As. Pac. J. Mol. Biol. & Biotech., Dec 2000 Vol. 8(2) : 101-106

The identification and distinction between Nipah virus and Hendra virus by using RT PCR, sequencing and restriction enzyme analysis

M. Maizan, A.R. Mohd. Ali and S.H. Sharifah*

Veterinary Research Institute, 59, Jalan Sultan Golan Shah 31400, Ipoh Perak

Received 28 August 2000 / Accepted 12 December 2000

Abstract.

A cytopathogenic infectious agent isolated from the lungs of dead and sick pigs with severe pneumonia during the 1998/1999 Nipah virus outbreak was used for the development of a RT-PCR, for the rapid diagnosis of Nipah virus infection. Oligonucleotide primers were constructed and reverse transcriptase polymerase chain reaction (RT-PCR) and nested PCR were established for the detection of Nipah and Hendra viruses. The primary and nested PCR based on the primers of specific regions within the N genes of both viruses amplified a 397 bp and a 300 bp product respectively. The nucleotide sequence of the amplified 397 bp fragments of the 1998 pig isolate and the naturally occurring Nipah virus from the buffy coat of blood of pigs from a recently identified farm was compared with the published N gene sequences of the Nipah and Hendra viruses. The sequences of the 397 bp PCR product derived from tissue culture infected cells and buffy coats showed 99% and 70.8% homology with Nipah and Hendra virus respectively. The amplified products were further identified as Hendra or Nipah virus by restriction enzyme analysis using EcoRV.

Key Words: Nipah virus, RT PCR, sequencing, restriction enzyme analysis