Duplex nested RT-PCR for detection of Nipah virus RNA from urine specimens of bats

doi:10.1016/j.jviromet.2006.11.023

Journal of Virological Methods

Volume 141, Issue 1, April 2007, Pages 97-101

https://doi.org/10.1016/j.jviromet.2006.11.023 Get rights and content

Abstract

A method for duplex nested RT-PCR (nRT-PCR) with internal control (IC) for the detection of Nipah virus RNA is described. Incorporation of IC RNA distinguished false and true negative results. The extrinsic RNA was added directly to the PCR master mix and co-amplified with virus specific RNA in a duplex reaction to determine the presence of PCR inhibitor. Limit of detection was affected minimally when IC was added. Of 53 pooled urine samples collected from fruit bats (Pteropus lylei), 16 were validated by the presence of IC band on gel electrophoresis. Seven of these were also Nipah virus RNA positive. The remaining 37 samples were considered invalid. Twenty-two urine samples became valid after dilution of 1:5 and re-examined; two were Nipah virus RNA positive. These nine positive results were confirmed by sequencing of heminested PCR products. The result indicated that at least two different Nipah strains circulated in this bat species from Thailand. This method should be useful for surveillance for Nipah virus infection in animals in a country where a biosecurity level (BSL) 4 laboratory is not available. PCR inhibitors were present in a significant number of bat urine samples. The technique described in this study should improve reliability of surveillance statistics.

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Acknowledgments

This work was supported by a grant from the Thailand Research Fund (grant DGB4880001) and the Thai Red Cross Society. SW and TH received funding support from the National Center for Genetic Engineering and Biotechnology, National Science and Technology Development, Thailand. We also acknowledge the help from the Division of Research Affair, Faculty of Medicine, Chulalongkorn University in the preparation of this manuscript.

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Citation Excerpt :
A putative NiV also caused an outbreak of disease in horses and people in the Philippines in 2014 [18•]. There is further evidence for broader distribution of NiV in flying fox species (pteropid fruit bats) across their range [19,20], and there is growing evidence that viruses related to HeV and NiV circulate in non-pteropid fruit bats across the globe [21,22,23,24,25•]. Nipah virus was first identified in 1998 in Malaysia, where it caused an outbreak of respiratory and neurological disease in pigs and encephalitis in people (reviewed by Ref. [26]).
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Pteropus bats (P. hypomelanus, P. lylei and P. vampyrus in Malaysia, P. giganteus in India and Bangladesh) appear to be the natural reservoir for NiV. Indeed it is possible the virus has co-evolved for millennia with this host due to the lack of symptoms in infected bats [10,11]. Experimentally infected bats inoculated subcutaneously with high doses of virus show no clinical disease, despite seroconversion and the detection of a low level of shed virus in urine [12].
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Short communicationDuplex nested RT-PCR for detection of Nipah virus RNA from urine specimens of bats

Abstract

Section snippets

Acknowledgments

Science

Microbes Infect.

Microbes Infect.

Microbes Infect.

Microbes Infect.

J. Virol. Methods

Microbes Infect.

Institute for Epidemiology Disease Control and Research, Ministry of Health and Family Welfare, Government of Bangladesh; Clinical Sciences Division and Health Systems and Infectious Diseases Division, ICDDR, B. Nipah virus outbreak from date palm juice

Health Sci. Bull.

Short communication
Duplex nested RT-PCR for detection of Nipah virus RNA from urine specimens of bats