Detection and Isolation of Exotic Newcastle Disease Virus from Field-Collected Flies

Chakrabarti, Seemanti; King, Daniel J.; Afonso, Claudio; Swayne, David; Cardona, Carol J.; Kuney, Douglas R.; Gerry, Alec C.

RNA was extracted from AAF by using TRIzol LS according to manufacturerÕs instructions (Invitrogen, Carlsbad, CA); 750 l of TRIzol LS reagent was added to 250 l of allantoic ßuid. The ßuid was vortexed and incubated at room temperature for 7 min. RNA was separated into the aqueous phase with the addition of 200 l of chloroform, followed by precipitation with isopropanol. After one wash with 70% ethanol, RNA was dried and resuspended in RNase-free water. The 5Ј end of the ENDV fusion gene was ampliÞed by polymerase chain reaction (PCR) and sequenced using primers targeting a 635-bp fragment that included the fusion protein cleavage site (forward primer, 5Ј-GAG GTT ACC TCY ACY AAG CTR GAG A-3Ј; reverse primer, 5Ј-TCA TTA ACA AAY TGC TGC ATC TTC CCW AC-3Ј). These primers amplify the NDV genomic region between positions 4317 and 5084. Standard 50 l reverse transcription (RT)-PCR reactions were carried out using a kit (SuperScript III One Step RT-PCR, Invitrogen) with annealing temperature at 56ЊC. PCR-ampliÞed samples were separated on a 1% agarose gel, and the bands were excised and eluted using the QuickClean 5M gel extraction kit (GenScript Corp., Piscataway, NJ). Once the PCR products were cleaned, samples were quantiÞed using a standard spectrophotomer and sequenced. All double-stranded nucleotide-sequencing reactions were performed with ßuorescent dideoxynucleotide terminators in an automated sequencer (ABI 3700 automated sequencer, Applied Biosystems, Foster City, CA). Nucleotide sequence editing and analysis were conducted with the LaserGene sequence analysis software package (LaserGene, version 5.07, DNAStar, Inc., Madison, WI). The virus was sequenced and compared by Blast analysis (Altschul et al. 1990 ) to an END viral sequence [chicken/U.S.(AZ)/236498/03] obtained from commercial poultry infected with ENDV during the 2002 outbreak. It also was compared with other available GenBank ENDV sequences. Genomic sequences from viruses recovered from ßies at each collection site were deposited in GenBank (accession nos. EF424375 and EF424376).