Previous studies using wild-type Encephalomyocarditis virus (EMCV) and Mengo virus, which have long poly(C) tracts (61 to 146 C's) at the 5 nontranslated region of the genome, and variants of these viruses genetically engineered to truncate or substitute the poly(C) tracts have produced conflicting data on the role of the poly(C) tract in the virulence of these viruses. Analysis of the nucleotide sequence of an EMCV strain isolated from an aborted swine fetus (EMCV 30/87) revealed that the virus had a poly(C) tract that was 7-to 10-fold shorter than the poly(C) tracts of other EMCV strains and 4-fold shorter than that of Mengo virus. Subsequently, we investigated the virulence and pathogenesis of this naturally occurring short-poly(C)-tractcontaining virus in rodents, pigs, and nonhuman primates. Infection of C57BL/6 mice, pigs, and cynomolgus macaques resulted in similar EMCV 30/87 pathogenesis, with the heart and brain as the primary sites of infections in all three animals, but with different disease phenotypes. Sixteen percent of EMCV 30/87-infected pigs developed acute fatal cardiac failure, whereas the rest of the pigs were overtly asymptomatic for as long as 90 days postinfection (p.i.), despite extensive myocardial and central nervous system (CNS) pathological changes. In contrast, mice infected with >4 PFU of EMCV 30/87 developed acute encephalitis that resulted in the death of all animals (n ‫؍‬ 25) between days 2 and 7 p.i. EMCV 30/87-infected macaques remained overtly asymptomatic for 45 days, despite extensive myocardial and CNS pathological changes and viral persistence in more than 50% of the animals. The short poly(C) tract in EMCV 30/87 (CUC 5 UC 8 ) was comparable to that of strain 2887A/91 (C 10 UCUC 3 UC 10 ), another recent porcine isolate.Encephalomyocarditis virus (EMCV) is a picornavirus belonging to the Cardiovirus genus that infects many animal species including pigs (17), rodents (45), cattle (41), elephants (13), raccoons (47), marsupials (38), baboons, macaques, chimpanzees, and humans (2,16,21,38,44,45). Rats and mice are the natural hosts of the virus, but pigs are the most commonly and severely infected domestic animals (5, 36). EMCV strains have been isolated from primates, pigs, and rodents (8,15,20,22,24,29). EMCV Rueckert (EMCV R/45), the prototype strain, was isolated from a 5-year-old chimpanzee that suffered acute fatal myocarditis in 1945 (15), whereas EMC-M/58 virus was isolated from a naturally infected domestic pig suffering from severe myocarditis (29). A number of EMC-M/58 variants, including EMC-D/58, EMC-B/58, PV2/58, and PV21/58, were generated in laboratories and used to study the pathogenesis of viral diabetes (6,7,18,25,46,49). Mengo virus, another cardiovirus that shares the same serotype as EMCV, was isolated from the cerebrospinal fluid of a paralyzed rhesus macaque in 1948 (8). EMCV 30/87 and 2887A/91 are more recent EMCV isolates obtained from aborted swine fetuses in the United States and Belgium, respectively, following natural EMCV outbreaks in domestic ...

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.

We recently demonstrated that pigs infected with porcine encephalomyocarditis virus (EMCV) develop a persistent infection (up to 90 days post-infection (PI)) in the heart and brain that is accompanied by virus-induced pathologic changes, and that EMCV productively infects human cardiomyocytes in vitro, suggesting that EMCV may pose a risk to humans following transplantation of pig tissues to humans (Brewer et al. J Virol 2001; 75: 11621-11629). In this report, we demonstrate that intra-abdominal of myocardial or pancreatic sections from acutely-EMCV infected pigs (2 days PI) in either non-mutant C57BL/6 or C57BL/6-RAG-1-/- mice that lack B or T lymphocytes, resulted in transmission of the virus and acute fatal disease in all mice. In recipient RAG-1-/- mice, fatal EMCV disease occurred within 2 days post-transplantation, and it was accompanied by high virus titers in brain, heart, liver, spleen, kidneys and skeletal muscle, whereas in non-mutant C57BL/6 mice, disease occurred 5 to 6 days post-transplantation and was accompanied by lower virus titers. Transplantation of myocardial or pancreatic tissues from chronically EMCV-infected pigs (21 and 50 days PI) did not induce clinical disease, but resulted in detection of EMCV RNA in the brain of recipient RAG-1-/- mice, no viral RNA was detected in non-mutant C57BL/6 mice. Intra-abdominal transplantation of uninfected porcine myocardial tissues into RAG-1-/- mice followed by intramuscular inoculation with EMCV induced acute clinical disease but did not result in transmission of virus to the xenograft. These results show that EMCV can be efficiently transmitted from pig myocardial and pancreatic tissues to mice, providing a model of pig-to-human viral xenozoonosis that can be used to develop and test prophylactic and therapeutic measures against such infection.

Although PICs are susceptible to human CVB-5, the infection does not appear to affect xenograft function in vitro or in vivo in the short term.

Previous studies demonstrated that porcine encephalomyocarditis virus (EMCV) caused acute and persistent infection in the myocardium, central nervous system, and spleen of non-human primates (cynomolgus macaques); and it productively infected primary human cardiomyocytes, suggesting that the virus may pose a risk in pig-to-human transplantation. Recently, transplantation of myocardial and pancreatic tissues from acutely infected pigs transmitted the virus to recipient mice, resulting in acute fatal EMCV disease. Here, we examined whether porcine islet cells (PICs), which are under clinical trial for treatment of type I diabetes in humans, are susceptible to porcine EMCV, and whether EMCV-infected PICs could function in vivo to reverse diabetes. PICs were infected with EMCV in vitro for 5 h, and resulting insulin production compared with that produced by uninfected PICs. Subsequently, infected PICs were transplanted intra-abdominally or under the kidney capsule of C57BL/6 mice, and both virus transmission and PIC function analyzed. PICs were highly susceptible to porcine EMCV, resulting in a 1500-fold increase in production of infectious virus within 5 h of inoculation and cytolysis that destroyed up to 50% of cells within 96 h. However, as long as they were viable, infected PICs produced insulin at levels comparable with uninfected PICs. Intra-abdominal transplantation of 2000 PICs, infected with one plaque forming unit (pfu) per cell of porcine EMCV, into C57BL/6 mice transmitted the virus resulting in acute fatal EMCV disease characterized by hind limb paresis and paralysis and acute respiratory distress in 40% of recipient mice. More importantly, transplantation of 2500 EMCV-infected PICs under the kidney capsule of diabetic C57BL/6 mice (glucose level > or =350 mg/dl) reversed diabetes in 83% of recipient mice (glucose level < or =170 mg/dl); however these mice succumbed to acute EMCV disease transmitted by the xenograft 5 days after transplantation. EMCV infection does not appear to affect insulin production by PICs, but infected xenografts can transmit the virus to recipient animals, resulting in severe disease.