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A highly divergent Encephalomyocarditis virus isolated from nonhuman primates in Singapore

Dawn Su-Yin Yeo1, Jing Er Lian2, Charlene J Fernandez3, Yueh-Nuo Lin3, Jasper Chin-Wen Liaw1, Moi-Lien Soh3, Elizabeth Ai-Sim Lim1, Kwai-Peng Chan4, Mah-Lee Ng5, Hwee-Cheng Tan6, Serena Oh7, Eng-Eong Ooi6 and Boon-Huan Tan189*

Author Affiliations

1 Defence Medical and Environmental Research Institute, DSO National Laboratories, #13-00, 27 Medical Drive, Buona 117510, Singapore

2 School of Biological Sciences, Nanyang Technological University, Nanyang, Singapore

3 Agri-Food and Veterinary Authority of Singapore, Singapore, Singapore

4 Department of Pathology, Singapore General Hospital, Changi, Singapore

5 Department of Microbiology, National University of Singapore, Singapore, Singapore

6 Duke-NUS GMS, National University of Singapore, Singapore, Singapore

7 Wildlife Reserves Singapore, Singapore, Singapore

8 Saw Swee Hock School of Public Health, National University of Singapore, Singapore, Singapore

9 Department of Civil and Environmental Engineering, National University of Singapore, Singapore, Singapore

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Virology Journal 2013, 10:248  doi:10.1186/1743-422X-10-248

Published: 2 August 2013

Abstract

Background

In 2001 and 2002, fatal myocarditis resulted in the sudden deaths of four, two adult and two juvenile, orang utans out of a cohort of 26 in the Singapore Zoological Gardens.

Methods

Of the four orang utans that underwent post-mortem examination, virus isolation was performed from the tissue homogenates of the heart and lung obtained from the two juvenile orang utans in Vero cell cultures. The tissue culture fluid was examined using electron microscopy. Reverse transcription and polymerase chain reaction with Encephalomyocarditis virus (EMCV)-specific primers targeting the gene regions of VP3/VP1 and 3D polymerase (3Dpol) confirmed the virus genus and species. The two EMCV isolates were sequenced and phylogenetic analyses of the virus genes performed. Serological testing on other animal species in the Singapore Zoological Gardens was also conducted.

Results

Electron microscopy of the two EMCV isolates, designated Sing-M100-02 and Sing-M105-02, revealed spherical viral particles of about 20 to 30 nm, consistent with the size and morphology of members belonging to the family Picornaviridae. In addition, infected-Vero cells showed positive immunoflorescence staining with antiserum to EMCV. Sequencing of the viral genome showed that the two EMCV isolates were 99.9% identical at the nucleotide level, indicating a similar source of origin. When compared with existing EMCV sequences in the VP1 and 3Dpol gene regions, the nucleotide divergence were at a maximum of 38.8% and 23.6% respectively, while the amino acid divergence were at a maximum of 33.9% and 11.3% respectively. Phylogenetic analyses of VP1 and 3Dpol genes further grouped the Sing-M100-02 and Sing-M105-02 isolates to themselves, away from existing EMCV lineages. This strongly suggested that Sing-M100-02 and Sing-M105-02 isolates are highly divergent variants of EMCV. Apart from the two deceased orang utans, a serological survey conducted among other zoo animals showed that a number of other animal species had neutralizing antibodies to Sing-M105-02 isolate, indicating that the EMCV variant has a relatively wide host range.

Conclusions

The etiological agent responsible for the fatal myocarditis cases among two of the four orang utans in the Singapore Zoological Gardens was a highly divergent variant of EMCV. This is the first report of an EMCV infection in Singapore and South East Asia.

Keywords:
Encephalomyocarditis virus; Variant virus; Fatal acute myocarditis; Orang utan