Between July 2001 and January 2002, four (3 Bornean, Pongo pygmaeus and 1 Sumatran, Pongo abelii) orang utans, two (male and female) adults and two (male and female) juveniles, from a cohort of 26 in the Singapore Zoological Gardens died suddenly. Post-mortem examination of the four orang utans revealed pathology characterized by multifocal myocarditis, pulmonary congestion and edema, hydropericardium, hydrothorax and Ascites.
A literature review revealed several reports describing Encephalomyocarditis virus (EMCV) as the etiological agent of viral myocarditis in captive primates. These primates included semi-wild bonobos , baboons , chimpanzees , lemurs , rhesus macques , and orang utans [3, 6]. EMCV infection was also implicated in the deaths of various animals in Audubon Park Zoo, New Orleans, in 1985 ; and in elephants in a Florida zoo in 1997 . Outside the United States, sporadic outbreaks of EMCV involving a variety of zoo animals had occurred in Taronga Zoo in Australia from 1987 to 1995 ; in free-living elephants in Kruger National Park, South Africa between 1993 and 1994 [9, 10]; and in an Italian zoo affecting 15 different primates between 2006 and 2008 . In addition, EMCV infections were reported in Russia from monkeys bred from the Sukhumi Breeding Center in 1974; and in the Adler Breeding Center, since 2001 . EMCV has also been recognized as a porcine pathogen, with EMCV infections in European pigs associated with sudden deaths and reproductive failure [12–16]. In Asia, EMCV was isolated from pigs in South Korea and implicated as the cause of reproductive failure in pigs in Taiwan [17, 18]. Apart from EMCV, there has been one other report describing Coxsackie virus B4 as the etiological cause of fatal myocarditis in a female orang utan at the Okinawan Zoo in 1999 .
Although the potential for EMCV to cross the species barrier has been demonstrated as seen from the various zoo outbreaks described above, human cases have fortunately been rare. Sporadic human EMCV infections and disease have been documented by virus isolation from different specimen types such as serum, stool samples, cerebrospinal fluid and throat washings [20, 21]. A recent study describing the etiology of acute febrile disease in locations across South America concluded that there is evidence supporting a role for EMCV in human infection and febrile illness . However, the extent of the effect of EMCV on human health is still largely unknown because the disease is so infrequent in humans.
We hypothesized that the etiological agent behind the deaths of the orang utans in the Singapore Zoological Gardens was either a Coxsackie virus B, or more likely, an EMCV. Although both viruses are members of the same family, Picornaviridae, they belong to different genera as Coxsackie virus B is an Enterovirus, and EMCV, a Cardiovirus. The Picornaviridae family is one of the largest and most diverse families of RNA viruses, and includes etiological agents that are responsible for a wide variety of human and animal diseases .
The picornavirions are small, non-enveloped and spherical, with a diameter of about 20 to 30 nm. Picornaviruses have a single-stranded, positive sense RNA genome that is between 7.2 and 9 kb in length and contain a large opening reading frame (ORF). The ORF encodes for a polyprotein that comprises both non-structural and structural elements divided into three primary precursor molecules, namely P1, P2 and P3, encoding for 11 distinct proteins. The structural proteins VP4, VP2, VP3 and VP1 make up the viral capsid and are encoded in the P1 region towards the 5′-end of the genome. Non-structural proteins are derived from the P2 and P3 regions and are encoded towards the 3′-end of the genome, the largest of which is the RNA-dependent RNA polymerase (3Dpol). In addition, cardioviruses code for an L (Leader) protein at the N-terminus of their polyproteins. The genomic RNA also contains a highly structured 5′-UTR (untranslated region) that includes an internal ribosome entry site (IRES) from which viral protein translation is initiated in a cap-independent manner. The shorter 3′-UTR terminates with a heterogeneous poly(A) tail that is known to be involved in the binding process of the viral replicase complex.
In this paper, we described the cell culture isolation and identification of two viral isolates obtained post-mortem from the two juvenile Bornean orang utans. The viral genomes of the two isolates were >95% sequenced, with the complete genome sequence lacking about 200 nucleotides (nt) from the 5′ end. Phylogenetic and sequence analyses suggested that the newly isolated viruses are highly divergent variants of EMCV and possibly a new serotype of the virus. This is the first report of EMCV infection in Singapore and South East Asia and its molecular characterization.