Porcine encephalomyocarditis virus persists in pig myocardium and infects human myocardial cells

Abstract

Recent advances toward using pig tissues in human transplantation have made it necessary to determine the risk of transmitting zoonotic viruses from pigs to humans or vice versa. We investigated the suitability of the porcine encephalomyocarditis virus (EMCV) model for such studies by determining its ability to persist in pigs, escape detection by routine serological methods, and infect human cells. Intraperitoneal inoculation of 5-week-old pigs with EMCV-30, a strain isolated from commercial pigs, resulted in acute cellular degeneration, infiltration of lymphocytes, and apoptosis in myocardium in 13 of 15 (86.7%) pigs during the acute phase of disease (3 to 21 days postinfection), followed by less-severe lymphocytic infiltration and apoptosis in 5 of 10 (50%) pigs during the chronic phase of the disease (day 45 to 90 postinfection). In the brain, lymphocytic infiltration, neuronal degeneration, and gliosis were observed in 26 to 33% of pigs in the acute phase of disease whereas perivascular cuffing was the predominant feature during chronic disease. EMCV antigens and RNA were demonstrated in the myocardium and brain during the chronic phase of disease. Analysis of 100 commercial pigs that were negative for EMCV antibodies identified two pig hearts positive for EMCV RNA. Porcine EMCV productively infected primary human cardiomyocytes as demonstrated by immunostaining using a monoclonal antibody specific for EMCV RNA polymerase, which is expressed only in productively infected cells, and by a one-step growth curve that showed production of 100 to 1,000 PFU of virus per cell within 6 h. The findings that porcine EMCV can persist in pig myocardium and can infect human myocardial cells make it an important infectious agent to screen for in pig-to-human cardiac transplants and a good model for xenozoonosis.

FIG. 1

EMCV-induced pathologic changes in the heart and brain. Five-week-old pigs were intraperitoneally inoculated with 2.9 × 10⁸ PFU of EMCV-30, and histopathologic changes were, analyzed at days 7, 21, 45, and 90 p.i. Micrographs show acute inflammatory and degenerative changes in the myocardium at 7 days p.i. (A), an inflammatory and fibrotic myocardial lesion at 21 days p.i. (B), myocardial lymphocyte infiltration at 90 days p.i. (C), and perivascular cuffing in the cerebral cortex at 90 days p.i. (D). Heart and brain sections were embedded in paraffin, and 4-μm-thick sections were stained with hematoxylin and eosin. Magnification, ×200 for panels A through C and ×400 for panel D.

FIG. 2

Scatter plot showing pathology scores of heart tissues from EMCV-infected pigs. Five-week-old pigs were intraperitoneally inoculated with 2.9 × 10⁸ PFU of EMCV-30, and histopathologic changes were analyzed at 3 (n = 4), 7 (n = 5), 21(n = 6), 45 (n = 5), and 90 (n = 5) days p.i. Each heart tissue section was given a score between 0 (no pathology) and 4 (necrosis on days 3 and 7, or fibrosis on days 21, 45, and 90) based on the extent of pathology as described in Materials and Methods. Solid lines represent the mean score at each time point (3 at day 3, 2.7 at day 7, 1.6 at day 21, 1.4 at day 45, and 1.2 at day 90 p.i.). The data indicate that EMCV, causes acute damage to the heart tissue, but in some pigs it can establish persistent infection.

FIG. 3

EMCV-induced apoptosis in pig myocardium. Frozen sections of the heart were immunostained to identify cells undergoing apoptosis using the in situ end-labeling peroxidase-based detection system. Extensive apoptosis (brown-stained cells indicated by arrows) was observed at day 7 p.i., and fewer apoptotic cells were detectable at days 45 and 90 p.i. Uninfected myocardial sections were negative. Sections were lightly counterstained with hematoxylin. Magnification, ×100.

FIG. 4

Localization of EMCV RNA and antigens in the hearts and brains of chronically EMCV-infected pigs. EMCV RNA was localized by in situ hybridization using a 309-bp ³⁵S-labeled VP2-specific probe in the myocardium (A) and brain (B) of a pig infected for 90 days. Black grains (arrows) indicate viral RNA-positive cells. (C) Demonstration of EMCV antigens (brown staining, indicated by arrows) in the myocardium of a 90-day-infected pig by immunohistochemistry using a monoclonal antibody specific for EMCV RNA polymerase. Magnification, ×268 for panels A and B and ×134 for panel C.

FIG. 5

Detection of EMCV RNA in pig tissues by RT-PCR. Five-week-old pigs were intraperitoneally inoculated with 2.9 × 10⁸ PFU of EMCV-30, and the brain, heart, kidney, liver, spleen, and skeletal muscle were tested by nested RT-PCR for EMCV RNA using VP1- or VP2-specific primer sets at days 7, 21, 45, and 90 p.i. (A) Agarose DNA gel showing RT-PCR products specific for VP1 (436 bp) and VP2 (390 bp) in the heart (lanes 2), liver (lanes 4), spleen (lanes 5), and skeletal muscle (lanes 6) of a pig infected for 90 days. The brain was positive with VP1 primers (lane 1) but negative with VP2 primers (lane not shown), whereas the kidney was negative with both VP1 and VP2 primers. (B) Presence of EMCV RNA in hearts of seronegative commercial pigs. Hearts were obtained at the time of slaughter and tested by nested RT-PCR. The gel shows that pig 67 was positive for VP1 RNA whereas the rest of the pigs (animals 61 to 66 and 68 to 70) were negative. Two of the 100 pig hearts analyzed (from 10 different herds) were positive.

FIG. 6

Detection of EMCV-specific IgG in sera from experimentally infected and commercial pigs. EMCV-specific IgGs were determined by ELISA. (A) Virus-specific IgG levels in sera of pigs infected for 7, 21, 45, and 90 days. Data are means (± standard errors) of A₄₀₅ readings from four to five serum samples at each time point, performed at serum dilutions between 1:125 and 1:128,000. (B) Profile of virus-specific IgG levels during acute (day 7 to 21) and chronic (day 45 to 90) infection determined at a 1:500 serum dilution, showing that antibody levels peaked at day 21 p.i. before decreasing to low levels at days 45 and 90 p.i. (C) Analysis of EMCV-specific IgGs in sera from 10 commercial pig herds obtained from an EMCV-free farm over a 10-month period. Each herd is represented by the mean (± standard error) A₄₀₅ reading of serum samples from 10 pigs. Sera from pigs experimentally infected with EMCV for 21 days were used as a positive control.

FIG. 7

EMCV antigens and RNA in primary human myocardial cells. Primary human cardiomyocytes obtained from Clonetics were inoculated with 3 to 5 PFU of EMCV-30 per cell, for 7 h. Cells were harvested and immunostained using a anti-EMCV RNA polymerase (3D protein) monoclonal antibody. Infected human cardiomyocytes were positive, as shown by the brown staining in the cytoplasm (B), whereas uninfected cells were negative (A). In addition, in situ hybridization using a 309-bp ³⁵S-labeled probe specific for the VP2 gene of EMCV was used to localize viral RNA in the infected cells. Large amounts of viral RNA were observed in human cardiomyocytes (black grains) infected with EMCV (D), whereas uninfected cells hybridized with the VP2 cDNA probe were negative (C). Cells were lightly counterstained with hematoxylin. Magnification, ×400.

FIG. 8

Growth curve demonstrating productive infection of primary human cardiomyocytes by porcine EMCV. Confluent primary human cardiomyocytes in 12-well tissue culture plates were inoculated with 10 PFU of EMCV-30 per cell for 1, 2, 4, 6, 8, or 16 h before harvesting. Infected cells were washed to remove unattached virus, and four wells were harvested at each time point for a plaque assay (3.1 × 10⁵ cells/well). Cells were freeze-thawed and sonicated to release intracellular virus and clarified by centrifugation, and serial 10-fold dilutions were added to confluent HeLa cells for plaque development. Results are expressed as the mean (± standard error) number of plaques per 6.2 × 10⁵ cells (from two wells) of four samples per time point. They show a 4-h lag phase followed by a 2-h exponential phase characterized by rapid production of infectious EMCV particles. The limit of detection for the test was 2 PFU per 6.2 × 10⁵ cells.