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West Nile virus and North America: an unfoldingstory

A. Glaser

Animal Health Diagnostic Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 14852,United States of America

SummaryBefore the introduction of the West Nile virus (WNV) into the United States ofAmerica (USA) in 1999, conditions in North America were ideal for an arboviralepidemic. Such factors as the large, susceptible and non-immune animal andhuman populations, the presence of competent vectors, increasing internationaltravel and commerce, existing methods for rapid dissemination and an ill-prepared animal and public health infrastructure all combined to create theessential elements for a severe animal and public health crisis – the ‘perfectmicrobial storm’. The introduction of WNV into New York City was the final factor,serving as the catalyst to initiate one of the most significant epidemics in theUSA. The spread of WNV across the country resulted in very large populationsof wildlife, equines and people being exposed and infected.The epidemic is still not fully understood and its character continues to changeand adapt. The recent recognition of a number of non-vector modes oftransmission has revealed the disease as a greater threat and more difficult tocontrol than first thought. West Nile virus gives every indication that it willbecome a permanent part of the ‘medical landscape’ of the USA, continuing tothreaten wildlife, domestic animals and humans as a now endemic disease. Thispaper discusses the features of this extraordinary epidemic, and emphasises theneed for an integrated surveillance system, greater diagnostic capacity andimproved control strategies.

KeywordsEmerging disease – Epizootic – Flaviviruses – New York City – North America – Review– West Nile virus.

IntroductionThe introduction of West Nile virus (WNV) into the New York metropolitan area in 1999 has provided a uniqueopportunity to follow the establishment of an introducedflavivirus into a new geographic location. The ‘perfect storm’metaphor which is the foundation of this issue of the Reviewis a fitting description for the history of WNV. North Americahas large, pristine, non-immune populations of human andanimal hosts, multiple efficient vectors and a favourableenvironment for disease transmission and spread. The introduction of West Nile virus became the catalyst andfinal element to initiate this microbial ‘storm’ for whichscientists know the beginning, but not the end of the story.The aggressiveness with which WNV became integrated into

the diverse ecosystems within the Americas and the severityof the epizootics in terms of human and animal morbidityand mortality continually surprised, dismayed and frustratedpublic and animal health communities as they worked toeducate the public, detect virus emergence, diagnose clinicalcases and try to slow epizootic transmission. Each year hasled to new information about the host range, modes oftransmission and clinical manifestations of infection. Oldparadigms were continually discarded as new informationbecame available through the co-ordinated efforts of publichealth agencies, the medical community, wildlife officials,veterinarians, animal health agencies, basic researchers andindustry in a broad coalition of disease surveillance,

reporting and research initiatives established in the wake ofthe virus. While the effort to establish a surveillanceinfrastructure was huge, nothing prepared the human andanimal health community for the rapidity of the geographicmigration of the virus or the escalating severity of theepizootic as it occurred at the leading edge of this migration.

This article will review aspects of the North Americanencounter with and response to WNV emergence. It is notintended to be a comprehensive review of WNV biology andpathogenesis. However, it will attempt to provide anepidemiological perspective that focuses on the key factorsdriving this emerging zoonotic disease, which continues tochange and develop. Several comprehensive and informativereviews dealing with the epidemiology and ecology of WNV,and the human illness caused by it, have recently beenpublished (5, 8, 13, 14, 25, 27, 28, 38) and the reader isdirected to these for additional information. This paper helpsto describe WNV on the North American continent andserves to complement other papers in this issue of theReview, which observe WNV in other geographic settings.

The first yearIn an era of heightened awareness of bioterrorism, WNVexposed significant weaknesses in the surveillancemechanisms for zoonotic infectious disease emergencewithin the United States of America (USA). As the epizooticunfolded in the summer and autumn of 1999, two parallelinvestigations were being conducted, which wouldeventually converge (64). One was the investigation of acluster of meningo-encephalitis cases in humans withassociated neuromuscular weakness, originally diagnosedas St Louis encephalitis on the basis of serological assays.The other was an investigation of avian mortality bywildlife officials and a veterinary pathologist at the Bronx Zoo (New York). The combined efforts of multipleparticipants in the medical, wildlife and veterinary fields,as well as in government and private laboratories, led tothe isolation and identification of WNV as the cause ofboth human and avian illness and mortality (9, 45). A laterinvestigation of a cluster of neurological illnesses amongequines on Long Island revealed additional WNV activity,which occurred at the same time as the morbidity andmortality in humans (66, 82).

The mechanism by which WNV was introduced into theNew York metropolitan area is not known. The fact that thevirus was nearly identical to the viruses widely circulatingin Israel from 1997 to 2000 provides circumstantialevidence that the virus identified in New York Cityoriginated in that area (10, 34, 45). The extensive traveland commercial activity associated with New York Cityprovides a plausible means for its delivery.

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Establishing surveillanceProgrammes for arbovirus surveillance in many areas of thecountry had disappeared or were on the brink ofdissolution. The re-establishment of surveillancecapabilities required the rebuilding of mosquitosurveillance programmes and laboratory infrastructureswithin the public and animal health arenas. One of the most challenging aspects of the epizootic wasproviding the testing services required to support massive surveillance programmes and real-time diagnostictesting for clinical cases, as well as efficiently integrating all animal and human surveillance data. The surveillance programmes established after 1999 haveled to the accumulation of data that document themovement of WNV across the continent. Integratedsurveillance activities involving the testing of mosquitoes,dead birds, equines and humans were instituted regionally from the East to the West between 1999 and 2004. A year-to-year summary of WNV movement in the USA andCanada is depicted in Figures 1 and 2. Within the USA, data from surveillance activities are co-operatively collected in a system called ArboNET (57).Summary information is updated twice weekly anddisplayed on a publicly accessible website (http://www.cdc.gov/ncidod/dvbid/westnile/ surv&control.htm).In collaboration with the Centers for Disease Control andPrevention (CDC), the United States Geologic Surveymaintains interactive maps of WNV activity, withinformation available for most areas at the county level (http://westnilemaps.usgs.gov/ index.html). InCanada, Health Canada maintains a website with updated information about WNV activity in humans andsubmitted wild birds (http://www.hc-sc.gc.ca/english/westnile/).

Surveillance activities include the following:– dead bird sightings reported by the general public– testing subsets of dead birds– mosquito trapping and testing– sentinel bird testing– equine testing– human testing.

Dead bird sightings were generally the most sensitiveindicator of the presence of the virus in a geographic area,but equine cases were the first indication of virus transmission in several regions in the Mid-western and Central West USA in 2002 and 2003. Ingeneral, surveillance of dead birds gave the earliest warning of WNV, followed by seroconversion of sentinel hosts, mosquito surveillanceand the identification of veterinary cases, with humancases being the least sensitive indicator of the presence ofthe virus.

Fig. 1The geographical spread of West Nile virus in the United States of AmericaSource: website of the United States Centers for Disease Control and Prevention

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Host range and clinical signsWest Nile virus has an extremely broad host range,replicating in birds, reptiles, amphibians, mammals,mosquitoes and ticks. Mortality in avians has beendocumented in 198 species (38), with mortalityapproaching 100% in American crows (Corvus brachyrhynchos) in some locations. Amongmammals, clinical illness following WNV infection has

been detected most frequently in humans and horses.Morbidity in humans is evenly distributed, irrespective ofage group or sex, but increasing age is associated with ahigher likelihood of developing encephalitis or meningitis(12, 17, 64, 72). A comprehensive review of clinical illnessin humans has recently been published (28) and will notbe dealt with further here.

Fig. 2The geographical spread of West Nile virus in Canada from 2001 to 2003Source: website of Health Canada

Clinical signs in equines have included the following:– acute onset ataxia– muscle fasciculation– weakness– flaccid paralysis of one or more limbs– recumbency– fever– blindness (66, 67, 82).

Animals presenting with more severe neurologicalabnormalities generally have the gravest prognosis. A mortality rate of approximately 30% has been consistentfrom year to year in horses with clinical signs followinginfection. The availability of WNV vaccines for horses (60,65) has probably substantially contributed to the decreasesin equine morbidity and mortality during 2003 and 2004.

While bird mortality is a useful surveillance tool, mostpublic health laboratories accepted only corvid species fortesting. This, combined with the strain on testing capacityin wildlife and veterinary laboratories, has limited theinformation available on wildlife clinical illness andmortality due to WNV. In 1999, an investigationconducted on diverse avian and mammalian species in acaptive population at the epicentre of the initial epizooticrevealed broad susceptibility to infection, with 34% ofbirds and 8% of the mammals tested found to haveantibodies against WNV (53). Of the mammalian speciestested, one species – two Indian rhinoceroses (Rhinoceros unicornis) – had clinical illness, but only onerhinoceros demonstrated seroconversion. Signs of thedisease consisted of the following:– partial anorexia– depression– lip droop– lethargy.

Among the avian species tested, 22% of birds also hadclinical signs. These signs were often non-specific andincluded the following:– anorexia– weakness– depression– weight loss– recumbency– being found dead with no preceding abnormal signsnoted.

Some birds exhibited neurological abnormalities, such as:

– ataxia– tremors– disorientation– circling

– impaired vision– abnormal head and neck posture.

Similar signs were reported in a group of eleven birdsidentified as WNV cases in 2002 (18). The most commonclinical signs reported were anorexia, depression, thesudden onset of recumbency, mild ataxia and tetraparesisand, less frequently noted, signs of tremors, nystagmus,seizures and sudden death. More than 280 species of birdswere reported to the CDC as WNV casualties from 1999 toNovember 2004, and a comprehensive review of the avianspecies experiencing morbidity or mortality due to WNVinfection has recently been published (38).

In addition to diverse avian species, WNV has causedmorbidity and mortality in alligators and crocodiles (59, 80), squirrels (30, 37, 41, 42), reindeer (Rangifer tarandus) (68), canids (50, 51), sheep (Ovis sp.)(88), alpacas (Lama pacos) (43, 88), a rabbit (Oryctolagus cuniculus), an eastern chipmunk (Tamias striatus) (42) and a cat (39), among others.

The effects of WNV on wildlife populations remain largelyunquantified. Birds of the Corvid family havedemonstrated high morbidity and mortality (42, 54, 87, 89). In one instance, where a population ofcrows was tracked, a confirmed death rate of 68% due toWNV infection was identified (89), and other studiessuggest significant short-term impacts on crow populations(20, 56). Collective data from surveys of breeding birdsand an annual winter bird census of a variety of passerinespecies indicate local declines in areas documented ashaving a high level of WNV epizootic activity, but no wide-ranging declines can be attributed to the virus (58). Aserosurvey of black bears (Ursus americanus) in New Jersey has confirmed exposureto and survival of WNV infection in this species (23).There is little question that WNV is capable of infecting alarge variety of bird and mammal populations. The lack ofsystematically collected data on wildlife morbidity andmortality and the scarcity of diagnostic resources todetermine causality ensure that drawing any empiricalconclusion about the effects of WNV on wildlifepopulations is a challenging proposition. The long-termeffect of WNV on wildlife species and ecosystems has yetto be determined.

Modes of transmissionThe most common exposure to WNV is through the bite ofan infected mosquito. The virus has been identified in 43 species of mosquito throughout the USA (28), althoughnot all of these are competent vectors (26, 75, 76, 83, 84, 85). The predominant species involvedin WNV transmission vary according to geographical

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location. In the northeast, Culex pipiens, C. restuans and C. salinarius were the species most frequently found to testpositive for virus (42, 63). Of these, C. restuans and C. salinarius have demonstrated vector competence (75).The recent discovery of hybrid Culex species feeding onboth birds and mammals may indicate that these play asignificant role as bridge vectors between animal and insecthosts in the northeast USA (Fig. 3) (24).

issue for blood banks after 23 cases of WNV infection werelinked to transfusion with donated blood products (70).Routine screening of blood donors for WNV by nucleicacid amplification assays (reverse-transcription polymerasechain reaction or RT-PCR) in small pools was rapidlyinstituted after 2002. As a result of this more rigorousscreening in 2003, more than 800 WNV-positive blooddonations were removed from the blood supply, but sixcases of transfusion-associated WNV were identified, eachassociated with a single donor with low viraemia (16).Screening of individual samples was begun in areas withactive transmission in 2004. A total of 195 virus-positiveblood donors were identified and one case of transfusion-associated transmission was reported in Arizona (15).

Several experiments in mammals and birds havedemonstrated the establishment of infection after oralexposure to the virus (2, 40). Cats were productively infectedafter ingesting WNV-infected mice (2) and 5 avian speciesout of 16 which were challenged orally with the virusbecame infected (40). Oral exposure to horse meat fromWNV-infected animals was the hypothesised route ofinfection in a group of farmed alligators in 2002 (59).

The possibility of faecal shedding of WNV from infectedbirds was considered after careful histopathological studiesand immuno-histochemical localisation of the virus in thegastro-intestinal and renal epithelium, performed in thefirst year of the epizootic (79). The amount of antigendetected in these tissues suggested the likelihood ofsignificant faecal shedding of the virus and a potentialalternative exposure route for other animals in theenvironment through aerosol or oral exposure. Directtransmission of the virus from infected birds to others inthe same cage (or ‘cage mates’) has now beenexperimentally demonstrated for five species out of the 16 tested (40, 49, 55). Experimental infections in crowsdemonstrated direct transmission from the infected birdsto their control cage mates (40, 54). Horizontaltransmission was relatively efficient, with five of sevencontrols becoming infected in one study (54), and five outof five contact controls becoming infected in the other(40). Experimental infection in chickens indicated thatnon-insect-mediated transmission was much less efficientin this species, with no detectable transmission in onestudy (77) and only one out of 16 contact birds becominginfected in another (49). Horizontal transmission of WNVhas also been demonstrated in blue jays (Cyanocitta cristata), black-billed magpies (Pica pica) andring-bill gulls (Larus delawarensis) (40). Both experimentaland field evidence indicate that horizontal transmissionalso occurs among geese (3, 81). Rough estimates of thequantities of cloacal shedding of WNV in several species ofexperimentally infected birds were obtained frommeasuring the virus on cloacal swabs (40). Most birds shedsome virus at a low titre, but the passerines, owls andcorvids that were tested excreted more than six logs of

Maintenancevector

mosquitoes

Humans, Horses

?

“Bridge vector”mosquitoes

Primarycycle

Birds Birds

Maintenancevector

mosquitoes

Fig. 3A schematic illustration of the West Nile virus transmissioncycleAdapted from (71)

In Florida, Culiseta melanura, Culex salinarius, C. nigrapalpus and C. quinquefaciatus were the mostfrequently identified seropositive mosquitoes, all of whichdisplay vector competence. In the west, C. tarsalis is aprominent species with efficient vector competence andindiscriminate feeding behaviour (84). The widespreaddistribution of this last Culex species probably contributedto the intense epizootic transmission seen in the westernstates in 2003.

As the epizootic evolved, the theory that the only mode ofWNV transmission was through the bite of an infectedmosquito to a mammal or bird was being challenged.Several new modes of transmission in humans wereidentified and described, including the following:

– infection through contaminated blood products (29, 71)

– organ transplantation (33)

– maternal transmission through breast milk (16) andintrauterine transmission (27).

Occupational exposure was also described throughpercutaneous exposure to WNV-contaminated laboratory‘sharps’ (1).

Within the human health field, identification ofasymptomatic infected individuals has become a crucial

virus while chickens shed two logs or less (49, 77). Theability to transmit virus to environmental contacts isprobably related to the amount of virus shed. This issupported by the fact that chickens, which shed a smallamount of virus, have a low in-contact transmission rate, incomparison to the high in-contact transmission rate ofcorvids, which shed much more virus. It is not knownwhat role alternative routes of infection may play in virusamplification cycles in the wild.

Molecular epidemiologyWest Nile virus was introduced into a setting that permittedwidespread and efficient replication in an astonishingly largevariety of host species. In that kind of environment, it mightbe expected that selective adaptation would occur.Nucleotide sequence analysis of early isolates from the EastCoast indicated that they were nearly identical (48). Analysisof additional isolates from New York, collected from 2002 to2003, revealed the presence of a virus segregating to adifferent clade emerging in 2002 (22). Strains belonging tothis more recently identified clade have a shorter extrinsicincubation period in mosquito hosts and appear to have aselective advantage, as they have become the dominantvariants in 2003 in New York State. Additional isolatesanalysed in Texas show phenotypic differences in plaquemorphology and neuro-invasiveness in mice, but not inneurovirulence (19). The emergence of a new dominantclade of viruses in at least one geographic region suggeststhat adaptation of WNV will be a dynamic and unpredictableprocess, and that molecular and phenotypic evaluation ofisolates must continue.

Diagnostic testingDiagnostic evaluation of samples involves identification ofboth the antibody and the antigen. Many recent advanceshave been made in the commercial production of rapidtesting methodologies. Rapid assays for the detection ofantigen include RT-PCR assays (10, 35, 44, 46, 47, 62, 69,78) and a commercially available antigen-capture assay (32, 74). The RT-PCR assays are all relatively sensitive,although it is difficult to compare them directly. For assayswith defined copy number controls, detection limits of 20 to 40 copies of ribonucleic acid can routinely beobtained. Virus can be detected in the tissues, especially inavian kidneys, brains and hearts (42). Virus can also bedetected on oral-pharyngeal or cloacal swabs from WNV-infected birds and this has become a valuable surveillancetool (52, 89).

Standard antibody detection tests include thehaemagglutination inhibition test (4) and plaque reductionneutralisation test (PRNT). The former assay suffers from

non-specific reactivity in some samples and will generallydetect antibodies against related flaviviruses. The PRNT ismore specific and, in most instances, can differentiate amongantibody reactivity to closely related viruses. Exposure tomore than one related flavivirus, or distance in time from theinitial infection, generally results in a broader serumneutralising response with a loss of the ability to identify theinfecting virus. The PRNT requires biosafety level 3 facilitiesin the USA, which limits the availability of the test.Immunoglobulin M (IgM) capture enzyme-linkedimmunosorbent assays (ELISAs) have been developed forhumans, equines, canines and chickens, and are useful fordetermining recent exposure to the virus. The fact that IgMpersists in humans complicates test interpretation (11, 12),but it is more transient in equines (66, 67) and canines (A.Glaser, unpublished findings). Samples that test positive forthe presence of antibody by IgM-capture ELISA must thenhave that antibody confirmed as being specific to eitherWNV or St Louis encephalitis virus, with a labour-intensivePRNT. Fortunately, a blocking ELISA test has been developedthat offers species-independent use and the ability to discernthe origin of antibody reactivity (6, 7, 36). A broadly reactiveIgG-capture ELISA for bird serum has also been developed.This assay performed well when tested with serum from 23species (12 orders) of birds (21), but specificity needs to bedetermined using the PRNT. Finally, the production ofexpressed protein antigens has led to the development of agrowing array of antibody test platforms, which shouldgreatly facilitate rapid serological diagnosis of flavivirusinfections and/or exposure (31, 61, 73, 86).

The epidemiology and biological characteristics of WNV inNorth America continue to be explored and elucidated. Thesustained level of WNV activity since 1999, the complexityof the epidemiology of transmission, the high levels ofviraemia in wildlife reservoirs, the large number of hosts andspecies of mosquitoes found infected, and the observation ofnew modes of transmission all suggest that WNV is a greaterpublic and animal health threat in North America, andparticularly in the USA, than scientists initially appreciated.The scope, scale, intensity and consequences of thisepidemic in the USA are unprecedented for an arbovirus.

West Nile virus is a remarkable example of an emergingzoonosis that involves the dynamic interface of wildlife,domestic animals and humans. North America, with its non-immune hosts, competent vectors and opportunities forrapid transmission, created an ideal setting for the ‘perfectmicrobial storm’ to occur. Today, the storm ‘continues torage’ and the prospect of controlling it in the near future isunlikely. West Nile virus will remain in North America and,with continuous ecological changes, adaptation of the virusand the potential expansion of its host range, the WNV storywill continue to unfold, often in unpredictable ways.

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A. Glaser

ResumenAntes de que el virus West Nile (VWN) penetrara en los Estados Unidos deAmérica, cosa que sucedió en 1999, América del Norte ofrecía condicionesidóneas para que surgiera una epidemia arbovírica. La combinación de factorestales como la existencia de grandes poblaciones animales y humanas sensiblesy no inmunizadas, la presencia de vectores competentes, la intensificación delos viajes y el comercio internacionales, los métodos existentes para una rápidapropagación y la poca preparación de las infraestructuras sanitarias yzoosanitarias generó la coyuntura necesaria para que se desatara una gravecrisis zoosanitaria y de salud pública: el perfecto ‘huracán microbiano’. Laintroducción del VWN en la ciudad de Nueva York fue el último factor, la espoleta

La historia del virus West Nile y América del Norte sale a la luz

A. Glaser

RésuméAvant l’introduction du virus West Nile aux États-Unis d’Amérique en 1999,l’Amérique du Nord réunissait toutes les conditions pour qu’une épidémie àarbovirus y fasse son apparition. L’existence de populations animales ethumaines nombreuses, susceptibles et non immunisées, la présence devecteurs compétents, l’accroissement des échanges et des déplacementsinternationaux, la facilité des méthodes favorisant une dissémination rapide et lapiètre préparation des infrastructures de santé animale et publique sont autantde facteurs qui ont conflué vers une situation de crise zoosanitaire et de santépublique d’une grande gravité, véritable « cyclone microbien ». L’introduction duvirus West Nile dans la ville de New York a constitué le dernier facteurcatalyseur en déclenchant l’une des plus importantes épidémies ayant jamaisaffecté les États-Unis d’Amérique. La propagation du virus dans tout le pays s’esttraduite par l’exposition et l’infection de vastes segments des populationsd’animaux sauvages, d’équidés et d’êtres humains.À l’heure actuelle, l’épidémie n’a pas encore été entièrement élucidée, tout encontinuant à évoluer et à s’adapter. La reconnaissance récente de certainsmodes de transmission non vectoriels a mis en évidence que la maladiereprésente une menace plus grave et difficile à maîtriser qu’on ne l’avaitsupposé dans un premier temps. Le virus West Nile semble voué à jouer un rôlepermanent dans le « paysage médical » des États-Unis d’Amérique, ens’installant durablement, et désormais sous forme endémique, aussi bien dans lafaune sauvage que chez les animaux domestiques et la population humaine.L’auteur examine les caractéristiques de cette extraordinaire maladie et metl’accent sur la nécessité de disposer d’un système de surveillance intégré, d’unecapacité diagnostique renforcée et de stratégies de prophylaxie améliorées.

Mots-clésAmérique du Nord – Épizootie – Étude – Flavivirus – Maladie émergente – New York –Virus West Nile.

Le virus West Nile et l’Amérique du Nord : une histoire qui sedévoile

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que encendió una de las más importantes epidemias vividas en los EstadosUnidos de América. La diseminación del virus por el país provocó la exposicióne infección de inmensas poblaciones de animales salvajes, equinos y personas.Aún no se entiende completamente la epidemia, que sigue cambiando yadaptándose. El reciente descubrimiento de una serie de mecanismos detransmisión no vectoriales ha puesto de relieve que la enfermedad plantea unaamenaza más grave y difícil de controlar de lo que en un principio se pensaba.Todo parece indicar que el virus West Nile va a instalarse definitivamente en el‘paisaje médico’ estadounidense y que seguirá amenazando a la fauna salvaje ydoméstica, así como al ser humano, como enfermedad a partir de ahoraendémica. El autor examina las características de esta extraordinaria epidemia,y recalca la necesidad de contar con un sistema de vigilancia integrado, másmedios de diagnóstico y mejores estrategias de control.

Palabras claveAmérica del Norte – Enfermedad emergente – Epizootia – Estudio – Flavivirus – Nueva York – Virus West Nile.

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