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JMD 2005, Vol. 7, No. 2
Copyright © 2005 American Society for Investigative Pathology & Association for Molecular Pathology

Molecular Typing of West Nile Virus, Dengue, and St. Louis Encephalitis Using Multiplex Sequencing

Thuraiayah Vinayagamoorthy*, Kirk Mulatz*, Michael Drebot{dagger} and Roger Hodkinson*

From Bio-ID Diagnostic Inc., * Saskatoon, Saskatchewan; and the National Microbiology Laboratory, {dagger} Canadian Science Centre for Human and Animal Health, Winnipeg, Manitoba, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We report the development of an assay to simultaneously identify three of the clinically important flaviviruses (West Nile Virus, Dengue, and St. Louis encephalitis). This assay is based on the nucleotide sequence variations within a 266-bp region of the non-structural protein 5. Further, based on the nucleotide variations in the same region of the non-structural protein 5, four of the present Dengue serotypes were identified. To identify some of the subtypes of WNV we have developed a second assay using multiplex sequencing technology. The format of the result of this assay is an electropherogram of two genomic segments of the WNV genome: a 48-nucleotide sequence from the anchored core protein C and a 45-nucleotide sequence coding for the non-structural proteins (proteinase and putative helicase genes).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
West Nile virus (WNV) is an RNA virus that belongs to the Flaviviridae family of viruses that also includes the Japanese/St. Louis encephalitis, Dengue, and yellow fever viruses.1 WNV has a range of animal hosts,2, 3 is transmitted mainly by a mosquito vector,4 and in severe infections produces various neurological disorders including paralysis.5 Recently there have been more than 3500 cases and 200 deaths due to WNV in North America and many more in rest of the world.6, 7, 8, 9, 10 Although the common route of infection via mosquito bites is associated with low fatality,11 previous studies have established that other potential routes, including infection through blood12, 13 and organ transplants,14 often carry a worse prognosis.

Presently, the presumptive diagnosis of WNV is mainly based on clinical symptoms and a definitive diagnosis is established by serology5, 8 Diagnostic procedures such as antigen capture (ELISA)15 have been developed for viral identification, but due to the low sensitivity of these methods, antibody detection continues to be the assay of choice when carrying out human case investigations. Nucleic acid-based assays14, 16, 17, 18 have been used to identify the viral genome in clinical samples such as cerebrospinal fluid and blood samples.

Various studies19, 20, 21, 22 have identified genetic variations among different isolates of WNV, and in some cases the presence of these variants have created difficulty in establishing identity. Hence, there is a need for an accurate assay with highest specificity to identify WNV subtypes. Furthermore, since infection with other flaviviruses (Dengue, St. Louis encephalitis) presents similar clinical symptoms, it is also necessary to be able to test for the other flaviviruses for optimal patient care.

Genome sequencing is increasingly accepted as the reference method for identification of viruses. Our assay includes generating a 266-bp amplicon that is common to WNV, Dengue, and St. Louis encephalitis. This amplicon was sequenced using the chain termination sequencing method. To distinguish between WNV serotypes, we generated two amplicons from separate regions of the WNV genome and sequenced them simultaneously.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Multiplex Sequencing Method (MultiGEN)
MultiGEN is a platform technology that allows for simultaneous determination of nucleotide sequences from multiple genomes or multiple segments from the same genome or a combination of both.23 This technology consists of three steps: The first is preparation of total nucleic acid from the sample. This could be DNA, RNA, or both. The second step is simultaneous amplification of all target genomes. This can be carried out by any one of present target amplification methods including PCR. The third step is simultaneous sequencing short stretches (20–40 nucleotides) at the 3' end of all amplicons. The molecular weight of sequencing primer (WNVseq2) is higher than the molecular weight of the longest truncated species generated by sequencing primer (WNVseq1). The sequences generated were identified by BLAST search (Figure 1)Go . The test protocol will vary based on the type of genomic targets to be analyzed (DNA, RNA, or both), the number of targets (2–20 are feasible), and the expected copy number of the targets in the samples.



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  		Figure 1. Stages of the MultiGEN process and the basic scientific principles. These include: Multiplex amplification, Multiplex sequencing, electrophoresis and computer analyzed electropherogram that is ready for BLAST search.

 
Sample Preparation
All clinical isolates were propagated in Vero E6 cells. Total RNA was extracted using the RNeasy Mini Kit (Qiagen). 100 µl of the cell lysates containing WNV, 12,040 pfu/µl; SLE, 420 pfu/µl; Dengue 1, 840 pfu/µl; Dengue 2, 840 pfu/µl; Dengue 3, 420 pfu/µl, and Dengue 4, 980 pfu/µl were extracted separately. The reagents of the kit were prepared according to manufacturer’s instructions, and 350 µl of RLT buffer and 250 µl of 100% ethanol were added to 100 µl of viral lysate. The sample was transferred to the spin column and centrifuged at 8000 rcf for 30 seconds. The sample was washed on the spin column three times, once with 700 µl of RW1 buffer and twice with 500 µl of RPE buffer. After each wash the spin column was centrifuged at 8000 rcf for 30 seconds. After washing, the column was thoroughly dried with a 1-minute centrifugation at 8000 rcf. Finally, the sample was eluted from the column by adding 50 µl of RNase-free water to the column and centrifuging for 1 minute at 8000 rcf. The RNA was further concentrated by re-applying the eluate to the column and centrifuging a second time at 8000 rcf for 1 minute. RNA/DNA concentrations were determined using a spectrophotometer (Ultrospec 3000, Pharmacia Biotech, Cambridge, UK).

RT-PCR
The SuperScript One-Step RT-PCR with Platinum Taq kit (Invitrogen) was used for individual RT-PCR. The reaction volume was 50 µl, which consisted of 25 µl 2X Reaction mix, 200 nmol/L each primer, 1 µl RT/Platinum mix Taq, and 10 µl of RNA extract. The tubes were placed in a thermocycler, GeneAmp 2400 (Applied Biosystems) and amplified according to the thermocycling profile shown in Table 1Go .


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  		Table 1. Primers and Thermocycle Profiles

 
Gel Electrophoresis
The amplicons were visualized by loading 2 µl of 6X loading buffer, 5 µl of TBE buffer, and 5 µl of the PCR product onto a 2% agarose gel and applying 110 V for 50 minutes. The gel was then stained for 15 minutes in ethidium bromide (5 µg/ml in TBE) solution and then destained in distilled water for 15 minutes.

Cycle Sequencing
Amplicons were sequenced by cycle sequencing using ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) on a GeneAmp 2400 thermocycler (PE Applied Biosystems) using the thermocycler profile in Table 1Go . Unincorporated dye terminators were removed using Centricep chromatography columns (Princeton, NJ). The samples were then dried and re-suspended in 20 µl of ABI PRISM Template Suppression Reagent. Samples were analyzed by capillary electrophoresis using the ABI PRISM Genetic Analyzer 310. The 47 cm x 50 µm uncoated capillary was filled with a Performance Optimized Polymer 6 (acrylamide/urea polymer) and heated to 50°C. 20 µl of the sequencing mixture was pipetted into a 0.2-ml microfuge tube provided by the manufacturer (Applied Biosystems). Samples were drawn into the capillary by electrokinetic injection at 2 kV for 50 to 200 seconds. The electrophoresis was carried out at 15 kV for 20 minutes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Selection of Target Regions and Primer Design
West Nile virus, Dengue, and St. Louis encephalitis genomic nucleotide sequences were obtained from GenBank (NIH) and aligned using GeneDoc version 3.1(http://www.psc.edu/biomed/genedoc) and GenBank BLAST. Based on this alignment, a segment common to all three flaviviral genomes was selected. This segment carried a core region showing nucleotide variations among WNV, Dengue, and St. Louis encephalitis, and sequences that could distinguish between various serotypes of Dengue and conserved flanking regions. The primers were designed using Oligo 6 Software version, 6.65 (Molecular Biology Insights) within conserved regions flanking the core region. Based on the nucleotide sequences within the amplicon WNV, Dengue and St. Louis encephalitis could be identified (Figure 2)Go . Furthermore, comparing the equivalent nucleotide sequences, the four Dengue serotypes could be identified (Figure 2)Go . The same approach was used to design two sets of PCR primers within the WNV genome to generate two amplicons (250 bp from the anchored core protein region and 396 bp from the proteinase and putative helicase regions). To sequence the 3' end of these two amplicons, two modified (Bio-ID Diagnostic Inc) sequencing primers (Table 1)Go were designed for simultaneous sequencing.



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  		Figure 2. Nucleotide sequences from two selected regions from the 266-bp amplicon. A: Signature nucleotide sequences that identify WNV and St. Louis encephalitis. B: Signature nucleotide sequences that identify Dengue types 1–4.

 
Detection of WNV, Dengue, and St. Louis Encephalitis
Total RNA was prepared from six viral isolates 10 µl of these RNA extracts were amplified separately using one step RT-PCR with Flav1/Flav2 primer set (Table 1)Go , with all six generating an amplicon of 266 bp from the non-structural protein 5 region. These amplicons were sequenced separately using Flav1 primer. The nucleotide sequences generated are shown in Figure 3Go . Various regions (highlighted in yellow) on each electropherogram (Figure 3)Go were subjected to BLAST search. Based on the alignments from BLAST searches they were identified as follows: Figure 3Go . WNV (A), Dengue subtypes (B-E), St. Louis encephalitis (F). The black boxes highlighting nucleotide sequences (Figure 3Go , electropherogram regions of samples B to E) denote regions that are conserved among the serotypes of Dengue and can be used to distinguish between the different serotypes. All of the sequences were confirmed by BLAST searches.



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  		Figure 3. Electropherograms showing nucleotide sequences from six RNA extracts analyzed. A: WNV; B-E: Dengue serotypes; F: St. Louis encephalitis.

 
Detection of WNV
Of the six samples tested, two produced amplicons (Figure 4B)Go using WNV1 and WNV2 PCR primer sets (Table 1)Go ; the other four and the negative control did not (Table 2)Go . The amplicons produced from these two samples were sequenced (separately) at their respective 3' end (Figure 4A)Go using two modified (Bio-ID Diagnostic Inc) sequencing primers WNV1seq, WNV2seq (Table 1)Go . The truncated molecular species from these two targets were then separated in a single capillary on an ABI 310 Genetic Analyzer. Of the two samples sequenced, only one produced an electropherogram. The nucleotide sequence from the electropherogram produced (Figure 4C)Go were BLAST searched and the sequences were found to be in 100% alignment with West Nile virus sequence subtype NY99 (Table 3)Go . The experiment was repeated with the same sample of WNV total RNA extracts (15 ng) spiked with 15 ng of human genomic DNA, and the MultiGEN analysis produced the expected WNV nucleotide sequences (data not shown). As an extension of this assay, the expected amplicons were generated using 500 pg of the original WNV extract corresponding to 1852 viral particles.



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  		Figure 4. A: Region of the WNV genome from which two amplicons were generated, positioning of the sequencing primers, and the electropherograms produced from two samples. B: Photograph of an ethidium bromide stained 2% agarose gel showing the DNA bands of the two amplicons.

 

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  		Table 2. PCR and Sequencing Results

 

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  		Table 3. Nucleotide Variation among WNV Isolates

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ability of MultiGEN technology to simultaneously generate nucleotide sequences from multiple viral targets raises the parameters of identification to its highest standard. Reverse transcriptase lacks 3'-5' proofreading and hence are prone to error in replication.24 This inherent ability to create progeny variation gives the virus an enhanced opportunity to survive the host immune response from previous infections.25 WNV RNA binding proteins have been identified26 and these may be associated with WNV replication. Using infected C3H/RV mouse cells, it has been shown27 that mutations at two unique spots in WNV RNA affects the efficiency of the production of virus progeny, and suggests that the WNV genome carries a conserved 3'-terminal secondary structure that is a polymerase recognition and binding site.28 Furthermore, truncation of the 2'-5' oligoadenylate synthetase L1 isoform point mutation in mouse has been correlated with the restriction of WNV replication.29 Such events create two challenges for effective management of epidemics. 1) Genetic changes in the RNA recognition motif (RRM) could affect the pathogenicity of the virus. Determination of nucleotide sequences in the relevant motif will indicate the change in pathogenicity. 2) If the mutational changes are within annealing regions of the target amplification primers (with PCR or sequencing) this will alter the melting temperature (Tm) of primer/target binding and hence affect target amplification. As the assay in this report targets two genomic regions, this increases the chance of obtaining a positive result even if such mutations should occur. Even though sample number 2 generated an amplicon with only WNV PCR primers, it failed to produce an electropherogram with the WNV sequencing primer. However, the same sample when tested with PCR primers for WNV, Dengue and St. Louis encephalitis was found to generate an electropherogram that definitively identified sample 2 as Dengue. Hence use of MultiGEN technology was able to prevent the potentially false identification, had PCR been used alone.

In summary, the reality of present international travel greatly facilitates the rapid global spread of the gene pool of microbial pathogens. Such challenge to global public health calls for the most accurate method for both rapid pathogen identification and monitoring the spread of the epidemic. MultiGEN technology removes practical limitations that are associated with conventional sequencing method being used for viral identifications.


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  		Table 4. Sequence Variations among Flaviviruses

 

   Footnotes
 
Address reprint requests to Thuraiayah Vinayagamoorthy, No. 1, 410 Downey Road, Innovation Place, Saskatoon, Saskatchewan, Canada S7N 4N1. E-mail: moorthy{at}bio-id-diagnostic.com

Accepted for publication August 18, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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