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Volume 8, Number 5—May 2002

Dispatch

Phylogenetic Analysis of a Human Isolate from the 2000 Israel West Nile virus Epidemic

Thomas Briese*Comments to Author , Andrew Rambaut†, Melissa Pathmajeyan*, Jihad Bishara‡, Miriam Weinberger‡, Silvio Pitlik‡, and W. Ian Lipkin*
Author affiliations: *University of California at Irvine, Irvine, California, USA; †University of Oxford, Oxford, UK; ‡Rabin Medical Center–Beilinson Campus, Petah Tikva, Israel;

Main Article

Table

Real Time reverse transcription-polymerase chain reaction (RT-PCR) analysis of RNA extracts from 2000 Israel West Nile patient specimens using primer set NY1999-NS5

NS5 standard NY1999 Armored
RNA    Armored
   RNA extract NY1999 specimens NS5 standard ISR2000 ISR2000 specimens
Amounta CTb Dil.c d Amount Dil.e Amountd Patient no. CT Amount Amountf CT Sample CT Amountg
2.5x106 16.4 1:101 4.5x106 1:101 4.4x105 1 29.3 6.9x102 2.5x106 16.5 cereb. 31.1 1.4x102
2.5x105 20.0 1:102 3.4x105 1:102 4.0x104 2 25.6 7.3x103 2.5x105 20.0 cortex 36.4h 1.9x100
2.5x104 23.6 1:103 3.1x104 1:103 4.3x103 3 30.0 4.6x102 2.5x104 23.1
2.5x103 27.2 1:104 3.1x103 1:104 6.0x102 5 34.8 2.3 x101 2.5x103 26.7
2.5x102 31.1 1:105 3.4x102 1:105 9.8x101 2.5x102 30.3
2.5x101 34.9 1:106 3.1x101 1:10 2.1x100 2.5x101 34.1
2.5x100 36.8h 1:107 3.0x100 h 1:107 n.di 2.5x100 37.3 h
0 >45 0 0 0 0 0 >45

a Plasmid DNA p88-D-21 was quantitated spectrophotometrically, and dilutions containing the indicated copy number of target sequence were added to each polymerase chain reaction (PCR) assay.
b CT , Cycle number at which signal crosses threshold.
c Armored RNA West Nile virus (HNY1999) standard (Ambion, Austin, TX) was diluted 1:10, boiled, reverse transcribed, and then diluted to result in amounts per PCR assay equivalent to the indicated dilution of the stock (5 μL).
d Amount calculated based on calibration curve obtained with NS5 Standard NY1999 (column 1).
e Dilutions of Armored RNA West Nile virus (HNY1999) standard (Ambion) were extracted with TRI-Reagent (Molecular Research Center, Cincinnati, OH) and then subjected to RT-PCR to result in amounts per assay equivalent to the indicated dilution of the stock (5 μL).
f Plasmid DNA pISR-Dfrag-D6 was quantitated spectrophotometrically, and dilutions containing the indicated copy number of target sequence were added to each PCR assay.
g Amount calculated based on calibration curve obtained with NS5 Standard ISR2000 (column 6).
h Poisson effects take place at low template concentration; duplicate assay deviations: 36.2 / 37.4, NY1999; 6.0 x100 / 0, armored RNA; 37.4 / 37.1, ISR2000; and 36.4 / >45, cortex.
i n.d. = not determined.

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