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A highly divergent Encephalomyocarditis virus isolated from nonhuman primates in Singapore.

Yeo DS, Lian JE, Fernandez CJ, Lin YN, Liaw JC, Soh ML, Lim EA, Chan KP, Ng ML, Tan HC, Oh S, Ooi EE, Tan BH - Virol. J. (2013)

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

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.

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Immunoflorescence staining of Vero cells infected with (a) Sing-M105-02 isolate, (b) Sing-M100-02 isolate, and (c) EMCV(Aust) strain. The infected cells were allowed to react with porcine polyclonal antibodies raised against EMCV. The virus-infected Vero cells stained greenish yellow in the cytoplasm (), and at the perinuclear region (), indicating the presence of virus antigens. (d) Uninfected Vero cells stained red with counter stain. Magnification at x60.
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Figure 2: Immunoflorescence staining of Vero cells infected with (a) Sing-M105-02 isolate, (b) Sing-M100-02 isolate, and (c) EMCV(Aust) strain. The infected cells were allowed to react with porcine polyclonal antibodies raised against EMCV. The virus-infected Vero cells stained greenish yellow in the cytoplasm (), and at the perinuclear region (), indicating the presence of virus antigens. (d) Uninfected Vero cells stained red with counter stain. Magnification at x60.

Mentions: A second passage of the virus isolates in Vero cells resulted in rapid and advanced CPE a day after inoculation. Sing-M105-02 (Figure 2a) and Sing-M100-02 (Figure 2b) virus-infected Vero cells reacted to polyclonal antiserum raised to EMCV with specific green and yellow fluorescence staining in the cytoplasm. The perinuclear regions were stained more intensely. These results were consistent with observations made for EMCV(Aust)-infected Vero cells (Figure 2c), indicating that the Sing-M105-02 and Sing-M100-02 viruses could be EMCV. Cytoplasmic areas appeared red with counter stain Evans Blue, and no green fluorescence was observed in uninfected Vero cells (Figure 2d).


A highly divergent Encephalomyocarditis virus isolated from nonhuman primates in Singapore.

Yeo DS, Lian JE, Fernandez CJ, Lin YN, Liaw JC, Soh ML, Lim EA, Chan KP, Ng ML, Tan HC, Oh S, Ooi EE, Tan BH - Virol. J. (2013)

Immunoflorescence staining of Vero cells infected with (a) Sing-M105-02 isolate, (b) Sing-M100-02 isolate, and (c) EMCV(Aust) strain. The infected cells were allowed to react with porcine polyclonal antibodies raised against EMCV. The virus-infected Vero cells stained greenish yellow in the cytoplasm (), and at the perinuclear region (), indicating the presence of virus antigens. (d) Uninfected Vero cells stained red with counter stain. Magnification at x60.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750836&req=5

Figure 2: Immunoflorescence staining of Vero cells infected with (a) Sing-M105-02 isolate, (b) Sing-M100-02 isolate, and (c) EMCV(Aust) strain. The infected cells were allowed to react with porcine polyclonal antibodies raised against EMCV. The virus-infected Vero cells stained greenish yellow in the cytoplasm (), and at the perinuclear region (), indicating the presence of virus antigens. (d) Uninfected Vero cells stained red with counter stain. Magnification at x60.
Mentions: A second passage of the virus isolates in Vero cells resulted in rapid and advanced CPE a day after inoculation. Sing-M105-02 (Figure 2a) and Sing-M100-02 (Figure 2b) virus-infected Vero cells reacted to polyclonal antiserum raised to EMCV with specific green and yellow fluorescence staining in the cytoplasm. The perinuclear regions were stained more intensely. These results were consistent with observations made for EMCV(Aust)-infected Vero cells (Figure 2c), indicating that the Sing-M105-02 and Sing-M100-02 viruses could be EMCV. Cytoplasmic areas appeared red with counter stain Evans Blue, and no green fluorescence was observed in uninfected Vero cells (Figure 2d).

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

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.

Show MeSH
Related in: MedlinePlus