A highly divergent Encephalomyocarditis virus isolated from nonhuman primates in Singapore

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 [1], baboons [2], chimpanzees [3], lemurs [4], rhesus macques [5], and orang utans [3, 6]. EMCV infection was also implicated in the deaths of various animals in Audubon Park Zoo, New Orleans, in 1985 [7]; and in elephants in a Florida zoo in 1997 [8]. 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 [3]; 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 [4]. 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 [11]. 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 [19].

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 [22]. 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[23], 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 [23].

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.

The virus outbreak in the Singapore Zoological Gardens took place over an estimated period of 8 months, and occurred in orang utans of various ages, as well as sporadically among other zoo animals. This is the first report of an EMCV infection in Singapore. There were altogether four deaths in the orang utan cohort, caused by acute myocarditis. Other animals, two zebras, one spider monkey, one guanaco, and two capybaras also died of similar clinical signs. One adult orang utan displayed respiratory problems but recovered with intensive supportive therapy.

From the two juvenile orang utans that died, viruses were isolated successfully from the heart (designated Sing-M105-02) and lung (designated Sing-M100-02) homogenates in Vero cells. Virus isolation was not performed on the two autopsied adult orang utans. The viral agents isolated in Vero cells were examined under transmission electron microscopy and the viruses showed similar morphology to the picornaviruses (Figure 1). In addition, virus-infected Vero cells showed positive immunofluorescence to polyclonal antiserum raised to EMCV (Figure 2). Our preliminary characterization of the Sing-M105-02 and Sing-M100-02 viruses indicated that the etiological agent in the Singapore zoo outbreak was an EMCV.

EMCV-specific primers targeting the VP3/VP1 capsid and 3Dpol genes were able to PCR-amplify products from the viral isolates. These primer sets had been tested on a wide variety of EMCV isolates from different geographic locations, different host species, and at different times [26‐28]. Structural proteins such as the VP1 capsid, have been shown to be suitable for phylogenetic analyses within different genera and species of picornaviruses [24, 33‐41]. Furthermore, analysis using the VP1 capsid could lead to the discovery of a novel variant such as the discovery reported for Hepatitis A [42]. Analysis at the VP1 capsid region revealed a maximum of 38.8% nt and 33.9% aa divergence between the Sing-M105-02 and Sing-M100-02 isolates and existing EMCV strains (Table 3). In addition, aa alignment of Sing-M105-02 and Sing-M100-02 against other EMCV strains showed considerable variation in two putative neutralization antigenic sites in VP1 (Figure 3), further supporting our hypothesis that these viral isolates can represent a new divergent group of EMCV and possibly a new serotype in the EMCV species.

The picornaviral polymerases or 3Dpol genes, on the other hand, are well conserved during evolution, and have been used as a common marker for comparisons between genera in phylogenetic studies [43‐45]. They can also be used to determine the degree of variation between the newly isolated viruses with existing EMCV strains. For example in Foot-and-Mouth-Disease viruses, high sequence divergence found in the 3Dpol regions is an indication of the virus strains diverging from the same isolate [46]. Sequence comparison for Sing-M105-02 and Sing-M100-02 isolates with existing EMCV strains showed a maximum divergence of 24.6% and 11.3% at the nt and aa levels respectively (Table 3), suggesting that they are indeed highly divergent variants of EMCV.

In addition, the phylogenetic analyses based on complete virus gene sequences (as compared to partial gene sequences) afforded us increased confidence that the Sing-M105-02 and Sing-M100-02 isolates constituted a divergent group of EMCV variants, as they clustered distinctly away from the other lineages of EMCV strains (Figure 4). This high degree of divergence within the EMCV species is perhaps not surprisingly as viruses with RNA genomes are generally reported to have a high rate of mutation [47‐49].

Our serological survey of the animal population further suggested that there were at least two virus strains circulating in the Singapore Zoo; the first, an EMCV(Aust)-like virus, and the second, a variant of EMCV. High seropositive rates for the EMCV variant was found in capybaras, chimpanzees, orang utans and two rats caught in baited traps. In addition, neutralizing antibodies to EMCV(Aust) were also found in all except one of the chimpanzee, one orang utan and one of the rats, indicating prior exposure to single or dual infections with the viruses. Although no attempt was made to recover the virus from these animals, we postulated that the EMCV(Aust)-like virus could have been the parental virus from which Sing-M105-02 and Sing-M100-02 viruses gradually evolved. Rats have been considered to be the primary host reservoir and disseminators of EMCV, and they have been implicated in other zoological outbreaks of EMCV [2‐4, 7, 9]. The rats could also have initiated the sporadic infections, by spreading both the parental and its variant virus in the zoo. However, this hypothesis remains to be investigated.

The etiological agent responsible for the outbreak for 2 of the 4 cases of fatal myocarditis in orang utans at the Singapore Zoological Gardens was an EMCV. High divergence in nt sequences of the VP1 capsid and 3Dpol genes as compared to other known EMCV strains, indicated that the outbreak was caused by a highly divergent variant of EMCV and possibly a new serotype of the virus. This is the first report of an EMCV infection in Singapore and South East Asia.