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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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Phylogenetic trees of the full genes of the (A) VP1 capsid, (B) 3D polymerase (3Dpol), and (C) open reading frame (ORF) of Encephalomyocarditis viruses (EMCV) and Theiloviruses based on their nucleotide sequence. Isolate names are followed by their GenBank accession number. The EMCV isolates generated in this study, Sing-M100-02 and Sing-M105-02, are highlighted in a shaded box. Percentage bootstrap values (1000 trials) of the major nodes are shown. Trees were constructed from CLUSTAL W method in the program MegAlign (DNASTAR, Lasergene Version 8), and viewed with TreeView 1.6.6.
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Figure 4: Phylogenetic trees of the full genes of the (A) VP1 capsid, (B) 3D polymerase (3Dpol), and (C) open reading frame (ORF) of Encephalomyocarditis viruses (EMCV) and Theiloviruses based on their nucleotide sequence. Isolate names are followed by their GenBank accession number. The EMCV isolates generated in this study, Sing-M100-02 and Sing-M105-02, are highlighted in a shaded box. Percentage bootstrap values (1000 trials) of the major nodes are shown. Trees were constructed from CLUSTAL W method in the program MegAlign (DNASTAR, Lasergene Version 8), and viewed with TreeView 1.6.6.

Mentions: We included fully sequenced EMCV and Theilovirus strains in our phylogenetic trees to demonstrate the divergence of the 2 species in the genus Cardiovirus, as well as to emphasize the diversity and clustering of various EMCV strains (Figure 4). Our phylogenetic analyses showed branching of the EMCV strains into four main lineages, A, B, C and D, at the nt level in the VP1 and 3Dpol regions, as well as for the entire ORF (Figure 4). Lineages A, B and C concurred with similar clusterings of EMCV strains as described previously [10,26,32]. However, the Sing-M105-02 and Sing-M100-02 viruses clustered separately as a distinct group by themselves in lineage D (Figure 4). A recent report of a second serotype of EMCV, isolate RD1338 [29], also clustered by itself in lineage E, highly distinct from the Sing-M105-02 and Sing-M100-02 viruses (lineage D) and other EMCV in lineages A, B and C. Our phylogenetic studies further demonstrated that Sing-M105-02 and Sing-M100 viruses are highly divergent strains of EMCV.


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)

Phylogenetic trees of the full genes of the (A) VP1 capsid, (B) 3D polymerase (3Dpol), and (C) open reading frame (ORF) of Encephalomyocarditis viruses (EMCV) and Theiloviruses based on their nucleotide sequence. Isolate names are followed by their GenBank accession number. The EMCV isolates generated in this study, Sing-M100-02 and Sing-M105-02, are highlighted in a shaded box. Percentage bootstrap values (1000 trials) of the major nodes are shown. Trees were constructed from CLUSTAL W method in the program MegAlign (DNASTAR, Lasergene Version 8), and viewed with TreeView 1.6.6.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Phylogenetic trees of the full genes of the (A) VP1 capsid, (B) 3D polymerase (3Dpol), and (C) open reading frame (ORF) of Encephalomyocarditis viruses (EMCV) and Theiloviruses based on their nucleotide sequence. Isolate names are followed by their GenBank accession number. The EMCV isolates generated in this study, Sing-M100-02 and Sing-M105-02, are highlighted in a shaded box. Percentage bootstrap values (1000 trials) of the major nodes are shown. Trees were constructed from CLUSTAL W method in the program MegAlign (DNASTAR, Lasergene Version 8), and viewed with TreeView 1.6.6.
Mentions: We included fully sequenced EMCV and Theilovirus strains in our phylogenetic trees to demonstrate the divergence of the 2 species in the genus Cardiovirus, as well as to emphasize the diversity and clustering of various EMCV strains (Figure 4). Our phylogenetic analyses showed branching of the EMCV strains into four main lineages, A, B, C and D, at the nt level in the VP1 and 3Dpol regions, as well as for the entire ORF (Figure 4). Lineages A, B and C concurred with similar clusterings of EMCV strains as described previously [10,26,32]. However, the Sing-M105-02 and Sing-M100-02 viruses clustered separately as a distinct group by themselves in lineage D (Figure 4). A recent report of a second serotype of EMCV, isolate RD1338 [29], also clustered by itself in lineage E, highly distinct from the Sing-M105-02 and Sing-M100-02 viruses (lineage D) and other EMCV in lineages A, B and C. Our phylogenetic studies further demonstrated that Sing-M105-02 and Sing-M100 viruses are highly divergent strains of EMCV.

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