Assessment of a Novel Real-Time Pan-Flavivirus RT-Polymerase Chain Reaction

Abstract

Outbreaks of West Nile virus (WNV) have occurred intermittently in regions around the Mediterranean coast, and the virus may have become established in Northern Italy and Romania, with reported intermittent outbreaks in Spain, Hungary, and France. WNV has also spread rapidly throughout the Americas since its introduction into New York in 1999. This capacity to emerge in new geographical locations and to spread rapidly together with the current increase in incidence of other flaviviruses such as tick-borne encephalitis virus, dengue virus, and Usutu virus has prompted us to design a novel pan-flavivirus RT-polymerase chain reaction for the purpose of surveillance for a range of flaviviruses. The assay utilizes degenerate primers targeting the flavivirus NS5 gene (RNA-dependent RNA polymerase) and detects a range of flaviviruses, including WNV. A small panel of WNV bird samples obtained from the United States has been shown to be detected using this assay. The amplicon generated is of sufficient size to provide sequence data to confirm the identity of the virus detected and undertake limited phylogenetic analysis. Testing using this assay has shown its ability to detect a range of tick-borne flaviviruses, particularly louping ill virus that is endemic in areas of the United Kingdom. The assay has been used to survey 160 bird samples and 1000 mosquito samples from the United Kingdom and found no evidence for WNV.

Introduction

V iruses belonging to the Flavivirus genus are transmitted by arthropod vectors. A number of viruses within the group have repeatedly demonstrated an ability to cause infection beyond what has traditionally been considered their geographical range. This has in part been enabled by human activities and challenges public and animal health authorities to remain vigilant for the emergence of exotic arthropod-borne viruses. West Nile virus (WNV) is a good example of both the gradual establishment of a virus in an area and the dramatic translocation of a virus to a new continent. The former is exemplified by the repeated emergence of WN virus around the Mediterranean basin, and the latter by its appearance in New York in 1999 and its rapid spread through both North and South America over the following decade. WNV was first isolated from a human with febrile illness in Uganda (Smithburn et al. 1940). It was subsequently reported throughout Africa in an ecological cycle involving mosquitoes and birds. Humans and equine species were occasional dead-end hosts often manifest as encephalitic disease. From the 1950s onward outbreaks of West Nile encephalitis among humans and horses were reported in Israel and then subsequently France (Zeller and Shuffenecker 2004), Romania (Ceianu et al. 2001), Hungary (Bakonyi et al. 2006, Krisztalovics et al. 2008), and Italy (Autorino et al. 2002). A further outbreak occurred in Italy in 2008 with cases in animals and humans, which persisted into 2009 (Barzon et al. 2009). Other reports suggest that the virus is also present in the Iberian peninsula (Connell et al. 2007, Jimenez-Clavero et al. 2007). While improved surveillance and diagnostic capacity has enabled detection of WN virus, these reports and many others documenting the presence of disease in both mammalian and avian hosts indicate that the virus has become established in southern Europe. This in turn presents both a warning and a challenge for those countries to the north to prepare for potential incursions of this virus. A further consideration is the presence of tick-borne encephalitis virus (TBEV) throughout much of Europe (Mansfield et al. 2009) and the mosquito-borne flavivirus, Usutu virus, which has also emerged in Central Europe (Weissenbock et al. 2002).

To date, only one flavivirus is known to be endemic in the United Kingdom, louping ill virus (LIV). This virus is present within Ixodes ricinus populations in moorland areas of United Kingdom and is manifest as encephalitic disease of sheep (Gritsun et al. 2003). A number of reports have suggested serological evidence for West Nile and Usutu virus in the United Kingdom (Buckley et al. 2003, Buckley et al. 2006). However, these reports have not been confirmed by isolation of virus or evidence of disease in horses, avian species, or humans. Subsequent surveillance in birds between 2001 and 2006 failed to demonstrate any circulating WNV activity in British species (Phipps et al. 2008).

The ability to detect a number of flaviviruses in a single assay offers economies of scale and effort. As a result, a large number of attempts have been made to develop such assays using polymerase chain reaction (PCR) technology (Table 1). All but one has targeted the nonstructural protein genes of the flavivirus genome as these regions show less sequence variation. The majority have targeted the NS5 gene as this shows most conservation across the flavivirus genome. To maximize the number of flaviviruses detected, we have used degenerate bases within both flanking primers that produce an amplicon of approximately 200 bp to ensure efficient real-time amplification and detection. The assay developed has been assessed against a range of zoonotic flaviviruses and used to screen panels of mosquito, avian, and livestock samples, particularly for the presence of WNV.

Table 1.

Summary of Universal Primers Used to Detect Flaviviruses

Reference	Location	Amplicon (bp)	Tested against
Fulop et al. 1993	NS5	989	14 flaviviruses
Chow et al. 1993	NS3	470	Dengue and others
Chang et al. 1994	3′ nontranslated regions	411–761	Den (1–4), YFV, JEV
Puri et al. 1994	ENV	983–1001	Den, WN, SLE, Kun
Pierre et al. 1994	NS5/3′NC	600	Dengue (1–4), WNV, JEV, YFV & Zika virus
Meiyu et al. 1997	NS1	413	Dengue, JE, Langat, Powassan
Figueiredo et al. 1998	NS5/3′NC	550–650	Den (1,2,4), Cacipacore, Iguape, Ilheus, Rocio, SLE, YFV
Kuno et al. 1998	NS5	Various	All known flaviviruses
Scaramozzino et al. 2001	NS5		8 flaviruses (3 Den, 2 WNV)
Sanchez-Seco et al. 2005	NS5	143	JEV, MVE, SLE, TBEV, WNV, DEN (1–4), YFV
Ayers et al. 2006	NS5	854–863	Dengue (1–4), YFV, WNV, JEV, MVE, SLE, Usutu
Chao et al. 2007	NS5	261	Dengue (1–4), YFV, JEV, SLEV
Dyer et al. 2007	Pre-M, capsid, NS5	Various	Dengue (1–4), TBEV, WNV, SLE
Moureau et al. 2007	NS5	269–272	51 flaviviruses + 3 tentative
Maher-Sturgess et al. 2008	NS5	800	60 flaviviruses (50 species)

Den, Dengue virus; WNV, West Nile virus; JEV, Japanese encephalitis virus; YFV, Yellow fever virus; SLE, St. Louis encephalitis virus; MVE, Murray Valley encephalitis virus; TBEV, tick-borne encephalitis virus.

Methods

Viruses/RNA

WNV (strain NY99), Sindbis virus, TBEV, Louping ill virus, and Usutu virus were provided by the Centre for Ecology and Hydrology, Oxford, United Kingdom. WNV (Goose Israel 1998) was provided by the Kimron Veterinary Institute, Israel. The panel of TBEV isolates from the Czech Republic was provided by Institute of Parasitology, University of South Bohemia, Czech Republic. RNA for Rift Valley fever virus was provided by the Health Protection Agency (HPA), United Kingdom. RNA for Dengue virus and Japanese encephalitis virus were provided by the University of Liverpool, United Kingdom. Louping ill isolates from the United Kingdom were submitted from Veterinary Laboratories Agency (VLA) regional laboratories at Penrith and Aberystwyth. Pooled mosquito samples were collected from locations within southern England and provided by the HPA, United Kingdom. RNA samples prepared from WNV infected bird organs were provided by the United States Geological Survey (USGS) National Wildlife Centre. Bird organ samples and ruminant serum samples were provided by the Veterinary Laboratories Agency regional network. RNA was extracted from mouse brain homogenates or cell lysates using TriZol (Invitrogen) following the manufacturer's protocol.

Flavivirus plaque assay

Briefly, a 10-fold dilution series of virus was prepared in serum-free Eagle's minimal essential medium (Earles salts) with penicillin/streptomycin/mycostatin (EMEM-WOS) in a 96-well plate, and transferred to a 12-well plate coated with Vero C1008 cells. After incubation at 37°C/5% CO₂ for 1 h, 1.5 mL of warmed 1.5% carboxymethylcellulose in EMEM overlay was then added to each well, and the plates were further incubated at 37°C/5% CO₂ for 4 days. Cells were fixed with 10% neutral buffered formalin for 3 h, and stained with 2.3% crystal violet (Sigma). Plaques were counted for each well, and plaque forming units per milliliter (PFU/mL) was calculated.

Primer design

Primers for detection of all flaviviruses were designed by alignment of 21 nonstructural protein 5 (NS5) sequences obtained from GenBank. Sequences of the following viruses were used: Gadgets Gully virus, Kyasanur Forest disease virus, Negishi virus, Omsk hemorrhagic fever virus (strain Kubrin), Powassan virus, Russian spring–summer encephalitis virus, louping ill virus, WNV, Dengue virus types 1 and 2, Usutu virus, and a number of TBEVs. The sequences of both sense and antisense primers, and their location on the WNV genome are given in Table 2. The amplicon from this primer pair is located toward the end of the NS5 coding sequence (Fig. 1) and produces an amplicon of the expected size of 203 bp. Extensive use was made of degenerate bases to broaden the ability of the primer pair to detect as wide a range of viruses as possible.

FIG. 1.

Schematic representation of the flavivirus genome showing the binding sites for primers Flavi-For and Flavi-Rev (arrows). NS represents nonstructural genes, and NC represents noncoding regions.

Table 2.

Pan-Flavivirus Primers Designed in This Study

Primer	Sequence (5′–3′)	Binding site on West Nile NS5 (AF196835)
Flavi-For (Sense)	GCMATHTGGTWCATGTGG	9103–9120
Flavi-Rev (Antisense)	GTRTCCCAKCCDGCNGTRTC	9283–9305

M = A+C; W = A+T; R = A+G; K = G+T; D = G+A+T; N = A+C+G+T.

Amplification method and product detection

Single-tube amplification was conducted using the QuantiTect kit (Qiagen), which includes Sybr green, with additional Quantiscript reverse transcriptase (Qiagen). One microliter of target RNA and 10 pmol of both primers were added to QuantiTect mastermix. The conditions used were reverse transcription at 50°C for 30 min followed by 15 min denaturation at 95°C followed by 40 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The reaction was completed by determination of the dissociation curve of all amplicons generated. All amplifications were performed using the MX3000p PCR system (Stratagene) and analyzed using MXPRO software (Stratagene). Positive amplifications show a fluorescence increase statistically higher than background expressed as a threshold cycle number (Ct).

Detection of WNV by TaqMan RT-PCR

WNV-specific primers and probe directed to the envelope-coding sequence (Lanciotti et al. 2000) were used to detect genomic RNA in organ samples obtained from infected U.S. birds (see above). The reactions were undertaken in a single-tube format using a modification of the lyssavirus assay described by Wakeley and co-workers (2005).

Sequencing/analysis (alignment & phylogenetic tree)

The identity of amplicons generated was confirmed by sequencing as previously described (Johnson et al. 2004). Sequences were edited using DNAstar package (Lasergene) and phylogenetic analysis was carried out using the PHYLIP program (version 3.5).

Results

Detection of arboviruses by pan-flavivirus primers

Assay specificity was assessed by analysis of a small panel of flaviviruses including two representative viral isolates of WNV. Real-time RT-PCR using the primer pair Flavi-For and Flavi-Rev successfully detected eight flaviviruses and failed to detect two mosquito-transmitted viruses unrelated to the Flavivirus genus (Table 3). Amplification curves (Fig. 2A) and dissociation curves (Fig. 2B) demonstrate the amplification of WNV isolates.

FIG. 2.

Detection of West Nile virus lineages 1 and 2 using pan-flavivirus primers. (A) Real-time amplification of two strains of West Nile (circles and squares) and a no-template control (triangles). (B) Dissociation curves of West Nile virus–positive samples and a no-template control.

Table 3.

Detection of Arboviruses by Pan-Flavi Primers Flavi-For/Flavi-Rev

Virus	Ct	Confirmed by gel	Confirmed by sequencing
West Nile virus (NY99)	21.11	+	+
West Nile virus (Isr98)	20.9	+	+
Louping ill virus	13.04	+	+
Tick-borne encephalitis virus	18.42	+	+
Usutu virus	29.8	+	+
Japanese encephalitis virus	32.6	+	+
Dengue virus	19.52	+	+
Sindbis virus	No Ct	NA	NA
Rift Valley fever virus	No Ct	NA	NA

NA, not applicable.

Detection of WNV RNA samples by pan-flavivirus primers

A further panel of 20 WNV RNA samples prepared from bird organ samples (pooled kidney and spleen) in the United States was assessed using this assay (Table 4). In each case WNV was detected in each sample although with a higher Ct than a WNV-specific RT-PCR.

Table 4.

Detection of West Nile Virus in RNA Isolated from Kidney/Spleen Samples Obtained from United States Birds

Sample no.	Species (state)	WNV-specific RT-PCR	Pan-flavivirus RT-PCR
1	Blue Jay (GA)	+(12.8)	+(28.5)
2	Blue Jay (AL)	+(13.2)	+(31.16)
3	American Crow (NY)	+(14.5)	+(36.18)
4	American Crow (NY)	+(14.0)	+(31.2)
5	Blue Jay (OH)	+(15.2)	+(33.8)
6	American Crow (NY)	+(14.1)	+(32.3)
7	Field Sparrow (NY)	+(12.5)	+(35.1)
8	American Crow (WI)	+(13.7)	+(31.0)
9	Blue Jay (AL)	+(13.1)	+(30.5)
10	American Crow (OH)	+(13.6)	+(31.4)
11	American Crow (OH)	+(14.6)	+(32.7)
12	American Crow (WI)	+(15.2)	+(33.3)
13	Blue Jay (OH)	+(14.7)	+(32.6)
14	Blue Jay (OH)	+(13.4)	+(30.4)
15	American Crow (NY)	+(15.3)	+(31.1)
16	American Crow (WI)	+(13.7)	+(30.2)
17	American Crow (MI)	+(15.0)	+(33.0)
18	American Crow (IL)	+(13.9)	+(32.4)
19	American Crow (OH)	+(14.9)	+(33.9)
20	Blue Jay (AL)	+(14.8)	+(32.9)

PCR, polymerase chain reaction.

Detection of TBEV by pan-flavivirus primers

To assess the ability of this assay to detect other flaviviruses in panels of samples, we used it to detect TBEV in a panel of 17 samples from the Czech Republic. Irrespective of the quantity of virus in each sample, as assessed by measurement of PFU, the assay detected flavivirus in each sample, including three samples where no PFU were detected (Table 5). The envelope protein of these isolates was sequenced to confirm their identity as TBEV isolates originating from the Czech Republic (data not shown).

Table 5.

Detection of Tick-Borne Encephalitis Virus from the Czech Republic

Isolate	Year	PFU/mL	Ct
Arb 13	1985	23,000	17.53
Arb 16	1986	26,000,000	19.67
Arb 17	No data	7,400,000	17.93
Arb 18	1989	Not measurable	37.6
Arb 19	No data	8,100,000	19.34
Arb 20	1990	180,000	17.67
Arb 21	1987	59,000,000	17.94
Arb 22	1989	Not measurable	37.34
Arb 23	1986	660,000	17.97
Arb 24	1986	Not measurable	17.55
Arb 25	1990	660,000	19.22
Arb 26	No data	810,000	16.48
Arb 27	1986	85,000,000	17.62
Arb 28	1986	1,400,000	17.93
Arb 29	1990	1,500,000	18.24
Arb 30	1990	140,000	18.14
Arb 31	1987	150,000,000	17.18

PFU/mL, plaque forming units per milliliter.

Detection of louping ill virus field samples by pan-flavivirus primers

A key feature of this assay is its ability to detect the U.K. endemic flavivirus, Louping Ill virus (LIV). To assess this, samples were submitted by VLA regional laboratories in LIV endemic areas (Cumbria/Wales). RNA was extracted from spinal cord material from ewes showing neurological disease and in which disease had been confirmed by immunohistochemistry for LIV envelope protein. In three brain samples, LIV genomic RNA was detected in real-time with Ct values of 27.9, 23.7, and 38.8. Each amplicon had a dissociation temperature of 83.5 and could be detected at the expected size on an agarose gel (Fig. 3A). DNA sequencing produced sequences with strong homology to a laboratory strain of LIV but with some base substitutions reflecting their isolation from different geographical locations. This has been demonstrated in a larger study of regional variations of LIV (McGuire et al. 1998).

FIG. 3.

Detection of Louping Ill virus using pan-flavivirus primers. (A) Agarose gel detection of LIV amplicons (arrow). (B) Sequence alignment of LIV derived from the amplicons generated by primers Flavi-For and Flavi-Rev.

Surveillance for flaviviruses in U.K. avian and mammalian samples

This assay was used to assess three panels of samples obtained within the United Kingdom: 1000 mosquito samples collected and previously shown to be negative for WNV; one hundred and sixty bird brain samples collected from locations around England; and one hundred blood samples collected from sheep and cattle that were obtained for Anaplasma phagocytophila screening. In each case the assay returned negative results for flaviviruses.

Discussion

The ability of flaviviruses, particularly those transmitted by mosquitoes, to expand from their existing geographic range or to suddenly appear in new regions suggests that continued vigilance for these viruses is required (Gould et al. 2006). Global examples of flavivirus expansion include Dengue virus, yellow fever virus, and Japanese encephalitis virus. In a European setting, WNV and Usutu virus are examples of this phenomenon. Molecular techniques that detect a range of potential exotic flaviviruses should enhance surveillance. Factors such as the establishment of WNV in southern Europe, climate change, mosquito competence and density, wildlife population increases, and human activity may increase the probability that incursions of exotic flaviviruses will occur (Gale et al. 2009a, 2009b). Bird migration has also been reported as a means of WNV introduction (Malkinson et al. 2002) and many bird species migrate from Africa, across the Mediterranean to the United Kingdom. The assay described in this study is capable of detecting a range of flaviviruses using degenerate primers targeting the NS5 gene. The amplicon generated by this approach is small enough to enable efficient real-time detection of viruses through the incorporation of Sybr green, but of sufficient size to derive enough sequence data for further analysis of the PCR product as shown by the alignment of LIV sequences (Fig. 3). There is only one endemic flavivirus present in the United Kingdom, LIV, and this can be detected by the assay described in this study (Fig. 3). For detection of WNV, sensitivity appears to be the main limitation of this assay in direct comparison with WNV-specific TaqMan RT-PCR assays. This is illustrated in the difference in Ct values observed in a direct comparison between the two real-time RT-PCR assays used in this study (Table 4). Difference in threshold value is likely to result from the use of degenerate bases within the flanking primers and has been observed previously in the detection of WNV (Lanciotti et al. 2003). The main benefit of this assay is to provide wide coverage for a range of flaviviruses where a single virus cannot be specified. Sensitive, specific assays should be used where a single virus is suspected. The relatively small size of the amplicon suggests that inclusion of TaqMan probes would be one obvious development, an approach adopted by Dyer and co-workers (2007). An alternative might be more precise measurement of the dissociation curves generated from the amplicons of different viruses. Further validation of this assay will be required to assess its sensitivity for each flavivirus and to confirm that the sequence derived for each will provide sufficient phylogenetic discrimination. Preliminary sequence derived from this study identifies viruses to the species level, but further assessment is required to provide discrimination at the lineage level.

Two articles have reported serological evidence that West Nile has entered the United Kingdom (Buckley et al. 2003, 2006). Further surveillance has failed to detect this virus (Phipps et al. 2008); however, it is only through continued surveillance using detection methods such as those described in this study that will provide evidence for the presence or absence of flaviviruses within the United Kingdom. The sample surveys in this report have relatively low power and require larger sample sizes to enable a definitive assessment for the presence or absence for non-LIV flaviviruses in the United Kingdom. Such studies are being devised and it is only through continued surveillance that the methods and skills required to respond to such an outbreak are retained and refined by diagnostic laboratories.

Footnotes

Acknowledgments

The authors would like to thank the following for contributions of viruses and reagents: Libor Grubhopper and Daniel Ruzek (Institute of Parasitology, University of South Bohemia, Czech Republic), Mertyn Malkinson (Kimron Veterinary Institute, Israel), Ernie Gould (formerly Centre of Ecology and Hydrology, United Kingdom), Hon Ip (USGS National Wildlife Health Centre), Rebecca Mearns, Alexandra Schock (VLA), Richard Vipond, Jane Burton, Mark Outlaw (HPA, United Kingdom), Tom Solomon, and Sareen Galbraith (University of Liverpool, United Kingdom). The authors would also like to thank Kristy Murray (University of Texas School of Public Health) for useful discussions on WNV surveillance. Funding for this work was provided by Defra (Grant SE4106 and ED1600), EU FP7 coordinating action “ArboZooNet” International Network for Capacity Building for the Control of Emerging Viral Vector Borne Zoonotic Diseases and through an internal investment grant (TD0067).

Disclosure Statement

No competing financial interests exist.

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