Pathogenicity and molecular analysis of an encephalomyocarditis virus isolate from mideastern China

Abstract

Encephalomyocarditis virus (EMCV) can infect many host species and cause acute myocarditis and sudden death in preweaned piglets. In this study, an EMCV strain (NJ08) was isolated from newborn pigs with clinical signs on a pig farm in mideastern China. It was identified by indirect immunofluorescence assay and reverse-transcription polymerase chain reaction. Experiments showed that the isolate could cause severe clinical symptoms and pathological changes in mice but no obvious clinical and pathological changes in commercial piglets. Complete genomic sequencing showed that the NJ08 strain was 78.3% to 100% identical with other isolates in regions coding for various proteins. Phylogenetic analysis showed that the NJ08 isolate belonged to subgroup Ia. This study confirmed that an EMCV isolate from pigs could be fatal to mice and provided new epidemiologic data on EMCV in China.

Encephalomyocarditis virus (EMCV) belongs to the genus Cardiovirus in the family Picornaviridae. It has been recognized worldwide as a pathogen infecting many species, including pigs, rodents, cattle, baboons, macaques, chimpanzees, bonobos, and humans (1–4). Usually rats and mice are the natural hosts of EMCV, and pigs are considered the domestic animal most commonly and severely infected (5). The virus is a single-stranded, positive-sense RNA approximately 7.8 kb in length that contains a large open reading frame (ORF) (6). It is covalently linked at the 5′ end to virion protein, genome-linked, and in the 5′ untranslated region (UTR), which contains the internal ribosome entry site and a long homo-polymeric cytosine tract (7). The 3′-UTR terminates with a short, heterogeneous poly(A) tail. Although EMCV has been isolated in the south and north of China (8), it has not been reported in the mid-east, and its pathogenicity and molecular epidemiology have not been deeply understood.

Three clinically ill newborn pigs from 1 pig farm in mideastern China that died suddenly before weaning were diagnostically studied in August 2008. The clinical signs had included anorexia, rapid breathing, listlessness, trembling, and staggering or paralysis. Necropsy showed no obvious gross lesions except for excessive pericardial fluid and white necrotic areas in the heart muscle. For virus isolation, heart and brain tissue were homogenized with Dulbecco’s modified Eagle’s medium (DMEM), frozen and thawed 3 times, and centrifuged at 10 000 × g for 10 min at 4°C. The supernatant was passed through a 0.22-nm filter and inoculated into BHK-21 cells. The cultures were examined daily for cytopathic effect. After 3 passages, 1 cytopathic isolate, characterized by cell rounding, pyknosis, and degeneration of the cell monolayer, was detected 24 h after inoculation. It was named NJ08 and stored at −20°C for further characterization.

The virus titer was 10^9.2 TCID₅₀ (median tissue culture infective dose) per milliliter. Indirect immunofluorescence assay (IFA) showed that the isolate reacted strongly with monoclonal antibody against EMCV VP1 protein (made in our laboratory by vaccination of BALB/c mice with recombinant VP1 protein). For further identification of the isolate, total RNA was extracted from the cell cultures with TRIzol reagent (Invitrogen, Carlsbad, California, USA). Reverse transcription was carried out with M-MLV (Promega, Madison, Wisconsin, USA) in a 25-μL reaction mixture containing 2 μg of RNA. The VP1 gene was amplified with use of the primers VP1-f (5′-GCTGGATCCGGAGTAGAAAACGCTGAAAATCTCAC-3′) and VP1-r (5′-GGCAAGCTTTTACTCTAGCATCAAGACTCCAG-3′) and cloned into pMD-18T vector for sequencing with the T7 promoter primer (Invitrogen). The polymerase chain reaction (PCR) product was 831 base pairs (bp) in length. Sequence alignment indicated that the NJ08 isolate matched isolates in GenBank.

The following experimental activities were approved by the Institutional Animal Care and Ethics Committee of Nanjing Agricultural University (permit IACECNAU20100609) and met the International Guiding Principles for Biomedical Research Involving Animals.

To determine the pathogenicity of the isolate, fourteen 50-day-old BALB/c mice free of EMCV were divided into 2 groups. Each mouse in group 1 was inoculated with 0.1 mL of 10^8.0 TCID₅₀/mL EMCV NJ08 by the celiac route. The mice in group 2 were inoculated with 0.1 mL of DMEM medium and served as negative controls. The clinical status of the mice was observed daily for 21 d. The mice in group 1 showed symptoms and signs as early as 12 h after inoculation and died 3 to 6 d after inoculation. Signs of depression and nerve effects included hunched posture, lethargy, and hindlimb paralysis. Cerebrovascular engorgement, hemorrhagic pneumonia, and petechial cardionecrosis were observed at necropsy. Heart and brain tissue sections were fixed in 10% neutral buffered formalin and stained with hematoxylin and eosin for histopathological examination, and IFA was performed to detect EMCV antigen in the brain tissue, as previously described (9), with the use of monoclonal antibody against EMCV VP1 protein.

All of the infected mice showed evidence of encephalitis and meningitis, including perivascular infiltration with mononuclear cells and some neuronal degeneration with necrosis (Figure 1A), as well as signs of myocarditis, including focal or diffuse accumulation of mononuclear cells, edema, and degeneration of the myocardial fibers with necrosis (Figure 1B). Moreover, EMCV antigen was detected in the brain of all the infected mice, presenting in the cytoplasm (Figure 1C): the brownish-yellow positive signal was distributed in different layers of cells, especially the granular and external pyramidal layer. No lesions were observed in the control group.

Figure 1.

Inflammation and degenerative changes in heart (A) and brain (B) tissue of mice infected with encephalomyocarditis virus (EMCV) (hematoxylin and eosin). Indirect immunohistochemistry assay demonstrates EMCV antigen in brain tissue (C): red arrow points at brown positive signal. Original magnification × 400.

Five 21-d-old suckling piglets free of EMCV [negative by reverse-transcription PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA)] were inoculated with 3 mL of 10^9.0 TCID₅₀/mL EMCV NJ08 by both intramuscular and intranasal routes and observed daily for 21 d. Although the piglets did not show clinical signs or visible gross lesions after inoculation, EMCV was detected by RT-PCR in blood samples collected on days 2 and 3 after inoculation from all 5 piglets. As well, serum antibody to EMCV was detected by ELISA with purified EMCV NJ08 antigen in blood samples collected on days 14 and 21 after inoculation from all 5 piglets. Five control suckling piglets inoculated with 3 mL of DMEM and housed in another room remained healthy and showed no evidence of EMCV infection and no antibody to EMCV in the blood.

These findings indicate that the EMCV NJ08 isolate could infect piglets but not cause clinical signs. Further investigation is necessary to determine the pathogenicity of the isolate in newborn pigs and progeny sows. The variable clinical appearance of EMCV in domestic pigs in various countries might be explained by differences in the pathogenicity of various strains (10,11).

To understand the genetic relationships between NJ08 and other isolates and the evolution of EMCV, the genome of the virus was sequenced completely. Primers for PCR (Table I) were designed on the basis of the sequence of EMCV strain BJC3 (GenBank accession number DQ464062); 5 fragments were amplified with AccuPrime Pfx DNA polymerase (Invitrogen). In addition, the 5′- and 3′-UTRs of EMCV NJ08 were amplified by the 5′ and 3′ Full RACE Core sets (TaKaRa Biotechnology Company, Dalian, China) according to the manufacturer’s instructions. The 5′-phosphorylated primers and the 3′ RACE adapter primer were used as reverse primers to synthesize 1st-strand cDNA, which was used as the template for amplification. The PCR products were subjected to gel electrophoresis, purified with a purification kit (TaKaRa), and cloned into the pMD18-T vector (TaKaRa) according to the manufacturer’s instructions. Three positive clones for each fragment were sequenced. After the sequence of each fragment was assembled, the complete genome sequence of NJ08 was obtained. The phylogenetic relationships were studied with the use of MEGA4 software (http://www.megasoftware.net) according to the nucleotide sequences of EMCV NJ08 (GenBank no. HM641897) and other known EMCV isolates worldwide, including Chinese isolates GX0602 (FJ604853), GXLC (FJ897755), BJC3 (DQ464062), and HB1 (DQ464063), Korean isolates CBNU (DQ517424), K3 (EU780148), and K11 (EU780149), European isolates BEL-2887A (AF356822), PV21 (X74312), and Mengo-M (L22089), and American isolates EMCV-R (M81861), PEC9 (DQ288856), D variant (M37588), EMCV-B (M22457), EMCV-D (M22458), and Mengo Rz-pMwt (DQ294633). Phylogenetic trees were constructed with MEGA4 by the neighbor-joining method. Reliability was assessed by bootstrap analysis with 1000 replicates. The results showed that the NJ08 genome is 7724 bp in length, with a 5′-UTR of 714 nt, a 3′-UTR of 131 nt, and a large ORF that encoded a polyprotein of 2292 amino acids. The isolate belongs to subgroup Ia together with all the tested isolates from pigs and mice except for the European isolate Mengo-M and 4 of the 6 American isolates (D variant, EMCV-B, EMCV-D, and Mengo Rz-pMwt) (Figure 2). Since domestic pigs and mice live in the same environment, it could be that the virus spreads between these 2 species.

Table I.

Oligonucleotide primers for amplifying the complete genome of the NJ08 strain of encephalomyocarditis virus (EMCV)

Primer	Sequence (5′ → 3′)	Positiona	Product [size (number of base pairs)]
EV1F	ATTGTATGGGATCTGATCTGGGG	603–625	F1 (1526)
EV1R	GGTCTTGACGGCTGTGTTGG	2109–2128
EV2F	TGCTGGTACCTGGTATTCTA	1936–1955	F2 (1580)
EV2R	ATCTAACCTCCAAACCTCTA	3496–3515
EV3F	GATTTCGGCACTCTGTTCTT	3260–3279	F3 (2146)
EV3R	AGTTACGCTCCTGTACTTTCG	5385–5405
EV4F	GACGAGGTCAGTTTCCATTCC	5294–5314	F4 (1002)
EV4R	CAACGGTGGGGCGTAGTGCT	6277–6296
EV5F	GGCGGTAGTGAATGCCTTTG	6196–6215	F5 (1316)
EV5R	CCTGCTTACCAGAATGAACG	7492–7511
5′-GSP1	GAGGAGGAGTTATTCTTGTCTGAGG	942–966	5′-UTR
5′-GSP2	GGGGCCTAGACGTTTTTTAACCTC	659–682
5′-RACE outer primer	CATGGCTACATGCTGACAGCCTA
5′-RACE inner primer	CGCGGATCCACAGCCTACTGATCAGTCGATG
3′-GSP1	ATGTTGTCATACTATCGTCCAGG	7430–7452	3′-UTR
3′-RACE outer primer	TACCGTCGTTCCACTAGTGATTT

UTR — untranslated region.

^aBased on the sequence of EMCV strain BJC3 (GenBank accession number DQ464062).

Figure 2.

Phylogenetic analysis of EMCV isolates based on complete genome sequences. The numbers at the nodes represent bootstrap values greater than 50% of 1000 replicates. The scale bar represents 0.005 inferred substitutions per site.

To detect any differences in the deduced ORF amino acid sequences between NJ08 and the other tested isolates, the sequences were analyzed and aligned by means of DNAStar software (DNAStar, Madison, Wisconsin, USA). The results indicated that the ORFs of NJ08 encoded 11 proteins that were similar to those reported previously for other EMCV strains: VP1 to VP4, 2A to 2C, 3A to 3D, and a virus-leading protein (L protein) (12,13). Sequence comparisons revealed 78.3% to 100% identity of the proteins with those of the other 15 isolates (Table II). The most conserved proteins were VP2 and 3D, and the most variable proteins were VP1 and 2A. However, previous studies indicated that the predominant antigen epitopes of EMCV were located in the VP1 and VP3 proteins (11). This discrepancy might be due to the impact of selection pressure. Thus, the molecular epidemiology and the variation in VP1 of new EMCV isolates should be monitored.

Table II.

Percentage similarity in deduced amino acid sequence of the open reading frames of NJ08 and other encephalomyocarditis virus isolates encoding various proteins

	Protein; sequence similarity (%)
Strain	VP4	VP2	VP3	VP1	2A	2B	2C	3A	3B	3C	3D
BEL-2887A	100	100	100	98.6	99.4	100	99.8	98.9	100	100	100
CBNU	100	100	100	99.3	99.4	100	100	100	100	99.5	100
EMCV-R	100	100	100	98.9	99.4	100	99.9	100	100	99.5	100
GX0602	98.6	98.8	100	97.8	97.9	100	99.9	96.6	100	99.0	99.8
GXLC	100	99.6	100	98.9	98.1	100	100	100	100	100	99.8
HB1	100	100	99.6	99.6	98.7	100	99.9	100	100	100	99.6
BJC3	98.6	100	99.1	98.6	98.7	99.3	99.8	100	100	100	99.8
K3	100	100	99.6	99.6	98.7	100	99.9	100	100	100	99.6
K11	100	99.6	99.6	97.5	99.4	100	99.9	100	100	99.5	99.8
PEC9	100	99.6	99.1	98.2	98.1	100	99.7	100	100	99.5	99.6
PV21	100	99.2	100	99.3	98.6	100	99.9	98.9	100	99.0	100
D variant	98.6	98.8	100	98.9	99.4	99.3	99.7	98.9	95.0	100	100
EMCV-B	95.7	98.0	99.1	97.1	86.6	96.3	99.0	92.0	85.0	92.7	96.7
EMCV-D	95.7	98.4	99.1	96.8	86.0	96.3	98.7	93.2	85.0	92.2	96.5
Mengo Rz-pMwt	95.7	98.4	99.1	97.1	86.6	96.3	98.7	93.2	85.0	92.2	96.5
Mengo-M	97.1	97.7	97.8	96.4	78.3	97.1	97.8	90.9	85.0	88.0	93.3

In summary, this study confirmed the presence of EMCV in a pig herd in mideastern China. The isolate, NJ08, has high pathogenicity in mice and is closely related in genomic sequence to the tested Korean, European, and other Chinese isolates. These findings provide new epidemiologic data on EMCV in China.