Development of a SYBR Green I-based real-time PCR for detection of elephant endotheliotropic herpesvirus 1 infection in Asian elephants (Elephas maximus)

The Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand.
Journal of virological methods (Impact Factor: 1.78). 06/2012; 185(1):160-5. DOI: 10.1016/j.jviromet.2012.06.005
Source: PubMed


Elephant endotheliotropic herpesvirus 1 (EEHV1) can cause fatal hemorrhagic disease in Asian elephants (Elephas maximus). Several studies have described this virus as a major threat to young Asian elephants. A SYBR Green I-based real-time polymerase chain reaction (PCR) was developed to identify EEHV1 on trunk swabs and necropsied tissues. Two of 29 (6.9%) trunk swab samples from healthy Asian elephants were positive for EEHV1. The viruses were analyzed and classified as EEHV1A based on 231 nucleotides of the terminase gene. Necropsied spleen and heart tissue showed the highest level and second highest levels of DNA virus copy accumulation, respectively. The detection limit of the test was 276 copies/μl of DNA. There was no cross-reaction with other mammalian herpesviruses, such as herpes simplex virus 1 and equine herpesvirus 2. Inter- and intra-assay showed low coefficients of variation values indicating the reproducibility of the test. The results indicated that the test can be practically used for epidemiological study, clinical diagnosis, and management and control of EEHV1.

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    • "At present, PCR combined with nucleotide sequencing has been available for routine diagnosis and strain identification (Richman et al., 2000; Fickel et al., 2001; Garner et al., 2009; Latimer et al., 2011; Atkins et al., 2013). Recently, real-time PCR has developed as an alternative technique for strain identification (Stanton et al., 2010; Hardman et al., 2012; Sariya et al., 2012; Stanton et al., 2012). The aim of the study was to characterize the partial genome of EEHV detected from captive Asian elephants in Thailand from 2007 to 2013. "
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    ABSTRACT: The study was aimed at characterizing elephant endotheliotropic herpesvirus (EEHV) that was detected in captive Asian elephants in Thailand from 2007 to 2013. Six tissue samples of dead elephants and two EDTA blood samples of surviving elephants in Thailand showed clinical signs or had lesions of the viral infection. Samples were extracted for DNA amplification using a PCR technique with strain specific primers based on terminase and DNA polymerase genes. Six samples gave positive amplicons for EEHV1 specific primers and two samples gave positive amplicons for EEHV3/4 specific primers. Nucleotide sequencing analysis was assured for strain identification. Five out of the six samples from EEHV1 PCR were positive for the EEHV1A strain and one sample was positive for the EEHV1B strain. The two samples of EEHV3/4 PCR positive products were revealed to be of the EEHV4 strain based on the sequencing of the partial terminase gene. Three strains of the EEHV including EEHV1A, EEHV1B and EEHV4 have been detected in Asian elephants in Thailand from 2007 to 2013. This study revealed the first EEHV1B isolate that has been detected in a captive Asian elephant in Thailand.
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    • "While the observed EEHV1 miRNA(s) are unlikely to represent targets for intervention in EEHV-induced disease, they may have potential as diagnostics and we were indeed able to readily detect miR-E3-5p expression in in vivo-derived samples, including whole blood, by RT-PCR (Table 2). Many EEHV1 infections appear to be sub-clinical and there is considerable interest in developing diagnostic tests for EEHV1 to prevent infection of naïve animals (Hardman et al., 2012; Sariya et al., 2012; Stanton et al., 2010). "
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    ABSTRACT: Elephant endotheliotropic herpesvirus 1 (EEHV1), a member of the Betaherpesvirinae subfamily, has recently emerged as an important viral pathogen of Asian elephants that can cause a severe, often fatal, hemorrhagic disease. EEHV1 does not replicate in culture and little is currently known about the molecular biology of this emerging pathogen, with the notable exception of its genomic DNA sequence. Here, we have used small RNA deep sequencing to determine whether EEHV1, like other human and murine betaherpesviruses, expresses viral microRNAs in infected tissues in vivo. Our data provide evidence supporting the existence of at least three novel viral microRNAs encoded by EEHV1 and one of these, miR-E3-5p, is shown to repress target mRNA expression. Moreover, miR-E3-5p expression was readily detectable in tissue samples derived from two infected elephants, including in whole blood. These data shed new light on the biology of EEHV1 and identify small RNAs that have the potential to be useful in the diagnosis of sub-clinical infections in captive Asian and African elephants.
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