Changing Patterns of West Nile Virus Transmission

Review article

Changing patterns of West Nile virus transmission:altered vector competence and host susceptibility

Aaron C. BRAULT*,**

Center for Vector-Borne Diseases and Department of Pathology, Microbiology and Immunology,School of Veterinary Medicine, University of California, 5327 VM3A,

One Shields Ave, Davis, CA 95616, USA

(Received 4 February 2009; accepted 29 April 2009)

Abstract West Nile virus (WNV) is a flavivirus (Flaviviridae) transmitted between Culex spp.mosquitoes and avian hosts. The virus has dramatically expanded its geographic range in the past ten years.Increases in global commerce, climate change, ecological factors and the emergence of novel viralgenotypes likely play significant roles in the emergence of this virus; however, the exact mechanism andrelative importance of each is uncertain. Previously WNV was primarily associated with febrile illness ofchildren in endemic areas, but it was identified as a cause of neurological disease in humans in 1994. Thismodulation in disease presentation could be the result of the emergence of a more virulent genotype as wellas the progression of the virus into areas in which the age structure of immunologically nave individualsmakes them more susceptible to severe neurological disease. Since its introduction to North America in1999, a novel WNV genotype has been identified that has been demonstrated to disseminate more rapidlyand with greater efficiency at elevated temperatures than the originally introduced strain, indicating thepotential importance of temperature as a selective criteria for the emergence of WNV genotypes withincreased vectorial capacity. Even prior to the North American introduction, a mutation associated withincreased replication in avian hosts, identified to be under adaptive evolutionary pressure, has beenidentified, indicating that adaptation for increased replication within vertebrate hosts could play a role inincreased transmission efficiency. Although stable in its evolutionary structure, WNV has demonstrated thecapacity for rapidly adapting to both vertebrate hosts and invertebrate vectors and will likely continue toexploit novel ecological niches as it adapts to novel transmission foci.

West Nile virus / vector competence / temperature / host competence / virus-host interaction

Table of contents

1. Introduction........................................................................................................................................... 21.1. Viral classification ....................................................................................................................... 21.2. Structure and replication.............................................................................................................. 21.3. Molecular epidemiology of WNV............................................................................................... 31.4. Disease and distribution............................................................................................................... 31.5. Transmission of WNV................................................................................................................. 41.6. Use of crow mortality to track WNV activity ............................................................................ 61.7. Virulence and immunity studies with WNV............................................................................... 6

* Corresponding author: [email protected], [email protected]** Current address: Division of Vector-Borne Infectious Diseases, Centers for Disease Control andPrevention, 3150 Rampart Rd/Foothills Campus, Fort Collins, CO 80521, USA

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1. INTRODUCTION

The purpose of this review is to presentsome of the potential factors that have beenassociated with the emergence of West Nilevirus (WNV) from an endemic virus, generallyassociated with childhood disease, to an agentthat has rapidly expanded its geographic range,becoming established in the New World withnovel, more serious disease manifestations.Ecological, virological, epidemiological andentomological avenues are explored to addressthis phenomenon.

1.1. Viral classification

West Nile virus is a member of the genusFlavivirus within the family Flaviviridae [59]and was first isolated from a human experienc-ing a febrile syndrome in the West Nile districtof Uganda in 1937 [142]. Serological crossreactivity has grouped WNV within the Japa-nese encephalitis (JE) virus serocomplex [27].In addition to WNV, the JE serocomplexincludes three other viral species respon-sible for human disease including Japaneseencephalitis (JEV), St. Louis encephalitis(SLEV) and Murray Valley encephalitis(MVEV) viruses [94].

Despite the name serocomplex, classifica-tion of these viruses is based on a combinationof serological cross reactivity, nucleotidesequence data, and vector association [59].The WNV species contains one viral subtype(Kunjin), comprised of isolates from Australia

and Malaysia [157]. West Nile viruses have apandemic distribution having been isolated onevery continent with the exception of Antarctica[57]. Despite the fact that serological assays dis-tinguish WNV isolates from India as well asKunjin from other WNV, only Kunjin is classi-fied as a distinct viral subtype [156]. Nucleotidesequence data has subsequently demonstratedKunjin viruses to have a greater genetic identitywith newly emergent WNV genotypes than OldWorld strains including those from India [115,135]. Altered serological reactivity is mostlikely due to the lack of an additional glycosyl-ation motif within the envelope glycoprotein ofKunjin viruses [1].

1.2. Structure and replication

West Nile virus is comprised of an envel-oped spherical virion with a diameter ofapproximately 50 nm. The virus has a linear,single-stranded, message-sense genome ofapproximately 11 Kb with a 50(7-methyguano-sine) cap and no polyadenylation at the 30endof the genome [129]. The 50 and 30 non-codingregions (NCR) have conserved nucleotide ele-ments that form stem-loop structures necessaryfor viral RNA transcription, translation andpackaging [25, 72, 139]. Viral proteins aretranslated as a single polyprotein that is post-translationally cleaved by host and viral prote-ases to produce 3 structural (C, prM and E)and 7 nonstructural proteins (NS1, NS2A,NS2B, NS3, NS4A, NS4B and NS5). The 50

third of the genome encodes the structural

2. Factors associated with West Nile viral emergence............................................................................. 62.1. Virological factors associated with emergence ........................................................................... 6

2.1.1. Emergence of avian virulence ......................................................................................... 62.1.2. Genotype associated with avian virulence ...................................................................... 72.1.3. Increased virulence as a factor for increased transmission............................................. 82.1.4. Viral adaptation for replication at higher temperature.................................................... 9

2.2. Environmental factors associated with emergence...................................................................... 102.2.1. Climate perturbation ........................................................................................................ 102.2.2. Species diversity, species behavior and transmission dynamics..................................... 102.2.3. Role of heterologous flaviviruses on WNV transmission .............................................. 112.2.4. Alternation of transmission patterns................................................................................ 11

3. Conclusion ............................................................................................................................................ 12

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proteins necessary for encapsidation of the viralRNA and the surface proteins required for viralreceptor interaction and fusion with host cells(Fig. 1) [76]. The remainder of the genomeencodes the nonstructural proteins that formthe viral replication complex for the generationof negative-sense template RNA as well aspositive-sense genomic RNA [161]. The largestof the NS proteins, NS3 and NS5, have beencharacterized for interactions and replicaseactivity; however, the function of the remainingNS proteins is largely unknown. The NS5protein has both RNA polymerase and methyl-transferase motifs necessary for viral replica-tion. Helicase and serine protease domainshave been described for the NS3 protein[129]. Interestingly, the NS1 protein is a secre-tory nonstructural protein that does not have adesignated function in RNA replication or asa control element [162].

1.3. Molecular epidemiology of WNV

Phylogenetic trees based on nucleotidesequence data indicate the existence of fivepotentially distinct genetic lineages of WNVthat have diverged by up to 29% at the nucleo-tide level [18, 89]. Sequence analysis of viralstrains from North America, Africa, the MiddleEast, Asia, and Australia (Kunjin) constitutelineage 1 based on structural [18, 63, 89] ornonstructural gene regions [14, 29]. Phyloge-netic analyses clearly indicate that Kunjin is amember of lineage 1 and that the second line-age WNV, restricted to a sub-Saharan Africandistribution, are more distantly related to otherlineage 1 WNV than to Kunjin virus [135].Lineage 3 WNV is represented by a CzechRepublic mosquito isolate (Rabensburg; RabV)

[11], lineage 4 is comprised of Dermacentorspp. tick isolates from the Russian Caucasus[117], and lineage 5 is represented by a separategenotype from India [21]. Phylogenetic group-ing of isolates does not correlate with geo-graphic distribution, indicating the potentialimportance of birds in viral dispersal [18].

All of the major outbreaks of human enceph-alitis have been associated with viruses geneti-cally categorized as lineage 1, making animportant correlation between viral geneticsand disease phenotype. The preponderance ofgenetic evidence suggests that the introductionof WNV into North America occurred fromthe Middle East. Complete genomic sequencingof an isolate from an outbreak in geese in Israelin 1998 and NY99 strains of WNV group themin the same genetic clade with a 99.7% sharednucleotide identity [29, 90]. The identificationof bird carcasses found to be positive forWNV from a number of species in Israel pro-vides evidence that naturally occurring bird vir-ulent strains could be circulating within theMiddle East and were introduced into NorthAmerica [95].

1.4. Disease and distribution

Clinical disease in humans ranges from mildfever to the development of severe neurologicaldisease [143]; however, retrospective serologicalstudies have indicated that the majority ofWNVinfections are actually asymptomatic [6, 154].West Nile fever is the most common clinicalmanifestation and is characterized by the devel-opment of high fever, chills, rash, headache,myalgia and nausea. Symptoms typically abatewithin 35 days of onset and result in lifetimeimmunity. WNV infection also may result in

Figure 1. Genomic organization of the WNV genome. Three structural proteins (C, prM, E) and sevennonstructural proteins (NS1, 2A, 2B, 3, 4A, 4B and 5) are translated as a single polyprotein directly fromthe 11 Kb positive-sense RNA genome.

Factors impacting West Nile virus circulation Vet. Res. (2009) 40:43

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the development of severe neurological symp-toms that include encephalitis, meningitis, mye-litis or combinations of these symptoms [58, 98].Acute flaccid paralysis of the limbs and respira-tory muscles has been described in North Amer-ica [8] and Romania [28].

Elderly individuals are at a greater risk fordeveloping severe neurological disease, withyounger patients more commonly developingWN fever [106]. It is not readily clear whetherthe more severe neurological symptoms are theresult of increased virulence of the introducedstrain or a reflection on the immunologicallynave status of the population whose age struc-ture might be more predisposed to severe dis-ease manifestations. Postulated mechanisms ofviral entry to the CNS and subsequent develop-ment of more severe disease include impairedstructural elements of the blood-brain barrier(BBB) or impaired immunity with increasedage [143]; however, data generated with micro-vascular endothelial cells indicates the mainte-nance of BBB integrity following infectionwith WNV, indicating the potential of free virusto pass this barrier. Furthermore, increased celladhesion molecule expression observed in thisstudy indicated the potential for infectedimmune cells to also traverse the BBB [158].

West Nile virus has been found to be ende-mic to Europe [80], Africa [45, 101], Asiaand Australasia [69]. Within its geographicrange, outbreaks of febrile disease in humanshave occurred sporadically with well docu-mented outbreaks being reported in Egypt[149], France [64] and South Africa [68] withoutbreaks occurring in 2008 in Romania[116], Hungary [84] and Italy [93, 133]. Likein the USA [4, 5, 7], recent disease outbreaksoccurring in Romania [154], Italy [10, 104],Russia [92] and Israel [30] have been associatedwith the severe disease symptomology. Since itsintroduction to the USA in 1999, a total of28 975 human cases including 1 124 deaths(through 2008) have been documented(www.cdc.gov).

West Nile virus has demonstrated a widehost range, infecting, for example, equines, alli-gators [62], dogs [9], sheep [71], llamas [86],alpacas [35, 87]; however, few animals consis-tently demonstrate overt disease. The attack rate

for equines is approximately 5% with an esti-mated 1 in 3 horses that demonstrate encephali-tis succumbing to the disease [10, 26]. Otherthan birds, some squirrel [51, 60, 78, 111,113], chipmunk [112] and rabbit species[153], and potentially alligators [79] most serveas dead-end hosts in which circulating viremiasare not sufficient for infection of mosquitovectors.

1.5. Transmission of WNV

Since its identification in 1937 from theWest Nile district in Uganda [142], WNV trans-mission cycles have been extensively describedin Egypt [149], South Africa [68], Israel [109]and Pakistan [3, 55, 121]. These maintenancecycles have all included ornithophilic mosquitospecies and mostly passerine avian hosts(Fig. 2) that maintain sufficient viremias forthe infection of subsequent mosquitoes. Isola-tions have been made most extensively frommosquitoes of the genus Culex, not only inEgypt [148] but also throughout its pandemicdistribution [121]. In South Africa, althoughisolations have been made from a wide rangeof mosquitoes, Culex univittatus is the onlymosquito vector that is efficiently infected attiters that are present in birds infected withWNV. The role of alternative mosquito popula-tions in viral transmission is presumably quitelow [66, 67]. In endemic transmission foci ofAustralia, Culex annulirostris is the major mos-quito vector [53], while the Culex vishnui com-plex and Culex tritaeniorhynchus have beenimplicated with transmission in India andPakistan [3, 102]. Evidence of seroconversionsof birds in endemic foci [81, 100, 165] and vire-mia [164] and antibody [22] production follow-ing experimental inoculation of bird species hasincriminated avian hosts as the vertebrate reser-voir for WNV throughout its distribution. Addi-tionally, certain mammalian species have beenfound to, in some cases, develop sufficient vire-mia for infection of mosquito vectors [112, 113,132]; however, the role that these hosts play inthe transmission dynamics of WNV are cur-rently uncertain but of significant interest dueto the potential infection of mammalophagicmosquito vectors.



Bird mortality has been a unique epidemio-logical finding for WNV transmission in Israel[19] and North America [131], having neverbefore been reported during outbreaks of WNfever in Europe [134] the Middle East [105]or with endemic transmission in Australia [53]or South Africa [68] (Fig. 2). In contrastto Old World transmission, isolations of WNVhave been made from numerous species ofNorth American mosquitoes representing 11genera. Mosquitoes most commonly infectedin the eastern USA have been members of theCulex pipiens species complex, and this speciescomplex was also associated withtransmission during a human urban epidemic

in Romania in 1996 [154]. However, isolationshave been made from marginally competentvectors such as the catholic vectors Aedes albo-pictus and Aedes vexans [155].

In North America, West Nile virus rapidlyexpanded its geographic range from four statesin 1999 to all contiguous 48 and Canada by2004 [120]. With this westward transition,WNV has adopted Culex tarsalis as its predom-inant rural vector in western North America.The mechanism for viral dispersal has not beenexperimentally addressed; however, the extre-mely high viremia titers present within both res-ident and migratory bird species (> 150)indicate the probable role of some of these bird

Figure 2. Transmission cycles of WNV. Transmission occurs between ornithophilic mosquito species (Culexspp.) and passeriform avian hosts. Birds typically manifest high viremias capable of exceeding oral infectionthresholds for many mosquito vectors. Humans and equines fail to generate sufficient viremia for theinfection of mosquito hosts and are deemed dead end or tangential hosts. Red: Avian mortality was firstidentified in a migratory stork and subsequently in commercially farmed geese in the late 1990s and hassubsequently been associated with widespread mortality among numerous North American avian species (inparticular, corvids). (A color version of this figure is available at www.vetres.org.)



taxa in viral dissemination [131] with the poten-tial for limited local dispersal by mosquitoes[118].

1.6. Use of crow mortality to trackWNV activity

Deaths in American crow (AMCR) wereespecially prevalent in New York City in1999 [4] and monitoring of crow mortalityhas been adopted as an epidemiological indica-tor for the tracking of WNV transmission in theUSA and has proven useful in predictingincreased risk for human infections [39, 40,65]. Dead crows with confirmed WNV infec-tions preceded the first human cases by threemonths in New York in 2000. Despite this fact,there is no knowledge regarding the geneticand/or pathological mechanism(s) of suscepti-bility for WNV in crows or any other birdspecies.

1.7. Virulence and immunity studies with WNV

West Nile virus neurovirulence has beenstudied in primate [114] as well as mouse [13,52, 160] and hamster models [166]. Age-relatedresistance to neuroinvasion is typical of mos-quito-borne flaviviruses [42, 48]; however,intraperitoneal infection of 1516 week-oldmice and adult hamsters with the newly intro-duced North American genotype induces fatalencephalitis [13]. This finding is in contrast toa study in which WNV encephalitis was rarein mice older than 7 weeks of age followinginoculation with a WNV strain from Egypt[160]. Genetic mapping studies have indicatedthe importance of the IFN-inducible 20-50-oli-goadenylate synthetase genes with resistanceto WNV infection [99].

Development of encephalitis is identified ator near the time of clearance of virus from theperipheral circulation concurrent with the pro-duction of neutralizing antibody in a hamstermodel of WNV encephalitis. This model hasalso demonstrated that hematogenous spreadof the virus across the blood-brain barrier isthe most likely route of entry to the brain asthe olfactory ganglia were not reported to havebecome infected [166]. Intranasal inoculation is

a highly inefficient mode of inducing neurovi-rulence in mice, further indicating that infectionof the olfactory bulb is likely not the most effi-cient mechanism of viral entry to the brain [13,52, 110]. Inoculation of mice with differentWNV strains has demonstrated a correlationbetween viral genotype and neurotropism[13]; however, genotypes with increased neu-rotropism do not correlate with increased bind-ing to either human or mouse brain receptorpreparations [12, 107]. Unlike AMCR that havegenerated titers of up to 10 log10 PFU/mLserum and uniformly succumb to infectionwithin 5 days of infection [83], hamstershave peak viremias of approximately 5 log10TCID50/mL of serum, have approximately50% survival rates and typically die 714 daysafter infection with the NY99 WNV genotype[166]. Antibody development may play animportant role in protecting hamsters frompotentially fatal encephalitis, since peripheralviral titers dissipate as circulating neutralizingantibody is produced [151, 166]. Immunizationof hamsters with heterologous flaviviruses suchas Japanese encephalitis and St. Louis encepha-litis viruses has demonstrated a protective effectagainst lethal encephalitis [152]. Administrationof anti-flaviviral antibodies within 46 dayspost-infection has also been demonstrated tobe efficacious for preventing flaviviral encepha-litis in a mouse model [130]. This time framecorrelates with viral invasion of neural tissues[52, 130, 166]. Intraperitoneal inoculation ofWNV in a hamster model also demonstratesthat serum neutralizing antibody developmentwithin five days of inoculation correlates withdecreased peripheral viral circulation [166].

2. FACTORS ASSOCIATED WITHWEST NILE VIRAL EMERGENCE

2.1. Virological factors associatedwith emergence

2.1.1. Emergence of avian virulence

An Egyptian strain, Ar-248, was isolatedfrom a moribund pigeon (Columba livia) squabin a field study conducted from 1952-1954.



This strain produced morbidity and mortality inexperimentally infected hooded crows (Corvuscorone) and house sparrows (Passer domesti-cus) [164]. High seroprevalence rates withinhooded crow populations in Egypt coupled withthe rapid deaths of the experimentally inocu-lated bird (some within 1 to 2 days of infec-tion), indicate the possibility that mortalitycould have been the result of experimentalhandling of the birds rather than natural viru-lence from infection [149].

No reports of natural WNV-associated mor-tality in adult birds occurred during the first sixdecades since the discovery of the virus in 1937[57]. However, significant avian morbidity andmortality have been recent hallmarks of WNVoutbreaks in Israel [19, 147] and North America[17]. In 1998, WNV was identified in tissuestaken from dead migratory storks in Israel[97] and later that year, an outbreak withingoose farms across Israel was identified.Affected flocks had severe mortality rates ingoslings between 3 and 8 weeks of age [96].Experimental infection of goslings with viruseslater isolated from North America demonstratedsimilar mortality and resulted in viremias thatwere sufficient to infect mosquito vectors[147]. Evaluation of the virulence of WNV iso-lated from New York in 1999 demonstratedmortality in 8 of 25 species experimentallyinfected. The most susceptible species werethe blue jay (Cyanocitta cristata), commongrackle (Quiscalus quiscula), house finch(Carpodacus mexicanus), AMCR (Corvusbrachyrhynchos), and house sparrow (Passerdomesticus). Corvids (including AMCR, bluejays andmagpies) all developed circulating vire-mias in excess of 8 log10 PFU/mL blood and insome cases exceeded 10 log10 PFU/mL blood[83]. Necropsy tissues taken from naturally[145] and experimentally infected [83] AMCRhave demonstrated that the North AmericanWNV is not limited to infection of particularorgan systems, with virus being identified inthe brain, heart, kidney, lungs, gonads, spleen,liver, intestines, esophagus and skin.

High mortality rates in AMCR and the quan-tity of virus identified in various organs havebeen factors for the adoption of crow mortalityas a surveillance tool to identify areas with

recent WNV activity [39, 40]. WNV infectionwith the NY99 genotype has been identifiedin more than 150 species of birds representingover 20 bird families in North America, causingmorbidity in some cases but not necessarilymortality [82, 83, 136]. Chickens, for example,demonstrate no mortality following infec-tion with WNV, yet titers as high as 5 log10PFU/mL of serum have been identified andvirus can be isolated from myocardium, spleen,kidney, lung, and intestine. Additionally, histo-pathological examination has identified myo-cardial necrosis, nephritis, and pneumonitismost notably in young birds [136].

The genetic changes observed in differentWNV strains can modulate their virulencepotential in avian species [23, 91]. Kunjinviruses have demonstrated low peak viremiathresholds compared to alternative lineage 1WNV strains in both AMCR [23] and HOSP[91]. Other lineage 1 WNV, closely related tobut different from the highly avian virulentNY99 WNV strain, have also demonstratedreduced viremia and subsequently loweredmortality rates in AMCR following experimen-tal infection [77]. These data in accordance withthe finding that WNV strains circulating inIsrael that are genetically most similar to theNY99 strain have been associated with avianvirulence [19] indicate that viral genetics playa crucial role in host susceptibility to lethalinfection.

2.1.2. Genotype associated with avian virulence

Positive selection modeling of WNV repre-senting members of all of the lineages has identi-fied a single genetic loci (NS3-249) to be underadaptive evolution [24]. This residue lies withinthe helicase domain of theNS3 protein in an areanot associated directlywithRNAbindingorATPhydrolysis; however, incorporation of a NY99substitution (NS3-T249P) into a closely relatedlineage 1 WNV that elicited a low level of viru-lence in AMCR was sufficient to elicit a viremiaand mortality response in crows indistin-guishable from the NY99 strain. Interestingly,this avian virulence mutation, NS3-249P, hasemerged on at least three independent occasions(Fig. 3). In each case, the viruses with this



substitutionhavebeenassociatedwithhumandis-ease outbreaks; however, only the Egyptian 1951isolate [164] as well as viruses from the Israeli/NorthAmerican clades [89] have been associatedwith avian virulence. The preponderance of thesedata indicated the potential importance of avianviremia for the emergence of lineage I WNVand suggests the potential for the emergence ofalternative WNV genotypes following smallgenetic changes. Interestingly, several WNVlineages contain the NS3-249P substitution buthave not been associated with avian virulence or

the emergence of disease outbreaks indicatingthe possible limitations that particular WNVgenetic backbones could impose on emergencepotential of alternative genotypes as well as thepotential role of the NS3-249P for increased rep-lication in a wider array of avian species.

2.1.3. Increased virulence as a factorfor increased transmission

A number of avian species are highlysusceptible to WNV infection, exhibiting high

Figure 3. Maximum likelihood phylogenetic tree of 21 complete genomes of WNV. Viruses are groupedaccording to four described lineages ofWNV (excluding the lineage 5 Indian group) and the genetic residue ateach NS3-249 site is indicated. The three independent emergences of the NS3-249P genotype has beendenoted on the tree. In each case an NS3-249Pro genotype emerged (designated in bold black) from apredecessor genotype (Thr; designated in grey). The asterisks represent nodes with bootstrap support values> 95%.



viremias and associated mortality rates, anddecreases in abundance [88]. Although it couldbe concluded that this resultant reduction of thebreeding population would diminish transmis-sion, high avian mortality is postulated topotentially increase transmission in three ways:(i) survival of previously infected and nowimmune birds could dilute the effect of subse-quent transmission events [50], (ii) mortalityin birds is associated with high peripheral vire-mia leading to the efficient infection of moder-ately susceptible mosquito vectors [23, 77, 83],and (iii) increased pathological manifestationscould favor increased infection of mosquitoesdue to reduced anti-mosquito defensive behav-ior of moribund avian hosts [33] as well asincreased attractiveness to mosquitoes as aresult of hyperthermia [77]. The time periodinwhich avian hosts such as AMCR are themostviremic coincides with maximal viremia andmoribund birds, presenting with elevated vire-mias on the ground could result in increasedefficiency for infectivity of contact with vectormosquitoes, thereby increasing transmissionpotential of WNV to humans or equines [108].Furthermore, it has been hypothesized that vec-tor-borne agents have the potential for toleranceof higher levels of mortality. This has been theo-rized to result from theneed for disseminated rep-lication for the infection of arthropod vectors thatoften results in more serious clinical manifesta-tionswithin the vertebrate host. The transmissionof vector-borne viruses by mobile vectors hasalso been postulated to compensate for anydampening of transmission due to the lack ofmobility of moribund vertebrate hosts [34, 43].

In contrast to WNV transmission in NorthAmerica in which low oral infection rates ofCulex spp. vectors are likely offset by high vire-mias in avian hosts, limited avian mortality andlow oral infection thresholds of Cx. univittatusand Cx. tritaeniorhynchus vectors are transmis-sion hallmarks within endemic foci. Although afew studies have demonstrated geographic dif-ferences in susceptibility of Old World endemicmosquito populations [2, 56], vector compe-tence experiments with North American mos-quitoes have indicated considerable temporaland spatial variability for oral susceptibilitywith no discernable pattern that could be linked

to amplification [125]. These data takentogether indicate the importance of avian ampli-fication by invasive WNV for infection of mar-ginally susceptible North American Culex spp.while low oral infection thresholds of specificvectors can allow for transmission in theabsence of high avian viremias within endemictransmission. These differing evolutionary strat-egies are also evident with a closely relatedflavivirus, SLEV. Comparatively, despite thefact that they utilize the same avian hosts andmosquito vectors, SLEV has an approximately10-fold greater oral susceptibility than WNVbut produces low magnitude viremias and isless virulent in birds [122].

2.1.4. Viral adaptation for replicationat higher temperature

One factor that has classically defined thelimit of the host range utilized by an RNA virushas been its ability to replicate at elevated tem-perature [36, 61]. Arboviruses such as WNVhave a wide range of temperatures in whichthey must replicate in order to be propagatedwithin both vertebrate and invertebrate hosts.An arbovirus such as dengue that replicates inprimates is incapable of replicating at tempera-tures above 39 C. This could be an importantfactor in the inability of these viruses to utilizebirds (that have higher body temperatures) asvertebrate hosts [85]. Much of our knowledgein reference to replication at alternative temper-atures is the result of the generation of temper-ature sensitive (ts) viral mutants for theidentification of the functional role of viral pro-teins in the replication process [140] or thedevelopment of mutants that have impaired rep-lication capacity [20, 167] for the developmentof vaccines. Replication of arboviruses in a twohost system can moderate the amount of selec-tion that can take place in either the vertebrateor invertebrate host [159]. Evidence also sug-gests that in vitro selection for increased repli-cation in vertebrates can result in a decreasedability to replicate in mosquito tissues [54].The temperature spectrum for which differentarboviruses can replicate (and subsequentlythe hosts that they are capable of utilizing) ispoorly understood.



Previous studies have defined the potentialimportance of both temperature constraintswithin avian hosts [77] as well as mosquito vec-tors for WNV transmission. Degree-day modelshave identified the replication limit of WNV inmosquito vector to be constrained by tempera-tures below 14 C and further demonstratedthat the strain of WNV introduced to NorthAmerican requires warmer temperatures for dis-semination and subsequent transmission [124]than a WNV strains from sub-Saharan Africa[31]. Further data has demonstrated that thegenotype of WNV that has predominated inNorth America since 2002, the WN02 geno-type, seems to disseminate more rapidly andat a more efficient level at elevated temperaturesthan the prototype NY99 North American strain[75, 103]. This shortening of the extrinsic incu-bation period (EIP), the time between a mos-quito imbibing an infectious bloodmeal andbeing capable of transmitting virus by bite,could have been the factor by which this geno-type of virus has been selected. Analyses oftemperature patterns in the USA have demon-strated an association between above-normaltemperatures and epidemics of WNV in north-ern latitudes [124]. These data coupled withthe finding of increased efficiency of vector dis-semination at elevated temperatures of the pre-dominant WNV genotype indicate temperatureas a potential important selection criterion forWNV evolution.

2.2. Environmental factors associatedwith emergence

2.2.1. Climate perturbation

Climate change can have a direct effect onWNV amplification rates in poikilothermicmosquito vectors as external temperature hasbeen documented to have an inverse relation-ship to the duration of the EIP [124]. Addition-ally, increases in transmission season can resultas well as expansion of transmission to moreextreme latitudes and elevations. Althoughincreasing temperature is negatively correlatedwith longevity of mosquitoes, in most instancesthe increased dissemination and transmissionrates actually result in increased transmission

efficiency. Patterns of WNV transmission aresignificantly linked to the abundance of vectorpopulations. It has been determined that heavyrainfall in the spring and warm, dry tempera-tures during the summer are optimal for Culexspp. population increases and are positively cor-related with WNV transmission [120, 126, 137,138].

2.2.2. Species diversity, species behaviorand transmission dynamics

One explanation for the lack of WNV-associated disease in Latin America is theincreased avian species diversity in the tropics.It is hypothesized that increased species diver-sity could negatively impact transmission ofWNV if there was a high proportion of incom-petent hosts. One study has made such a con-nection by identifying a negative correlationbetween non-passerine species diversity andthe force of WNV transmission, presumablyowing to a dilution effect of vertebrate hostswith incompetent non-passerine avian species[44]. Other studies have suggested the impor-tance of super-spreaders or certain avian spe-cies such as the American robin (Turdusmigratorius) in temperate habitats that appearto not only be highly susceptible to infectionwith WNV but also are preferential sourcesfor bloodmeals [73]. These concepts willrequire further investigation as a number ofcompeting theories for the reduced impact ofWNV in Latin America have been proposedincluding the circulation of WNV strains withreduced virulence phenotypes [15] and thepotential inhibitory role of acquired immunityto heterologous flavivirus circulation inMexico, Central and South America [47].

Shifts in mosquito host selection have beendocumented and associated with the potentialfor increased infection. One study indicated thatCulex pipiens mosquitoes in the northeasternUSA shift their feeding behavior from highlycompetent American robins to mammals andhumans in the late summer to early fall, coin-ciding with the emigration of this avian species[74]. This host switching has also beenobserved with Culex tarsalis in the westernUSA [150] and Cx. nigripalpus in Florida



[38], indicating the importance of feedingbehavior in the tangential transmission ofWNV. Utilizing microsatellite markers to genet-ically distinguish Nearctic and Palearctic Culexspp. mosquitoes, one study inferred thatPalearctic European species limit their feedingto avian hosts while North American Nearcticspecies feed indiscriminately upon avian andmammalian hosts [49] and concluded that thisdifferential feeding behavior was the explana-tion for the lack of persistent WNV activity inEurope. However, a number of other studies[144] have provided evidence that PalearcticEuropean Culex spp. feed on both mammalianand avian hosts, which contradicts the conclu-sion that feeding behavior could modulate epi-demiological patterns.

Previously, WNV infection in AMCR hasbeen uniformly lethal. However, the detectionof seropositive AMCR has been reported, indi-cating that survival of individual birds in thesehighly susceptible avian species can occur[163]. Resistance to lethal infection could beassociated with lower peak viremia, thus lead-ing to selection for WNV genotypes withreduced oral infective thresholds for mosqui-toes. As such, the effect that these modulationsin vertebrate host population susceptibilitycould have on a concomitant selection forincreased oral susceptibility of mosquito vectorsshould be monitored closely.

2.2.3. Role of heterologous flaviviruses on WNVtransmission

The effect that the presence of heterologousflaviviruses has had on WNV transmission is asubject of some debate; however, it is apparentthat increased avian herd immunity and mortal-ity has had a detrimental effect on the transmis-sion of, for instance, SLEV in southwesterntransmission foci [127]. Cross-protection withWNV has been demonstrated to precludeHouse finches (HOFI) from mounting detect-able viremia following SLEV challenge. Con-versely, inoculation of HOFI with SLEV priorto WNV infection substantially reduces WNVviremia to levels below that associated with oralinfection in most mosquito vectors [46, 123].The potential effect of selective pressures from

heterologous flaviviral immunity is currentlynot known. Heterologous infection of vertebratehosts and mosquito vectors with alternative fla-viviruses could also result in potential recombi-nation events as well as block mosquitoinfectivity through barriers to super-infection.Barriers to super-infection have been describedpreviously for arthropod-borne viruses [16, 32,37, 41, 70, 141, 146]. For example, Aedes tri-seriatus mosquitoes experimentally infectedwith LaCrosse virus (LACV) have been shownto be resistant to infection with another closelyrelated bunyavirus, Snowshoe Hare virus(SSHV), approximately two days post-infection[16]. A resistance period for dual infection wasidentified for ticks co-infected with Thogotovirus (THOV) for a period ranging between24 h and 10 days of the initial infection withan alternative THOV strain [32]. An Aedes alb-opictus cell (C6/36) model further demonstratedthat the inhibition of the secondarily infectingalphavirus is sequence specific. Alternative al-phaviruses were blocked from super-infectionof C6/36 cells, while unrelated bunyavirus orflaviviruses could establish infection in alphavi-ruses-infected C6/36 cells within the time per-iod that these cells were resistant to infectionwith heterologous alphaviruses [37, 70]. Themechanism for this super-infection exclusionhas not been identified; however, it has beenhypothesized that competitive exclusionthrough template scavenging during RNA repli-cation as well incompatible interactionsbetween viral proteins exclude replication ofthe secondarily infecting virus. Inhibitoryeffects on the super-infecting virus have beenidentified in the processes of binding to the cel-lular surface receptors, low pH fusion with theendocytic vesicle, viral uncoating, viral replica-tion as well as viral maturation and budding[141].

2.2.4. Alternation of transmission patterns

Since WNV was introduced into the USA in1999, an identified pattern has been recognizedin which the season of introduction is character-ized by mild activity with few human cases.This is followed by an epidemic year character-ized by large numbers of cases, high infection



rates in mosquitoes with subsequent avian sero-conversions and mortality. The third season ofactivity has been referred to as the subsidenceyear in which human cases and enzootic indi-cators drop precipitously, presumably owing toincreased herd immunity and avian depopula-tion of highly competent amplification hosts[119]. Decreased avian herd immunity resultingfrom the presence of nave hatchlings as well aswaning immunity in after-hatch year birds isthen conducive for continued transmission dur-ing subsequent seasons. In contrast, areas lack-ing super spreaders or highly susceptiblecorvids have failed to amplify enzootic trans-mission to outbreak levels [127], whereas otherareas have experienced recurring epidemic leveltransmission during sequential seasons [128].The identification of more severe neurologicalsyndromes in North America could be the resultof an increased virulence of the WNV strainintroduced as well as age-related exposure ratesin a completely nave population. Within ende-mic areas of WNV or SLEV transmission inwhich youth exposure rates are high, neuroin-vasive disease is uncommon. While the viru-lence of the imported strains deserves furtherattention, the role of a virgin soil effectshould not be overlooked as an explanationfor the altered disease phenotypes observed inNorth America.

3. CONCLUSION

Since its introduction to North America,WNV has undergone a dramatic increase ingeographic range and has demonstrated agreater association with a more severe neuro-logical clinical presentation. Undoubtedly anumber of ecological, virological and socialfactors have contributed to this emergence.Virological factors highlighted in this reviewhave included genetic adaptation for increasedreplication in avian hosts as well as increaseddissemination in mosquito vectors at elevatedtemperatures. As with other RNA viruses, thepresence of an error-prone polymerase thatlacks proofreading function coupled with thelarge population sizes occurring in both infectedavian hosts and mosquito vectors allow for the

rapid selection of novel genotypes. Novel eco-logical niches that have been created by cli-matic perturbations and social or demographicchanges assuredly will result in the emergenceof novel genotypes to occupy these habitatsand thus perpetuate the continually changinglandscape of WNV epidemiology.

Acknowledgements. I would like to thank Dr EddieHolmes for the construction of the Phylogenetic treepresented in Figure 3 as well as Dr Bill Reisen forhis many helpful suggestions.

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Changing Patterns of West Nile Virus Transmission