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Lineage 2 West Nile Virus as Cause of Fatal Neurologic Disease in Horses, South Africa
research-article
Author(s):
Marietjie Venter
,
Stacey Human ,
Dewald Zaayman ,
Gertruida H. Gerdes ,
June Williams ,
Johan Steyl ,
Patricia A. Leman ,
Janusz Tadeusz Paweska ,
Hildegard Setzkorn ,
Gavin Rous ,
Sue Murray ,
Rissa Parker ,
Cynthia Donnellan ,
Robert Swanepoel
Publication date (Print):
June 2009
Journal:
Emerging Infectious Diseases
Publisher:
Centers for Disease Control and Prevention
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Abstract
Lineage 2 WNV may be missed as a cause of neurologic infections in horses and humans in this region.
Abstract
Serologic evidence suggests that West Nile virus (WNV) is widely distributed in horses in southern Africa. However, because few neurologic cases have been reported, endemic lineage 2 strains were postulated to be nonpathogenic in horses. Recent evidence suggests that highly neuroinvasive lineage 2 strains exist in humans and mice. To determine whether neurologic cases are being missed in South Africa, we tested 80 serum or brain specimens from horses with unexplained fever (n = 48) and/or neurologic signs (n = 32) for WNV. From March 2007 through June 2008, using reverse transcription–PCR (RT-PCR) and immunoglobulin (Ig) M ELISA, we found WNV RNA or IgM in 7/32 horses with acute neurologic disease; 5 horses died or were euthanized. In 5/7 horses, no other pathogen was detected. DNA sequencing for all 5 RT-PCR–positive cases showed the virus belonged to lineage 2. WNV lineage 2 may cause neurologic disease in horses in South Africa.
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Most cited references34
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Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States.
R S Lanciotti, J. T. Roehrig, V. Deubel … (1999)
In late summer 1999, an outbreak of human encephalitis occurred in the northeastern United States that was concurrent with extensive mortality in crows (Corvus species) as well as the deaths of several exotic birds at a zoological park in the same area. Complete genome sequencing of a flavivirus isolated from the brain of a dead Chilean flamingo (Phoenicopterus chilensis), together with partial sequence analysis of envelope glycoprotein (E-glycoprotein) genes amplified from several other species including mosquitoes and two fatal human cases, revealed that West Nile (WN) virus circulated in natural transmission cycles and was responsible for the human disease. Antigenic mapping with E-glycoprotein-specific monoclonal antibodies and E-glycoprotein phylogenetic analysis confirmed these viruses as WN. This North American WN virus was most closely related to a WN virus isolated from a dead goose in Israel in 1998.
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Lineage 1 and 2 Strains of Encephalitic West Nile Virus, Central Europe
Tamás Bakonyi, Éva Ivanics, Károly Erdélyi … (2006)
Geographically, West Nile virus (WNV) is the most widespread member of the Japanese encephalitis virus (JEV) complex within the genus Flavivirus and the family Flaviviridae. The first strain (B 956) was isolated from a human patient in the West Nile district of Uganda in 1937; later the virus was also detected in several mosquito species, horses, humans, and other hosts in Africa, Europe, Asia, and Australia (where it has been named Kunjin virus) ( 1 – 3 ). WNV was introduced into the United States in 1999, and it spread quickly over large parts of North America and reached Mexico ( 4 – 7 ). The clinical impact of WNV varies in different regions. In the Old World, WNV causes relatively mild infections with influenzalike symptoms or no apparent disease ( 2 ); encephalitis and fatalities in the human population, horses, or poultry are spasmodic ( 3 , 8 , 9 ). In the New World, WNV exhibits increased virulence among the local wild bird populations and causes more frequent severe central nervous system symptoms and deaths in humans and horses ( 6 , 10 ). Although exactly how WNV was introduced into New York is unclear, phylogenetic comparison of the viral nucleic acid sequences has shown a close relationship between the American WNV isolates and strains isolated from encephalitic geese and storks in Israel in 1998 ( 11 – 13 ). Experimental infections of rodents indicated that the neurovirulence of WNV correlates with its genotype, and the North American strains are highly neurovirulent for mice ( 14 ). WNV shows relatively high levels of sequence diversity. Comprehensive studies on the phylogenetic relatedness of WNV strains show that they form at least 2 main lineages ( 15 – 17 ). Lineage 1 is composed of WNV strains from different geographic regions, and it is subdivided into at least 3 clades. Clade A contains strains from Europe, Africa, the Middle East, and America; clade B represents the Australian (Kunjin) strains; and clade C contains Indian WNV isolates. Lineage 2 contains the B 956 prototype strain and other strains isolated so far exclusively in sub-Saharan Africa and Madagascar. In addition to the 2 major WNV lineages, we recently proposed 2 lineages for viruses that exhibited considerable genetic differences to the known WNV lineages: lineage 3 consists of a virus strain isolated from Culex pipiens mosquitoes at the Czech Republic/Austria border (named Rabensburg virus), and lineage 4 consists of a unique virus isolated in the Caucasus. These 2 viruses, however, may also be considered independent flaviviruses within the JEV complex ( 18 ). WNV has been known to be present in central Europe for a long time. Seroprevalence in humans was reported in several countries, including Hungary, and WNV strains were isolated from mosquitoes, humans, migrating birds, and rodents during the last 30 years ( 3 ). Until 2003, however, WNV infections in Hungary have never been associated with clinical symptoms, although a severe outbreak of West Nile encephalitis in humans was reported in 1996 and 1997 in neighboring Romania. In late summer 2003, an outbreak of encephalitis emerged in a Hungarian goose flock, resulting in a 14% death rate among 6-week-old geese (Anser anser domesticus). Based on histopathologic alterations, serologic investigations, and nucleic acid detection by reverse transcription–polymerase chain reaction (RT-PCR), WNV was diagnosed as the cause of the disease ( 19 ). Chronologically and geographically related to the outbreak in geese, a serologically confirmed WNV outbreak was also observed in humans, which involved 14 cases of mild encephalitis and meningitis ( 20 ). One year later, in August 2004, a goshawk (Accipiter gentilis) fledgling showed central nervous system symptoms and died in a national park in southeastern Hungary. When histopathologic methods and RT-PCR were used, WNV antigen and nucleic acid were detected in the organs of the bird. Furthermore, the virus was isolated after injection of suckling mice. Here we report the sequencing and phylogenetic results of these 2 encephalitic WNV strains that emerged recently in central Europe. Materials and Methods Brain specimens from one 6-week-old goose, which died during the encephalitis outbreak in a Hungarian goose flock, and brain samples from a goshawk, which also died from encephalitis, were used for WNV nucleic acid determination. The brain samples were homogenized in ceramic mortars by using sterile quartz sand, and the homogenates were suspended in RNase-free distilled water. Samples were stored at –80°C until nucleic acid extraction was performed. Viral RNA was extracted from 140 μL of brain homogenates by using the QIAamp viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. First, a universal JEV-group specific oligonucleotide primer pair designed on the nonstructural protein 5 (NS5) and 3´-untranslated regions (UTR) of WNV (forward primer: 5´-GARTGGATGACVACRGAAGACATGCT-3´ and reverse primer: 5´-GGGGTCTCCTCTAACCTCTAGTCCTT-3´ [21]; ) was applied on the RNA extracts in a continuous RT-PCR system employing the QIAGEN OneStep RT-PCR Kit (Qiagen). Each 25-μL reaction mixture contained 5 μL of 5× buffer (final MgCl2 concentration 2.5 mmol/L), 0.4 mmol/L of each deoxynucleoside triphosphate, 10 U RNasin RNase Inhibitor (Promega, Madison, WI, USA), 20 pmol of the genomic and reverse primers, 1 μL enzyme mix (containing Omniscript and Sensiscript Reverse Transcriptases and HotStarTaq DNA polymerase) and 2.5 μL template RNA. Reverse transcription was carried out at 50°C for 30 min, followed by a denaturation step at 95°C for 15 min. Thereafter, the cDNA was amplified in 40 cycles of heat denaturation at 94°C for 40 s, primer annealing at 57°C for 50 s, and DNA extension at 72°C for 1 min, and the reaction was completed by a final extension for 7 min at 72°C. Reactions were performed in a Perkin-Elmer GeneAmp PCR System 2400 thermocycler (Wellesley, MA, USA) and in a Hybaid PCR Sprint thermocycler (Thermo Electron Corporation, Waltham, MA, USA). After RT-PCR, 10 μL of the amplicons was subjected to electrophoresis in a 1.2% Tris acetate-EDTA-agarose gel at 5 V/cm for 80 min. The gel was stained with ethidium bromide; bands were visualized under UV light and photographed with a Kodak DS Electrophoresis Documentation and Analysis System using the Kodak Digital Science 1D software program (Eastman Kodak Company, Rochester, NY, USA). Product sizes were determined with reference to a 100-bp DNA ladder (Promega). Where clear PCR products of the previously calculated sizes were observed, the fragments were excised from the gel, and DNA was extracted by using the QIAquick Gel Extraction Kit (Qiagen). Fluorescence-based direct sequencing was performed in both directions on PCR products. Sequencing of PCR products was carried out with the ABI Prism Big Dye Terminator cycle sequencing ready reaction kit (Perkin-Elmer), according to the manufacturer's instructions, and an ABI Prism 310 genetic analyzer (Perkin-Elmer) automated sequencing system. Nucleotide sequences were identified by Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/blast) search against gene bank databases. Based on the sequence information obtained from the amplification products, complete WNV sequences that exhibited the highest nucleotide identities with the Hungarian genotypes were selected from the GenBank database to design primers that amplify overlapping RT-PCR products covering the entire genome of the strains. Oligonucleotide primers were designed with the help of the Primer Designer 4 for Windows 95 (Scientific and Educational Software, Version 4.10; Microsoft, Redmond, WA, USA) and were synthesized by GibcoBRL Life Technologies, Ltd. (Paisley, Scotland, UK). Detailed information on all primers is in the Tables A1 and A2. PCR amplification products were directly sequenced in both directions; the sequences were compiled and aligned to complete genome sequences of selected representatives of WNV lineages 1a, 1b, 2, and putative lineages 3 and 4 (listed in Table). Phylogenetic analysis was performed by using the modified neighbor-joining method (ClustalX [22]; ), and trees were constructed to demonstrate the relationship between the Hungarian WNVs and other WNV strains (Figure). Figure Phylogenetic tree based on the complete nucleotide sequences of selected West Nile virus strains demonstrating the genetic relatedness of these strains (abbreviations are listed in Table). Boxes indicate different lineages and clades. The Hungarian strains reported in this article are highlighted with gray background). RabV, Rabensburg virus; JEV, Japanese encephalitis virus. Scale bar depicts degree of relatedness. Table West Nile virus strains included in the phylogenetic analysis Name Code Accession no. Isolation Year Host Origin Lineage, clade WNV HNY1999 NY99a AF202541 1999 Human New York, USA 1a WNV NY99flamingo38299 NY99b AF196835 1999 Flamingo New York, USA 1a WNV IS98STD Is98 AF481864 1998 Stork Israel 1a WNV goose-Hungary/03 Hu03 DQ118127 2003 Goose Hungary 1a WNV Italy1998Equine It98 AF404757 1998 Horse Italy 1a WNV RO9750 Ro96 AF260969 1996 Culex pipiens Romania 1a WNV VLG4 Rus99a AF317203 1999 Human Volgograd, Russia 1a WNV LEIV-Vlg99-27889 Rus99b AY277252 1999 Human Volgograd, Russia 1a WNV PaH001 Tu97 AY268133 1997 Human Tunisia 1a WNV PaAn001 Fr00 AY268132 2000 Horse France 1a WNV Eg 101 Eg51 AF260968 1951 Human Egypt 1a WNV Chin-01 Chin01 AY490240 1950s ? Russia 1a WNV Kunjin MRM61C Kunjin D00246 1960 Cx. annulirostris Australia 1b WNV Sarafend Sarafend AY688948 Laboratory strain 2 WNV B956 (WNFCG) Ug37 NC_001563 1937 Human Uganda 2 WNV goshawk-Hungary/04 Hu04 DQ116961 2004 Goshawk Hungary 2 Rabensburg virus (97-103) RabV AY765264 1997 Cx. pipiens Czech R. 3? WNV LEIV-Krnd88-190 Rus98 AY277251 1998 Dermacentor marginatus Caucasus, Russia (Georgia?) 4? The nucleotide sequences of the Hungarian WNV strains goose-Hungary/03 (Hu03) and goshawk-Hungary/04 (Hu04) were submitted to the GenBank database. They are available under accession numbers DQ118127 and DQ116961, respectively. Results In this study, the complete genome sequences of WNV strains derived from a 6-week-old goose, which died in 2003 during an outbreak of encephalitis in a Hungarian goose flock (strain goose-Hungary/03), and from a goshawk, which also died from encephalitis in the same region 1 year later (strain goshawk-Hungary/04), were determined, aligned, and phylogenetically analyzed. The genome of the goose-Hungary/03 strain is composed of 10,969 nucleotides (nt) and contains 1 open reading frame between nucleotide positions 97 and 10,398, coding for a 3,433 amino acid (aa)–long putative polyprotein precursor. The complete genomic sequence of the virus was subjected to a BLAST search against gene bank databases. The highest identity rates (98% at the nucleotide and 99% at the amino acid level) were found with WNV strains isolated in 1998 in Israel and in 1999 in the United States. In addition, phylogenetic analysis was performed to indicate the relationships between the Hungarian goose–derived WNV strain and selected representatives of WNV clades and clusters. The resulting phylogenetic tree (Figure) confirmed the results of the BLAST search, i.e., the Hungarian goose–derived WNV strain is clustering close to the previously mentioned WNV strains isolated in the United States and Israel, which belong to lineage 1a of WNV. Other European WNV strains (isolated in Italy, France, and Romania) are more distant to the Hungarian strain; they form a separate cluster consisting of a Romanian/Russian and a French/Italian subcluster. The complete nucleotide sequence of the goshawk-Hungary/04 WNV strain is composed of 11,028 nt and contains 1 open reading frame between nucleotide positions 97 and 10,401, coding for a 3,434-aa putative polyprotein precursor. In BLAST search, the strain showed the highest (96% nt and 99% aa) identity to the WNV prototype strain B 956. Consequently, as the phylogram also indicates (Figure), this virus belongs to lineage 2 of WNV. Alignments of the available partial sequences from the E protein coding regions of other representatives of this cluster showed even higher identities (97%–98% nt and 100% aa) with WNV strains isolated in central Africa in 1972 (AnB3507, AF001563) and in 1983 (HB83P55, AF001557), respectively ( 15 ). More recently (in early August 2005), additional lethal cases of encephalitis occurred in birds of prey in the same place in which the goshawk died of West Nile encephalitis in 2004, involving up to a total of 3 goshawks and 2 sparrow hawks (A. nisus); 2 of the goshawks and 1 sparrow hawk died. Preliminary investigations detected WNV-specific nucleic acid in the brains of the birds. The partial nucleotide sequence of the 2005 virus (1,000 bp at the NS5´–3´-UTR regions) showed 99.9% identity with the goshawk-Hungary/04 strain (only 1 substitution at nucleotide position 9,376 [g→a] has been observed, which did not influence the putative amino acid sequence). Additional observation of the outbreak and investigations of the cases are in progress. Discussion The primary aim of our investigations was to show the genetic relatedness of the WNV strains detected in Hungary in the last 2 years and to estimate their clinical and epidemiologic impact. The phylogenetic analysis emphasizes the close genetic relationship of the goose-Hungary/03 strain with a WNV strain isolated in Israel in 1998 and the WNV strain introduced in New York in 1999, since the 3 WNVs form 1 single cluster within clade 1a of lineage 1. These strains caused outbreaks in birds, humans, and horses. Previous European WNV isolates exhibited lower identity values, e.g., the strain that was responsible for the Romanian outbreak(s) in 1996 and 1997 showed only 96% nt identity with the Hungarian goose-2003 strain, and in the phylogenetic tree the other European isolates form a separate cluster consisting of 2 subclusters (Figure). The earliest representatives of the Israel/USA/goose-Hungary/03 cluster were reported by Malkinson et al. ( 23 ) from ill and dead white storks (Ciconia ciconia) in Israel in 1998. These storks, however, had hatched in central Europe, and during their autumn migration southwards, strong winds had blown them off course, from their usual route to Africa, to southern Israel. Malkinson et al. suspected that these birds introduced the neurovirulent genotype of WNV to Israel from their hatching place. The wetlands of southeastern Hungary are foraging and nesting habitats for storks and many other wild bird species, and the goose farm, where the WNV outbreak occurred in 2003, is located in this region. These facts, together with the close phylogenetic relatedness of the Israeli/US/Hungarian WNV strains, strongly support the theory that storks carried the neurovirulent WNV strain from central Europe (that is, from Hungary) to Israel, which sheds new light on the introduction of WNV to New York. This virus could have originated in Israel (which is the generally accepted although not proven theory) or central Europe. In both cases, however, the virus seems to have its true origin in Europe. In a recent publication, Lvov et al. suggested that WNV could have been introduced into New York by ships traveling from Black Sea ports ( 24 ). When a WNV infection was detected in 2004 in a goshawk fledgling, which died from encephalitis in the same region of Hungary in which the outbreak in geese and humans occurred during the previous year, we anticipated a WNV strain more or less identical to the genotype detected there in 2003. The genomic sequence of this strain was not closely related to the sequence of the WNV strain detected in geese in the year before, however, but belonged to the group of central African lineage 2 WNV strains. A closely related strain from this cluster (ArB3573, AF001565, and AF458349) was identified as a neuroinvasive strain of WNV in a mouse model ( 14 ). To our knowledge, this report is the first on the emergence of a lineage 2 WNV strain outside Africa. Migratory birds that had overwintered in central Africa probably introduced this exotic strain to the wetlands of Hungary. On the other hand, as the goshawk is not a migratory species, and infection occurred in August, the African WNV strain must have already successfully adapted to local mosquito vectors. Consequently, this neurotropic, exotic WNV strain may become a resident pathogen in Europe with all the possible public health consequences. Our results indicate that the WNV strains that emerged in 2 consecutive years and caused avian deaths in Hungary are epidemiologically unrelated. Genetically distinct WNV strains are circulating simultaneously yet independently in local birds and thus most likely also in local mosquito populations within the same region. They cause sporadic cases of encephalitis and also raise the possibility of spreading to other European countries or even to other continents, as happened in 1999 with another WNV strain, which resulted in a public health catastrophe in America. In addition to the above 2 novel WNVs, we recently characterized another novel flavivirus of so far unknown human pathogenicity named Rabensburg virus, which has been isolated from Culex pipiens mosquitoes in 1997 and 1999 at the Czech Republic–Austria border, only a few hundred kilometers from the region where the Hungarian WNVs emerged. After the entire genome was sequenced, Rabensburg virus turned out to represent either a new (third) lineage of WNV or a novel flavivirus of the JEV group ( 18 ). Thus, several distinct WNV strains seem to circulate in central Europe. In 2001 another flavivirus of the JEV group, Usutu virus, which has never previously been observed outside Africa, emerged in Austria and resulted in deaths in several species of birds, especially Eurasian blackbirds (Turdus merula) ( 21 ). This virus became a resident pathogen in Austria and continues to disperse and cause deaths in blackbirds and other species of birds ( 25 , 26 ). The snowy winter and rainy spring of 2005 resulted in serious floods in the area in which the Hungarian WNV strains were identified. Since the floodplains and polders were under water, the conditions for mosquito development were ideal. The summer was also very rainy, which resulted in more floods in the region and continuous mosquito gradation. The most recent data imply that the lineage 2 WNV strain may have overwintered in Hungary, causing several clinical cases of encephalitis in Accipiter species in 2005 as well. The routine diagnostic techniques in most of the European public health and veterinary laboratories are designed to detect lineage 1 WNV strains. In a recent PCR external quality assurance multicenter test, <40% of the involved laboratories could detect lineage 2 WNV strains (Matthias Niedrig, pers. comm.). Therefore, a major goal of this article is to increase the scientific and public awareness of this potential public health threat for Europe and, perhaps, America. Furthermore, comprehensive investigations on the occurrence, ecology, and epidemiology of the different WNV strains circulating in central Europe, as well as the development of monitoring and surveillance programs, must be of highest priority. One may also speculate on environmental factors, such as climate change or global warming, that may have enhanced the recent emergence of viruses, which had previously been restricted to Africa, in new habitats and continents. Improved observation, reporting, and detection methods have also contributed to the apparent increasing emergence of these viruses.
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Virology, Pathology, and Clinical Manifestations of West Nile Virus Disease
Edward B. Hayes, James J. Sejvar, Sherif R. Zaki … (2005)
The impressive spread of West Nile virus (WNV) in the Western Hemisphere after its detection in 1999 during an outbreak of encephalitis in New York City has caused >16,000 human disease cases and >660 deaths in North America. Research on the signs, symptoms, and pathogenesis of WNV disease has greatly intensified in the past 5 years. The number of recognized cases of flaccid paralysis due to WNV infection has increased substantially, and research into prognosis and possible therapy has expanded. Genetic variation of the virus has been further characterized and continues to be explored. The pathology and pathogenesis of WNV disease have been described more completely than ever before. Several strategies are being pursued to develop effective vaccines to prevent WNV disease. This article highlights new information about the virology, clinical manifestations, laboratory diagnosis, pathology, and prognosis of WNV illness in humans. The expanded knowledge about WNV disease provides a new platform for future development of diagnostic tests, therapy, and vaccine development. Characteristics of West Nile Virus WNV is an arbovirus in the family Flaviridae. Its spherical, enveloped capsid has a diameter of ≈50 nm and contains single-stranded RNA that encodes the capsid (C), envelope (E), and premembrane (prM) proteins, as well as 7 nonstructural proteins that likely contribute to viral replication. The virus has 2 genetic lineages: lineage 1 strains are found in North America, Europe, Africa, Asia, and Australia; lineage 2 strains have been isolated only in sub-Saharan Africa and Madagascar. Lineage 1 strains have been further divided into 4 clades: Kunjin, Indian, A, and B (which includes an Indian isolate) (1). The isolates in clade B, which includes strains from the United States, are all virulent in mice; lineage 2 and other clades in lineage 1 comprise both virulent and attenuated strains (1). Differences in pathogenicity may be related to nucleotides that code for specific regions in the prM, E, or nonstructural proteins of the virus (1,2). WNV strains from the United States are closely related to strains from Israel, with 99.7% homology in nucleotide sequences, indicating that the strains in the United States almost certainly originated from the Middle East (3). The strain isolated in New York in 1999 is more virulent in American crows (Corvus brachyrynchos) than strains from Kenya and Australia (Kunjin virus, a subtype of WNV), and both the New York strain and the Kenyan strain experimentally killed house sparrows whereas the Australian strain did not (4). Two genetic variants of the North American WNV strain were isolated in Texas in 2002; the major variant differed from the New York 1999 isolate by 0.18% of nucleotides, and the minor variant by 0.35% (1). The 2 variants differed from each other by 0.5% of nucleotides, and their neuroinvasiveness in mice was similar to that of the New York 1999 isolate. In 2003, attenuated WNV strains were found in birds in Texas and Mexico, providing the first evidence of phenotypic variation of WNV strains in the Western Hemisphere (2,5). The reduced neuroinvasiveness and smaller plaque size of the Texas strains may be due to mutations in nonstructural proteins that result in lower levels of viremia; the attenuated strain from Mexico had a mutation in the E protein (2,5). Pathogenesis WNV is thought to replicate at the site of inoculation and then spread to lymph nodes and the bloodstream (6). Viral penetration of the central nervous system appears to follow stimulation of toll-like receptors and increased levels of tumor necrosis factor-α, which increases permeability of the blood-brain barrier (7). WNV directly infects neurons, particularly in deep nuclei and gray matter of the brain, brainstem, and spinal cord (8–10). Collateral destruction of bystander nerve cells may contribute to paralysis (11). Immune-mediated tissue damage may also contribute to pathologic changes in some cases (12). Genetic susceptibility for severe disease in mice has been postulated to involve a deficiency in production of 2´–5´-oligoadenylate synthetase, but this genetic susceptibility has not been elucidated in humans (10). Although most nonfatal WNV infections appear to be cleared by the host immune response, the virus may persist in some vertebrate hosts (10,13). Clinical Manifestations The clinical spectrum of symptomatic WNV infection in humans has been further defined during the North American epidemics. About 80% of human infections are apparently asymptomatic (14). Of those persons in whom symptoms develop, most have self-limited West Nile fever (WNF), characterized by the acute onset of fever, headache, fatigue, malaise, muscle pain, and weakness; gastrointestinal symptoms and a transient macular rash on the trunk and extremities are sometimes reported (15,16). A recent follow-up study of WNF patients who sought medical attention found that difficulty concentrating and neck pain or stiffness were also prominent symptoms, and that fatigue and muscle weakness frequently lasted for ≈1 month after onset (16). Of the 98 patients interviewed, 31% were hospitalized, 79% missed school or work because of their illness, and the median time before patients felt fully recovered was 60 days. These patients probably represent the most severe WNF, but even without neurologic manifestations, WNV infection clearly can cause a notable public health problem, Additional nonneurologic clinical manifestations that may rarely occur during WNV infection include hepatitis, pancreatitis, myocarditis, rhabdomyolysis, orchitis, and ocular manifestations (17–24). Chorioretinitis may be more common than previously thought; a study in Tunisia found that 69% of 29 patients hospitalized with WNV disease had chorioretinitis (24). Cardiac dysrhythmias have been observed in some North American patients (Centers for Disease Control and Prevention [CDC], unpub. data) (22). Neuroinvasive disease develops in 4-fold higher than titers to other epidemiologically relevant flaviviruses included in the assay. However, PRNT may not discriminate between WNV infection and other flaviviral infections in patients with previous flavivirus exposure, because the neutralizing antibody in such cases may broadly cross-react to several related flaviviruses. WNV infection can also be diagnosed by detecting virus in CSF, serum, or tissues by isolation or nucleic acid amplification tests (NATs). WNV is best isolated in cell culture or suckling mice and identified by indirect immunofluorescence assay with specific monoclonal antibodies or by reverse transcriptase–polymerase chain reaction (RT-PCR). However, WNV is rarely isolated from the blood of patients with neuroinvasive WNV disease because viremia levels are typically low or absent by the time neurologic symptoms develop. Real-time RT-PCR and nucleic acid sequence-based amplification are the most sensitive NATs, able to detect ≥50 viral RNA copies per mL (≈0.1 PFU/mL), which is ≈1,000-fold more sensitive than culture (39). WNV can be detected in serum by NAT if the specimen is obtained early in infection and is readily detected by NAT, isolation, or IHC staining in brain tissue from persons with fatal cases. The sensitivity of RT-PCR among 28 patients with serologically confirmed neuroinvasive WNV disease was 57% in CSF and 14% in serum (40). The diagnosis of WNV encephalitis can be supported histopathologically, and there is no pathognomonic lesion. Differential diagnoses include arboviral and other viral encephalitides, rickettsial infections, and various noninfectious diseases. When serum samples and frozen tissues are not available, IHC testing of formalin-fixed tissues with specific monoclonal and polyclonal antibodies is particularly useful. Prognosis The clinical course of WNF ranges from a mild febrile illness of several days' duration to debilitating fatigue, aching, and weakness that may last for weeks or months (16,29,41). Although cases of meningitis without alteration of the patient's mental status or other focal neurologic features have a favorable prognosis, persistent headaches and fatigue may be reported (29). Patients with WNV encephalitis or focal neurologic manifestations often have persistent neurologic deficits for months or years (28,29). Of 35 patients hospitalized with WNV disease in New York, only 13 (37%) reported full recovery in physical, cognitive, and functional abilities 12 months after illness onset (41). Many patients with WNV-associated poliomyelitislike syndrome do not recover, but some improvement in limb strength may occur over time (42,43). The overall case-fatality rate for neuroinvasive WNV disease is ≈9% (26). Clinical Management Management of severe WNV illness remains supportive. Patients with severe meningeal symptoms often require pain control for headaches and antiemetic therapy and rehydration for associated nausea and vomiting. Patients with severe encephalitis should be observed for development of elevated intracranial pressure and seizures, and patients with encephalitis or paralysis must be monitored for inability to protect the airway. Acute neuromuscular respiratory failure may develop rapidly, particularly in patients with prominent bulbar signs; prolonged ventilatory support may be required (22,30,34). Ribavirin, interferon-α, WNV-specific immunoglobulin, and antisense gene–targeted compounds have all been considered as specific treatments for WNV disease, but no rigorously conducted clinical trials have been completed. Nonspecific immunoglobulin and plasmapheresis should be considered for patients with Guillain-Barré syndrome but are not indicated for patients with paralysis due to damage of anterior horn cells (30). Vaccine Development Two vaccines are available for vaccinating equines: an inactivated WNV vaccine and a recombinant vaccine that uses canarypox virus to express WNV antigens (44,45). An inactivated vaccine is also being studied for use in humans (46). A chimeric live virus vaccine incorporating the genetic sequences for E and prM antigens into a 17-D yellow fever virus backbone has been shown to be efficacious in hamsters and is undergoing initial clinical trials in humans (46). Another chimeric vaccine incorporating WNV genetic sequences into a backbone of attenuated serotype-4 dengue virus–induced protective immunity in monkeys (44). A DNA vaccine that elicits expression of WNV E and prM antigens has been used in mice, horses, and birds (44). Vaccination of crows with Kunjin virus, a subtype of WNV, protected against WNV, and a DNA vector, which elicited expression of attenuated Kunjin virus, provided protective immunity against WNV in mice (46). Future Directions Since the 1990s, WNV has gained notoriety as a cause of severe neuroinvasive disease in humans. As WNV isolates and genetic sequences accumulate over an increasing geographic and clinical range, the virus shows signs of genetic modifications that likely interact with host factors in causing different patterns of neuroinvasiveness and neurovirulence. Several areas warrant research focus over the next few years. More efficient diagnostic assays will help with both clinical diagnosis and disease surveillance. Improved knowledge about the pathogenesis and natural history of WNV disease is crucial to developing effective treatment, and promising therapies need to be carefully evaluated in controlled clinical trials. Given the focal distribution of WNV outbreaks, and the uncertain distribution of future cases of WNV disease, prospective clinical studies need to be designed with the flexibility to gather information from widely dispersed and changing locations. The development of a safe and effective vaccine for humans is a clear priority for prevention, and the public health strategies and recommendations for vaccination deserve careful thought. Given the relatively low incidence of WNV neuroinvasive disease and the focal occurrence of WNV epidemics thus far, vaccination will likely require targeting to higher risk groups to approach the cost-effectiveness of many recommended public health prevention strategies.
Author and article information
Journal
Journal ID (nlm-ta): Emerg Infect Dis
Journal ID (publisher-id): EID
Title:
Emerging Infectious Diseases
Publisher:
Centers for Disease Control and Prevention
ISSN
(Print):
1080-6040
ISSN
(Electronic):
1080-6059
Publication date
(Print):
June
2009
Volume: 15
Issue: 6
Pages: 877-884
Affiliations
[1]University of Pretoria, Pretoria, South Africa (M. Venter, S. Human, D. Zaayman, J.
Williams, J. Steyl, C. Donnellan)
Author notes
Address for correspondence: Marietjie Venter, Department of Medical Virology, Faculty
of Health Sciences, University of Pretoria/NHLS Tswhane Academic Division, PO Box
2034, Pretoria 0001, South Africa; email:
marietjie.venter@
123456up.ac.za
Article
Publisher ID: 08-1515DOI: 10.3201/eid1506.081515
PMC ID: 2727306
PubMed ID: 19523285
SO-VID: 3e5ddb89-2115-4c10-af9c-1ca8a47405c1
History
Categories
Subject:
Research
ScienceOpen disciplines: Infectious disease & Microbiology