MJA: Indentifying isolates of Japanese encephalitis virus

2: Identification of isolates of Japanese encephalitis virus

Isolate identification was performed by immunofluorescence of infected C6/36 cells using a panel of monoclonal antibodies (6B6C-1*, 6B4A-10*, M-E6** , M2-8C4** and KUN 2-2B2**), and confirmed by nucleotide sequence of the prM gene and a 500-nucleotide sequence encompassing the N-terminus of the NS5 gene and part of the untranslated region (UTR) of the virus. Two primer pairs were used: one pair was designed to amplify the prM gene (primer PrMf: CGT TGA ATC CAG CCA AGC CTC; primer PrMb: CTG GCA AGC TTG GCA GTT GTC); and the other pair was designed to amplify the NS5-3'UTR.¹⁰ Both primer pairs were used in a direct reverse transcriptase polymerase chain reaction (RT-PCR) on virus isolates by a previously described protocol. ¹¹

The resulting PCR products were precipitated by addition of 20 µL 4 mol/L NH ₄ CH ₃ COO and 40 µL propan-2-ol. The DNA was washed once with 70% ethanol, resuspended in 20 µL of water, and used in two sequencing reactions (one for each primer used in the PCR step) according to the PRISM kit protocol (Perkin Elmer Applied Biosystems Division, San Francisco, Calif, USA), and sequenced with an ABI 373A automated sequencer (Perkin Elmer Applied Biosystems Division). Sequences thus obtained were aligned with published sequences ^12-15 and dendrograms created (see Figure 1).

Figure 1: Dendrogram showing the nucleotide-sequence homology of the prM gene of different isolates of JE virus; the JE viruses isolated from the Torres Strait are designated NO and FU. Numbers above the branches of the endrogram represent average nucleotide homology between branches.

* Supplied by Dr N Karabatsos, Centers for Disease Control and Prevention, USA.
**Supplied by Dr R A Hall, University of Queensland, Brisbane, QLD.

The Medical Journal of Australia eMJA