Jpn. J. Infect. Dis., 57, 129-130, 2004
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Laboratory and Epidemiology Communications
Newly Developed TaqMan Assay to Detect West Nile Viruses in a Wide Range of Viral Strains
Shuzo Usuku*, Yuzo Noguchi and Tomohiko Takasaki1
Department of Testing and Research, Yokohama City Institute of Health, Kanagawa 235-0012 and 1Department of Virology I, National Institute of Infectious Diseases, Tokyo 162-8640
Communicated by Tatsuo Miyamura
(Accepted May 27, 2004)
*Corresponding author: Mailing address: Yokohama City Institute of Health, Takigashira 1-2-17, Isogo-ku, Yokohama City, Kanagawa 235-0012, Japan. Tel: +81-45-754-9804, Fax: +81-45-754-2210, E-mail: sh00-usuku@city.yokohama.jp
In the United States during 2003, 9,862 people were reported to be infected with West Nile virus (WNV) to the Centers for Disease Control and Prevention, and 264 of these people died of related encephalitis and meningitis (http://www.cdc.gov/ncidod/dvbid/westnile/surv&controlCaseCount03_detailed.htm). There is the possibility of WNV infection even in Japan in the near future.
Genetically, WNV can be divided into two lineages. NY99, prevalent in the United States in a 1999 survey of avian- and mosquito samples, belongs to lineage 1 (1). Lanciotte et al. (2) developed a TaqMan Reverse Transcriptase (RT)-PCR assay based on the sequence of the NY99 strain. The 3'NC primers and probe assay detects 0.1 PFU/5 ml of sample of NY99 strain and reacts with other six WNV strains. Two main WNV strains, g2266 (lineage 1) and FCG (lineage 2), are available in local public laboratories in Japan. However, they are genetically distinct from the NY99 strain. The 3'NC primers and probe assay cannot detect the FCG- and g2266 strains used as respective control strains in real time polymerase chain reaction (real time PCR) in a Japanese local laboratory. It is thus necessary to develop new primers and probe sets to detect various WNV strains in order to screen avian and mosquito samples.
We developed a new TaqMan RT-PCR assay to detect both lineages 1 and 2 WNVs including strains of NY99, Eg101, Kunjin (OR393), g2266, and FCG. To develop the TaqＭan primers and probe, we performed multiple alignments using 12 WNV complete sequences (NY99, IS-98 STD, RO97-50, Italy-98, FCG, etc.) submitted to GenBank and used the nucleocapside (cap) coding region, which is highly conserved. Primers and probe are shown in Table 1, and the length of these amplicons was 70 bp. We also confirmed the specificity of this assay by using negative controls of Japanese encephalitis virus and dengue virus, respectively (Table 2).
To estimate the sensitivity of this assay, we performed 10-fold serial dilution samples of NY99, Eg101, Kunjin, and FCG strains using the WNV cap probe and primers. Table 2 shows the sensitivity of our cap primers and probe set. Compared with that of the 3'NC primers and probe, our cap primers and probe has a lower sensitivity to the NY99 strain. Further, our cap primers and probe detected a wider range of strains than did the 3'NC primers and probe, and has sufficient sensitivity for use in WNV-screening of field collected avian and mosquito samples. Our cap primers and probe will be useful for WNV surveillance.
We thank Dr. David W. Smith, Arbovirus Research and Surveillance Group, Department of Microbiology, University of Western Australia for providing the initial stock of Kunjin virus (OR393 strain).
REFERENCES
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