Isolation, Molecular and Phylogenetic Analysis of Porcine Encephalomyocarditis Virus Strain HLJ in China

Yin Xue-ting, Liu Peng, Zou Meng-meng, Liu Hao-yun, Huang Xiao-dan, Ren Yu-dong, and Li Guang-xing*

1 College of Veterinary Medicine, Heilongjiang Key Laboratory for Animal and Comparative Medicine, Northeast Agricultural University, Harbin 150030, China

2 College of Electrical and Information, Northeast Agricultural University, Harbin 150030, China

Abstract: Encephalomyocarditis virus (EMCV) is a positive single-stranded small RNA virus without envelope, which can infect a variety of mammals. Swines 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. In this study, a strain of EMCV was isolated from a swine aborted fetus in northeast China. It was identified by reverse transcriptase polymerase chain reaction (RT-PCR), electron microscopic observation and indirect immunofluorescence assay. The subsequent results showed that the virus titer of HLJ strain grew to 8.3 lgTCID50 on baby hamster kidney 21 (BHK-21) cells. And HLJ strain caused the specific cytopathic effect (CPE) on BHK-21 cells and severe pathological changes in mice. Complete genome sequencing and multiple sequence alignment showed that the homology between HLJ strain and other isolates worldwide was 71.5%-99.7%. Phylogenetic analysis showed that EMCV isolates fell into five clusters: lineage I, II, III, IV and V, based on the nucleotide sequences of the entire open reading frame (ORF) and VP1 gene. HLJ isolate was grouped into lineage I. The analyses of amino acid mutation sites of VP1 protein showed that the amino acids at positions 20 and 54 in VP1 junction were unique to HLJ strain. The isolation of HLJ strain enriched the epidemiological database of EMCV.

Key words: Encephalomyocarditis virus, isolation, molecular and identification, phylogenetic analysis

Introduction

Encephalomyocarditis virus (EMCV) is an animal pathogen, which is characterized by encephalitis, myocarditis or sudden death (Baggenet al., 2019; Franciscoet al., 2019). EMCV was first diagnosed in Florida in 1945 from a gibbon and then isolated and investigated in many countries (Helwig and Schmidt, 1945; Murnaneet al., 1960). Since the epidemic of EMCV, researchers reported that it can cause a variety of animal diseases in a variety of hosts, including livestocks, rodents and wild animals (Billinis, 2009; Canelliet al., 2010; Larueet al., 2003; Mauriceet al., 2005; Zhanget al., 2018). Besides piglets are susceptible to EMCV, rodent mice and rats are natural hosts of EMCV. The virus can cause acute myocarditis or even acute death in piglets with a mortality rate of 100% (Liet al., 2019b). In mice, EMCV caused mainly myocarditis, neurovirulence and diabetes (Cerutiset al., 1989; Noguchiet al., 2019; Yoon, 1990).

EMCV belongs to the genusCardiovirusof the familyPicornaviruses. The virus particle is icosahedral symmetrical, non-encapsulated and about 27 nm in diameter, with a bare viral capsid on the periphery (van der Greinet al., 2019). EMCV is a single-stranded RNA virus (Bazzoneet al., 2019). The full-length of the genome encodes a large ORF of about 7.8 kb flanked by the 5' UTR and 3' UTR of the untranslated region (Liuet al., 2017). 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 (Ghassemiet al., 2017). 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 (Liet al., 2019a). The proteins encoded by ORF contain non-structural and structural proteins, which are divided into three precursor molecules P1, P2 and P3. The genome encodes a total of 11 different proteins. 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 regions are mainly viral genome replication related proteins and polyprotein cleavage related proteins (Carmodyet al., 2018; Luoet al., 2017).

In recent years, EMCV infection in swine industry is increasing at an alarming rate. In addition, it also infects human beings. This study described the isolation, genomic sequencing and bioinformatics analysis of an EMC virus, HLJ isolate, from a swine aborted fetus. The strain had 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 would provide references for the prevention and control of EMCV in the future.

Materials and Methods

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 three times, followed by clarification through a 0.22-μm filter, and 1% antibiotic was added. Confluent 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℃ with 5% CO2. 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℃. 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 (TCID50)/100 µL.

Electron microscopy (EM) and indirect immunofluorescence 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℃. The resulting pellet was resuspended in 500 µL of 0.01 mol · L-1PBS (pH 7.2-7.4), 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 fixed 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-specific rabbit polyclonal antibody (1 : 200 dilution) for 1 h and incubated with fluorescein isothiocyanate (FITC) labeled goat anti-rabbit IgG (1 : 100 dilution; Sigma-Aldrich) for 1 h at 37℃, then re-stained the nucleus with DAPI (Beyotime, China). It was observed under an inverted fluorescence microscope. EMCV-specific rabbit polyclonal antibody was prepared in our laboratory.

Mice and infection experiment

Female Kunming mice, aged from 4 to 6 weeks, were intraperitoneally infected with 200 μL HLJ strain at the concentration of 2×104TCID50(I.P.) or Dulbecco modified Eagle medium (DMEM, Gibco, USA). The 25 mice were randomly divided into two groups. One group of 20 mice was each inoculated with the virus and the other group of five 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.

Immunofluorescence of fresh frozen tissues 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 fixation, indirect immunofluorescence experiment was carried out. The sections were treated with EMCV-specific polyclonal antibody and FITCconjugated goat anti-rabbit IgG, then re-stained the nucleus with DAPI. After sealing, the sections were observed under positive fluorescence microscope. In addition, brain and myocardial tissue samples were fixed with 10% formalin, embedded in paraffin, sliced on Leica RM 2125RT slicing machine, dewaxed and hematoxylin-eosin (HE) staining, according to the standard procedure.

Nucleotide sequence analysis

EMCV-specific primers were designed based on the ORF of BJC3 strain (DQ464062). The whole genome of HLJ stain was amplified and sequenced using a set of specific primers (data not displayed). DNA fragments corresponding to the complete nucleotide sequences of HLJ strain were amplified in sections, ligated with 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 SeqMan program included with DNASTAR software. Multiple sequence alignment based on 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 ORFs andVP1 genes using the distance-based neighbor-joining method in 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.

Table 1 Encephalomyocarditis virus isolates used in this study

Results

Virus isolation and characterization

An EMCV strain named HLJ was isolated from the heart tissue of the swine aborted fetus from breeding farm in Heilongjiang Province of China (collected in March 2016) which had shown positive RT-PCR results (data not displayed). After three passages on BHK-21 cells, the virus titer of HLJ strain grew to 108.3TCID50on BHK-21 cells (Fig. 1A). And HLJ strain caused specific 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 confirmed by IFA with EMCV-specific polyclonal antibody. BHK-21 cells inoculated with HLJ strain showed specific green fluorescence (Fig. 2B).

Fig. 1 Virus isolation and titration

Fig. 2 Electron micrographs and immunofluorescence detection

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. 3Ab). The mice in the negative control group did not have any clinical symptoms and all survived (Fig. 3Aa). After autopsy, the heart of the infected mice was soft, and there were white punctate necrotic foci on the surface (Fig. 3Bd). In addition, the brain was congested and swollen(Fig. 3Bb), 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 infiltration, obvious satellite phenomenons, nerve phagocytosis and microglial nodules, and typical vascular sheath phenomenon around blood vessels. Myocardial tissues showed diffuse inflammatory cell infiltration, local cell degeneration and necrosis, hemorrhage and disintegration of myocardial fibers (Fig. 3C). Indirect immunofluorescence results showed that green fluorescence 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.

Fig. 3 Characteristics of HLJ strain virus in vivo

Whole genome sequencing and genomic characterization of HLJ strain

Five fragments covering the complete genome of HLJ strain were amplified 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 was MH191297. The full-genome sequence of HLJ strain was 7 746 bp long, with a 5' UTR of 717 bp, a 3' UTR of 138 bp, and encoded 2 292 amino acids.

The ORF of 6 879 bp encoded 11 proteins which were similar to those of the previous reported EMCV strains. HLJ strain showed high sequence homology (99.2%-99.7%) with BD2, BJC3, EMCV-R, FJ13, GS01, HB1, GX0602, HN13, JZ1202, JZ1203, pEC9, YM13 and YY13, and lower sequence homology (71.5%-84.6%) with EMCV-B, EMCV-D, D variant, EMCV-30, Mengo-M, Rz-pMwt, RD 1338, PV2, Sing-M100-02 and Sing-M105-02 (data not displayed). The non-structural protein was more conservative than the structural protein coding region, and 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 was the most conserved protein of all the proteins.

Phylogenetic analysis of HLJ strain and structural analysis of VP1 protein

The results of phylogenetic analysis showed that HLJ strain and other reference strains were divided into five clusters (lineage I, II, III, IV and V), based on the nucleotide sequences of ORF andVP1 gene. HLJ isolate was grouped into lineage I. Lineage I comprised 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 ORF nucleotide sequences. HLJ strain was most closely related to HN13 strain in phylogenetic analysis, based on the nucleotide sequences ofVP1 gene (Fig. 4). Structural analysis of VP1 protein of HLJ strain showed that it had two new mutations at 20 and 54 amino acid positions compared with other strains (Table 2).

Table 2 Amino acid mutation site of VP1 protein of HLJ strain

Fig. 4 Phylogenetic relationship between HLJ strain and other EMCV strains

Discussion

EMCV displayed a wide spectrum of host and disease, as it was able to infect nonhuman primates, swine, boars, rodents and elephants, and human infections had also been reported (Obersteet al., 2009). Pigs were susceptible animals that could easily lead to myocarditis, reproductive failure and high mortality in pregnant sows, fetuses and weaned piglets (Liuet al., 2017). EMCV infection had been detected in many swine farms by etiological and serological methods (Fenget al., 2015; Yuanet al., 2014). Therefore, the isolation and identification of endemic strains and genome-wide sequence analysis could enrich the accumulation of EMCV molecular epidemiology and bioinformatics research data.

In this study, HLJ strain was isolated from a swine aborted fetus, and it was continuously propagated and identified by cell culture. It was clearly proved that HLJ strain had phenotypic and genetic stability in cell culture through a series of studies. Also, the results of animal regression experiments showed that HLJ strain had strong pathogenicity to mice, which was consistent with the results of previous studies (Canelliet al., 2010; Carocciet al., 2011). The amplification of the whole genome sequence was the basis for further studies on the virus. Fanget al. (2015) used the strategy of segmented cloning to construct an infectious clone of NJ08 strain. Next, it was planned to use the segmented cloning strategy to amplify the full-length of HLJ strain and construct an infectious clone for further studies on the pathogenic mechanism of EMCV.

The results of genome-wide homology analyses showed that HLJ strain was highly homologous to the domestic swine source and mouse source. It was suggested that it could infect a variety of hosts, and there might be cross-infection, in which rodents had a wide range of activities, high mobility and other characteristics that might be major factors in the spread of EMCV (Mauriceet al., 2016). Nucleotide and deduced amino acid homology analyses of each gene showed that 2A protein was the least conserved protein of non-structural proteins. Some studies had shown that 2A protein could still infect cells after the deletion of 127 amino acids and 12 amino acid mutations, but had no pathogenicity to mice, indicating that 2A protein was related to the pathogenicity of mice (Carocciet al., 2011; Napthineet al., 2019). This might be the method of developing attenuated live vector vaccine.

Comprehensive phylogenetic analyses showed that HLJ strain was located in lineage I, which was closely related to wild boar HN13 or swine GS01 strain. It might 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 affected the genetic evolution of EMCV. Therefore, the geographical location of genetic variation of EMCV occupied the main factors, followed by transmission host, and there was cross infection. In addition, the mutation of EMCV VP1 protein was also the research direction. It had been reported that amino acid mutations of VP1 protein could cause pathogenic changes in mice (Zhuet al., 2011). Therefore, the results showed two new mutation points of VP1 protein of HLJ strain that might cause pathogenic changes.

Conclusions

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 a swine aborted fetus. The virus had high pathogenicity and lethality to mice. Phylogenetic analyses showed that HLJ strain was located in lineage I, with all the Chinese isolates and a few American isolates. Geographical location was the main factor affecting the genetic variation of EMCV. Isolation and identification of EMCV HLJ strain and molecular biology analyses could lay a theoretical and material basis for the development of new and efficient approaches for the disease prevention.