The untranslated regions (UTR) are involved in translation and viral RNA replication and likely play an important role in genome packaging. Both the 5' UTR and the 3' UTR in the WNV genome form highly conserved secondary and tertiary structures, some elements of which are similar among mosquito-borne flaviviruses. The cyclization of the flavivirus genome is necessary for viral RNA replication. In addition to base pairing between 5′-3′ UAR and 5′-3′ CS specific sequences involved in cyclization, a third stretch of nucleotides was identified to form a double-stranded region between the 5′ and 3′ UTRs (Friebe & Harris, 2010). Different functional regions have been described inside the 5'UTR and 3′UTR of flaviviruses based on such factors as nucleotide content, degree of sequence conservation, occurrence of repeated sequence motifs, and predicted secondary structure (Gritsun & Gould, 2007; Markoff, 2003; Proutski et al., 1997; Tajima et al., 2006). The 5′ end of the WNV genomic RNA has a type I cap structure (m7GpppAmp) mediating cap-dependent translation. The 5'UTR contains two functional elements, the stem-loop A (SLA) and capsidcoding region hairpin (cHP) essential for RNA replication. The 3'UTR is generally divided into three regions based on the differences in the level of conservation: (1) the variable region is located immediately after the ORF; (2) the intermediate region has a moderate level of conservation and contains several hairpin motifs; (3) the conserved 3'-terminal region contains a cyclization sequences and stable stem-loop structure (Bryant et al., 2005; Markoff, 2003). These regions are believed to contain sequences that confer identity of the flaviviruses as demonstrated by attempts to exchange portions of the 3'UTR between WNV and dengue virus (DENV) that resulted in chimeric viruses which were unable to replicate (Yu et al., 2008).

WNV is maintained in nature by transmission between mosquitoes and birds, but it can also infect humans, other mammals (Beasley, 2005; Petersen & Marfin, 2002) and reptiles (Klenk et al., 2004) by mosquito bite (Fig.2). *Culex spp.* mosquitoes are the main vectors of WNV, although the virus has also been found in at least 43 other mosquito species (Granwehr et al., 2004; Higgs et al., 2004; Petersen et al., 2001). WNV can be transmitted vertically and overwinter in hibernating female mosquitoes, providing the mechanisms for viral persistence and reemergence each spring (Nasci et. al, 2001). WNV has spread within many bird species, including crows, magpies, and jays, house sparrows, house finches, grackles, and others representing 63 species, 30 families and 14 orders (Kramer & Bernard, 2001). They are all primarily competent reservoirs for WNV infection. By contrast, mammals

polymerase (Beasley, 2005; Egloff et al., 2002).

including humans and horses are "dead-end" hosts in this enzootic cycle. They do not develop prolonged high-level viremia, so the concentration of the virus in blood is insufficient to infect a feeding mosquito. Most human infections are asymptomatic (~80%). The severity of symptomatic cases ranges from flu-like illness (~20% of infections) to severe neurological disease (~ 1%) (Hayes & Gubler, 2006). Additional modes of transmission were identified in 2002, including human-to-human by blood transfusion, breast-feeding, transplacental transmission, and by organ transplants extending the impact of WNV to blood safety and other areas of public health worldwide (Austgen et al., 2004; Pealer et al., 2003; Sbrana et al., 2005).

Fig. 2. Scheme of WNV transmission cycle. The maintenance of WNV in nature depends on an enzootic cycle involving many avian and mosquito species. Humans and other animals are incidental hosts that can become infected by WNV-infected mosquito bites. The virus can be also transmitted from human to human by blood transfusion and by solid organ transplantation.

Historically, since its isolation in Uganda in 1937, WNV outbreaks occurred in Africa, Europe, the Middle East, and caused a rare and mild febrile illness in humans and horses. A significant geographical expansion occurred starting from 1999 when the virus was introduced into North America. First detected in the U.S. in 1999, WNV has become endemic, causing yearly summer outbreaks. In 2002, the virus spread westward and the number of reported human cases increased dramatically. The North American epidemics of 2002 and 2003 represent the largest WNV outbreaks ever reported. WNV now is the most widespread arbovirus in the world (Kramer et al., 2008). The spread of the virus and intensity of the outbreak was correlated with the appearance of a new genotype with higher virulence and ability to disseminate in mosquitoes (Beasley, 2005). From the first outbreak in 1999 through 2010, WNV is estimated to have infected ~4 million humans in the US, causing over 30,000 serious illnesses, including 12,729 neuroinvasive disease cases with 1,206 deaths reported to the CDC (http://www.cdc.gov/ncidod/dvbid/westnile/). The virus has also been detected in the continental U.S. and in several areas of Canada, Central and South America, and the Caribbean. The persistence of WNV indicates that it has become endemic

Application of a Microarray-Based Assay for the Study of Genetic Diversity of West Nile Virus 161

The monitoring and surveillance of pathogens is highly dependent on the capability of detection technology to simultaneously monitor multiple genomic signatures specific for different genetic variants of the pathogen. One of the approaches that enable this type of analysis is microarray technology. Generally, nucleotide microarrays are microscopic slides loaded with hundreds or thousands of pathogen-specific probes (DNA fragments or synthetic oligonucleotides) which can specifically hybridize with the target molecules to produce either quantitative (gene expression) or qualitative (diagnostic) data.  Microarrays have the unique potential to simultaneously detect and identify defined pathogens, as well as to detect mutations within the complete viral genomes and target areas of a pathogen's genome. It provides a significant advantage for the field of clinical microbiology and molecular epidemiological studies. Printed microarrays historically were the first arrays utilized for detection of mutations in many research laboratories and they are so-called because of the "printing" of the probes onto the surface of a glass microscope slide

Fig. 3. Schematic outline of a printed microarray experiment. Microarray probes are spotted onto a surface of glass slide. In this example, the target sample and quality control sample (QC) with different fluorescent labeling were mixed and hybridized to the microarray probes. The efficiency of hybridization is monitored through measurement of the

fluorescent signal from each spot by using a laser microarray scanner equipped with two lasers: 632 nm and 543 nm for excitation of Cy5 and Cy3 dyes respectively. The obtained

fluorescent images are analyzed using specific computer software.

**1.2 Basics of nucleotide microarrays**

(Fig. 3).

in the Western Hemisphere (Kramer et al., 2008). The pattern of yearly reoccurring outbreaks in North America differs from that of sporadic outbreaks observed in Europe and Africa. However, in recent years, WNV epidemics in humans and horses have become more frequent in several Southern European countries, and these epidemics could potentially be associated with an emergence of new viral genotypes (Chevalier et al. 2011). The speed with which the virus spread over the world triggered great interest and prompted a detailed investigation of the genetic evolution of the virus in search of the cause of its rapid adaptability.

Based on phylogenetic analyses, WNV has been initially divided into two major genetic lineages (Lanciotti et al., 2002). Lineage 1 included viruses circulating in Europe, Israel, United States, India, Russia, and Australia, while the Lineage 2 contained strains that circulated in sub-Saharan Africa and Madagascar. Lineage 1 was further divided into 3 subclades: 1a (including strains from Africa, Europe, US, Middle East, and Russia), 1b (Kunjin strain from Australia), and 1c (India) (Lanciotti et al., 2002). WNV has now been reported to have at least five distinct lineages based on phylogenetic analysis of all known full WNV genome sequences of viral isolates that correlates well with the geographical points of their isolation from various regions around the world including the U.S. (Davis et al., 2005; Herring et al., 2007; Grinev et al., 2008a; McMullen, 2011), Europe and Mediterranean (Parreira et al., 2007), and Africa (Botha et al., 2008).

In 2001 a new WNV genotype, named WN02, emerged in the US. The new genotype became prevalent in 2002, eventually displacing the ancestor genotype NY99, which is believed to have been introduced to the New World from the Middle East (Davis et al., 2005; Herring et al., 2007; Lanciotti et al., 1999). When compared to the WN99 genotype, the WN02 genotype possesses a few fixed silent nucleotide mutations and one amino acid substitution in the E protein (E-V159A). The highest rate of nucleotide sequence divergence among viruses isolated from 2002-2010 varies in the range of 0.4% - 0.6% (Davis et al., 2005; Ebel et al., 2004; Grinev et al., 2008a; McMullen et al., 2011). It is noteworthy that 80% of the nucleotide changes are observed in the structural regions represented by U<->C transitions; 75% among them are silent mutations (Grinev et al., 2008a). The possible explanation for the rapid displacement of the WN99 genotype by the new dominant genotype WN02 is due to the ability of the new viruses to more efficiently proliferate in domestic mosquitoes (Jerzak et al., 2005; Moudy et al., 2007). Phylogenetic analysis of modern WNV isolates demonstrates the existence of at least two subtypes of WN02 genotype co-circulating in North America. In addition to the common E-V159A amino acid substitution two other substitutions have become fixed in the significant part of North American WNV population: NS4a-A85T and NS5-K318R. Positive selection of these two amino acid substitutions potentially could impact viral fitness, phenotype and virulence (McMullen et al., 2011). As indicated by sequence analysis of new WNV genetic variants isolated in different areas of the US, the virus continues to diverge from the precursor isolate. Thus, changes in the WNV genome and viral proteins have the potential to negatively affect the sensitivity of screening and diagnostic assays currently used for virus detection, and to impact the development of vaccines and potential antiviral therapeutic agents. Therefore, development of new methods able to rapidly detect the emergence of WNV genetic variants is critical for epidemiological surveillance.

### **1.2 Basics of nucleotide microarrays**

Viral Genomes – Molecular Structure,

160 Diversity, Gene Expression Mechanisms and Host-Virus Interactions

in the Western Hemisphere (Kramer et al., 2008). The pattern of yearly reoccurring outbreaks in North America differs from that of sporadic outbreaks observed in Europe and Africa. However, in recent years, WNV epidemics in humans and horses have become more frequent in several Southern European countries, and these epidemics could potentially be associated with an emergence of new viral genotypes (Chevalier et al. 2011). The speed with which the virus spread over the world triggered great interest and prompted a detailed investigation of the genetic evolution of the virus in search of the cause of its rapid

Based on phylogenetic analyses, WNV has been initially divided into two major genetic lineages (Lanciotti et al., 2002). Lineage 1 included viruses circulating in Europe, Israel, United States, India, Russia, and Australia, while the Lineage 2 contained strains that circulated in sub-Saharan Africa and Madagascar. Lineage 1 was further divided into 3 subclades: 1a (including strains from Africa, Europe, US, Middle East, and Russia), 1b (Kunjin strain from Australia), and 1c (India) (Lanciotti et al., 2002). WNV has now been reported to have at least five distinct lineages based on phylogenetic analysis of all known full WNV genome sequences of viral isolates that correlates well with the geographical points of their isolation from various regions around the world including the U.S. (Davis et al., 2005; Herring et al., 2007; Grinev et al., 2008a; McMullen, 2011), Europe and Mediterranean

In 2001 a new WNV genotype, named WN02, emerged in the US. The new genotype became prevalent in 2002, eventually displacing the ancestor genotype NY99, which is believed to have been introduced to the New World from the Middle East (Davis et al., 2005; Herring et al., 2007; Lanciotti et al., 1999). When compared to the WN99 genotype, the WN02 genotype possesses a few fixed silent nucleotide mutations and one amino acid substitution in the E protein (E-V159A). The highest rate of nucleotide sequence divergence among viruses isolated from 2002-2010 varies in the range of 0.4% - 0.6% (Davis et al., 2005; Ebel et al., 2004; Grinev et al., 2008a; McMullen et al., 2011). It is noteworthy that 80% of the nucleotide changes are observed in the structural regions represented by U<->C transitions; 75% among them are silent mutations (Grinev et al., 2008a). The possible explanation for the rapid displacement of the WN99 genotype by the new dominant genotype WN02 is due to the ability of the new viruses to more efficiently proliferate in domestic mosquitoes (Jerzak et al., 2005; Moudy et al., 2007). Phylogenetic analysis of modern WNV isolates demonstrates the existence of at least two subtypes of WN02 genotype co-circulating in North America. In addition to the common E-V159A amino acid substitution two other substitutions have become fixed in the significant part of North American WNV population: NS4a-A85T and NS5-K318R. Positive selection of these two amino acid substitutions potentially could impact viral fitness, phenotype and virulence (McMullen et al., 2011). As indicated by sequence analysis of new WNV genetic variants isolated in different areas of the US, the virus continues to diverge from the precursor isolate. Thus, changes in the WNV genome and viral proteins have the potential to negatively affect the sensitivity of screening and diagnostic assays currently used for virus detection, and to impact the development of vaccines and potential antiviral therapeutic agents. Therefore, development of new methods able to rapidly detect the emergence of WNV genetic

adaptability.

(Parreira et al., 2007), and Africa (Botha et al., 2008).

variants is critical for epidemiological surveillance.

The monitoring and surveillance of pathogens is highly dependent on the capability of detection technology to simultaneously monitor multiple genomic signatures specific for different genetic variants of the pathogen. One of the approaches that enable this type of analysis is microarray technology. Generally, nucleotide microarrays are microscopic slides loaded with hundreds or thousands of pathogen-specific probes (DNA fragments or synthetic oligonucleotides) which can specifically hybridize with the target molecules to produce either quantitative (gene expression) or qualitative (diagnostic) data.  Microarrays have the unique potential to simultaneously detect and identify defined pathogens, as well as to detect mutations within the complete viral genomes and target areas of a pathogen's genome. It provides a significant advantage for the field of clinical microbiology and molecular epidemiological studies. Printed microarrays historically were the first arrays utilized for detection of mutations in many research laboratories and they are so-called because of the "printing" of the probes onto the surface of a glass microscope slide (Fig. 3).

Fig. 3. Schematic outline of a printed microarray experiment. Microarray probes are spotted onto a surface of glass slide. In this example, the target sample and quality control sample (QC) with different fluorescent labeling were mixed and hybridized to the microarray probes. The efficiency of hybridization is monitored through measurement of the fluorescent signal from each spot by using a laser microarray scanner equipped with two lasers: 632 nm and 543 nm for excitation of Cy5 and Cy3 dyes respectively. The obtained fluorescent images are analyzed using specific computer software.

Application of a Microarray-Based Assay for the Study of Genetic Diversity of West Nile Virus 163

**Isolate ID Year Location Passage GenBank No.** FDA/HU-02 2002 NY FFP,P1-P3 AY646354 ARC10-02 2002 MI P1 AY795965 ARC12-02 2002 OH P1 DQ666453 ARC16-02 2002 IN P1 DQ666456 BSL5-03 2003 UT P1 DQ005530 BSL9-03 2003 TX P1 DQ666458 BSL62-03 2003 SD P2 DQ666460 RMS1-03 2003 MN P1 DQ666462 RMS2-03 2003 IN P1 DQ666463 RMS3-03 2003 IN P1 DQ666464 RMS4-03 2003 IA P1 DQ666465 BSL2-04 2004 AZ P1 DQ666467 BSL4-04 2004 AZ P2 DQ666468 BSL6-04 2004 AZ P1 DQ666469 BSL7-04 2004 AZ P2 DQ666470 BSL8-04 2004 AZ P2 DQ666471 GCTX1 2005 TX P1 DQ666449 GCTX2 2005 TX P1 DQ666450 BSL2-05 2005 SD P1 DQ666452 BSL6-05 2005 AZ P1 DQ666472 BSL9-05 2005 TX P1 DQ666473 BSL10-05 2005 LA P1 DQ666474 BSL13-05 2005 AZ P1 DQ666451 **ARC140-07** 2007 ID P1 JF957168 **BSL2-09** 2009 NV P1 JF957175 **BSL5-09** 2009 AZ P1 JF957176 **BSL6-09** 2009 NV P1 JF957177 **BSL11-09** 2009 NV P1 JF957178 **BSL18-09** 2009 LA P1 JF957179 **BSL20-09** 2009 NV P1 JF957180 **BSL22-09** 2009 SD P1 JF957181 **BSL24-09** 2009 TX P1 JF957182 **BSL27-09** 2009 TX P1 JF957183 **CO7-09** 2009 CO P1 JF957184 Table 1. WNV isolates used for microarray assay validation. FFP indicates fresh frozen plasma sample. Passage P1 indicates first isolation in Vero cells; P2 and P3 indicates subsequent virus

passages. Isolates in boldface were used for the full genome array validation.

of the reference WNV strain NY99.

These samples were collected in different geographic locations of the continental U.S. from the 2002-2009 epidemic seasons under IRB approved informed consent. In addition to 23 previously published isolates, which were used for structural region investigation (Grinev et al., 2008b), 11 WNV isolates from 2007 and 2009 were used to conduct microarray analyses of their full genomes in order to detect emerged genetic differences in comparison with that

Chemically activated glass slides are commonly used for microarrays because they permit irreversible attachment of microarray probes, allow for efficient hybridization kinetics between probes and analyzed targets, and have a low background fluorescence level (Cheung et al., 1999). Printed arrays can be produced using double-stranded DNA (dsDNA) fragments or oligonucleotides. For dsDNA microarrays, the probes usually consist of PCR products (amplicons) obtained using primers designed from a known genomic sequence or shotgun library clones. The double-stranded probes must be denatured prior to hybridization, either using a special printing buffer or after immobilization on the glass surface (Tomiuk et al., 2001). These microarrays containing relatively long 200-800 bp dsDNA probes usually demonstrate a high sensitivity but are not very useful for detection of minor genetic differences (e.g., single nucleotide mutations) between the probe and target nucleic acid (Hager et al., 2006). In contrast to dsDNA microarray probes, the length of oligonucleotide probes usually ranges from 20 to 80 nucleotides. Use of shorter probe lengths increases the microarray specificity and enables efficient detection of minor genetic changes between the probe and target (Chou et al., 2004). For more efficient attachment of oligonucleotide probes to the functional groups on the surface of chemically activated glass slides (usually aldehyde, epoxy, and succinimide groups), the 5' or 3' end of the probes contain primary amino groups introduced during chemical synthesis.

Microarray technology has been used to study gene expression in clinical and biological samples, detect and genotype pathogens (Honma et al., 2007; Wade et al., 2004), detect single base pair mismatches (Anthony et al., 2003; Hacia et al., 1999), design genomic maps (Roerig et al., 2005), and study viral evolution (Cherkasova et al., 2003).

The methods based on combination of initial PCR amplification of target genetic material followed by hybridization of amplicons with specific microarray oligonucleotide probes allowed for reconstitution of instant mutation profiles and determination of evolutionary divergence of individual viral isolates (Neverov et al., 2006). The microarrays consisting of multiple individual short oligoprobes were shown to be an efficient and sensitive genetic method for detection of single point mutations in viral and bacterial genomes (Chizhikov et al., 2002; Grinev et al., 2008b; Laassri et al., 2003, 2005, 2007; Volokhov et al., 2002). Microarray assays can also help simultaneously detect and identify the genotype and strain of common food-borne viruses without using PCR (Chen et al., 2011). In general, microarray technology can be easily implemented for detection and genotyping of any pathogen. Recently a pan-Microbial Detection Array was designed to detect all known viruses and bacteria (Gardner et al., 2010). Although further improvements, optimizations, and automation are still needed to fully implement the microarray technique in routine research and clinical practices, the potential role of these robust technologies in rapid diagnostics of multiple viral and bacterial pathogens is indisputable (Miller, 2009).

### **2. Material and methods**

### **2.1 Plasma samples**

The microarray development and evaluation study included the analysis of a total of 34 plasma specimens from blood donor units identified as positive for WNV by nucleic acid tests used to screen blood donations (Table 1).

162 Diversity, Gene Expression Mechanisms and Host-Virus Interactions

Chemically activated glass slides are commonly used for microarrays because they permit irreversible attachment of microarray probes, allow for efficient hybridization kinetics between probes and analyzed targets, and have a low background fluorescence level (Cheung et al., 1999). Printed arrays can be produced using double-stranded DNA (dsDNA) fragments or oligonucleotides. For dsDNA microarrays, the probes usually consist of PCR products (amplicons) obtained using primers designed from a known genomic sequence or shotgun library clones. The double-stranded probes must be denatured prior to hybridization, either using a special printing buffer or after immobilization on the glass surface (Tomiuk et al., 2001). These microarrays containing relatively long 200-800 bp dsDNA probes usually demonstrate a high sensitivity but are not very useful for detection of minor genetic differences (e.g., single nucleotide mutations) between the probe and target nucleic acid (Hager et al., 2006). In contrast to dsDNA microarray probes, the length of oligonucleotide probes usually ranges from 20 to 80 nucleotides. Use of shorter probe lengths increases the microarray specificity and enables efficient detection of minor genetic changes between the probe and target (Chou et al., 2004). For more efficient attachment of oligonucleotide probes to the functional groups on the surface of chemically activated glass slides (usually aldehyde, epoxy, and succinimide groups), the 5' or 3' end of the probes

Microarray technology has been used to study gene expression in clinical and biological samples, detect and genotype pathogens (Honma et al., 2007; Wade et al., 2004), detect single base pair mismatches (Anthony et al., 2003; Hacia et al., 1999), design genomic maps

The methods based on combination of initial PCR amplification of target genetic material followed by hybridization of amplicons with specific microarray oligonucleotide probes allowed for reconstitution of instant mutation profiles and determination of evolutionary divergence of individual viral isolates (Neverov et al., 2006). The microarrays consisting of multiple individual short oligoprobes were shown to be an efficient and sensitive genetic method for detection of single point mutations in viral and bacterial genomes (Chizhikov et al., 2002; Grinev et al., 2008b; Laassri et al., 2003, 2005, 2007; Volokhov et al., 2002). Microarray assays can also help simultaneously detect and identify the genotype and strain of common food-borne viruses without using PCR (Chen et al., 2011). In general, microarray technology can be easily implemented for detection and genotyping of any pathogen. Recently a pan-Microbial Detection Array was designed to detect all known viruses and bacteria (Gardner et al., 2010). Although further improvements, optimizations, and automation are still needed to fully implement the microarray technique in routine research and clinical practices, the potential role of these robust technologies in rapid diagnostics of

The microarray development and evaluation study included the analysis of a total of 34 plasma specimens from blood donor units identified as positive for WNV by nucleic acid

contain primary amino groups introduced during chemical synthesis.

(Roerig et al., 2005), and study viral evolution (Cherkasova et al., 2003).

multiple viral and bacterial pathogens is indisputable (Miller, 2009).

**2. Material and methods**

tests used to screen blood donations (Table 1).

**2.1 Plasma samples**

Table 1. WNV isolates used for microarray assay validation. FFP indicates fresh frozen plasma sample. Passage P1 indicates first isolation in Vero cells; P2 and P3 indicates subsequent virus passages. Isolates in boldface were used for the full genome array validation.

These samples were collected in different geographic locations of the continental U.S. from the 2002-2009 epidemic seasons under IRB approved informed consent. In addition to 23 previously published isolates, which were used for structural region investigation (Grinev et al., 2008b), 11 WNV isolates from 2007 and 2009 were used to conduct microarray analyses of their full genomes in order to detect emerged genetic differences in comparison with that of the reference WNV strain NY99.

Application of a Microarray-Based Assay for the Study of Genetic Diversity of West Nile Virus 165

Table 2. Forward and reverse primers used for PCR amplification of different regions of WNV genome. Numbers in brackets indicates the primer position in the NY99 genome. Reverse primers marked as '*m*' contained the T7 RNA polymerase promoter sequence.

### **2.2 Viral isolates**

West Nile virus isolation from tested plasma samples was performed using Vero cells. Vero cells were plated in T75 flasks and grown to 85% confluence in EMEM (GIBCO BRL, Gaithersburg, MD, USA) supplemented with 5% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 10µg/mL of penicillin/streptomycin (GIBCO). For viral isolation, growth medium was removed, 500 µl of each plasma sample were added to individual flasks and the total volume was adjusted to 5 ml with fresh medium. Vero cells were incubated with the viral inoculum for 2 hours, either at room temperature under gentle rocking or at 37oC with mixing every 10-15 min. After incubation, 10 ml of fresh medium were added, and the cultures were additionally incubated at 37 ºC in 5% CO2, and observed daily under phase microscopy for gross morphological degeneration i.e., cytopathic effect (CPE). Supernatants were harvested when extensive CPE was observed. Harvested supernatants were centrifuged to remove cell debris and aliquots were frozen at –80 ºC until further analysis.

### **2.3 RNA extraction**

Total RNA was extracted from 1-3 ml of plasma samples with Trizol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer protocol with additional step of ethanol precipitation. Viral RNA from 140 µl of infected Vero cell culture supernatants was extracted by using the QiaAMP viral RNA extraction kit (Qiagen, Valencia, CA), according to the manufacturer protocol. Each RNA sample was dissolved in 60 µl of RNase-free water and stored at -80◦C.

### **2.4 Reverse transcription**

Reverse transcription was performed in 20-40 µl reaction volume at 47◦C for 2 h using a mixture of specific reverse primers (Table 2) and SuperScript III (Invitrogen, Carlsbad, CA) reverse transcription system according to the manufacturer's instructions. 2-3 µl of the reaction mixture were used for the subsequent DNA amplification.

### **2.5 PCR amplification**

PCR fragments covering the entire structural region of the FDA-Hu2002 plasma sample were amplified by semi-nested PCR (Figure 4) from cDNA using the Hi-Fidelity PCR system (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Primers used for PCR amplification are shown in Table 2.

cDNA was amplified in the first round of PCR in a GeneAmp 9700 thermocycler (Applied Biosystems Inc., Foster City, CA) using the following protocol: denaturation at 94 °C for 30 s., 35 cycles of 30 s at 94 °C, 30 s at 50 °C, and 2 min at 68 °C. The final extension was carried out at 68 °C for 7 min. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen Valencia, CA), according to the manufacturer's protocol. 5 µl of the 1st round PCR product was used for the 2nd round of amplification with reverse primers containing the T7 RNA polymerase promoter sequence at the 5' ends tagged to the WNV sequence using the cycling program described above and the protocol for Hi-Fidelity PCR kit.

164 Diversity, Gene Expression Mechanisms and Host-Virus Interactions

West Nile virus isolation from tested plasma samples was performed using Vero cells. Vero cells were plated in T75 flasks and grown to 85% confluence in EMEM (GIBCO BRL, Gaithersburg, MD, USA) supplemented with 5% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 10µg/mL of penicillin/streptomycin (GIBCO). For viral isolation, growth medium was removed, 500 µl of each plasma sample were added to individual flasks and the total volume was adjusted to 5 ml with fresh medium. Vero cells were incubated with the viral inoculum for 2 hours, either at room temperature under gentle rocking or at 37oC with mixing every 10-15 min. After incubation, 10 ml of fresh medium were added, and the cultures were additionally incubated at 37 ºC in 5% CO2, and observed daily under phase microscopy for gross morphological degeneration i.e., cytopathic effect (CPE). Supernatants were harvested when extensive CPE was observed. Harvested supernatants were centrifuged to remove cell debris and aliquots were frozen at –80 ºC until further

Total RNA was extracted from 1-3 ml of plasma samples with Trizol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer protocol with additional step of ethanol precipitation. Viral RNA from 140 µl of infected Vero cell culture supernatants was extracted by using the QiaAMP viral RNA extraction kit (Qiagen, Valencia, CA), according to the manufacturer protocol. Each RNA sample was dissolved in 60 µl of RNase-free water

Reverse transcription was performed in 20-40 µl reaction volume at 47◦C for 2 h using a mixture of specific reverse primers (Table 2) and SuperScript III (Invitrogen, Carlsbad, CA) reverse transcription system according to the manufacturer's instructions. 2-3 µl of the

PCR fragments covering the entire structural region of the FDA-Hu2002 plasma sample were amplified by semi-nested PCR (Figure 4) from cDNA using the Hi-Fidelity PCR system (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Primers used for

cDNA was amplified in the first round of PCR in a GeneAmp 9700 thermocycler (Applied Biosystems Inc., Foster City, CA) using the following protocol: denaturation at 94 °C for 30 s., 35 cycles of 30 s at 94 °C, 30 s at 50 °C, and 2 min at 68 °C. The final extension was carried out at 68 °C for 7 min. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen Valencia, CA), according to the manufacturer's protocol. 5 µl of the 1st round PCR product was used for the 2nd round of amplification with reverse primers containing the T7 RNA polymerase promoter sequence at the 5' ends tagged to the WNV sequence using the

reaction mixture were used for the subsequent DNA amplification.

cycling program described above and the protocol for Hi-Fidelity PCR kit.

**2.2 Viral isolates**

analysis.

**2.3 RNA extraction**

and stored at -80◦C.

**2.4 Reverse transcription**

**2.5 PCR amplification**

PCR amplification are shown in Table 2.

Table 2. Forward and reverse primers used for PCR amplification of different regions of WNV genome. Numbers in brackets indicates the primer position in the NY99 genome. Reverse primers marked as '*m*' contained the T7 RNA polymerase promoter sequence.

Application of a Microarray-Based Assay for the Study of Genetic Diversity of West Nile Virus 167

mixed with the Cy5-QC oligonucleotide at a molar ratio of 10 to 1, followed by denaturing at 95°C for 2 min and rapid chilling on ice. The area of each array on the slide was covered with a separate 20×9-mm glass cover-slip containing two rails of 35µm (±10µm) in height (Erie Scientific, Portsmouth, NH) that created a tiny "hybridization chamber" between the rail-elevated cover-slip and microarray slide. Each hybridization mixture (approximately 5-7 µl) was gently loaded by pipette into this space; the slides were placed in a hybridization cassette (Tele-Chem International, Inc., Sunnyvale, CA) to minimize sample evaporation and hybridized for 1 hour at 50°C. After hybridization, the slides were washed twice for 5 min with 2× SSC with 0.1% SDS pre-warmed to 50°C, then once for 2 min with 0.2× SSC buffer and once for 1 min with 0.1× SSC buffer all at room temperature followed by

Fig. 4. Scheme of preparation of Cy3 fluorescently labeled RNA samples. The Cy3 image shows the design of array and the results of hybridization experiment. Each microarray slide contained five identical arrays for analysis of five different RNA samples including the

 **NY99 S1 S2 S3 S4**

**Hybridization and scan**

**RNA synthesis RNA synthesis**

**Labeling Cy3 Labeling Cy3**

**R-T7**

**+ Cy5 QC**

 **Vero supernatant**

**RNA isolation**

**One Step RT-PCR**

The fluorescent images of processed microarray slides were generated using GenePix 4100 (Axon Instruments) and ScanArray 5000 (Perkin Elmer) scanners equipped with two lasers

reference WNV isolate NY99 and four clinical samples (S1-S4).

**2.9 Microarray scanning and image analysis**

centrifugation at 1000 rpm for 5 min to remove any traces of the buffer.

**F**

**Cultivation**

**F R**

**Plasma sample**

**RNA isolation**

**cDNA synthesis**

**1st round PCR**

**2nd round PCR**

Viral RNA samples isolated from Vero cell culture supernatants were amplified using forward and T7 tagged reverse primers (Table 2) using the OneStep RT-PCR Kit (Qiagen Valencia, CA), according to the manufacturer's Q-Solution protocol with the 40 cycle program as recommended. PCR products were separated by electrophoresis in 0.8% agarose gel prepared in 1×TAE buffer containing 0.2 mg/ml of ethidium bromide. The stained DNA fragments were excised under UV light. PCR products were purified using the MinElute Gel Extraction Kit (Qiagen Valencia, CA), according to the manufacturer's protocol, and stored at -20 °C. DNA fragments were used for the subsequent RNA synthesis using T7 RNA polymerase.

### **2.6 Preparation of fluorescently labeled WNV RNA for hybridization**

The scheme for preparation of fluorescently labeled RNA samples is shown in Figure 4. Single-stranded RNA (ssRNA) samples used for hybridization were synthesized by T7 polymerase-driven transcription of the PCR products using the MEGA script T7 High Yield Transcription Kit (Ambion, Austin, TX) according to the manufacturer's instructions. The MICROMAX ASAP RNA Labeling Kit (Perkin Elmer, Boston, MA) was used to incorporate Cy3 fluorophore into the ssRNA molecules. Fluorescently labeled ssRNA samples were purified from unincorporated dye using the Centrisep Spin Columns (Princeton Separations, Adelphia, NJ), dried under vacuum, and solubilized in the MICROMAX Hybridization Buffer III at a final concentration of 0.5-1.0 µM. The Cy5 antisense-QC oligonucleotide was prepared by 5'-end labeling with indocarbocyanine (Cy5)-dCTP during synthesis. The Cy5-antisense QC oligonucleotide was purified by high performance liquid chromatography (HPLC).

### **2.7 Microarray oligoprobe design and microchip fabrication**

Oligonucleotide probes (oligoprobes) were designed on the basis of the nucleotide sequence of the reference strain NY99 (GenBank accession no.: AF196835) using the OligoScan software. A total of 1274 oligoprobes overlapping by half-lengths, with melting temperatures around 50°C, were designed for microarray-based detection of single point mutations in the entire WNV genome. Each oligoprobe spotting mixture contained 20 µM specific oligoprobe and 1 µM quality control (QC) oligonucleotide in 1× printing buffer (150 mM sodium phosphate, pH 8.5). The probes were used to print five identical arrays (each array contained triplicate set of oligoprobes) per each amine-binding glass slide (CodeLink, Amersham Biosciences, Piscataway, NJ) using a contact microspotting robot PIXSYS 5500 (Cartesian Technologies, Inc.) and a ChipMaker microspotting device equipped with CMP-7 pins delivering approximately 2 to 3 nl of a spotting mixture per spot (Tele-Chem International Inc.). Normally, the size of spots did not exceed 250 μm in diameter. After printing slides were processed and stored according to the manufacturer's protocol. Thus, each microarray slide could be used for simultaneous analysis of five RNA samples. The array layout is shown in Fig. 4. The quality of each lot of printed slides was tested by hybridizing the last printed slide with Cy5-labeled antisense QC oligonucleotide.

### **2.8 Hybridization conditions**

Hybridization between microarray oligoprobes and fluorescently labeled ssRNA samples was performed for 1 hour at 50°C. Before hybridization, Cy3-labeled ssRNA samples was

166 Diversity, Gene Expression Mechanisms and Host-Virus Interactions

Viral RNA samples isolated from Vero cell culture supernatants were amplified using forward and T7 tagged reverse primers (Table 2) using the OneStep RT-PCR Kit (Qiagen Valencia, CA), according to the manufacturer's Q-Solution protocol with the 40 cycle program as recommended. PCR products were separated by electrophoresis in 0.8% agarose gel prepared in 1×TAE buffer containing 0.2 mg/ml of ethidium bromide. The stained DNA fragments were excised under UV light. PCR products were purified using the MinElute Gel Extraction Kit (Qiagen Valencia, CA), according to the manufacturer's protocol, and stored at -20 °C. DNA fragments were used for the subsequent RNA synthesis using T7 RNA polymerase.

The scheme for preparation of fluorescently labeled RNA samples is shown in Figure 4. Single-stranded RNA (ssRNA) samples used for hybridization were synthesized by T7 polymerase-driven transcription of the PCR products using the MEGA script T7 High Yield Transcription Kit (Ambion, Austin, TX) according to the manufacturer's instructions. The MICROMAX ASAP RNA Labeling Kit (Perkin Elmer, Boston, MA) was used to incorporate Cy3 fluorophore into the ssRNA molecules. Fluorescently labeled ssRNA samples were purified from unincorporated dye using the Centrisep Spin Columns (Princeton Separations, Adelphia, NJ), dried under vacuum, and solubilized in the MICROMAX Hybridization Buffer III at a final concentration of 0.5-1.0 µM. The Cy5 antisense-QC oligonucleotide was prepared by 5'-end labeling with indocarbocyanine (Cy5)-dCTP during synthesis. The Cy5-antisense QC oligonucleotide was purified by high performance liquid

Oligonucleotide probes (oligoprobes) were designed on the basis of the nucleotide sequence of the reference strain NY99 (GenBank accession no.: AF196835) using the OligoScan software. A total of 1274 oligoprobes overlapping by half-lengths, with melting temperatures around 50°C, were designed for microarray-based detection of single point mutations in the entire WNV genome. Each oligoprobe spotting mixture contained 20 µM specific oligoprobe and 1 µM quality control (QC) oligonucleotide in 1× printing buffer (150 mM sodium phosphate, pH 8.5). The probes were used to print five identical arrays (each array contained triplicate set of oligoprobes) per each amine-binding glass slide (CodeLink, Amersham Biosciences, Piscataway, NJ) using a contact microspotting robot PIXSYS 5500 (Cartesian Technologies, Inc.) and a ChipMaker microspotting device equipped with CMP-7 pins delivering approximately 2 to 3 nl of a spotting mixture per spot (Tele-Chem International Inc.). Normally, the size of spots did not exceed 250 μm in diameter. After printing slides were processed and stored according to the manufacturer's protocol. Thus, each microarray slide could be used for simultaneous analysis of five RNA samples. The array layout is shown in Fig. 4. The quality of each lot of printed slides was tested by

hybridizing the last printed slide with Cy5-labeled antisense QC oligonucleotide.

Hybridization between microarray oligoprobes and fluorescently labeled ssRNA samples was performed for 1 hour at 50°C. Before hybridization, Cy3-labeled ssRNA samples was

**2.6 Preparation of fluorescently labeled WNV RNA for hybridization**

**2.7 Microarray oligoprobe design and microchip fabrication**

chromatography (HPLC).

**2.8 Hybridization conditions**

mixed with the Cy5-QC oligonucleotide at a molar ratio of 10 to 1, followed by denaturing at 95°C for 2 min and rapid chilling on ice. The area of each array on the slide was covered with a separate 20×9-mm glass cover-slip containing two rails of 35µm (±10µm) in height (Erie Scientific, Portsmouth, NH) that created a tiny "hybridization chamber" between the rail-elevated cover-slip and microarray slide. Each hybridization mixture (approximately 5-7 µl) was gently loaded by pipette into this space; the slides were placed in a hybridization cassette (Tele-Chem International, Inc., Sunnyvale, CA) to minimize sample evaporation and hybridized for 1 hour at 50°C. After hybridization, the slides were washed twice for 5 min with 2× SSC with 0.1% SDS pre-warmed to 50°C, then once for 2 min with 0.2× SSC buffer and once for 1 min with 0.1× SSC buffer all at room temperature followed by centrifugation at 1000 rpm for 5 min to remove any traces of the buffer.

Fig. 4. Scheme of preparation of Cy3 fluorescently labeled RNA samples. The Cy3 image shows the design of array and the results of hybridization experiment. Each microarray slide contained five identical arrays for analysis of five different RNA samples including the reference WNV isolate NY99 and four clinical samples (S1-S4).

### **2.9 Microarray scanning and image analysis**

The fluorescent images of processed microarray slides were generated using GenePix 4100 (Axon Instruments) and ScanArray 5000 (Perkin Elmer) scanners equipped with two lasers

Application of a Microarray-Based Assay for the Study of Genetic Diversity of West Nile Virus 169

hybridization target that also contributes to the difference in sensitivity-specificity balance

Fig. 5. Scheme of the WNV microarray assay. Five arrays containing 1274 oligoprobes overlapped by half-lengths that covered the entire genome of WNV are shown. The Cy3 hybridization images show the layout of the printed arrays, each oligoprobe was spotted in triplicate. The analysis of the hybridization profile of WNV isolate ID140 from the 2007 U.S. epidemic is shown as a chart of hybridization signal ratios (y-axis) between the reference

isolate NY99 and the tested isolate. Oligoprobe numbers are shown on the x-axis.

mismatches for most of the spots of the array.

The oligoprobes were designed to have similar thermodynamic characteristics to ensure uniform hybridization signals from all microarray probes. The design of the microarray probes was performed using the OligoScan software, which is capable of selecting multiple oligonucleotide probes with similar thermodynamic features. The microarray contained overlapping oligoprobes of 15-26 bp in length with melting temperatures around 50°C, which was previously shown to be optimal for the detection of single nucleotide mutations and deletions-insertions (Laassri et al., 2005, 2007). The predicted melting temperatures of the oligoprobes varied from 49.7°C to 52.6°C. Therefore, an optimization of hybridization temperature that would provide efficient hybridization and high specificity for each spot on the array is required. We evaluated three hybridization temperatures: 47, 50 and 53°C. During the optimization process we determined that hybridization and washing of the microarrays at 50°C resulted in better discrimination between perfect matches and

The synthetic 5'-aminated oligoprobes were printed on CodeLink Activated slides, previously shown to be suitable slides for detection of single nucleotide mismatches (Laassri et al., 2007). Five arrays containing triplicate sets of oligoprobes were printed on each slide

for each oligoprobe and its targets.

operating at 632 nm (for excitation of Cy5 dye) and 543 nm (for excitation of Cy3 dye). Images were analyzed using ScanArray Express 2.1 (Perkin Elmer) and GenePix 3.0 (Axon Instruments) software. Each spot was defined by manual positioning of a grid over the array image. Atypical and empty spots were manually flagged and excluded from further analysis. Background fluorescence readings obtained from the region surrounding each spot were subtracted, and the net value of the Cy3 fluorescence signal from each oligoprobe was divided by the Cy5 signal value from the QC probe of the same spot to minimize the effect of spot size variation on resulting data. Data files generated by ScanArray Express and GenePix software were exported into MS Excel. To identify positions in the WNV genome where mutations occurred, the intensities of fluorescent signals from each array spot obtained for tested WNV isolates were compared with that of the reference NY99 isolate. The fluorescent signal ratio values between the reference NY99 and the tested sample were normalized using a linear regression model. A signal intensity ratio threshold from reference spots, specific to each microarray printing lot, was defined as an average ratio plus two standard deviation values. Any spots showing a ratio greater than the threshold value for a particular printing lot (i.e. considerably lower signal than reference isolate NY99) potentially indicated the presence of a mutation in the genomic area covered by the oligoprobe of that spot.

### **2.10 Validation of the microarray results**

Validation of the microarrays was performed by comparing microarray results with the sequencing data obtained for the tested WNV isolate. Direct sequencing of PCR products was described previously by Grinev et al. (2008a).

### **3. Results and discussion**

### **3.1 Optimization of the WNV microarray assay**

The objective of this study was to develop and assess the feasibility of a DNA microarray approach for rapid throughput detection of spontaneous nucleotide mutations in the genome of WNV. A microarray containing a set of 1274 oligoprobes overlapping by half of their lengths (overlapping factor = 2) was developed and evaluated by assessing the ability of microarray to detect all identified mutations in 34 clinical isolates of WNV that were previously sequenced in our laboratory (Fig. 5).

In fact, optimization of an oligonucleotide microarray assay is a multi-parametric task. Thus, the fluorescent signal from the microarray oligoprobes hybridized to the fluorescently labeled target is known to depend on several factors including the sequence of oligoprobes, the character of mutations, and the position of the mismatched nucleotide in the oligoprobe, as well as the propensity of the RNA hybridization target to form a secondary structure (Relogio et al., 2002; Liu et al., 2005; Naiser et al., 2008). The goal of the optimization process was to determine the hybridization conditions which would enable the sensitive and specific hybridization of the target RNA to the vast majority of oligoprobes composing the WNV microarray. In our study, we optimized the temperature and time of hybridization, the stringency of post-hybridization washing conditions, the image scanning settings (PMT and laser power) to achieve the most efficient discrimination for each probe. It should be noted that the different oligoprobes have slightly different melting temperatures with the

168 Diversity, Gene Expression Mechanisms and Host-Virus Interactions

operating at 632 nm (for excitation of Cy5 dye) and 543 nm (for excitation of Cy3 dye). Images were analyzed using ScanArray Express 2.1 (Perkin Elmer) and GenePix 3.0 (Axon Instruments) software. Each spot was defined by manual positioning of a grid over the array image. Atypical and empty spots were manually flagged and excluded from further analysis. Background fluorescence readings obtained from the region surrounding each spot were subtracted, and the net value of the Cy3 fluorescence signal from each oligoprobe was divided by the Cy5 signal value from the QC probe of the same spot to minimize the effect of spot size variation on resulting data. Data files generated by ScanArray Express and GenePix software were exported into MS Excel. To identify positions in the WNV genome where mutations occurred, the intensities of fluorescent signals from each array spot obtained for tested WNV isolates were compared with that of the reference NY99 isolate. The fluorescent signal ratio values between the reference NY99 and the tested sample were normalized using a linear regression model. A signal intensity ratio threshold from reference spots, specific to each microarray printing lot, was defined as an average ratio plus two standard deviation values. Any spots showing a ratio greater than the threshold value for a particular printing lot (i.e. considerably lower signal than reference isolate NY99) potentially indicated the presence of a mutation in the genomic area covered by the

Validation of the microarrays was performed by comparing microarray results with the sequencing data obtained for the tested WNV isolate. Direct sequencing of PCR products

The objective of this study was to develop and assess the feasibility of a DNA microarray approach for rapid throughput detection of spontaneous nucleotide mutations in the genome of WNV. A microarray containing a set of 1274 oligoprobes overlapping by half of their lengths (overlapping factor = 2) was developed and evaluated by assessing the ability of microarray to detect all identified mutations in 34 clinical isolates of WNV that were

In fact, optimization of an oligonucleotide microarray assay is a multi-parametric task. Thus, the fluorescent signal from the microarray oligoprobes hybridized to the fluorescently labeled target is known to depend on several factors including the sequence of oligoprobes, the character of mutations, and the position of the mismatched nucleotide in the oligoprobe, as well as the propensity of the RNA hybridization target to form a secondary structure (Relogio et al., 2002; Liu et al., 2005; Naiser et al., 2008). The goal of the optimization process was to determine the hybridization conditions which would enable the sensitive and specific hybridization of the target RNA to the vast majority of oligoprobes composing the WNV microarray. In our study, we optimized the temperature and time of hybridization, the stringency of post-hybridization washing conditions, the image scanning settings (PMT and laser power) to achieve the most efficient discrimination for each probe. It should be noted that the different oligoprobes have slightly different melting temperatures with the

oligoprobe of that spot.

**3. Results and discussion**

**2.10 Validation of the microarray results**

was described previously by Grinev et al. (2008a).

**3.1 Optimization of the WNV microarray assay**

previously sequenced in our laboratory (Fig. 5).

hybridization target that also contributes to the difference in sensitivity-specificity balance for each oligoprobe and its targets.

Fig. 5. Scheme of the WNV microarray assay. Five arrays containing 1274 oligoprobes overlapped by half-lengths that covered the entire genome of WNV are shown. The Cy3 hybridization images show the layout of the printed arrays, each oligoprobe was spotted in triplicate. The analysis of the hybridization profile of WNV isolate ID140 from the 2007 U.S. epidemic is shown as a chart of hybridization signal ratios (y-axis) between the reference isolate NY99 and the tested isolate. Oligoprobe numbers are shown on the x-axis.

The oligoprobes were designed to have similar thermodynamic characteristics to ensure uniform hybridization signals from all microarray probes. The design of the microarray probes was performed using the OligoScan software, which is capable of selecting multiple oligonucleotide probes with similar thermodynamic features. The microarray contained overlapping oligoprobes of 15-26 bp in length with melting temperatures around 50°C, which was previously shown to be optimal for the detection of single nucleotide mutations and deletions-insertions (Laassri et al., 2005, 2007). The predicted melting temperatures of the oligoprobes varied from 49.7°C to 52.6°C. Therefore, an optimization of hybridization temperature that would provide efficient hybridization and high specificity for each spot on the array is required. We evaluated three hybridization temperatures: 47, 50 and 53°C. During the optimization process we determined that hybridization and washing of the microarrays at 50°C resulted in better discrimination between perfect matches and mismatches for most of the spots of the array.

The synthetic 5'-aminated oligoprobes were printed on CodeLink Activated slides, previously shown to be suitable slides for detection of single nucleotide mismatches (Laassri et al., 2007). Five arrays containing triplicate sets of oligoprobes were printed on each slide

Application of a Microarray-Based Assay for the Study of Genetic Diversity of West Nile Virus 171

overlapping templates covering the full length of the larger template. The use of a hybridization RNA target of approximately 2500 nucleotides covering the entire structural region of WNV resulted in a twofold reduction of some hybridization signals when compared to the use of a mixture of three overlapping RNA templates in the equivalent

molar concentrations. The typical results of this experiment are shown in Figure 6.

Fig. 6. The analysis of microarray hybridization is shown as a chart that contains the oligoprobe numbers on the x-axis. The y-axis shows the signal ratio values between the isolate NY99 and two isolates from 2005: BSL13-05; and GCTX2-05. The results for hybridization using a mixture of three overlapping RNAs covering the whole structural region of WNV and 5'UTR are shown in blue. The results for hybridization using the one long RNA covering the whole structural region of WNV and 5'UTR are shown in red.

Consequently, mixtures of three RNA templates (instead of one long RNA target) were normally used for hybridization with microarray to ensure the high efficiency of mutation discrimination. The initial use of in-house printed arrays represented a convenient model for the development and optimization of mutation-detecting microarrays. However, the low densities of in-house printed arrays usually force the use of a substantial number of slides for a single hybridization experiment. It is quite laborious and relatively expensive. After optimization of the microarray system including the set of oligoprobes and hybridization conditions, a high density array can be prepared using a well developed system like the widely used GeneChip produced by Affymetrix, which relies on *in situ* synthesis of all oligoprobes covering the entire WNV genome in a single array. In contrast to the printed oligonucleotide arrays described above, the oligonucleotide probes for high-density arrays are synthesized directly on the surface of the microarray, which is usually a small quartz wafer. Because *in situ*-synthesized probes normally form a very tiny spots, multiple overlapping probes for each target may be included to improve sensitivity, specificity, and statistical accuracy. On the other hand, the use of a single array and mixture of RNA probes

to allow for simultaneous analysis of four target samples in each hybridization experiment. One array on each separate slide was always hybridized with the fluorescently labeled RNA prepared using the reference WN-NY99 strain while other four arrays were used for hybridization with fluorescently labeled RNAs prepared from the WNV isolates to be tested. To assess the reproducibility of microarray fabrication, all hybridizations were repeated twice using different slides. The hybridization temperature and time, post-hybridization washing and the detector setting were optimized. The MS Excel worksheet was developed as a result of analysis of each scan image and data obtained from triplicates of each oligoprobe averaged by the median. The values from each microarray spot were normalized by the signal from the quality controls. The normalized signals from the reference array were then divided by the relevant signals obtained from the sample array. The occurrence of mismatches between oligoprobe and fluorescently labeled RNA target resulted in significant reduction of the hybridization signal in comparison with a perfect matching pair. Thus, the monitoring of the ratios of hybridization signals from unknown samples and the reference samples could be used as a tool for detection of spontaneous mutations in the target region of the WNV genome. The scheme of microarray experiments is shown on Figure 5.

DNA microarrays containing short oligonucleotide probes (15-25 nt) provide a greater discrimination power compared to microarrays composed of larger oligonucleotides or PCR-amplified DNA fragments. The strongest signal ratios between perfect matched and mismatched sequences were observed when mutations were located near the center of an oligoprobe, and the shortest probes always had better discriminatory power (Urakawa et al., 2003). Although the microarray hybridization method provides limited information about the position of mutations in the analyzed genomic region when compared to sequencing, it has the significant advantage of allowing the identification of "hot spots" where random mutations occur within short (a few nucleotides) areas. It also may allow for the detection of those mutations even when they occur at relatively low levels (up to 1%) as in the case of mixtures of quasispecies that cannot be detected by traditional direct sequencing methods (Cherkasova et al., 2003; Leberre at al., 2007). The efficiency of microarrays for identification and discrimination of closely related bacteria and viruses has been previously demonstrated (Chizhikov et al., 2002; Hsia et al., 2007; Laassri et al., 2003; Nordström et al., 2005; Volokhov et al., 2002; Wade et al., 2004). The use of oligonucleotide microchips for screening of random mutations is based on the ability of microarrays to identify the presence of singlenucleotide mutations in the hybridization template (Hacia et al., 1999; Urakawa et al., 2003).

### **3.2 Optimization of hybridization probes**

In general, single-base-pair discrimination can be achieved by optimization of hybridization or array washing conditions. However, optimization of washing conditions does not guarantee the equal efficiency of mismatch detection for all designed oligoprobes, which is likely to be caused by nature and position of the particular mutation or even formation of the secondary and tertiary structures in the hybridization mixture. The increase of size of the RNA template tends to increase the complexity of the secondary structure of the RNA molecule, and may affect the efficiency of hybridization of the RNA template with the microarray oligoprobes. Therefore, the RNA template may be subdivided into shorter

170 Diversity, Gene Expression Mechanisms and Host-Virus Interactions

to allow for simultaneous analysis of four target samples in each hybridization experiment. One array on each separate slide was always hybridized with the fluorescently labeled RNA prepared using the reference WN-NY99 strain while other four arrays were used for hybridization with fluorescently labeled RNAs prepared from the WNV isolates to be tested. To assess the reproducibility of microarray fabrication, all hybridizations were repeated twice using different slides. The hybridization temperature and time, post-hybridization washing and the detector setting were optimized. The MS Excel worksheet was developed as a result of analysis of each scan image and data obtained from triplicates of each oligoprobe averaged by the median. The values from each microarray spot were normalized by the signal from the quality controls. The normalized signals from the reference array were then divided by the relevant signals obtained from the sample array. The occurrence of mismatches between oligoprobe and fluorescently labeled RNA target resulted in significant reduction of the hybridization signal in comparison with a perfect matching pair. Thus, the monitoring of the ratios of hybridization signals from unknown samples and the reference samples could be used as a tool for detection of spontaneous mutations in the target region

of the WNV genome. The scheme of microarray experiments is shown on Figure 5.

**3.2 Optimization of hybridization probes**

DNA microarray technology provides an opportunity to perform parallel nucleic acid hybridization with a large number of immobilized oligonucleotides on a small surface area. DNA microarrays containing short oligonucleotide probes (15-25 nt) provide a greater discrimination power compared to microarrays composed of larger oligonucleotides or PCR-amplified DNA fragments. The strongest signal ratios between perfect matched and mismatched sequences were observed when mutations were located near the center of an oligoprobe, and the shortest probes always had better discriminatory power (Urakawa et al., 2003). Although the microarray hybridization method provides limited information about the position of mutations in the analyzed genomic region when compared to sequencing, it has the significant advantage of allowing the identification of "hot spots" where random mutations occur within short (a few nucleotides) areas. It also may allow for the detection of those mutations even when they occur at relatively low levels (up to 1%) as in the case of mixtures of quasispecies that cannot be detected by traditional direct sequencing methods (Cherkasova et al., 2003; Leberre at al., 2007). The efficiency of microarrays for identification and discrimination of closely related bacteria and viruses has been previously demonstrated (Chizhikov et al., 2002; Hsia et al., 2007; Laassri et al., 2003; Nordström et al., 2005; Volokhov et al., 2002; Wade et al., 2004). The use of oligonucleotide microchips for screening of random mutations is based on the ability of microarrays to identify the presence of singlenucleotide mutations in the hybridization template (Hacia et al., 1999; Urakawa et al., 2003).

In general, single-base-pair discrimination can be achieved by optimization of hybridization or array washing conditions. However, optimization of washing conditions does not guarantee the equal efficiency of mismatch detection for all designed oligoprobes, which is likely to be caused by nature and position of the particular mutation or even formation of the secondary and tertiary structures in the hybridization mixture. The increase of size of the RNA template tends to increase the complexity of the secondary structure of the RNA molecule, and may affect the efficiency of hybridization of the RNA template with the microarray oligoprobes. Therefore, the RNA template may be subdivided into shorter overlapping templates covering the full length of the larger template. The use of a hybridization RNA target of approximately 2500 nucleotides covering the entire structural region of WNV resulted in a twofold reduction of some hybridization signals when compared to the use of a mixture of three overlapping RNA templates in the equivalent molar concentrations. The typical results of this experiment are shown in Figure 6.

Fig. 6. The analysis of microarray hybridization is shown as a chart that contains the oligoprobe numbers on the x-axis. The y-axis shows the signal ratio values between the isolate NY99 and two isolates from 2005: BSL13-05; and GCTX2-05. The results for hybridization using a mixture of three overlapping RNAs covering the whole structural region of WNV and 5'UTR are shown in blue. The results for hybridization using the one long RNA covering the whole structural region of WNV and 5'UTR are shown in red.

Consequently, mixtures of three RNA templates (instead of one long RNA target) were normally used for hybridization with microarray to ensure the high efficiency of mutation discrimination. The initial use of in-house printed arrays represented a convenient model for the development and optimization of mutation-detecting microarrays. However, the low densities of in-house printed arrays usually force the use of a substantial number of slides for a single hybridization experiment. It is quite laborious and relatively expensive. After optimization of the microarray system including the set of oligoprobes and hybridization conditions, a high density array can be prepared using a well developed system like the widely used GeneChip produced by Affymetrix, which relies on *in situ* synthesis of all oligoprobes covering the entire WNV genome in a single array. In contrast to the printed oligonucleotide arrays described above, the oligonucleotide probes for high-density arrays are synthesized directly on the surface of the microarray, which is usually a small quartz wafer. Because *in situ*-synthesized probes normally form a very tiny spots, multiple overlapping probes for each target may be included to improve sensitivity, specificity, and statistical accuracy. On the other hand, the use of a single array and mixture of RNA probes

Application of a Microarray-Based Assay for the Study of Genetic Diversity of West Nile Virus 173

Table 3. Fixed nucleotide mutations and ratios of hybridization signals normalized against

23 WNV isolates from 2002-2005 epidemic seasons we used to evaluate the first array containing oligoprobes covering the structural region. Fluorescent signal ratios for all mutations ranged from 4.4 to 85.5 depending on the position of the mismatch within the oligoprobe and the character of the mismatched nucleotide (Grinev et al., 2008b). Table 3 shows the positions of identified nucleotide mutations and ratios of hybridization signals from the respective hybridization templates normalized against the reference isolate NY99 for 24 fixed mutations determined in the complete genomes of isolates from 2007 and 2009 epidemics. All 11 completely sequenced isolates from this study shared 12 nucleotide mutations including a non-silent mutation in Env T1442C. Ten more common mutations were detected in 4 isolates ARC140-07, BSL5-09, BSL6-09 and BSL11-09. The signal ratios for these mutations varied in the range 3.1-73.9. The fluorescence signal ratios produced by single nucleotide mutations in all hybridization experiments exceeded the experimentally determined cut-off threshold value ranging from 0.2 to 2.1 when compared to that of completely matched pairs. The ratio value over 2.1 can be considered as an indication on the potential mutation in a specific genomic region pointing to the need for additional analysis of this region by sequence analysis. This approach has the advantage of substantially reducing the numbers and length of sequences required for proper surveillance studies. The results of our study showed that the WNV microarray was able to unambiguously detect all mutations in the viral genome previously identified

the reference isolate NY99. Unique mutations are not shown.

by routine sequencing analysis.

in a single experiment requires additional optimization of hybridization conditions. We found a loss of some signals while using a template composed of a mixture of 15 overlapping RNAs covering the entire WNV genome, probably due to formation of double stranded molecules between complementary parts of the WNV genome such as the cyclization sequences in the 5' and 3' UTRs and tight secondary structure formation. The usage of a number of shorter RNAs produced by multiplex PCR of a target region as a hybridization template might reduce this negative effect and improve the signal, as would chemical defragmentation of the long RNA probe.

### **3.3 Evaluation of genetic stability of West Nile Virus after isolation in Vero cell culture**

Our study required WNV isolation from human plasma samples in Vero cell cultures because of the low concentration of WNV and limited volume of starting material available from many of the specimens. It was shown previously that genetic changes in flaviviruses can be induced by consecutive passages in Vero cell cultures. For example, multiple passage of Dengue virus (DENV), which is closely related to WNV, in Vero cells has resulted in emergence of mutants with amino acid changes in E and occasionally in prM, but not in C. Some nucleotide mutations were detected after the first five passages (Lee et al., 1997). Further studies showed that three nucleotide mutations (two in E and one in NS1 proteins) that resulted in two amino acid alterations (one each in E and NS1) emerged in the DENV genome after 20 continuous passages in Vero cells. Additional passage of the virus for 30 passages caused four nucleotide changes (two each in E and NS1) that resulted in three amino acid substitutions (one in E and two in NS1) (Chen et al., 2003).

In order to investigate whether viral isolation using three consecutive passages in Vero cells resulted in genetic changes in the WNV genome, we cultivated the isolate FDA/HU-02 and compared the sequence and microarray results for the three passages (P1, P2 and P3). Total RNA samples were isolated from aliquots of the original plasma and from each of the passages 1-3. RNA samples were reverse transcribed and amplified by PCR using WNVspecific primers designed to cover the complete structural region of the virus, followed by PCR product purification, direct sequencing, and microarray hybridization. We found no significant changes in fluorescent signal ratio values obtained in one slide microarray hybridization experiment for each original plasma sample and the RNA isolated from each of the Vero cell culture passages. We also performed comparative sequence analysis of the aforementioned viral samples. There was no difference between the genomic sequence obtained from the isolate FDA-Hu2002 (P1) and two passages (P2 and P3) when compared to the genomic sequence obtained from RNA extracted from the original plasma sample. These results are in good concordance with the genetic stability data previously published for the chimeric Dengue and Yellow Fever-Dengue vaccine candidates passaged 10-20 times in Vero cells (Guirakhoo et al., 2004; Butrapet et al., 2006). Therefore we assume that WNV isolates generated from human specimens by a few passages in Vero cells represented the original virus, and that the structural region was not changed during a low number of serial passages in Vero cell cultures.

### **3.4 Evaluation of oligonucleotide microarray using clinical WNV isolates**

The ability of microarray to detect mutations in the target regions was evaluated by testing 34 previously sequenced WNV isolates obtained in the course of the 2002 - 2009 US epidemics.

172 Diversity, Gene Expression Mechanisms and Host-Virus Interactions

in a single experiment requires additional optimization of hybridization conditions. We found a loss of some signals while using a template composed of a mixture of 15 overlapping RNAs covering the entire WNV genome, probably due to formation of double stranded molecules between complementary parts of the WNV genome such as the cyclization sequences in the 5' and 3' UTRs and tight secondary structure formation. The usage of a number of shorter RNAs produced by multiplex PCR of a target region as a hybridization template might reduce this negative effect and improve the signal, as would

**3.3 Evaluation of genetic stability of West Nile Virus after isolation in Vero cell culture**  Our study required WNV isolation from human plasma samples in Vero cell cultures because of the low concentration of WNV and limited volume of starting material available from many of the specimens. It was shown previously that genetic changes in flaviviruses can be induced by consecutive passages in Vero cell cultures. For example, multiple passage of Dengue virus (DENV), which is closely related to WNV, in Vero cells has resulted in emergence of mutants with amino acid changes in E and occasionally in prM, but not in C. Some nucleotide mutations were detected after the first five passages (Lee et al., 1997). Further studies showed that three nucleotide mutations (two in E and one in NS1 proteins) that resulted in two amino acid alterations (one each in E and NS1) emerged in the DENV genome after 20 continuous passages in Vero cells. Additional passage of the virus for 30 passages caused four nucleotide changes (two each in E and NS1) that resulted in three

In order to investigate whether viral isolation using three consecutive passages in Vero cells resulted in genetic changes in the WNV genome, we cultivated the isolate FDA/HU-02 and compared the sequence and microarray results for the three passages (P1, P2 and P3). Total RNA samples were isolated from aliquots of the original plasma and from each of the passages 1-3. RNA samples were reverse transcribed and amplified by PCR using WNVspecific primers designed to cover the complete structural region of the virus, followed by PCR product purification, direct sequencing, and microarray hybridization. We found no significant changes in fluorescent signal ratio values obtained in one slide microarray hybridization experiment for each original plasma sample and the RNA isolated from each of the Vero cell culture passages. We also performed comparative sequence analysis of the aforementioned viral samples. There was no difference between the genomic sequence obtained from the isolate FDA-Hu2002 (P1) and two passages (P2 and P3) when compared to the genomic sequence obtained from RNA extracted from the original plasma sample. These results are in good concordance with the genetic stability data previously published for the chimeric Dengue and Yellow Fever-Dengue vaccine candidates passaged 10-20 times in Vero cells (Guirakhoo et al., 2004; Butrapet et al., 2006). Therefore we assume that WNV isolates generated from human specimens by a few passages in Vero cells represented the original virus, and that the structural region was not changed during a low number of serial

amino acid substitutions (one in E and two in NS1) (Chen et al., 2003).

**3.4 Evaluation of oligonucleotide microarray using clinical WNV isolates**

The ability of microarray to detect mutations in the target regions was evaluated by testing 34 previously sequenced WNV isolates obtained in the course of the 2002 - 2009 US epidemics.

chemical defragmentation of the long RNA probe.

passages in Vero cell cultures.

Table 3. Fixed nucleotide mutations and ratios of hybridization signals normalized against the reference isolate NY99. Unique mutations are not shown.

23 WNV isolates from 2002-2005 epidemic seasons we used to evaluate the first array containing oligoprobes covering the structural region. Fluorescent signal ratios for all mutations ranged from 4.4 to 85.5 depending on the position of the mismatch within the oligoprobe and the character of the mismatched nucleotide (Grinev et al., 2008b). Table 3 shows the positions of identified nucleotide mutations and ratios of hybridization signals from the respective hybridization templates normalized against the reference isolate NY99 for 24 fixed mutations determined in the complete genomes of isolates from 2007 and 2009 epidemics. All 11 completely sequenced isolates from this study shared 12 nucleotide mutations including a non-silent mutation in Env T1442C. Ten more common mutations were detected in 4 isolates ARC140-07, BSL5-09, BSL6-09 and BSL11-09. The signal ratios for these mutations varied in the range 3.1-73.9. The fluorescence signal ratios produced by single nucleotide mutations in all hybridization experiments exceeded the experimentally determined cut-off threshold value ranging from 0.2 to 2.1 when compared to that of completely matched pairs. The ratio value over 2.1 can be considered as an indication on the potential mutation in a specific genomic region pointing to the need for additional analysis of this region by sequence analysis. This approach has the advantage of substantially reducing the numbers and length of sequences required for proper surveillance studies. The results of our study showed that the WNV microarray was able to unambiguously detect all mutations in the viral genome previously identified by routine sequencing analysis.

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### **4. Conclusion**

Viral adaptation through fixation of spontaneous mutations is an important factor potentially associated with reoccurrence of WNV outbreaks in the New World. The emergence of new genetic variants of WNV raise issues of public health importance because they may affect the sensitivity of both screening and diagnostic assays, as well as the development of vaccines and drugs. We have developed and optimized a WNV microarray assay, which enabled simple monitoring of WNV genetic variability and rapid detection of any nucleotide mutation within the entire viral genome. Our microarray system potentially can serve as a high throughput, rapid and effective approach for the identification of WNV mutations, and characterization of circulating WNV genetic variants.

### **5. Acknowledgments**

We would like to thank Drs Caren Chancey, German Anez Gutierrez and Robert Duncan for helpful discussion and review of the manuscript, Dr Majid Laassri for technical assistance, and Dr Konstantin Chumakov for OligoScan software.

### **6. References**

174 Diversity, Gene Expression Mechanisms and Host-Virus Interactions

Viral adaptation through fixation of spontaneous mutations is an important factor potentially associated with reoccurrence of WNV outbreaks in the New World. The emergence of new genetic variants of WNV raise issues of public health importance because they may affect the sensitivity of both screening and diagnostic assays, as well as the development of vaccines and drugs. We have developed and optimized a WNV microarray assay, which enabled simple monitoring of WNV genetic variability and rapid detection of any nucleotide mutation within the entire viral genome. Our microarray system potentially can serve as a high throughput, rapid and effective approach for the identification of WNV

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**9**

*USA*

**Microarray Techniques for Evaluation of**

**Genetic Stability of Live Viral Vaccines**

Mones S. Abu-Asab3 and Konstantin Chumakov1

*3National Cancer Institute, National Institutes of Health, Bethesda*

Recent advances in biotechnology gave rise to a set of microarray technologies that became ubiquitous in research, medicine, and industry for tens of applications. Microarray technology has centered on providing platforms for analyzing, in a single experiment, tens or even hundreds of samples from different biologic sources. Its rapid and global adoption has been predicated on its simplicity and efficiency in quickly providing relevant data generated by simultaneous testing of biological samples with a large number of probes. In this chapter, we describe new microarray approaches that have considerably simplified the characterization of viral genes and genomes with specific emphasis on analysis of the

There are different types of vaccines that immunize against viruses: whole viral vaccines (either live attenuated or inactivated), subunit vaccines; purified or recombinant viral

Genetic instability and plasticity of genomes are inherent properties of viruses, especially RNA viruses, with many profound implications for their replication, evolution, and pathogenesis. Because of the presence of a large number of mutants, populations of viruses are often described as quasispecies (Domingo et al., 1985; Hansen et al., 2004). Most mutants are present at a relatively low level, making them difficult to detect using conventional

Genetic stability of live viral vaccines, including recombinant virus vaccines, is a key element of their safety and protective efficacy. Assessment of genetic stability is an important part of pre-licensure evaluation and quality control of a live viral vaccine, both during its manufacturing and after its administration. Spontaneous mutations easily emerge during viral replication and accumulation of mutants must be identified to ensure

**1. Introduction**

genetic stability of live viral vaccines.

antigen vaccines, and DNA vaccines.

sequencing methods.

the safety of live vaccines.

Majid Laassri1, Elena Cherkasova2,

*1Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Rockville, 2National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda,*

### **Microarray Techniques for Evaluation of Genetic Stability of Live Viral Vaccines**

Majid Laassri1, Elena Cherkasova2,

Mones S. Abu-Asab3 and Konstantin Chumakov1 *1Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Rockville, 2National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, 3National Cancer Institute, National Institutes of Health, Bethesda USA*

### **1. Introduction**

Viral Genomes – Molecular Structure,

180 Diversity, Gene Expression Mechanisms and Host-Virus Interactions

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elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral

Recent advances in biotechnology gave rise to a set of microarray technologies that became ubiquitous in research, medicine, and industry for tens of applications. Microarray technology has centered on providing platforms for analyzing, in a single experiment, tens or even hundreds of samples from different biologic sources. Its rapid and global adoption has been predicated on its simplicity and efficiency in quickly providing relevant data generated by simultaneous testing of biological samples with a large number of probes. In this chapter, we describe new microarray approaches that have considerably simplified the characterization of viral genes and genomes with specific emphasis on analysis of the genetic stability of live viral vaccines.

There are different types of vaccines that immunize against viruses: whole viral vaccines (either live attenuated or inactivated), subunit vaccines; purified or recombinant viral antigen vaccines, and DNA vaccines.

Genetic instability and plasticity of genomes are inherent properties of viruses, especially RNA viruses, with many profound implications for their replication, evolution, and pathogenesis. Because of the presence of a large number of mutants, populations of viruses are often described as quasispecies (Domingo et al., 1985; Hansen et al., 2004). Most mutants are present at a relatively low level, making them difficult to detect using conventional sequencing methods.

Genetic stability of live viral vaccines, including recombinant virus vaccines, is a key element of their safety and protective efficacy. Assessment of genetic stability is an important part of pre-licensure evaluation and quality control of a live viral vaccine, both during its manufacturing and after its administration. Spontaneous mutations easily emerge during viral replication and accumulation of mutants must be identified to ensure the safety of live vaccines.

Microarray Techniques for Evaluation of Genetic Stability of Live Viral Vaccines 183

Additionally, increased yields of such viruses during vaccine production may help stabilize the genome by relieving selective pressure, thereby preventing random and potentially

Conventional assays of genetic stability of viruses by combined sequencing and sequence analysis are generally too insensitive to detect small proportion of mutant viruses in a quasispecies, and are laborious. Thus, sensitive high-throughput microarray techniques including microarray for resequencing and sequence heterogeneity (MARSH), microarray analysis of viral recombination (MAVR) assay, and microarray for quantitation of known virulent mutations (MQNVM) have been developed and applied as valuable tools to evaluate the genetic stability of live viral vaccines (Cherkasova et al., 2003; Laassri et al.,

These microarray approaches allow large-scale full-genome mutational screening of live viral vaccines from various sources including cell culture, humans, monkeys, and mice. They can be used to improve quality control and to accelerate development of safer and more effective vaccines. The study of genetic stability also contributes to our understanding

DNA microarray is a high-throughput hybridization technology used for quantitative and qualitative assessments of gene-expression, chromosomal aberrations, and mutations in molecular biology and biotechnology. It consists of an arrayed series of tens or thousands of micro-spots of oligonucleotides of specific DNA sequence, known as probes. Probes can be short regions of a gene or other DNA elements used to hybridize DNA or RNA samples (targets) under high-stringency conditions. Probe-target hybridizations are usually detected and quantified using fluorophore-labeled targets to determine the relative quantities of nucleic acid sequences in the target. Since an array can contain tens of thousands of probes, a microarray experiment enables analysis of many genes simultaneously. Therefore, arrays

In standard microarrays, the probes are attached via their engineered chemical group to a solid surface by a covalent bond to a chemical matrix (via epoxy - silane, amino-silane, lysine, polyacrylamide, or others). The solid surface can be glass, a silicon chip, or

DNA microarrays can be used to measure changes in expression level, to detect and quantify single nucleotide polymorphisms (SNPs), to genotype and resequence mutant genomes, or to determine recombinant nucleotide sequences. Microarrays vary in fabrication, operating protocols, accuracy, and efficiency. Additional factors affecting microarray experiments are experimental design and methods for analyzing the data.

Several approaches based on hybridization of viral probes with oligonucleotide microarrays have been applied for rapid analysis of genetic variations during the microevolution of viruses. Microarray for resequencing and sequence heterogeneity (MARSH) was used to

undesirable mutations from being passively selected through the "passenger effect".

2011; Laassri et al., 2005; Laassri et al., 2007).

of live viral vaccine evolution.

microscopic beads.

**2. DNA microarray: An overview**

have dramatically accelerated many types of investigations.

**3. Microarray for resequencing and sequence heterogeneity**

To ensure maximum genetic stability and to optimize genetic structure of prospective live vaccine strains, it is important to identify the mutations that accumulate both during manufacturing and replication in vaccine recipients. Incorporation of mutations that increase virus fitness and do not affect its attenuation into the genetic makeup of the new vaccine strain may increase its potency and contribute to genetic stability

Most new viral vaccines are produced by propagation in cell cultures that do not necessarily represent the natural substrate for the virus, raising the possibility of introducing undesirable mutations in the course of virus adaptation. RNA viral vaccines mutate easily upon passage in cell cultures, which can change the phenotype (Amexis et al., 2001), leading to increased pathogenicity. That occurred with pSPBNGA-GA, a live rabies virus recombinant vaccine candidate, which was obtained via reverse genetics (Dietzschold et al., 2004). Additionally, it was demonstrated that some deletions in HIV-1 vaccine strains can evolve into fast-replicating variants by multiplication of remaining sequence motifs, and their safety is therefore not guaranteed (Berkhout et al., 1999), and the presence of even a small fraction of viral mutants in an oral poliovirus vaccine can have negative effect on its safety (Chumakov et al., 1991), suggesting that genetic consistency must be carefully monitored to ensure that accumulated mutants do not adversely impair the safety and efficacy of the vaccine.

In addition, vaccines have been recently developed to serve as live viral vectors expressing heterologous host genes.  The recombination in genomes of chimeric viruses probably plays an important role in the reduction of viral fitness that leads to attenuation. This creates a selective pressure to accumulate mutants that restore viral fitness by adapting heterologous genomic parts to each other, potentially leading to a loss of attenuation. The accumulation of mutations and genetic stability of flaviviruses were previously reported (Dunster et al., 1999; Laassri et al., 2011; Pugachev et al., 2004; Pugachev et al., 2002; Pugachev et al., 2007).

Of paramount importance is the need to demonstrate the genetic stability of the recombinant construct and confirm the fidelity of the heterologous gene inserted into the vector genome (WHO, 1990). An important consideration for the licensure and use of any genetically engineered live vaccine is the stability of the vector and that of the recombinant construct. In addition, genetic stability is an important safety concern, since predictions of vaccine behavior rely heavily on the knowledge of the genetic makeup of the recombinant. If a recombinant vaccine is to be useful, it should undergo no substantial mutation either during production of the vaccine by passage of working seed or after administration to the target species.

Therefore, it is essential to identify genomic loci that are prone to mutations and determine their phenotypes. If mutations in unstable genomic loci increase virulence, then methods to prevent their emergence and control their presence in vaccine preparations must be developed. On the other hand, if the fitness-restoring mutants do not lead to de-attenuation, then it may be desirable to incorporate them into the genetic makeup of the vaccine strain. Additionally, increased yields of such viruses during vaccine production may help stabilize the genome by relieving selective pressure, thereby preventing random and potentially undesirable mutations from being passively selected through the "passenger effect".

Conventional assays of genetic stability of viruses by combined sequencing and sequence analysis are generally too insensitive to detect small proportion of mutant viruses in a quasispecies, and are laborious. Thus, sensitive high-throughput microarray techniques including microarray for resequencing and sequence heterogeneity (MARSH), microarray analysis of viral recombination (MAVR) assay, and microarray for quantitation of known virulent mutations (MQNVM) have been developed and applied as valuable tools to evaluate the genetic stability of live viral vaccines (Cherkasova et al., 2003; Laassri et al., 2011; Laassri et al., 2005; Laassri et al., 2007).

These microarray approaches allow large-scale full-genome mutational screening of live viral vaccines from various sources including cell culture, humans, monkeys, and mice. They can be used to improve quality control and to accelerate development of safer and more effective vaccines. The study of genetic stability also contributes to our understanding of live viral vaccine evolution.

### **2. DNA microarray: An overview**

Viral Genomes – Molecular Structure,

182 Diversity, Gene Expression Mechanisms and Host-Virus Interactions

To ensure maximum genetic stability and to optimize genetic structure of prospective live vaccine strains, it is important to identify the mutations that accumulate both during manufacturing and replication in vaccine recipients. Incorporation of mutations that increase virus fitness and do not affect its attenuation into the genetic makeup of the new

Most new viral vaccines are produced by propagation in cell cultures that do not necessarily represent the natural substrate for the virus, raising the possibility of introducing undesirable mutations in the course of virus adaptation. RNA viral vaccines mutate easily upon passage in cell cultures, which can change the phenotype (Amexis et al., 2001), leading to increased pathogenicity. That occurred with pSPBNGA-GA, a live rabies virus recombinant vaccine candidate, which was obtained via reverse genetics (Dietzschold et al., 2004). Additionally, it was demonstrated that some deletions in HIV-1 vaccine strains can evolve into fast-replicating variants by multiplication of remaining sequence motifs, and their safety is therefore not guaranteed (Berkhout et al., 1999), and the presence of even a small fraction of viral mutants in an oral poliovirus vaccine can have negative effect on its safety (Chumakov et al., 1991), suggesting that genetic consistency must be carefully monitored to ensure that accumulated mutants do not adversely impair the safety and

In addition, vaccines have been recently developed to serve as live viral vectors expressing heterologous host genes.  The recombination in genomes of chimeric viruses probably plays an important role in the reduction of viral fitness that leads to attenuation. This creates a selective pressure to accumulate mutants that restore viral fitness by adapting heterologous genomic parts to each other, potentially leading to a loss of attenuation. The accumulation of mutations and genetic stability of flaviviruses were previously reported (Dunster et al., 1999; Laassri et al.,

Of paramount importance is the need to demonstrate the genetic stability of the recombinant construct and confirm the fidelity of the heterologous gene inserted into the vector genome (WHO, 1990). An important consideration for the licensure and use of any genetically engineered live vaccine is the stability of the vector and that of the recombinant construct. In addition, genetic stability is an important safety concern, since predictions of vaccine behavior rely heavily on the knowledge of the genetic makeup of the recombinant. If a recombinant vaccine is to be useful, it should undergo no substantial mutation either during production of the vaccine by passage of working seed or after administration to the

Therefore, it is essential to identify genomic loci that are prone to mutations and determine their phenotypes. If mutations in unstable genomic loci increase virulence, then methods to prevent their emergence and control their presence in vaccine preparations must be developed. On the other hand, if the fitness-restoring mutants do not lead to de-attenuation, then it may be desirable to incorporate them into the genetic makeup of the vaccine strain.

vaccine strain may increase its potency and contribute to genetic stability

2011; Pugachev et al., 2004; Pugachev et al., 2002; Pugachev et al., 2007).

efficacy of the vaccine.

target species.

DNA microarray is a high-throughput hybridization technology used for quantitative and qualitative assessments of gene-expression, chromosomal aberrations, and mutations in molecular biology and biotechnology. It consists of an arrayed series of tens or thousands of micro-spots of oligonucleotides of specific DNA sequence, known as probes. Probes can be short regions of a gene or other DNA elements used to hybridize DNA or RNA samples (targets) under high-stringency conditions. Probe-target hybridizations are usually detected and quantified using fluorophore-labeled targets to determine the relative quantities of nucleic acid sequences in the target. Since an array can contain tens of thousands of probes, a microarray experiment enables analysis of many genes simultaneously. Therefore, arrays have dramatically accelerated many types of investigations.

In standard microarrays, the probes are attached via their engineered chemical group to a solid surface by a covalent bond to a chemical matrix (via epoxy - silane, amino-silane, lysine, polyacrylamide, or others). The solid surface can be glass, a silicon chip, or microscopic beads.

DNA microarrays can be used to measure changes in expression level, to detect and quantify single nucleotide polymorphisms (SNPs), to genotype and resequence mutant genomes, or to determine recombinant nucleotide sequences. Microarrays vary in fabrication, operating protocols, accuracy, and efficiency. Additional factors affecting microarray experiments are experimental design and methods for analyzing the data.

### **3. Microarray for resequencing and sequence heterogeneity**

Several approaches based on hybridization of viral probes with oligonucleotide microarrays have been applied for rapid analysis of genetic variations during the microevolution of viruses. Microarray for resequencing and sequence heterogeneity (MARSH) was used to

Microarray Techniques for Evaluation of Genetic Stability of Live Viral Vaccines 185

The MARSH assay was first used to analyze mutations that accumulate in the region coding for VP1, the most variable capsid protein of poliovirus (Cherkasova et al., 2003; Laassri et al., 2005). Later, this microarray approach was expanded to discriminate between vaccinia strains and to evaluate genetic stability of the vaccinia virus Ankara (MVA) B5R gene following propagation of a cloned isolate of MVA in Vero and MRC-5 cell lines (Laassri et al., 2007), to analyze the variability of the structural region of West Nile (WN) virus (Grinev et al., 2008), and to evaluate stability of the entire genome of a WN/Dengue 4 chimeric virus

The MARSH microarray approach facilitates rapid analysis of viral genes and genomes, and circumvents traditional more laborious methods. With the microarray method, many samples can be analyzed simultaneously within a few hours. Furthermore, test samples do not need to be cloned, thus preserving the natural composition of viral gene populations. This method permits large-scale full genome screening of viral isolates, useful for epidemiological surveillance, vaccine quality control, and analysis of genetic changes in

Overlapping oligonucleotides

Fluorescence ratio

Fig. 1. Schematic overview of the MARSH assay: (A) Oligonucleotide microarray containing short oligonucleotides overlapped at half-length covering the entire viral genome. Each microarray has four identical sub-arrays hybridized at the same time. (B) Images of 4 individual identical sub-arrays hybridized with control sample. (C) Ratio of hybridization signals from reference and test sample preparations was plotted to reveal peaks indicating the presence of mutations. The hybridization images and the ratio plot are cartoons given

Nucleotide location

5' 3'

Overlap factor 1/2

under study as a new candidate of WN vaccine (Laassri et al., 2011).

(A) Viral genome

viruses that may occur in response to drug treatment.

(B) (C)

for illustration purposes only.

identify mutations in vaccine viruses and their derivatives, revealing the degree of their evolutionary divergence and quantifying mutant genome proportions present.

The MARSH assay was based on the hybridization of fluorescently-labeled RNA produced from the virus genome with microarrays of oligonucleotide probes that are complementary to and cover the entire viral genome or specific genes. Quantitative comparison of hybridization data produced for a test sample with the data for homogeneous reference RNA reveal mutations that have emerged and accumulated during the replication of vaccine strains *in vitro* or *in vivo* (Figure 1).

MARSH (Figure 1) microchips were fabricated using a set of short oligonucleotides (Tm ~ 50°C) overlapping at half length, matching genomic sequences of virus strains, and covering a specific viral region of interest in the genome or the entire viral genome. Each oligonucleotide probe was synthesized with an aminolink group at its 5' end for immobilization on a specific platform and purified after automated synthesis. Microarrays were printed on sialylated (aldehyde-coated) glass slides by using a contact microspotting robot equipped with a microspotting pin. Each oligonucleotide probe was spotted several times within a single microarray for redundancy to increase the reliability of results.

For RNA viruses, cDNA was prepared with reverse transcriptase using a specific reverse primer at the 3' end region of the genome. Microarrays of immobilized oligonucleotide probes were hybridized with fluorescently-labeled RNA transcribed by T7 RNA polymerase from PCR-amplified viral cDNA. First, the viral genome was amplified using the specific primers (the reverse primer contains T7 promotor) to produce the needed DNA segments. RNA for hybridization was produced by *in vitro* transcription of the PCR products with a T7 RNA polymerase kit. Each RNA product (~10 µg) can be fluorescently labeled with a Cy3 RNA Labeling Kit. Labeled RNA samples were purified using spin columns.

Microarray hybridization was performed as follow: fluorescently-labeled RNA samples were vacuum-dried prior to hybridization, reconstituted in Hybridization Buffer, and denatured by incubation for one minute at 95°C. The final concentration of each fluorescent target in the hybridization solution should not exceed 0.1 M. An aliquot of the hybridization mixture (~10 l) was applied to the microarray area and covered with an individual plastic cover slip. Hybridization was performed in an incubation chamber for one hour at 45°C. Fluorescent images of processed microarray slides were captured using a ScanArray 5000, and the images were analyzed using ScanArray Express software.

In the experiments presented on Figure 1, each microarray contained 4 identical sub-arrays that were simultaneously hybridized in order to assess reproducibility of the hybridization results and to eliminate outlier data points. Hybridization signals from individual sub-array elements that differ from the average value were calculated for all 4 replicates of the oligonucleotide probes by more than two standard deviations were discarded; the number of such invalidated data points should not exceed 0.1%. Next, average values from the 4 sub-arrays were normalized by the total fluorescence signal from the entire array. Finally, normalized signals from the reference sample (homogeneous RNA) were divided by the respective normalized signals from the test samples, and the results were expressed as a fluorescence ratio (Figure 1C). Regions with no mutations should have ratios close to one, while test samples with mutations reduced hybridization with some oligonucleotide probes and therefore, produce ratios greater than one.

184 Diversity, Gene Expression Mechanisms and Host-Virus Interactions

identify mutations in vaccine viruses and their derivatives, revealing the degree of their

The MARSH assay was based on the hybridization of fluorescently-labeled RNA produced from the virus genome with microarrays of oligonucleotide probes that are complementary to and cover the entire viral genome or specific genes. Quantitative comparison of hybridization data produced for a test sample with the data for homogeneous reference RNA reveal mutations that have emerged and accumulated during the replication of vaccine

MARSH (Figure 1) microchips were fabricated using a set of short oligonucleotides (Tm ~ 50°C) overlapping at half length, matching genomic sequences of virus strains, and covering a specific viral region of interest in the genome or the entire viral genome. Each oligonucleotide probe was synthesized with an aminolink group at its 5' end for immobilization on a specific platform and purified after automated synthesis. Microarrays were printed on sialylated (aldehyde-coated) glass slides by using a contact microspotting robot equipped with a microspotting pin. Each oligonucleotide probe was spotted several

For RNA viruses, cDNA was prepared with reverse transcriptase using a specific reverse primer at the 3' end region of the genome. Microarrays of immobilized oligonucleotide probes were hybridized with fluorescently-labeled RNA transcribed by T7 RNA polymerase from PCR-amplified viral cDNA. First, the viral genome was amplified using the specific primers (the reverse primer contains T7 promotor) to produce the needed DNA segments. RNA for hybridization was produced by *in vitro* transcription of the PCR products with a T7 RNA polymerase kit. Each RNA product (~10 µg) can be fluorescently labeled with a Cy3