rst diagnosed in Florida in 1945 from a gibbon and then isolated and investigated in many countries [3,4]. Since the epidemic of EMCV, researchers reported that it can cause a variety of animal diseases in a variety of hosts including livestock, rodents and wild animals [5][6][7][8][9]. Besides piglets are susceptible to EMCV, rodent mice and rats are natural hosts of EMCV.
EMCV belongs to the genus Cardiovirus of the family Picornaviruses. The virus particle is icosahedral symmetrical, non-encapsulated and about 27 nm in diameter, with a bare viral capsid on the periphery [14]. EMCV is a single-stranded RNA virus [15]. The full length of the genome encodes a large ORF of about 7.8 kb anked by the 5' UTR and 3' UTR of the untranslated region [16]. The genomic structure is that 5' UTR connects to the guiding protein L, which inhibits the accumulation of stress particles produced by EMCV infection. Downstream is linked to a poly(C) tract and an internal ribosomal entry site (IRES) type II, EMCV structure without cap relies on IRES as the starting point of protein translation [17]. The 3' UTR is about 120 bp long, and the polyadenylation at the end forms a poly(A) structure, which functions to stabilize mRNA and initiate RNA replication, and is related to the infectivity of the virus [18]. The proteins encoded by ORF contain non-structural proteins and structural proteins, which are divided into three precursor molecules P1, P2 and P3. The genome encodes a total of 11 different proteins. The P1 region protein constitutes the virus capsid, which is the main antigenic site of the virus, and is closely related to the adsorption and pathogenicity of the virus. P2 and P3 region are mainly viral genome replication related proteins and polyprotein cleavage related proteins [19,20].
In recent years, EMCV infection in swine industry is increasing at an alarming rate. In addition, it also infects human beings. This study describes the isolation, genomic sequencing and bioinformatics analysis of an EMC virus, HLJ isolate, from an aborted fetus. The strain has high pathogenicity to mice.
The whole genome and deduced amino acid sequences of HLJ strain were compared with the nucleotide and amino acid sequences of typical EMCV strains from different countries. This study revealed the molecular evolution characteristics of HLJ strain and provided a reference for the prevention and control of EMCV in the future.

Virus isolation and titration
A piece of myocardium of aborted fetus was homogenized in phosphate-buffered saline (PBS) using a glass homogenizer. The suspension was repeatedly frozen and thawed 3 times, followed by clari cation through a 0.22-µm lter, and 1% antibiotic was added. Con uent BHK-21 cells in 6-well plates were inoculated with 300 µL of sample and 100 µL of medium. After 45 min, another 1.0 mL of complete growth medium was added to each well in the 6-well plate. Inoculated cells were incubated at 37 °C with 5% CO 2 . When a 70% CPE developed, the plates were subjected three times to freezing and thawing. The mixtures were centrifuged at 3,000 × g for 10 min at 4 °C. The supernatants were harvested for further propagation. Inoculated cells at each passage were tested using an RT-PCR assay. 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 median tissue culture infective dose (TCID 50 )/100 µL. Electron Microscopy (em) And Indirect Immuno uorescence Assay (ifa) Samples were prepared for negative staining examination by electron microscopy (EM). BHK-21 cells were infected with the third generation virus of HLJ isolate, frozen and thawed 48 h later, and centrifuged at 3,000 × g for 10 min. The supernatant was harvested and the virus was granulated from the supernatant by ultracentrifugation at 159,000 × g for 1.5 h at 4 °C. The resulting pellet was resuspended in 500 µl of 0.01 M PBS (pH 7.2 to 7.4), which was atomized onto coated EM grids. The grids were stained with 1% phosphotungstic acid (pH 7.0) and observed with a transmission electron microscope. mice were randomly divided into two groups. The one group of twenty mice was each inoculated with the virus and the other group of ve mice was inoculated with DMEM as negative control. The brain and heart tissues of mice that died of EMCV infection were harvested on the day of death. Negative control mice were euthanized at the end of the experiment.

Immuno uorescence Of Fresh Frozen Tissue And Histological Examination
The dying mice were dissected and their internal organs were examined. The fresh brain and myocardial tissue materials were immediately embedded in optimal cutting temperature compound and then made into frozen sections with a thickness of 6 µm. After acetone xation, indirect immuno uorescence experiment was carried out. The sections were treated with EMCV-speci c polyclonal antibody and FITCconjugated goat anti-rabbit IgG, then re-stained the nucleus with DAPI. After sealing, the sections were observed under positive uorescence microscope. In addition, brain and myocardial tissue samples were xed with 10% formalin, embedded in para n, sliced on Leica RM 2125RT slicing machine, dewaxed and hematoxylin-eosin (HE) staining according to the standard procedure.
Nucleotide Sequence Analysis EMCV-speci c primers were designed based on the ORF of the BJC3 strain (DQ464062). The whole genome of HLJ stain was ampli ed and sequenced using a set of speci c primers (Additional le 1: Table S1). DNA fragments corresponding to the complete nucleotide sequences of HLJ strain were ampli ed in sections, ligated with the pMD18-T vector (Takara, Japan) to construct a recombinant plasmid and the amplicons were sequenced commercially (JinWeizhi, Suzhou). Sequence assembly was competed by means of the SeqMan program included with the DNASTAR software. Multiple sequence alignment based on the ORFs, and each coding gene sequence of HLJ and other 23 EMCV reference strains (Table 1) was conducted using DNAStar software. Subsequent phylogenetic analysis was performed on the ORFs and VP1 genes using the distance-based neighbor-joining method in the MEGA5 software package. Bootstrap analysis was carried out on 1,000 replicate data sets. The amino acid sequence of VP1 protein of HLJ isolate was compared with those of different EMCV strains in China.  (Fig. 1A). And HLJ strain caused speci c cytopathic effects (CPE) within 24 h (Fig. 1B. b). After BHK-21 cells were infected with HLJ strain for 48 h, the ultra-thin sections were prepared. Under transmission electron microscope, it was found that the virus particles were located in the cytoplasm. The virions were arranged in a lattice ( Fig. 2A). The growth of the virus was con rmed by IFA with EMCVspeci c polyclonal antibody. The BHK-21 cells inoculated with HLJ strain showed speci c green uorescence (Fig. 2B).

Characteristics Of Hlj Strain Virus In Vivo
The results showed that some mice in the attack group began to show clinical symptoms on the 3rd day, such as depression, wrinkled fur, loss of appetite, tears, hunchback posture, abnormal gait, hindlimb paralysis and quadriplegia, and death ( Fig. 3A. b). The mice in the negative control group did not have any clinical symptoms and all survived ( Fig. 3A. a). After autopsy, the heart of the infected mice was soft, and there were white punctate necrotic foci on the surface (Fig. 3B. d). In addition, the brain was congested and swollen ( Fig. 3B. b), and there were no obvious pathological changes in other tissues. After HE staining, the brain of mice in the infected group was mainly characterized by extensive atrophy and necrosis of nerve cells in the cortical area, accompanied by extensive glial cell in ltration, obvious satellite phenomenon, nerve phagocytosis and microglial nodules, and typical vascular sheath phenomenon around blood vessels. Myocardial tissue showed diffuse in ammatory cell in ltration, local cell degeneration and necrosis, hemorrhage and disintegration of myocardial bers (Fig. 3C). Indirect immuno uorescence results showed that green uorescence signals could be detected in brain and heart tissues of the infected group (Fig. 3D). In conclusion, the virus was successfully isolated from the myocardium of dead aborted fetuses, and the virus was lethal to mice.

Whole Genome Sequencing And Genomic Characterization Of Hlj Strain
Five fragments covering the complete genome of HLJ strain were ampli ed by RT-PCR method and sequenced. After splicing the above sequencing results, the whole genome nucleic acid sequence of HLJ strain was obtained, and the accession number of GenBank is MH191297. The full-genome sequence of Sing-M105-02 (Additional le 2: Table S2). The non-structural protein was more conservative than the structural protein coding region, and the VP1 protein had the greatest variation in the structural protein.
On the other hand, 2A protein had the greatest variation in non-structural protein. The stable genomic structure of 3D protein is the most conserved protein of all proteins.

Phylogenetic analysis of HLJ strain and structural analysis of the VP1 protein
The results of phylogenetic analysis showed that, HLJ strain and other reference strains were divided into ve clusters (lineage I, II, III, IV, and V) based on the nucleotide sequences of ORF and VP1 gene. HLJ isolate was grouped into lineage I. Lineage I comprises all the Chinese isolates, American isolates pEC9, EMCV-R, and EMCV-30. HLJ strain was more closely related to GS01 strain in phylogenetic analysis based on the ORF nucleotide sequences. HLJ strain was most closely related to HN13 strain in phylogenetic analysis based on the nucleotide sequences of VP1 gene (Fig. 4). Structural analysis of the VP1 protein of HLJ strain showed that it had two new mutations at 20 and 54 amino acid positions compared with the other strains ( Table 2). Table 2 The amino acid mutation site of VP1 protein of HLJ strain

Discussion
EMCV displays a wide spectrum of host and disease, as it is able to infect nonhuman primates, swine, boars, rodents, and elephants, and human infections have also been reported [21]. Pigs are susceptible animals that can easily lead to myocarditis, reproductive failure and high mortality in pregnant sows, fetuses and weaned piglets [16]. EMCV infection has been detected in many swine farms by etiological and serological methods [22,23]. Therefore, the isolation and identi cation of endemic strains and genome-wide sequence analysis can enrich the accumulation of EMCV molecular epidemiology and bioinformatics research data.
In this study, HLJ strain was isolated from an aborted fetus, and it was continuously propagated and identi ed by cell culture. We clearly proved that HLJ strain has phenotypic and genetic stability in cell culture through a series of studies. Also, the results of animal regression experiment showed that HLJ strain had strong pathogenicity to mice, which was consistent with the results of previous studies [8,24]. The ampli cation of the whole genome sequence is the basis for further study of the virus. Fang and others used the strategy of segmented cloning to construct an infectious clone of NJ08 strain [25]. Next, we plan to use the segmented cloning strategy to amplify the full length of HLJ strain and construct an infectious clone for further study of the pathogenic mechanism of EMCV.
The results of genome-wide homology analysis showed that HLJ strain was highly homologous to the domestic swine source and mouse source. It is suggested that it can infect a variety of hosts, and there may be cross-infection, in which rodents have a wide range of activities, high mobility and other characteristics that may be major factors in the spread of EMCV [26]. Nucleotide and deduced amino acid homology analysis of each gene showed that 2A protein was the least conserved protein of nonstructural proteins. Some studies have shown that the method of continuous passage of attenuated virus has found that 2A protein can still infect cells after the deletion of 127 amino acids and 12 amino acid mutations, but has no pathogenicity to mice, indicating that 2A protein is related to the pathogenicity of mice [24,27]. This may be the method of developing attenuated live vector vaccine.
Comprehensive phylogenetic analysis showed that, HLJ strain was located in lineage I, which was closely related to wild boar HN13 or swine GS01 strain. It may be a genetic variation from wild boar to domestic boar. The two Singaporean orangutan EMCV isolates were grouped in lineage IV. All the western strains were in the lineage II, III, and V, indicating that geographical factors and hosts affect the genetic evolution of EMCV. Therefore, the geographical location of genetic variation of EMCV occupies the main factor, followed by transmission host, and there is cross infection. In addition, the mutation of EMCV VP1 protein is also the research direction that people have been studying. It has been reported that amino acid mutations of VP1 protein can cause pathogenic changes in mice [28]. So, our results showed two new mutation points of VP1 protein of HLJ strain that may cause pathogenic changes.

Conclusion
EMCV causes acute myocarditis and respiratory failure in piglets and reproductive disorders in pregnant sows. At present, there are no effective drugs and vaccines for the prevention and control of the disease. In this study, HLJ strain was isolated from an aborted fetus. The virus has high pathogenicity and lethality to mice. Phylogenetic analysis showed that HLJ strain was located in lineage I, with all the Chinese isolates and a few American isolates. Geographical location is the main factor affecting the genetic variation of EMCV. Isolation and identi cation of EMCV HLJ strain and molecular biology analysis can lay a theoretical and material basis for the development of new and e cient approaches for the disease prevention.