West Nile Virus Infection of Horses Javier Castillo-Olivares, James Wood

West Nile virus infection of horses Javier Castillo-Olivares, James Wood

To cite this version:

Javier Castillo-Olivares, James Wood. West Nile virus infection of horses. Veterinary Research, BioMed Central, 2004, 35 (4), pp.467-483. 10.1051/vetres:2004022. hal-00902791

HAL Id: hal-00902791 https://hal.archives-ouvertes.fr/hal-00902791 Submitted on 1 Jan 2004

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Vet. Res. 35 (2004) 467–483 467 © INRA, EDP Sciences, 2004 DOI: 10.1051/vetres:2004022 Review article

West Nile virus infection of horses

Javier CASTILLO-OLIVARES*, James WOOD

Centre for Preventive Medicine, Animal Health Trust, Newmarket, Suffolk CB8 7UU, United Kingdom

(Received 26 January 2004; accepted 1 March 2004)

Abstract – West Nile virus (WNV) is a flavivirus closely related to Japanese encephalitis and St. Louis encephalitis viruses that is primarily maintained in nature by transmission cycles between mosquitoes and birds. Occasionally, WNV infects and causes disease in other vertebrates, including humans and horses. West Nile virus has re-emerged as an important pathogen as several recent outbreaks of encephalomyelitis have been reported from different parts of Europe in addition to the large epidemic that has swept across North America. This review summarises the main features of WNV infection in the horse, with reference to complementary information from other species, highlighting the most recent scientific findings and identifying areas that require further research.

West Nile virus / flavivirus / horses / encephalitis

Table of contents

1. Introduction...... 468 2. Aetiology ...... 468 2.1. Virion structure ...... 468 2.2. The envelope protein E ...... 468 2.3. Virus replication in the cell ...... 469 3. Ecology ...... 470 4. Clinical signs in horses ...... 472 5. Epidemiology...... 472 6. Pathology in horses ...... 474 7. Pathogenesis...... 474 8. Diagnosis ...... 475 8.1. WNV detection ...... 475 8.2. Detection of antibody...... 476 9. Immune responses and vaccinates ...... 477 10. Prevention and control of West Nile encephalitis ...... 478 11. Conclusion ...... 479

* Corresponding author: [email protected] 468 J. Castillo-Olivares, J. Wood

1. INTRODUCTION core (CPC), Alfuy (ALF) and Yaounde (YAO) viruses [38]. West Nile virus (WNV) was first isolated in the West Nile district of Uganda in 2.1. Virion structure 1937 from the blood of a woman suffering from a mild febrile illness [83]. Since then, Consistent with the typical virion archi- sporadic and major outbreaks, mainly in tecture of flaviviruses, the WNV virion humans, but also in horses, have been comprises an icosahedral core composed of reported during the 1960’s in Africa, the multiple copies of a highly basic capsid pro- middle East and Europe (reviewed in [67]). tein (C) of 12 kDa. This is surrounded by a In the last decade, WNV has re-emerged as host-cell envelope modified by the inser- an important pathogen for humans and tion of two virus encoded proteins, the horses, as frequent outbreaks with increased major envelope protein E (53 kDa) and the proportion of neurological disease cases membrane protein M (8 kDa). The latter have been reported [23, 27, 39, 66, 98, 99]. derives from a precursor protein, prM (18– Indeed, outbreaks in Romania and Morocco 20 kDa), that is cleaved before virion in 1996, Tunisia in 1997, Italy in 1998, Rus- release from the infected cell [18, 31]. sia, United States and Israel in 1999, and The capsid encloses a single-stranded France, United States and Israel in 2000 RNA molecule with positive polarity which presented either an increase in the number of severe human cases, an increase in the lacks a polyadenylate tract at the 3’end. The severity of neurological disease in horses or genome contains a 5’ and a 3’ noncoding high bird mortality [14, 32]. In some instances region of 96 and 631 nucleotides respec- these three features were present, as was the tively, flanking one single open reading case in the USA outbreaks. West Nile virus- frame of 10 302 nucleotides encoding a poly- related disease in humans and horses is still protein. This is processed by viral and cel- being reported from the USA and lately lular proteases into three structural proteins confirmed human cases have been reported (C, E and M or pr-M) and 5 non-structural from France [56]. It has also been intro- proteins (NS1, NS2a / NS2b, NS3, NS4a / duced during 2003 in Canada [97], Mexico NS4b and NS5). The ends of the genome and the Caribbean region [14, 32]. Further- contain tertiary structures that play impor- more, recent serological investigations in tant regulatory functions in replication and birds in Britain suggest that a WN-like virus assembly. has been circulating amongst resident bird The non-structural proteins of flavivi- populations [21]. ruses have virus replication and assembly functions. Thus, NS1 and NS4A participate in virus replication, NS2A is involved in 2. AETIOLOGY assembly and virion release, NS3 and NS2B have proteolytic activities and NS5 acts as West Nile virus is a positive sense sin- an RNA-dependent RNA polymerase and gle-stranded RNA enveloped virus of the methyltransferase participating in the methyl- genus Flavivirus, family Flaviviridae [64]. ation of the 5’-cap structure [18]. Amongst the 12 Flavivirus sero-groups, classified according to cross-reactivity in 2.2. The envelope protein E virus neutralisation assays, WNV belongs to the Japanese encephalitis sero-complex Many biological properties of flavivi- group together with Japanese encephalitis ruses, such as tropism, cell binding, viru- (JE), Murray Valley encephalitis (MVE), St lence, haemagglutination and antigenicity, Louis encephalitis (SLE), Kunjin (KUN), are associated with the envelope protein E. Usutu (USU), Koutango (KOU), Cacipa- This protein contains 12 conserved cysteine West Nile virus infection in horses 469 residues involved in the formation of intramo- identified as antigens. For example, prM lecular disulphide bridges and forms head- specific monoclonal antibodies of dengue 3 to-tail rod-shaped curved homodimers which and dengue 4 virus with neutralising activ- do not protrude from the surface of the vir- ity have been obtained [46], and NS3 spe- ion [18]. The protein E of the flavivirus of cific monoclonal antibodies of dengue 1 tick-borne-encephalitis contains three anti- virus have shown some protective effect in genic domains. A central domain I (previ- passive immunisation experiments in mice ously referred to as domain C) is formed by [86]. Likewise, the prM and NS3 proteins the 50 N-terminal amino acids folded as an of WNV can be recognised by WNV-spe- eight-stranded β-barrel. This is flanked by cific mouse and horse antisera [29] and the the antigenic domain II (previously domain antigenic nature of WNV NS5 was demon- A), structured in two loops and containing a strated in a recent study [101] that shows highly conserved sequence among all flavi- that WNV human patient sera recognise viruses and possibly a fusion sequence, and this antigen consistently. a domain III (previously domain B) composed of seven antiparallel β-strands resem- The NS1 protein is a glycosylated protein that is expressed on the surface of bling the constant region of immunoglobu- infected cells and can also be secreted. Pol- lins and which contains important neutralising yclonal and monoclonal antibodies to the epitopes and cell binding receptors [64]. NS1 protein have identified type-, com- Recently, neutralising epitopes of WNV plex-, and flavivirus-specific epitopes [34], have been mapped to residues 307, 330 which are mainly conformation dependant and 332 of protein E which correspond to but are not virus neutralising. The NS1 pro- domain III [8]. tein of WNV has also been demonstrated to Antigenic relationships between flaviv- be antigenic and has been used as a diag- iruses have been studied using immune pol- nostic reagent in antibody capture ELISA yclonal antisera in virus neutralisation tests. procedures [44]. Monoclonal antibody studies have revealed the complexity of these relationships, show- ing the existence of flavivirus-group, sub- 2.3. Virus replication in the cell group, sero-complex, type-specific and strain- specific antigenic determinants [38]. The West Nile virus, like many other flaviv- identification and classification of WNV iruses, grows in a wide variety of primary isolates has been made using these reagents cells and continuous cell lines from mam- in virus neutralisation and indirect immun- malian (Vero cells, BHK-21, RK-13, SW-13) ofluorescence tests, but the analysis of and mosquito species (C6/36, Aedes albopic- nucleotide sequences encoding the protein tus, Aedes Aegypti cells) [64]. In vivo it can E is perhaps what has revealed more clearly grow in a variety of cells from different tis- the relationship between various WNV iso- sues depending on the host species. These lates. Thus, it has been found that WNV tissues include neurons, glial cells, and cells viruses fall into two distinct lineages [11, from spleen, liver, heart, lymph nodes and 78, 80]. Lineage 1 includes viruses isolated lung [24]. Virus replication takes place in outside and inside the African continent the perinuclear region of the rough endo- which have been associated with recent epi- plasmic reticulum (ER) where the newly demics of increased severity in humans and synthesised E, NS1 and prM are translo- horses, whereas lineage 2 comprises viruses cated to the ER lumen where prM and E het- that have only been found to circulate in erodimerise [30, 31, 100]. The immature enzootic cycles in birds in Africa. virions are transported through the secre- Apart from protein E, flavivirus proteins tory pathway to the cell membrane where prM, NS1, NS3 and NS5 have also been the final cleavage of the prM protein takes 470 J. Castillo-Olivares, J. Wood place by the action of furin. The virions must then feed again on susceptible hosts. are finally released by exocytosis or by bud- The temperature dependence of both mos- ding. quito reproduction and viral replication in Cell infection with WNV can result in insect vectors results in highly seasonal var- cell lysis, syncytia formation or may result iation in WNV transmission and disease in virus persistence. This phenomenon has outbreaks. In temperate regions such as been observed in vitro in neuroblastoma Europe, Canada and the northern states of cell lines infected with JE and in vivo in USA, most encephalitis cases are seen in many flavivirus related diseases [64] includ- late summer or autumn when insect num- ing WNV infections in monkeys where virus bers and temperatures are high [39]. was recovered up to five months post-infec- While experimental studies suggest that, tion [77], and in hamsters with viruses being as for many other arboviruses, transmission isolated up to day 53 post-challenge [103]. from infected mosquito to susceptible hosts is very efficient if feeding occurs [22, 69], transmission from vertebrate host to mos- 3. ECOLOGY quito is very dependent on the level of viraemia in the vertebrate. Duration and The natural cycle of all members of the titre of viraemia are typically both much JE antigenic complex of flaviviruses involves greater in birds than in mammals, but both birds as the main amplifying host and sev- vary hugely between avian species. Mam- eral species of mosquitoes as vectors. The malian species, including both horse and natural cycle of WNV typically involves man, are thought rarely to develop titres ornithophilic mosquitoes, particularly, but sufficient to infect mosquito species and so not exclusively, Culex species. The identity of the primary vectors and vertebrate host are unlikely to be able to sustain infectivity species is dependent on the geographic area cycles. Although they are typically referred and the levels of virus that are circulating to as “dead-end” hosts [47], occasional [47]. However, there is a vast range of both individuals, given sufficient numbers, may avian and mosquito species that can be in fact be able to infect mosquitoes. Modes infected by WNV [9, 12]. of transmission other than via insect vectors may occur and direct bird to bird spread During periods of adult mosquito blood may be possible under some circumstances feeding, WNV can be transmitted continu- [9], although again it is perhaps unlikely ously between mosquito vectors and avian that such a mechanism is important in the reservoir hosts. Infectious mosquitoes carry ecology of the disease. WNV in salivary glands and infect susceptible vertebrate hosts at feeding. Competent Identifying the avian hosts principally vertebrate hosts will sustain a viraemia, typ- responsible for amplifying WNV during ically for up to five days and, if the virus is periods of both epidemic and endemic viral to be transmitted on, other insect vectors activity is not straightforward. Serological must feed on these viraemic hosts during studies in themselves only serve to identify this period to become infected. In common which species are becoming infected and with many other arboviruses, a temperature cannot be used to determine major ampli- dependent “extrinsic period” then ensues, fying hosts. Not only must these hosts be during which virus must replicate and enter capable of sustaining high-level viraemia the salivary glands of the mosquitoes. Typ- for sufficient periods of time, they must also ically, this period is around two weeks dur- be fed on by sufficient numbers of compe- ing warm periods, but is sensitive to both tent insect vectors during this period. The temperature and humidity [9, 28]. After this relative importance of different mosquito period, if the cycle is to be maintained, suf- species in the transmission cycle, whilst ficient numbers of infected mosquitoes affected by the competence of each species West Nile virus infection in horses 471 to become infected with and transmit virus, between periods of continuous transmis- is also determined by host feeding prefer- sion. Presence of virus in hibernating [102] ences, longevity and contact rates between or overwintering mosquitoes [68], or contin- vector and competent host [9]. It is likely uous, but low level, transmission in verte- that of the (at least) 16 species of mosquito brate hosts have been proposed as possible reported to be virologically competent vec- means of persistence, but the mechanism(s) tors of WNV [47], far fewer are likely to be currently remain unknown [47]. Many important. authors have speculated on the role of Detailed studies of WNV have now sug- migratory birds in repeated re-introduction gested that transmission cycles of WNV in of WNV to temperate areas where transmis- both Europe and North America are typi- sion occurs sporadically, such as the Camargue cally maintained in passerine birds, partic- in southern France, where irregular epi- ularly the house sparrow (Passer domesti- demics in horses have been reported and cus) [47, 79], which is the only New World where there are large populations of migra- avian host in which a prolonged (5-6 day) tory birds from areas of endemic activity in and high titre viraemia has been reported Africa, [66, 67]. Migratory storks and other [48, 79]. However, care needs to be taken species may also be important in introduc- when interpreting and extrapolating these ing the infection to the Middle East [57] and results, as viraemia may be shorter and of a seropositive birds have also recently been lower titre when other strains of WNV are reported in the United Kingdom [21]. The considered [59]. Cx. pipiens, thought to be speed and pattern of spread across North the major insect vector of WNV in both America makes the role of migratory birds North America and Europe [9, 47], feed less likely than that from dispersal move- almost exclusively on passerine and columb- ments from non-migratory birds such as the iform birds [9]. Other Culex species, includ- house sparrow [79]. ing Cx. nigripalpus and Cx. tarsalis in North Transovarial transmission of WNV has America feed predominantly on birds in the been identified in Kenya in one species of early part of the transmission season, then mosquito [62] and has been demonstrated increasingly switch to mammalian hosts experimentally in a range of Culex and during the summer months; other Culex Aedes species mosquitoes [7], as well as in species feed indiscriminately on both avian north American Cx. pipiens [94]. It has been and mammalian hosts, some preferring suggested that this mode of transmission of multiple hosts [9]. These less discriminant WNV is probably unimportant as it is usu- species may function as important bridge ally inefficient in flaviviruses [9], but it vectors that spread infection to horses and does provide a source of WNV persistence. man from birds. Understanding of host The development of quantitative, biologi- feeding preferences of species involved in cally parameterised mathematical models transmission of WNV is critical to the under- describing transmission of WNV would standing of WNV ecology and has been lit- hugely inform evaluation of the signifi- tle researched, particularly in Europe; knowl- cance of such mechanisms and rates of edge of preferences in species involved in transmission. transmission amongst birds as well as to Following the introduction of WNV to man and horse is vital to inform control North America in 1999, avian mortality has strategies. been extensive [10] and crow deaths in the While WNV transmission may be main- USA have been one of the most sensitive tained by continuous circulation between sentinel systems for appearance and spread avian and mosquito species in tropical or of West Nile virus, as well as for both sub-tropical areas, different mechanisms may equine and human cases of disease [33, 49]. be important in more temperate regions Other than outbreaks in Israel in 1998 and 472 J. Castillo-Olivares, J. Wood

1999 involving mortality in geese [71], affected during these outbreaks presented reports of extensive avian mortality are with ataxia, dysmetria, abnormal mentation more or less unique to the Western hemi- ranging from somnolence to hyperexcita- sphere; the similarity of the viruses involved bility or even aggression, and hyperaesthe- in the North American and Israeli outbreaks sia. Some animals presented with facial [51] suggests strongly that this is likely to nerve paralysis, paresis of the tongue and be a feature of the virus strain involved. dysphagia resulting from deficits in cranial Current evidence suggests that monitoring nerves VII, XII and IX. avian mortality in areas where other strains A proportion of horses suffering from of virus are involved may not detect virus WNV infection do not recover and die spon- transmission. taneously or, more often, are euthanased on humane grounds. Mortality rates among clinically affected horses have been esti- 4. CLINICAL SIGNS IN HORSES mated around 38%, 57.1% and 42% during outbreaks in the USA in 2000, France in 2000 WNV infection in horses is usually not and Italy in 1998 respectively [24, 66, 74]. accompanied by presentation of clinical ill- In contrast to the human disease, severe ness. However, the latest outbreaks of neurological disease in horses does not WNV saw an increased proportion of neu- appear to occur preferentially in old indi- rological disease in both humans and horses viduals. [75]. Approximately 10% of horses and around 1% of humans infected with WNV Treatment guidelines for horses with WNV presented neurological disorders. These epi- encephalomyelitis can be found in recent demiological observations have been cor- publications [54, 78]. Treatment is aimed at roborated by experimental infections of reducing CNS inflammation, preventing equines [22] where only one animal out of self-inflicted injuries and providing fluid 12 displayed clear neurological symptoms. and nutritional care. Except for fever, clinical signs of WNV in horses are almost exclusively of neurological nature and reflect the pathology in the 5. EPIDEMIOLOGY central nervous system (CNS). These occur predominantly in the spinal cord, rhomben- Before 1994, WNV was not perceived as cephalon and mesencephalon being the cere- a serious public health problem. Rather, it bral cortex less often affected [24]. A transitory was regarded as a mosquito-borne infection febrile period might occur after infection of birds, which occasionally infected humans although this is not always observed in some and equines causing illness on rare occa- epidemics, e.g. outbreak in Italy 1998 [23]. sions. Indeed, the first epidemiological The most common symptoms are associ- studies, performed in the Nile delta in Egypt ated with spinal cord injury like ataxia, [87], demonstrated viral activity in birds paresis or paralysis of the limbs, which can without any epidemics in horses. A study in affect one, two (usually the hindlimbs) or all the same area [81] recorded an equine sero- four limbs, the latter usually progress to positivity rate of 54% with only one fatal recumbency. Often these signs are accom- case. Between 1950 and 1994, WNV was panied by skin fasciculations, muscle trem- isolated in different parts of Europe (Spain, ors and muscle rigidity. In addition to the Portugal, Russia, the former Czechoslovakia, above, the USA outbreaks saw a proportion Romania, France), Africa (South Africa, of horses displaying symptoms derived from Senegal, Kenya) and Asia (Israel, Iran, damage of the medulla oblongata, pons, India) but was rarely associated with clinical thalamus, the reticular formation, cerebel- disease in either horse or man. The principle lum and brain cortex [73, 74]. Thus, horses exception to this was the epidemic in the West Nile virus infection in horses 473

Camargue in the south east of France in were not reported from the WNV outbreaks 1962 and 1963 [43]. During this epidemic, of Italy and France, despite the equine cases. at least 80 horses were affected with ataxia In the late summer of 1999, WNV infec- and weakness and 25–30% died. Other wild tions were recorded for the first time in the Camargue horses were apparently also western hemisphere when cases in people affected but the number affected was never and horses occurred in New York. Over a reported. There was another important out- period of eight weeks 59 people had to be break in South Africa in 1974, in which hospitalised with severe neurological ill- 18 000 people were affected, although no ness, including seven deaths, in the New deaths were recorded; no information about York City metropolitan area [2, 4]. Simul- equine disease was reported [45, 59]. taneously, approximately 20 horses from Since the mid 1990’s, the number of out- Long Island were confirmed as WNV breaks and their severity has increased. encephalitis cases [92]. Later investiga- Human cases with deaths were reported tions indicated that in contact animals were from Algeria in 1994 [52], Romania in 1996 also seropositive to WNV (approximately 20%). As in Israel, there was substantial [93], Morocco in 1996 [88], Tunisia in 1997 avian mortality, in particular in crows, [91], the Democratic Republic of Congo in which in this case preceded the human and 1998 [70], Russia in 1999 [76] and in Israel horse disease reports. from 1998 to 2000 [57]. In the 1996 Morocco outbreak, WNV fever was also described in Despite the cold New York winter, the 94 horses of which 42 died. In the Roma- infection did not disappear and virus activ- nian and Russian epidemics a relatively ity was first detected in 2000 when WNV high number of people were affected clini- was isolated from a Red-tailed hawk in New York in February 2000, as well as from cally but there is little or no published infor- adult Culex mosquitoes overwintering in mation about horse disease. In the out- protected areas in January and February breaks in Israel, WNV infections were 2000 [1]. By the end of the year, the Centers reported in over 400 people, 325 of whom for Disease Control and Prevention had were hospitalised and 33 died. At least received reports of 21 human cases, 63 equine 75 horses were affected with encephalitis cases, 4 304 infected dead birds and 6 infected and 15 died, but, very unusually, there was other mammals [75] in a total of 7 other also extensive mortality in birds, particu- northeastern states. larly geese [6, 85]. WNV spread further to all the USA, In addition to these outbreaks, there were Mexico, Canada and the Caribbean during limited outbreaks restricted to horses in the next three years. Indeed, in 2001 WNV northern Italy in 1998 [23] and in France in was reported from 20 states. During this 2000 [66]. The activity in Italy during 1998 time, 738 horses were confirmed with resulted in 14 horses displaying neurologi- WNV by the laboratory, most of which cal signs. Follow up serological studies [5] (550) came from the state of Florida. revealed a 58% equine seroprevalence in The outbreak exploded in 2002 across the area of the outbreak, a far higher fre- the USA and into Canada and by the end quency than that for those with clinical of the year, WNV had been identified in signs. The clinical disease in horses described 44 states, being associated with over 4000 con- during the French outbreak in 2000 was in firmed encephalitis cases in people, includ- the Hérault and Gard department, close to ing 284 deaths, nearly 15 000 laboratory where the outbreak in 1962–1963 occurred. confirmed equine encephalitis cases and Approximately 131 equines developed neu- 16 500 dead birds [3]. Many equine cases rological disease, in 76 of which WNV were also diagnosed in five Canadian prov- infection was confirmed. WNV human cases inces [97]. 474 J. Castillo-Olivares, J. Wood

In 2003, WNV encephalitis was diag- experimental intraperitoneal infection [103]. nosed by USDA in more than 4 000 horses These animals often showed, in addition to in 41 states, with a preponderance of cases the lesions of the lower brain stem and spi- in Colorado, Montana, New Mexico and nal cord, inflammatory changes as well as Wyoming. In contrast to the equine situa- antigen staining in regions of the cerebral tion, there were more human cases in 2003, cortex and cerebellum. However, the extent with nearly 9 000 being diagnosed. One rea- of the inflammatory changes was not prom- son for the relative reduction in equine inent despite the frequent antigen staining cases compared to human cases could have of neurons. Abnormalities in extraneural been that widespread vaccination of horses tissues were not significant. was practiced. In addition to the continued Western hemisphere outbreak in 2003, further cases were diagnosed in France (Var 7. PATHOGENESIS department), where confirmed cases in three horses and six people and probable cases in Flavivirus infections are initiated after one horse and one person were reported virus inoculation into the skin by an [56] (S. Zientara personal communication), infected arthropod (mosquito or tick). The although detailed investigations do not cur- virus then replicates in local tissues and in rently appear available. regional lymph nodes and is transported via lymphatic vessels to the blood stream. Lang- erhans cells of the skin have been associated 6. PATHOLOGY IN HORSES with this virus transport to the lymph nodes in WNV infections in mice [42]. This virus The pathology of WNV in horses has replication and viraemia may seed infection been studied in natural cases during the epi- in extraneural tissues, increasing the virus demics of Italy in 1998 and the northeastern titres in blood and perhaps preceding the states of the USA in 1999 [23, 24]. As invasion of the CNS. Neuroinvasion path- briefly indicated earlier in the text, lesions ways for flaviviruses are not well defined, of WNV infection in the horse are predom- but may involve passive diffusion across inantly, if not exclusively, limited to the the capillary endothelium, virus replication CNS, and lesions affecting extraneural tis- in endothelial cells and budding of virus sues are rarely described, which contrasts into the CNS parenchyma, or retroaxonal with widespread lesions observed in many virus transport of infected neurons of the internal organs of WNV infected birds [84]. olfactory epithelium. The low titre and WNV causes polioencephalomyelitis in horses, short duration of viraemia in horses [22], as particularly evident in the lower brain stem opposed to birds, and failure to detect WNV and ventral horns of the spinal cord. The antigen in vascular endothelial cells [24] lesions are characterised by inflammatory make the first two neuroinvasion pathways changes accompanied by scant viral antigen unlikely. However, in the hamster model of staining. Lesions include perivascular cuffs WNV infection [103], West Nile antigen of lymphocytes and macrophages with fre- was not detected in the olfactory bulb of any quent haemorrhages, but generally in the of the infected hamsters. absence of viral infection of vessel walls; The mechanism of neurological damage scattered foci of microgliosis; and, in the is uncertain. In the WNV infection model in most severe cases, neuronal degeneration hamsters it was observed that many degen- with cytoplasmic swelling and chromatol- erating neurons underwent apoptosis but ysis. These lesions are less commonly this was not associated with inflammatory observed in the cortex of cerebellum and cells and the authors suggested cellular cerebrum. These observations contrast with damage was caused by the WNV infection. the findings observed in hamsters following In contrast, Cantile et al. [24] suggested that 475 J. Castillo-Olivares, J. Wood neurological damage in natural WNV infec- sistent infections of the CNS. It is not tion of horses has an immunopathological known whether this occurs in the horse. component since inflammatory changes were present in the absence of abundant detecta- ble WNV antigen. 8. DIAGNOSIS The outcome of WNV infection probably depends on host factors and virus strain. Laboratory procedures are necessary to It has been demonstrated in experimental confirm the presence of WNV due to the mouse models of Murray Valley encepha- subclinical nature of many WNV infections litis (MVE) that pathogenicity is dependent and the similar symptomatology of WNV on innate immune responses, especially encephalomyelitis to other equine neuro- type I interferon responses (IFN-α/β) [53]. logical syndromes. These include JE, alphavi- A deficit in these responses during the acute rus encephalitides (VEE, WEE, EEE), pro- phase of the infection results in increased tozoal meningoencephalitis, EHV-1 myelitis, virus replication in extraneural tissues with rabies and Borna Disease. Other less likely the subsequent rapid entry of virus into the causes of disease that may be considered in brain. Further work would be required to some cases include botulism, hypocalcae- elucidate whether a deficient innate immune mia, leukoencephalomalacia and hepatoen- response may result in the development of cephalopathy. equine neurological disease in WNV infec- The confirmation of WNV infection can tion. be made directly by identification of the Neurovirulence and neuroinvasiveness virus or indirectly by testing for antibodies of flaviviruses are typically associated with in clinical specimens which include, post- sequence variation of the protein E although mortem tissues, cerebrospinal fluid, whole other viral proteins such as NS1 and NS2b blood or serum. might also play a role. McMinn [60] reviewed comprehensively the molecular basis of fla- 8.1. WNV detection viviruses pathogenicity. Accordingly, it has been shown that recent WNV outbreaks of Detection of WNV in field cases in increased virulence are caused by strains of horses is hampered by the typically short lineage 1 rather than lineage 2, this classifi- duration and low level of the viraemia in cation being based on nucleotide sequences of horses. Negative virus detection test results the genome region encoding the E protein should thus never be regarded as evidence [51]. of absence of WNV. Persistent infections of flaviviruses, Virus isolation can be attempted from including WNV, have been documented cerebrospinal fluid (CSF), blood or tissues in vitro and in vivo [26, 50, 77, 95, 103]. in Vero, RK-13 cells [74] or mosquito cell Persistent WNV infections have been associ- lines. However, cytopathic effect is not always ated with the generation of defective interfer- evident, especially in mosquito cells, and ing particles, temperature sensitive mutants indirect immunofluorescence using a mon- and non-plaquing mutants [15–17, 19]. oclonal antibody (MAb) of well defined Whether persistent infections occur in vivo specificity is necessary to confirm the pres- during WNV infection of horses and these ence of and/or to identify virus isolates. genetic variations represent immune eva- Alternatively, the presence of the virus sion strategies employed by the virus is not can be confirmed by nucleic acid detection. known. Recurrent neurological disease in A sensitive and WNV-specific reverse tran- children previously exposed to JE and chronic scription and nested polymerase chain reac- progressive encephalitis after tick-borne tion method has been used successfully as encephalitis in humans may be due to per- a diagnostic tool in clinical samples from 476 J. Castillo-Olivares, J. Wood suspected cases of WNV encephalitis [41, (PRNT) have been preferred for serological 74]. This method detected WNV nucleic diagnosis and surveillance during recent acid in all brain tissues of serologically con- outbreaks of WNV fever. firmed cases of WNV encephalitis. Results The IgM antibody capture ELISA from plasma samples from some confirmed (MAC-ELISA) test is based on the capture WNV cases were negative in contrast to of IgM in horse serum or CSF by an anti- those collected from experimentally infected IgM antibody bound to a microtitre plate, horses during the first six days post-inocu- followed by demonstration of specific anti- lation. It is important to note that samples WNV antibodies. A positive result by the from natural clinical cases were collected MAC-ELISA test in a single sample has rel- after the onset of neurological signs, when evant diagnostic value since WNV-specific virus is unlikely to be present in blood. The IgM appear in the circulation by day 6–7 presentation of clinical signs in infected post-infection [22] and are believed to last hamsters also coincides with the end of the less than three months [73]. However, these viraemic phase [103]. Non-nested PCR antibodies have been detected for longer techniques, such as those in routine use for periods in human WNV infection and more diagnosis of infection in man and birds, are studies are necessary to understand better unsuitable for use in horses due to their rela- the kinetics of IgM responses in the horse. tive insensitivity at detecting low titres of virus. Likewise, the presence in CSF of IgM is Immunocytochemistry on CNS tissues highly indicative of a recent WNV infection (including cortex, cerebellum, brain stem of the CNS since IgM cannot cross the hae- and spinal cord) using WN-specific MAb, matoencephalic barrier. can also be used [24]. Detection of IgG by ELISA has been Antigen capture ELISA tests have been performed in serosurveillance studies in used to confirm the presence of WNV in Italy [5] and France [66]. ELISA plates are avian tissues and mosquito pools [40] but coated with viral antigen and the presence are unsuitable for use in horses due to the of serum antibodies are revealed by a per- low level of viraemia. oxidase labelled anti-horse IgG. This ELISA test is particularly valuable for serosurveil- 8.2. Detection of antibody lance as it is very sensitive and WNV IgG lasts for at least 15 months after infection Although haemagglutination inhibition [74]. However, a positive result on a single and complement fixation tests have been sample has limited clinical diagnostic value widely used in the past for the diagnosis of and it is necessary to demonstrate a recent flavivirus diseases [64], including Japanese infection by a four-fold increase in antibody encephalitis in horses (Manual of Standards titre on paired serum samples collected for Diagnostic Tests and Vaccines of the 14 days apart. Office International des Épizooties, 2000), and these tests are available for WNV [5, Both the IgM and IgG ELISA tests 72, 73], they are not widely used for labo- appear sensitive but they cross react with ratory diagnosis of WNV infection in the antibodies against other flaviviruses. The horse as they are laborious, time-consuming, more specific PRNT has been used success- slow and cross-reactive with other flavivi- fully as a diagnostic tool for WNV infection ruses. However, they can be valuable in in horses [22, 74]. areas free of circulating WNV-related fla- More recently, an epitope blocking viviruses and also have the advantage of ELISA test has been developed which can being species-independent. Instead, WNV- be used to detect WNV specific antibodies specific IgM and IgG capture ELISA tests in serum samples from birds and various and the plaque reduction neutralisation test mammalian species, including horses [13, 14]. West Nile virus infection in horses 477

A correct diagnosis depends, as always, otypic antibodies would interfere with the on careful interpretation of combined his- development of immunity. It is important to tory, clinical and laboratory data. study protection against different WNV strains or even against other flaviviruses. There are reports describing cross-protec- 9. IMMUNE RESPONSES tive efficacy of JE vaccine against WNV AND VACCINES infection in macaques [37] and that immunisation of mice with SL or JE viruses pro- As mentioned earlier, prM, E, NS1, NS3 vided protection against WNV encephalitis and NS5 are important antigens of flavivi- [90]. However, antibody-enhancement of ruses, including WNV. Both humoral and disease is a phenomenon observed in some cellular immune responses have been asso- flavivirus infection models. Thus, vaccina- ciated with protective immunity against tion against JE did not protect against but various flavivirus infections. Effector mech- enhanced MV encephalitis [20]. This phe- anisms of immunity against flaviviruses nomenon is due to the presence in serum comprise cytotoxic T lymphocytes, virus of sub-neutralising levels of heterologous neutralising antibody and possibly non- antibodies and has been described in vitro neutralising antibody responses and anti- [36] and recently in vivo [96]. Elucidating body-dependent cell mediated cytotoxicity whether this might occur in the context of (ADCC) (anti-NS1 antibodies have protec- WNV infections in the horse is of impor- tive efficacy) [64]. tance for the safety of vaccination. In par- Various vaccination strategies have been ticular, it would be important to know the devised to protect horses against WNV levels of protection that inactivated vac- infection [63]. These include inactivated cines prepared with North American strains vaccines, DNA vaccines, live attenuated provide against challenge with European vaccines and genetically modified live vac- viruses. cines. Most inactivated vaccines have the dis- Traditionally, inactivated vaccines against advantage of not eliciting cellular immu- flaviviruses have been prepared in mouse nity and rely on the stimulation of strong brain or tissue culture followed by inacti- antibody responses for protection. How- vation with formalin or betapropiolactone. ever, CTL responses have been detected A number of these vaccines have been used against other flaviviruses and it would be against other flavivirus diseases, including important to establish their relationship louping ill, JE, MV fever and tick-borne with virus clearance and/or immunopathol- encephalitis. An inactivated vaccine against ogy in the WNV infection of the horse. WNV in horses is available in North Amer- DNA and live vaccines have the capacity to ica and its experimental efficacy has been stimulate adaptive cellular immunity. The reported [69]. In this study, the vaccine potential of DNA vaccination for WNV has stimulated a virus neutralising antibody been demonstrated in mouse and horse response that persisted for 12 months after infection models [25, 29]. In this strategy, administration of a primary 2 doses course. a c-DNA copy of the prM/E coding region Protective efficacy of the vaccine was of WNV is inserted into the mammalian assessed by comparing the viraemia frequen- expression plasmid vector pCBJESS, a cies in vaccinated and control seronegative derivative of pCBamp modified to contain horses after a challenge at 12 months post- the JE virus signal sequence. Single immu- vaccination. Nine of 11 control animals nisation of mice resulted in the induction of developed viraemia, whereas this was only neutralising antibodies (titres of 1:320– detected in 1 of the 19 vaccinates. 1:640 on week 9 post-immunisation) and It is not known whether previous expo- 100% survival rate after mosquito or intra- sure to other flaviviruses and hence heter- peritoneal challenge on week 9. Similarly, 478 J. Castillo-Olivares, J. Wood horses developed neutralising antibodies sus macaques of chimeric YF/JE, YF/Dengue (1:5–1:40 titres on day 37 post-immunisa- and YF-17D showed that the recombinant tion) and were protected against viraemia viruses have lower neurovirulence than the when challenged by infected mosquitoes on YF-17D vaccine strain in both species and day 39 post-immunisation. that they induced self-limited viraemia in Prototype live attenuated WNV vaccines monkeys identical to those induced by the have been developed by repetitive passage YF-17D virus. A prototype YF/WNV vac- of virus in mosquito cells in the presence of cine has been constructed and shown to be a neutralising monoclonal antibody [55]. protective against WNV challenge of ham- This vaccine showed capacity to protect sters [89]. This vaccination approach is cur- against WNV infection in geese. The authors rently being explored in horses and humans. are not aware of extension of these studies to the horse. 10. PREVENTION AND CONTROL One possible disadvantage of develop- OF WEST NILE ENCEPHALITIS ing a live vaccine by repetitive virus passage in tissue culture is that the position of Strategies for prevention of West Nile the attenuating mutations cannot be con- encephalitis in endemic areas include vac- trolled and these mutations may revert to cination, reduction of virus circulation through the virulent variant once the virus infects measures influencing mosquito populations the natural host. The development of cDNA and reduction of contact with infected mos- infectious clones of WNV [82] allows the quito vectors through altered behaviour or attenuation of the virus by direct mutagen- management. esis introducing the mutations in regions of Thus far, the most widely used measure the genome that are known for their genetic has been vaccination of equidae in north stability, like the 3’ noncoding region. This America using the licensed whole virus approach has been used for Dengue and killed vaccine [69]. While cases of disease tick-borne encephalitis viruses [58, 61] by have been reported in horses that have introduction of modifications in the 3’ non- received the product, most had not com- coding region. pleted the initial course of two doses [35]. Another vaccination approach for WNV Use of this and other products, such as the is the use of chimeric yellow fever (YF) recently licenced recombinant Canarypox viruses expressing WNV antigens [63, 65]. WNV vaccine, will remain an important This strategy has been used for JE and Den- means of preventing the disease in horses. gue and the development of these vaccines There is a long history of environmental are in advanced clinical trials. The recom- insecticide use in the management of arbo- binant virus is constructed using the cDNA virus outbreaks, particularly in North Amer- backbone of the YF-17D vaccine strain ica, and undoubtedly their use is beneficial which is modified by replacing the YF prM/E during periods of intense challenge. How- coding region by that of the flavivirus for ever, it is unlikely that their use would be which the vaccine is developed. This immu- sanctioned merely to reduce equine disease nisation offers the advantages of live vaccines: outbreaks (they are principally a public stimulation of humoral as well as cellular health measure) and their short and long immunity (M and E are targeted by neutral- term environmental impact is uncertain. ising antibodies and by CTLs) with a single Other effective methods of reducing the dose, and long-lasting immunological mem- local populations of some mosquito species ory, with the added advantage of increased include control of standing water and safety. Thus, intracerebral inoculation of removal of larval breeding sites, but again, both adult and sucking mice and intracere- there is no information available on their bral and subcutaneous inoculations in rhe- specific efficacy against WNV transmission. West Nile virus infection in horses 479

An important area in West Nile preven- [3] Anonymous, Provisional Surveillance Sum- tion is management of horses to prevent mary of the West Nile Virus Epidemic – United States, January–November 2002, CDC exposure to infected mosquitoes. Stabling, MMWR Morb. Mortal. Wkly Rep. 52 (2003) particularly at night in insect proof stabling 1160–1160. may form an inexpensive, safe and effective [4] Asnis D.S., Conetta R., Teixeira A.A., Waldman means of preventing infection. The effec- G., Sampson B.A., The West Nile virus out- tiveness of stabling in non-insect proof sta- break of 1999 in New York: the Flushing Hos- bling is less clear, but probably depends on pital experience, Clin. Infect. Dis. 30 (2000) 413–418. specific local environmental factors such as [5] Autorino G.L., Battisti A., Deubel V., Ferrari the local species of mosquito, the availabil- G., Forletta R., Giovannini A., Lelli R., Murri ity of other hosts for mosquitoes to feed on S., Scicluna M.T., West Nile virus epidemic and the mosquito and horse population den- in horses, Tuscany region, Italy, Emerg. sities. Normal stabling could be enhanced Infect. Dis. 8 (2002) 1372–1378. by the use of insecticides within stables and [6] Banet-Noach C., Malkinson M., Brill A., topical insect repellents but the efficacy of Samina I., Yadin H., Weisman Y., Pokamunski S., King R., Deubel V., Stram Y., Phyloge- such measures has not been fully evaluated. netic relationships of West Nile viruses isolated from birds and horses in Israel from 1997 to 2001, Virus Genes 26 (2003) 135–141. 11. CONCLUSION [7] Baqar S., Hayes C.G., Murphy J.R., Watts D.M., Vertical transmission of West Nile West Nile infection has re-emerged as an virus by Culex and Aedes mosquitoes, Am. J. important zoonosis that has demonstrated a Trop. Med. Hyg. 48 (1993) 757–762. capacity to spread over an entire continent [8] Beasley D.W., Barrett A.D., Identification of in a few years, causing illness and deaths in neutralizing epitopes within structural domain III of the West Nile virus envelope protein, J. humans and horses. Its future course is Virol. 76 (2002) 13097–13100. unpredictable but it is expected that the lat- [9] Bernard K.A., Kramer L.D., West Nile Virus est improvements in laboratory diagnosis Activity in the United States, 2001, Viral will reveal important epidemiological fea- Immunol. 14 (2001) 319–338. tures of WNV. Other recent scientific [10] Bernard K.A., Maffei J.G., Jones S.A., advances have also contributed to a better Kaufman E.M., Ebel G.D., Dupuis A.P. 2nd, understanding of WNV and the disease but Ngo K.A., Nicholas D.C., Young D.M., Shi P.Y., Kulasekera V.L., Eidson M., White D.J., many aspects about immunity, pathogene- Stone W.B., Kramer L.D., West Nile virus sis and molecular basis of virulence, espe- infection in birds and mosquitoes, New York cially in the horse, are still unknown. State, 2000, Emerg. Infect. Dis. 7 (2001) 679– 685. [11] Berthet F.X., Zeller H.G., Drouet M.T., Rauzier ACKNOWLEDGEMENTS J., Digoutte J.P., Deubel V., Extensive nucleotide changes and deletions within the enve- The authors are very grateful to Dr Nick lope glycoprotein gene of Euro-African West Davis-Poynter, Dr Duncan Hannant and Dr Janet Nile viruses, J. Gen. Virol. 78 (1997) 2293– Daly for critically reviewing the manuscript. 2297. [12] Blitvich B.J., Marlenee N.L., Hall R.A., Calisher C.H., Bowen R.A., Roehrig J.T., Komar N., REFERENCES Langevin S.A., Beaty B.J., Epitope-blocking enzyme-linked immunosorbent assays for the [1] Anderson J.F., Andreadis T.G., Vossbrinck detection of serum antibodies to West Nile C.R., Tirrell S., Wakem E.M., French R.A., virus in multiple avian species, J. Clin. Micro- Garmendia A.E., Van Kruiningen H.J., Isola- biol. 41 (2003) 1041–1047. tion of West Nile virus from mosquitoes, [13] Blitvich B.J., Fernandez-Salas I., Contreras- crows, and a Cooper’s hawk in Connecticut, Cordero J.F., Marlenee N.L., Gonzalez-Rojas Science 286 (1999) 2331–2333. J.I., Komar N., Gubler D.J., Calisher C.H., [2] Anonymous, Outbreak of West Nile-like viral Beaty B.J., Serologic evidence of West Nile encephalitis–New York, 1999, MMWR Morb. virus infection in horses, Coahuila State, Mex- Mortal. Wkly Rep. 48 (1999) 845–849. ico, Emerg. Infect. Dis. 9 (2003) 853–856. 480 J. Castillo-Olivares, J. Wood

[14] Blitvich B.J., Bowen R.A., Marlenee N.L., mid DNA induces protective immunity and Hall R.A., Bunning M.L., Beaty B.J., Epitope- prevents Japanese encephalitis in mice, J. blocking enzyme-linked immunosorbent assays Virol. 74 (2000) 4244–4252. for detection of West Nile virus antibodies in [26] Chen L.K., Liao C.L., Lin C.G., Lai S.C., Liu domestic mammals, J. Clin. Microbiol. 41 C.I., Ma S.H., Huang Y.Y., Lin Y.L., Persist- (2003) 2676–2679. ence of Japanese encephalitis virus is associ- [15] Brinton M.A., Isolation of a replication-effi- ated with abnormal expression of the non- cient mutant of West Nile virus from a persist- structural protein NS1 in host cells, Virology ently infected genetically resistant mouse cell 217 (1996) 220–229. culture, J. Virol. 39 (1981) 413–421. [27] Chowers M.Y., Lang R., Nassar F., Ben- [16] Brinton M.A., Characterization of West Nile David D., Giladi M., Rubinshtein E., Itzhaki virus persistent infections in genetically resist- A., Mishal J., Siegman-Igra Y., Kitzes R., Pick ant and susceptible mouse cells. I. Generation N., Landau Z., Wolf D., Bin H., Mendelson E., of defective nonplaquing virus particles, Pitlik S.D., Weinberger M., Clinical charac- Virology 116 (1982) 84–98. teristics of the West Nile fever outbreak, [17] Brinton M.A., Analysis of extracellular West Israel, 2000, Emerg. Infect. Dis. 7 (2001) Nile virus particles produced by cell cultures 675–678. from genetically resistant and susceptible [28] Cornel A.J., Jupp P.G., Blackburn N.K., Envi- mice indicates enhanced amplification of ronmental temperature on the vector compe- defective interfering particles by resistant cul- tence of Culex univittatus (Diptera: culicidae) tures, J. Virol. 46 (1983) 860–870. for West Nile virus, J. Med. Entomol. 30 [18] Brinton M.A., The molecular biology of West (1993) 449–456. Nile virus: a new invader of the western hemisphere, Annu. Rev. Microbiol. 56 (2002) [29] Davis B.S., Chang G.J., Cropp B., Roehrig 371–402. J.T., Martin D.A., Mitchell C.J., Bowen R., Bunning M.L., West Nile virus recombinant [19] Brinton M.A., Davis J., Schaefer D., Charac- DNA vaccine protects mouse and horse from terization of West Nile virus persistent infec- virus challenge and expresses in vitro a non- tions in genetically resistant and susceptible infectious recombinant antigen that can be mouse cells. II. Generation of temperature- used in enzyme-linked immunosorbent assays, sensitive mutants, Virology 140 (1985) 152– J. Virol. 75 (2001) 4040–4047. 158. [30] Despres P., Frenkiel M.P., Deubel V., Differ- [20] Broom A.K., Wallace M.J., Mackenzie J.S., ences between cell membrane fusion activi- Smith D.W., Hall R.A., Immunisation with ties of two dengue type-1 isolates reflect mod- gamma globulin to murray valley encephalitis ifications of viral structure, Virology 196 virus and with an inactivated Japanese encepha- (1993) 209–219. litis virus vaccine as prophylaxis against aus- tralian encephalitis: evaluation in a mouse [31] Deubel V., Fiette L., Gounon P., Drouet M.T., model, J. Med. Virol. 61 (2000) 259–265. Khun H., Huerre M., Banet C., Malkinson M., Despres P., Variations in biological features [21] Buckley A., Dawson A., Moss S.R., Hinsley of West Nile viruses, Ann. N.Y. Acad. Sci. S.A., Bellamy P.E., Gould E.A., Serological 951 (2001) 195–206. evidence of West Nile virus, Usutu virus and Sindbis virus infection of birds in the UK, J. [32] Dupuis A.P. 2nd, Marra P.P., Kramer L.D., Gen. Virol. 84 (2003) 2807–2817. Serologic evidence of West Nile virus trans- [22] Bunning M.L., Bowen R.A., Cropp C.B., mission, Jamaica, West Indies, Emerg. Infect. Sullivan K.G., Davis B.S., Komar N., Godsey Dis. 9 (2003) 860–863. M.S., Baker D., Hettler D.L., Holmes D.A., [33] Eidson M., Komar N., Sorhage F., Nelson R., Biggerstaff B.J., Mitchell C.J., Experimental Talbot T., Mostashari F., Mc Lean R., Crow infection of horses with West Nile virus, deaths as a sentinel surveillance system for Emerg. Infect. Dis. 8 (2002) 380–386. West Nile virus in the Northeastern United [23] Cantile C., Di Guardo G., Eleni C., Arispici States, 1999, Emerg. Infect. Dis. 7 (2001) M., Clinical and neuropathological features of 615–620. West Nile virus equine encephalomyelitis in [34] Falconar A.K., Young P.R., Production of Italy, Equine Vet. J. 32 (2000) 31–35. dimer-specific and dengue virus group cross- [24] Cantile C., Del Piero F., Di Guardo G., Arispici reactive mouse monoclonal antibodies to the M., Pathologic and immunohistochemical dengue 2 virus non-structural glycoprotein findings in naturally occuring West Nile virus NS1, J. Gen. Virol. 72 (1991) 961–965. infection in horses, Vet. Pathol. 38 (2001) [35] Geiser S., Seitzinger A., Salazar P., Traub-Dargatz 414–421. J., Morley P., Salman M., Wilmont D., Steffen [25] Chang G.J., Hunt A.R., Davis B., A single D., Cunningham W., Economic impact of intramuscular injection of recombinant plas- West Nile virus on the Colorado and Nebraska West Nile virus infection in horses 481

equine industries: 2002, United States Depart- [46] Kaufman B.M., Summers P.L., Dubois D.R., ment of Agriculture , Animal and Plant Health Cohen W.H., Gentry M.K., Timchak R.L., Inspection Services – Veterinary Services Burke D.S., Eckels K.H., Monoclonal anti- Info Sheet N394.0403, 2003. bodies for dengue virus prM glycoprotein pro- [36] Gollins S.W., Porterfield J.S., Flavivirus tect mice against lethal dengue infection, Am. infection enhancement in macrophages: radi- J. Trop. Med. Hyg. 41 (1989) 576–580. oactive and biological studies on the effect of [47] Komar N., West Nile viral encephalitis, Rev. antibody on viral fate, J. Gen. Virol. 65 (1984) Sci. Tech. 19 (2000) 166–176. 1261–1272. [48] Komar N., Davis B., Bunning M.L., Hettler [37] Goverdhan M.K., Kulkarni A.B., Gupta A.K., D.L., Experimental infection of wild birds Tupe C.D., Rodrigues J.J., Two-way cross- with West Nile virus (New York 1999 strain), protection between West Nile and Japanese Am. J. Trop. Med. Hyg. 62 (2000) 229–230. encephalitis viruses in bonnet macaques, Acta [49] Komar N., Lanciotti R.S., Bowen R., Langevin Virol. 36 (1992) 277–283. S., Bunning M.L., Detection of West Nile [38] Heinz F.X., Purcell R.H., Gould E.A., Howard virus in oral and cloacal swabs collected from C.R., Houghton M., et al., Family: Flaviviridae, bird carcasses, Emerg. Infect. Dis. 8 (2002) in: Van Regenmortel M.H.V., Bishop D.H.L., 741–742. Carstens E.B., Estes M.K., Lemon S.M., Maniloff J., Mayo M.A., McGeoch D.J., Pringle [50] Lancaster M.U., Hodgetts S.I., Mackenzie C.R., Wickner R.B. (Eds.), Virus taxonomy: J.S., Urosevic N., Characterization of defec- 7th Report of the International Committee on tive viral RNA produced during persistent Taxonomy of Viruses, Academic Press, San infection of Vero cells with Murray Valley Diego, 1999, pp. 859–878. encephalitis virus, J. Virol. 72 (1998) 2474– 2482. [39] Hubalek Z., Halouzka J., West Nile fever-a reemerging mosquito-borne viral disease in [51] Lanciotti R.S., Roehrig J.T., Deubel V., Smith Europe, Emerg. Infect. Dis. 5 (1999) 643– J., Parker M., Steele K., Crise B., Volpe K.E., 650. Crabtree M.B., Scherret J.H., Hall R.A., Mac- Kenzie J.S., Cropp C.B., Panigrahy B., Ostlund [40] Hunt A.R., Hall R.A., Kerst A.J., Nasci R.S., E., Schmitt B., Malkinson M., Banet C., Savage H.M., Panella N.A., Gottfried K.L., Weissman J., Komar N., Savage H.M., Stone Burkhalter K.L., Roehrig J.T., Detection of W., McNamara T., Gubler D.J., Origin of the West Nile virus antigen in mosquitoes and West Nile virus responsible for an outbreak of avian tissues by a monoclonal antibody-based encephalitis in the northeastern United States, capture enzyme immunoassay, J. Clin. Micro- Science 286 (1999) 2333–2337. biol. 40 (2002) 2023–2030. [52] Le Guenno B., Bougermouh A., Azzam T., [41] Johnson D.J., Ostlund E.N., Pedersen D.D., Bouakaz R., West Nile: a deadly virus? Lancet Schmitt B.J., Detection of North American 348 (1996) 1315. West Nile virus in animal tissue by a reverse transcription-nested polymerase chain reac- [53] Lobigs M., Mullbacher A., Wang Y., Pavy M., tion assay, Emerg. Infect. Dis. 7 (2001) 739– Lee E., Role of type I and type II interferon 741. responses in recovery from infection with an encephalitic flavivirus, J. Gen. Virol. 84 [42] Johnston L.J., Halliday G.M., King N.J., (2003) 567–572. Langerhans cells migrate to local lymph nodes following cutaneous infection with an arbovi- [54] Long M.T., Ostlund E.N., Porter M.B., Crom rus, J. Invest. Dermatol. 114 (2000) 560–568. R.L., Equine West Nile Encephalitis: Epide- miologocal and Clinical Review for Practi- [43] Joubert L., Oudar J., Hannoun C., Beytout D., tioners, AAEP Proceedings 48 (2002) 1–6. Corniou B., Guillon J.C., Panthier R., Epide- miology of the West Nile virus: study of a [55] Lustig S., Olshevsky U., Ben-Nathan D., focus in Camargue. IV. Meningo-encephalo- Lachmi B.E., Malkinson M., Kobiler D., myelitis of the horse, Ann. Inst. Pasteur (Paris) Halevy M., A live attenuated West Nile virus 118 (1970) 239–247. strain as a potential veterinary vaccine, Viral [44] Jozan M., Evans R., McLean R., Hall R., Immunol. 13 (2000) 401–410. Tangredi B., Reed L., Scott J., Detection of [56] Mailles A., Zeller H., Durand J.-P., Zientara West Nile virus infection in birds in the United S., Goffette R., Gloaguen C., Armengaud A., States by blocking ELISA and immunohisto- Schaffner F., Hars J., Chodorge E., Barbat J., chemistry, Vector Borne Zoonotic Dis. 3 Human and equine West Nile virus infections (2003) 99–110. in France, August – September 2003, Euro- [45] Jupp P.G., The ecology of West Nile virus in surveillance Weekly Archives 7 (2003). South Africa and the occurrence of outbreaks [57] Malkinson M., Banet C., Weisman Y., in humans, Ann. N.Y. Acad. Sci. 951 (2001) Pokamunski S., King R., Drouet M.T., Deubel 143–152. V., Introduction of West Nile virus in the 482 J. Castillo-Olivares, J. Wood

Middle East by migrating White Storks, West Nile virus, Dev. Biol. (Basel) 114 (2003) Emerg. Infect. Dis. 8 (2002) 392–397. 221–227. [58] Mandl C.W., Holzmann H., Meixner T., [70] Nur Y.A., Groen J., Heuvelmans H., Tuynman Rauscher S., Stadler P.F., Allison S.L., Heinz W., Copra C., Osterhaus A.D., An outbreak of F.X., Spontaneous and engineered deletions West Nile fever among migrants in Kisangani, in the 3’ noncoding region of tick-borne Democratic Republic of Congo, Am. J. Trop. encephalitis virus: construction of highly Med. Hyg. 61 (1999) 885–888. attenuated mutants of a flavivirus, J. Virol. 72 [71] Office International des Epizooties, West Nile (1998) 2132–2140. fever in Israel in geese, OIE Dis. Info. 12 [59] McIntosh B.M., Dickinson D.B., McGillivray (1999) 166. G.M., Ecological studies on Sindbis and West [72] Omilabu S.A., Olaleye O.D., Aina Y., Nile viruses in South Africa. V. The response Fagbami A.H., West Nile Complement Fixing of birds to inoculation of virus, S. Afr. J. Med. antibodies in Nigerian domestic animals and Sci. 34 (1969) 77–82. humans, J. Hyg. Epidemiol. Microbiol. [60] McMinn P.C., The molecular basis of viru- Immunol. 34 (1990) 357–363. lence of the encephalitogenic flaviviruses, J. [73] Ostlund E.N., Andresen J.E., Andresen M., Gen. Virol. 78 (1997) 2711–2722. West Nile encephalitis, Vet. Clin. North Am. [61] Men R., Bray M., Clark D., Chanock R.M., Equine Pract. 16 (2000) 427–441. Lai C.J., Dengue type 4 virus mutants contain- [74] Ostlund E.N., Crom R.L., Pedersen D.D., ing deletions in the 3' noncoding region of the Johnson D.J., Williams W.O., Schmitt B.J., RNA genome: analysis of growth restriction Equine West Nile encephalitis, United States, in cell culture and altered viremia pattern and Emerg. Infect. Dis. 7 (2001) 665–669. immunogenicity in rhesus monkeys, J. Virol. 70 (1996) 3930–3937. [75] Petersen L.R., Roehrig J.T., West Nile virus: a reemerging global pathogen, Emerg. Infect. [62] Miller B.R., Nasci R.S., Godsey M.S., Savage Dis. 7 (2001) 611–614. H.M., Lutwama J.J., Lanciotti R.S., Peters C.J., First field evidence for natural vertical [76] Platonov A.E., Shipulin G.A., Shipulina O.Y., transmission of West Nile virus in Culex uni- Tyutyunnik E.N., Frolochkina T.I., Lanciotti vittatus complex mosquitoes from Rift Valley R.S., Yazyshina S., Platonova O.V., Obukhov Province, Kenya, Am. J. Trop. Med. Hyg. 62 I.L., Zhukov A.N., Vengerov Y.Y., Pokrovskii (2000) 240–246. V.I., Outbreak of West Nile virus infection, Volgograd Region, Russia, 1999, Emerg. [63] Monath T.P., Prospects for development of a Infect. Dis. 7 (2001) 128–132. vaccine against the West Nile virus, Ann. N.Y. Acad. Sci. 951 (2001) 1–12. [77] Pogodina V.V., Frolova M.P., Malenko G.V., Fokina G.I., Koreshkova G.V., Kiseleva L.L., [64] Monath T.P., Heinz F.X., Flaviviruses, in: Bochkova N.G., Ralph N.M., Study on West Fields B.N., Howley P.M. (Eds.), Fields Nile virus persistence in monkeys, Arch. Virology, Vol. 1, Lippincott-Raven Publish- Virol. 75 (1983) 71–86. ers, Philadelphia, 1996, pp. 961–1034. [78] Porter M.B., Long M.T., Getman L.M., [65] Monath T.P., Arroyo J., Miller C., Guirakhoo Giguere S., MacKay R.J., Lester G.D., Alleman F., West Nile Virus Vaccine. Current Drug A.R., Wamsley H.L., Franklin R.P., Jacks S., Targets – Infectious Disorders 1 (2001). Buergelt C.D., Detrisac C.J., West Nile virus [66] Murgue B., Murri S., Zientara S., Durand B., encephalomyelitis in horses: 46 cases (2001), Durand J.P., Zeller H., West Nile outbreak in J. Am. Vet. Med. Assoc. 222 (2003) 1241–1247. horses in southern France, 2000: the return [79] Rappole J.H., Hubalek Z., Migratory birds after 35 years, Emerg. Infect. Dis. 7 (2001) and West Nile virus, J. Appl. Microbiol. 94 692–696. (2003) 47S–58S. [67] Murgue B., Zeller H., Deubel V., The ecology [80] Savage H.M., Ceianu C., Nicolescu G., and epidemiology of West Nile virus in Africa, Karabatsos N., Lanciotti R., Vladimirescu A., Europe and Asia, Curr. Top. Microbiol. Laiv L., Ungureanu A., Romanca C., Tsai T.F., Immunol. 267 (2002) 195–221. Entomologic and avian investigations of an [68] Nasci R.S., Savage H.M., White D.J., Miller epidemic of West Nile fever in Romania in J.R., Cropp B.C., Godsey M.S., Kerst A.J., 1996, with serologic and molecular character- Bennett P., Gottfried K.L., Lanciotti R.S., ization of a virus isolate from mosquitoes, West Nile Virus in overwintering Culex mos- Am. J. Trop. Med. Hyg. 61 (1999) 600–611. quitoes, New York city, 2000, Emerg. Infect. [81] Schmidt J.R., Elmansoury H.K., Natural and Dis. 7 (2001) 1–3. Experimental infection of Egyptian equines [69] Ng T., Hathaway D., Jennings N., Champ D., with West Nile virus, Ann. Trop. Med. Para- Chiang Y.W., Chu H.J., Equine vaccine for sitol. 57 (1963) 415–427. West Nile virus infection in horses 483

[82] Shi P.Y., Genetic systems of West Nile virus [94] Turrell M.J., O'Guinn M., Dohm D.J., Jones and their potential applications, Curr. Opin. J.W., Vector competence of North American Investig. Drugs 4 (2003) 959–965. mosquitoes (Diptera:Culicidae) for West Nile virus, J. Med. Entomol. 38 (2001) 130–134. [83] Smithburn K.C., Hughes T.P., Burke A.W., Paul J.H., A neurotropic virus isolated from [95] Vlaycheva L.A., Chambers T.J., Neuroblas- the blood of a native of Uganda, Am. J. Trop. toma cell-adapted yellow fever 17D virus: Med. 20 (1940) 471–492. characterization of a viral variant associated with persistent infection and decreased virus [84] Steele K.E., Linn M.J., Schoepp R.J., Komar spread, J. Virol. 76 (2002) 6172–6184. N., Geisbert T.W., Manduca R.M., Calle P.P., Raphael B.L., Clippinger T.L., Larsen T., [96] Wallace M.J., Smith D.W., Broom A.K., Smith J., Lanciotti R.S., Panella N.A., McNa- Mackenzie J.S., Hall R.A., Shellam G.R., mara T.S., Pathology of fatal West Nile virus McMinn P.C., Antibody-dependent enhance- infections in native and exotic birds during ment of Murray Valley encephalitis virus vir- the 1999 outbreak in New York City, New ulence in mice, J. Gen. Virol. 84 (2003) York, Vet. Pathol. 37 (2000) 208–224. 1723–1728. [85] Steinman A., Banet C., Sutton G.A., Yadin H., [97] Weese J.S., Baird J.D., DeLay J., Kenney Hadar S., Brill A., Clinical signs of West Nile D.G., Staempfli H.R., Viel L., Parent J., virus encephalomyelitis in horses during the Smith-Maxie L., Poma R., West Nile virus outbreak in Israel in 2000, Vet. Rec. 151 encephalomyelitis in horses in Ontario: 28 (2002) 47–49. cases, Can. Vet. J. 44 (2003) 469–473. [86] Tan C.H., Yap E.H., Singh M., Deubel V., [98] Weinberger M., Pitlik S.D., Gandacu D., Lang Chan Y.C., Passive protection studies in mice R., Nassar F., Ben David D., Rubinstein E., with monoclonal antibodies directed against Izthaki A., Mishal J., Kitzes R., Siegman-Igra the non-structural protein NS3 of dengue 1 Y., Giladi M., Pick N., Mendelson E., Bin H., virus, J. Gen. Virol. 71 (1990) 745–749. Shohat T., West Nile fever outbreak, Israel, 2000: epidemiologic aspects, Emerg. Infect. [87] Taylor R.M., Work T.H., Hurlbut H.S., Rizk Dis. 7 (2001) 686–691. F., A study of the ecology of West Nile virus in Egypt, Am. J. Trop. Med. Hyg. 5 (1956) [99] Weiss D., Carr D., Kellachan J., Tan C., Phillips 579–620. M., Bresnitz E., Layton M., Clinical findings of West Nile virus infection in hospitalized [88] Tber-Aldelhaq A., West Nile fever in horses patients, New York and New Jersey, 2000, in Morocco, Bull. OIE 11 (1996) 867–869. Emerg. Infect. Dis. 7 (2001) 654–658. [89] Tesh R.B., Arroyo J., Travassos Da Rosa [100] Westaway E.G., Mackenzie J.M., Kenney A.P., Guzman H., Xiao S.Y., Monath T.P., M.T., Jones M.K., Khromykh A.A., Ultrastruc- Efficacy of killed virus vaccine, live attenu- ture of Kunjin virus-infected cells: colocali- ated chimeric virus vaccine, and passive immu- zation of NS1 and NS3 with double-stranded nization for prevention of West Nile virus RNA, and of NS2B with NS3, in virus- encephalitis in hamster model, Emerg. Infect. induced membrane structures, J. Virol. 71 Dis. 8 (2002) 1392–1397. (1997) 6650–6661. [90] Tesh R.B., Travassos da Rosa A.P., Guzman [101] Wong S.J., Boyle R.H., Demarest V.L., H., Araujo T.P., Xiao S.Y., Immunization with Woodmansee A.N., Kramer L.D., Li H., heterologous flaviviruses protective against Drebot M., Koski R.A., Fikrig E., Martin fatal West Nile encephalitis, Emerg. Infect. D.A., Shi P.Y., Immunoassay targeting non- Dis. 8 (2002) 245–251. structural protein 5 to differentiate West Nile [91] Triki H., Murri S., Le Guenno B., Bahri O., virus infection from dengue and St. Louis Hili K., Sidhom M., Dellagi K., West Nile encephalitis virus infections and from flaviv- viral meningo-encephalitis in Tunisia, Med. irus vaccination, J. Clin. Microbiol. 41 Trop. (Madr.) 61 (2001) 487–490. (2003) 4217–4223. [92] Trock S.C., Meade B.J., Glaser A.L., Ostlund [102] Work T.H., Hurlbut H.S., Taylor R.M., E.N., Lanciotti R.S., Cropp B.C., Kulasekera Indigenous wild birds of the Nile Delta as V., Kramer L.D., Komar N., West Nile virus potential West Nile virus circulating reser- outbreak among horses in New York State, voirs, Am. J. Trop. Med. Hyg. 4 (1955) 872– 1999 and 2000, Emerg. Infect. Dis. 7 (2001) 888. 745–747. [103] Xiao S.Y., Guzman H., Zhang H., Travassos [93] Tsai T.F., Popovici F., Cernescu C., Campbell da Rosa A.P., Tesh R.B., West Nile virus G.L., Nedelcu N.I., West Nile encephalitis infection in the golden hamster (Mesocrice- epidemic in southeastern Romania, Lancet tus auratus): a model for West Nile encepha- 352 (1998) 767–771. litis, Emerg. Infect. Dis. 7 (2001) 714–721.