Introductions of West Nile Virus Strains to Mexico.
West Nile virus (WNV), a mosquitoborne flavivirus for which birds serve as reservoir and amplification hosts, was introduced into New York in 1999 (1) and spread across the United States to California by 2003 (2). By 2002, serosurveys demonstrated WNV circulation in [greater than or equal to] 6 eastern Mexican states and along its northern border with the United States (3-5). This pattern of WNV appearance in Mexico suggested a southwesterly spread across the United States and into northeastern Mexico through Texas. However, in the spring of 2003, the first WNV isolate found in Mexico was obtained from a raven in the southeastern state of Tabasco (3). If WNV reached southern Mexico by incremental spread through northern Mexico from Texas, the index isolate would have been expected sooner and in a northern Mexican state. Phylogenetic analyses showed the raven isolate to be unexpectedly divergent from contemporary Texas strains, but exact relationships and a route of entry could not be determined by using premembrane and envelope glycoprotein (prM-E) sequences (3).
The divergence between the southern Mexican raven and Texas isolates suggested that WNV arrived in southern Mexico by an alternate route, perhaps the Caribbean. After its spread throughout the northeastern United States, WNV appeared abruptly in Florida in 2001, appearing to bypass several mid-Atlantic states. This pattern could be explained by spread of migratory birds (6); the Atlantic coast flyway overlaps both New York and Florida, while the Mississippi Valley flyway overlaps both Louisiana and the Yucatan Peninsula of Mexico. Longitudinal avian serosurveys that began in 2000 showed WNV seropositivity in at least 3 migratory and 2 resident bird species captured in the Yucatan Peninsula from 2002 to 2003 (7). Thus, migratory birds may have carried WNV from the southeastern United States into Mexico, either directly or through the Caribbean. Serosurveys have suggested WNV circulation among birds in various Caribbean islands since 2002 (8-10).
The possibility of a third WNV introduction into Mexico at the California border must also be considered. A 2003 horse isolate from the northern Mexican state of Nuevo Leon was closely related to Texas isolates from 2002 (11), based on its prM-E sequence. We do not know whether WNV reached California from Texas and the Midwest by crossing the Rocky Mountains or by traveling first into northern Mexico and subsequently spreading north from Baja California. The latter route is suggested by the geographic link with the first detection of WNV in southeastern California (2).
The reported incidence of human West Nile encephalitis is much greater on the US (California) than on the Mexican (Baja California and Sonora) side of the common border. Possible explanations for this discrepancy include differences in disease surveillance and reporting. Another possibility is that the WNV strains circulating in Mexico are attenuated compared to US strains, and the identification of a murine-attenuated glycosylation variant in Tabasco State (12) is consistent with this hypothesis.
To investigate possible routes of WNV entry into Mexico from the United States, 9 isolates from Mexico (all strains available) and 13 strains isolated in the United States from hypothetical points of introduction into Mexico (2 from Florida, 2 from Louisiana, 3 from Arizona, and 6 from California) were compared. Isolates from several northern Mexican states, 1 from Sonora, 1 from Tamaulipas, and 7 from Baja California, were obtained from a variety of birds and from a horse (Table, Figure 1) by injection of Vero cells. RNA was extracted from first or second Vero cell passages by using the QIAamp Viral RNA Mini-kit (Qiagen Inc, Valencia, CA, USA). Reverse transcription-polymerase chain reactions (RT-PCRs) were performed to amplify the complete WNV genome in 6 overlapping amplicons by using primers described previously (12). Amplicons were purified from agarose gels by using the QIAquick gel-extraction kit (Qiagen), and both strands were sequenced directly by using the PCR primers and the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied BioSystems, Foster City, CA, USA) with a 3100 Genetic Analyzer (Applied Biosystems).
[FIGURE 1 OMITTED]
Complete genomic sequences excluding the 5' and 3' terminal 20 nucleotides (representing primers incorporated into amplicons) were aligned with all homologous WNV sequences from the GenBank library by using ClustalW. Sequences were analyzed by using maximum parsimony and neighbor-joining programs implemented in the PAUP 4.0 software package (13) as well as Bayesian analysis using MRBAYES v3.0 (14) with 100,000 generations, a general time-reversible model with empirically estimated base frequencies, and either a codon position-specific (for the open reading frame alone) or a gamma distribution of substitution rates among nucleotide sites.
All phylogenetic trees placed the North American WNV isolates into monophyletic groups with strong bootstrap and Bayesian support values; the tree generated using the Bayesian analyses is presented in Figure 2. The Tabasco 2003 isolate grouped with 3 strains from 2001 and 2002 in Florida and Louisiana and more distantly with a New York isolate from 2000, with strong Bayesian probability and bootstrap support; inclusion of the New York grouse strain was weakly supported (bootstrap and Bayesian probability values <80%). In contrast, a 2004 Louisiana isolate and other recent strains from Texas were positioned basally to the large clade containing all northern Mexico, California, and Arizona isolates. This California/Arizona/northern Mexico group was highly conserved, with <0.5% nucleotide and 0.04% amino acid sequence divergence. The 2003 Tabasco strain was phylogenetically distinct from all other Mexico isolates, which grouped with the California and Arizona isolates. Surprisingly, despite the greater geographic distances between Tamaulipas and Baja California/Sonora, compared to the distance between Tamaulipas and Texas, the Tamaulipas WNV strains grouped more closely with strains from Baja California and Sonora than with those from Texas.
[FIGURE 2 OMITTED]
Compared to the Tabasco strain, the other Mexican isolates differed by 0.55%-0.66% nucleotide sequence divergence across the genome. The gene with the most sequence divergence was prM, with 0.72% 1.4% divergence from the Tabasco strain. However, the 5' untranslated region was more variable with 3.0%4.6% divergence. The most conserved gene was NS2B, with 0.0%-0.24% divergence from the Tabasco strain. The E gene, often used for phylogenetic analyses, had 0.46%-0.66% sequence divergence.
Of the Mexican WNV isolates, only the 2003 Tabasco raven isolate had the E-156 Pro residue, which ablates the N-linked glycosylation site found in most North American strains. In addition, 2 other WNV isolates (GenBank accession nos. AY490240 and AF260968) share this E-156 Pro residue despite their geographic diversity (China and Egypt, respectively) and their placement in different lineages. Although the paraphyletic nature of this Pro mutation suggests that it could be selected either during laboratory isolation or passage, its identification in the low-passage Tabasco isolate may indicate its presence in nature.
Our data support the hypothesis that WNV entered Mexico through at least 2 independent introductions. The introduction detected by the first virus isolation in May 2003 from a raven in Tabasco State probably occurred from a migratory bird that flew southward from the southeastern United States to the Gulf of Mexico or the Caribbean and bypassed northern Mexico. The isolation and sequencing of WNV isolates from islands in the Caribbean may shed further light on how WNV reached southern Mexico. However, the extreme genetic conservation of North American WNV strains may preclude identifying the exact routes of introduction. Independently, other WNV strains probably spread incrementally from the southwestern United States into northern Mexico. Both northward and southward movements of WNV between northern Mexico and California or Arizona may also occur.
Available WNV strains from Mexico indicate that the murine-attenuated, E-156 glycosylation-negative variant identified in Tabasco state may be limited in its distribution to southern Mexico, while the glycosylated variant typical of US strains is widespread in northern Mexico. However, our sampling was limited and may also be biased because many WNV isolates were from sick or dying animals; the attenuated E-156 Pro residue phenotype could be undersampled because relatively benign infections are rarely identified.
The epidemiology of WNV-associated disease in Mexico is puzzling. According to the Centers for Disease Control and Prevention, 2,470 human cases of WNV infection were confirmed during 2004 in the United States, with >80% of these from areas of California and Arizona bordering the northern Mexico states of Baja California and Sonora where many of our viral isolations were made. In contrast, only 7 human cases of WNV have been confirmed in Mexico. The cases occurred in the border states of Chihuahua (n = 4), Sonora (n = 1), and Nuevo Leon (n = 1) in 2003, and Sonora (n = 1) in 2004 (15). Our results of extremely low sequence divergence between the southwestern United States and northern Mexican WNV isolates indicate that this epidemiologic discrepancy is unlikely to be explained by genetic and phenotypic differences among WNV strains. The possibility that WNV circulating in Mexico has an attenuated phenotype was suggested by the murine-attenuating mutation in the Tabasco raven isolate (12). However, none of our northern Mexico isolates have the E-156-P attenuating mutation, and all appear extremely closely related to isolates made in southwestern areas of the United States with a high disease incidence.
Another possible explanation for the low incidence of WNV disease in Mexico is resistance in the Mexican human population, possibly because cross-protective immunity from other flavivirus infections such as dengue and St. Louis encephalitis viruses. Although St. Louis encephalitis virus is common in some areas of the continental United States, including California, dengue virus infections are rare; only 157 cases of dengue were reported in the northern states of Mexico in 2004:25 in Sonora; 21 in Nuevo Leon; 88 in Tamaulipas; 3 in Coahuila; 0 in Chihuahua; and 0 in Baja California (http://www.dgepi. salud.gob.mx/boletin/2004/sem52). Of all Mexican states, Baja California and Sonora adjacent to the US border have the lowest incidence of flaviviral infections. Human flavivirus serosurveys should be conducted in northern Mexico to further evaluate the possibility of cross-protective flavivirus immunity. Newer approaches to detect and identify flaviviral disease are also needed in Mexico to more accurately assess the impact of WNV.
We thank Igor Romero and many veterinarians from CPA-SAGARPA for field support; Emily N. Green for technical assistance; and Ronald C. Cheshier, Lilian Stark, and Barbara Cahoon-Young for providing virus isolates.
This research was supported by contract NO 1-AI25489 from the National Institutes of Health and by the California Mosquito Research Program. E.D. was supported by the T01/CCT622892 CDC Fellowship Training Grant in Vector-Borne Infectious Diseases. C.T.D. was supported by NIH T32 training grant AI 7256 in Emerging and Tropical Infectious Diseases.
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Eleanor Deardorff, * Jose G. Estrada-Franco, * Aaron C. Brault, ([dagger]) Roberto Navarro-Lopez, ([double dagger]) Arturo Campomanes-Cortes, ([double dagger]) Pedro Paz-Ramirez, ([double dagger]) Mario Solis-Hernandez, ([double dagger]) Wanichaya N. Ramey, ([dagger]) C. Todd Davis, * David W.C. Beasley, * Robert B. Tesh, * Alan D.T. Barrett, * and Scott C. Weaver *
* University of Texas Medical Branch, Galveston, Texas, USA; ([dagger]) University of California, Davis, California, USA; and ([double dagger]) Comision Mexico-Estados Unidos para la Prevencion de la Fiebre Aftosa y Otras Enfermedades Exoticas de los Animales, Mexico City, Mexico
Address for correspondence: Scott C. Weaver, Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609, USA; fax: 409-747-2415; email: firstname.lastname@example.org
Ms Deardorff is a graduate student in the experimental pathology program at the University of Texas Medical Branch. Her research interests include arbovirology, zoonoses, viral ecology, and evolution.
Table. West Nile virus isolates included in the phylogenetic analyses * GenBank No. Strain Year Host AB185914 (NY) I 1999 Not reported AB185915 (NY) II 1999 Not reported AB185916 (NY) III 1999 Not reported AB185917 (NY) IV 1999 Not reported AF196835 NY '99 1999 Flamingo AF202541 NY 1999 Human AF206518 2741 1999 Culex pipiens AF260967 NY99-eqhs 1999 Equine AF260968 Eg101 1951 Human AF260969 RO97-50 1996 Cx. pipiens AF317203 VLG-4 1999 Human AF404753 crow265 2000 Crow AF404754 MQ5488 2000 Cx. Pipiens AF404755 grouse3282 2000 Grouse AF404756 crow3356 2000 Crow AF404757 Italy equine 1998 Equine AF481864 IS-98 STD 1998 Store AF533540 US Hum. 1 2001 Human AY185911 V1151 2002 Mosquito AY262283 Kenya3829 1998 Mosquito AY268132 PaAn001 2000 Equine AY268133 PaH001 1997 Human AY277252 27889 2003 Human AY278441 Ast99-901 1999 Human AY278442 VIg00 27924 2000 Human AY289214 TVP 8533 2002 Human AY490240 Chin-01 2003 Not reported AY660002 TM171-03 2003 Raven AY701412 96-111 1996 Equine AY701413 04.05 2003 Equine AY712945 Bird 1153 2003 Bird AY712946 Bird 1171 2003 Bird AY712947 Bird 1461 2003 Bird AY712948 v4369 2003 Mosquito AYS42931 385-99 1999 Not reported D00246 Kunjin MRM61C 1960 Culex spp. M12294 Uganda WNFCG 1937 Human D0080070 TVP 9115# 2003# Grackle# DQ080069# TVP 9117# 2003# Horse# DQ080068# TVP 9218# 2003# Blue heron# DQ080067# TVP 9219# 2003# Green heron# DQ080066# TVP 9220# 2003# Cormorant# DQ080065# TVP 9221# 2003# Grackle# DQ080064# TVP 9222# 2003# Coot# DQ080063# TVP 9223# 2003# Pigeon# DQ080060# Cc# 2004# Raven# DQ080072# FL232# 2001# Catbird# DQ080071# FL234# 2002# Equine# DQ080062# TWN 165# 2002# Mosquito# DQ080061# TWN 496# 2004# Northern cardinal# DQ080051# AZ-03-1623# 2003# Cx. tarsalis# DQ080052# Az-03-1681# 2003# Cx. tarsalis# DQ080053# Az-03-1799# 2003# Cx. tarsalis# DQ080054# CA-03 GRLA# 2003# Cx. quinquefasciatus# DQ080056# CA-03 IMPR 102# 2003# Cx. tarsalis# DQ080056# CA-03 IMPR 1075# 2003# Cx. tarsalis# DQ080057# CA-03SO333081# 2003# Crow# DQ080058# CA-03SO334814# 2003# Crow# DQ080059# CA-04 04-7168# 2003# Yellow-billed magpie# GenBank No. Location ([dagger]) AB185914 New York AB185915 New York AB185916 New York AB185917 New York AF196835 New York AF202541 New York AF206518 Connecticut AF260967 New York AF260968 Egypt AF260969 Romania AF317203 Russia AF404753 Maryland AF404754 New Jersey AF404755 New York AF404756 New York AF404757 Italy AF481864 Israel AF533540 New York AY185911 Texas AY262283 Kenya AY268132 France AY268133 Tunisia AY277252 Russia AY278441 Russia AY278442 Russia AY289214 Beaumont, Texas AY490240 China AY660002 Tabasco AY701412 Morocco AY701413 Morocco AY712945 Harris Co., Texas AY712946 Harris Co., Texas AY712947 Harris Co., Texas AY712948 Harris Co., Texas AYS42931 New York D00246 Australia M12294 Uganda D0080070 Sonora, Mexico# DQ080069# Tamaulipas# DQ080068# Baja California, Mexico# DQ080067# Baja California, Mexico# DQ080066# Baja California, Mexico# DQ080065# Baja California, Mexico# DQ080064# Baja California, Mexico# DQ080063# Baja California, Mexico# DQ080060# Baja California, Mexico# DQ080072# Palm Beach Co., Florida# DQ080071# Sumter Co., Florida# DQ080062# Iberia Co., Louisiana# DQ080061# Iberia Co., Louisiana# DQ080051# Cohise Co., Arizona# DQ080052# Maricopoa Co., Arizona# DQ080053# Apache Co., Arizona# DQ080054# Los Angeles, California# DQ080056# Imperial Valley, California# DQ080056# Imperial Valley, California# DQ080057# Arcadia, California# DQ080058# Arcadia, California# DQ080059# Sacramento, California# Note: Newly sequenced strains are indicated with #. * Newly sequenced strains are printed in bold text. ([dagger]) Locality and state (Mexico and United States) or country of isolation.
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|Author:||Weaver, Scott C.|
|Publication:||Emerging Infectious Diseases|
|Date:||Feb 1, 2006|
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