West Nile virus in Mexico: evidence of widespread circulation since July 2002.

West Nile virus (WNV) antibodies were detected in horses from five Mexican states, and WNV was isolated from a Common Raven in the state of Tabasco. Phylogenetic studies indicate that this isolate, the first from Mexico, is related to strains from the central United States but has a relatively high degree of sequence divergence.

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During the summer of 2002, the Agricultural Ministry of Mexico (SAGARPA) received reports of encephalitis-like illness in horses from several different areas of Mexico, concurrent with reports of West Nile virus (WNV) encephalitis outbreaks in horses along the Texas border in the states of Coahuila, Tamaulipas, and Chihuahua. Other suspected cases were reported from several southern, tropical states. We report the results of an equine serosurvey conducted from July 2002 to March 2003 by the Office of Exotic Diseases of the Agricultural Ministry (CPA-SAGARPA). We also describe the first isolation of WNV in Mexico, in a Common Raven (Corvus corax) from the state of Tabasco.

The Republic of Mexico is divided by the Tropic of Cancer, with temperate, arid climate zones in the north and at higher elevations and humid, subtropical, and tropical climate zones in the south. Our study encompassed most of these climatic zones, as equine serum samples were collected from 3 border states, 1 state on the Tropic of Cancer, and 10 states south of the Tropic of Cancer (Figure 1). Sampled equine populations were chosen on the basis of a history of clinical encephalitis; medical history was provided by owners and corroborated by CPA-SAGARPA veterinarians. In total, 441 serum samples were analyzed for WNV antibodies.

[FIGURE 1 OMITTED]

Because most serum samples were collected late in the probable virus transmission season, all were first screened for immunoglobulin (Ig) G antibodies, using IgG enzyme-linked immunosorbent assays (ELISA) with a recombinant, envelope protein domain III antigen expressed and purified from Escherichia coli (D.W.C. Beasley, et al., submitted for pub.). Positive samples were confirmed by hemagglutination inhibition (HI) tests against WNV and St. Louis encephalitis virus (SLEV), by 90% plaque reduction neutralization tests (PRNT) against WNV (1), and by ELISA with WNV, SLEV, and Venezuelan equine encephalitis virus (VEEV) antigens and viruses. (The presence of several endemic arboviruses, including SLEV and VEEV, necessitated additional testing.) WNV infection was confirmed if the WNV antibody titer was [greater than or equal to] fourfold higher than the SLEV titer. To investigate evidence of recent WNV infection, 198 samples were also tested by using both IgG- and IgM-specific ELISA with WNV-infected cell culture antigens (2). Selected samples were tested by ELISA and PRNT for VEEV antibodies to determine if this virus was circulating in areas reporting equine encephalitis.

Results

A total of 441 equine serum samples from 14 states of Mexico were tested (Figure 1). WNV-specific antibodies were detected in 97 (22%) of the samples. These data probably overestimate the true equine seropositivity rate because sampling focused on herds with a history of clinical encephalitis. Representative data from 22 of the WNV-positive samples obtained in five different states are presented in Table 1. No evidence was obtained of SLEV infection in equines, but VEEV-specific antibodies were detected in serum samples from the states of Veracruz and Yucatain. (Horses are vaccinated against VEEV in the states of Chiapas and Oaxaca, so samples from these locations were not tested for VEEV antibodies.) The positive samples, several of which also contained WNV IgG, represent natural VEEV circulation and infection of horses, presumably with enzootic subtype IE strains (3,4).

On May 5, 2003, the CPA-SAGARPA received a report of a dead Common Raven from the El Yumka wildlife preserve in the city of Villahermosa, state of Tabasco. Although this species is native to Tabasco and other regions of Mexico, this bird was one of two ravens imported from the United States in 1999. A necropsy was performed, and virus isolation was attempted on tissue samples at the CPA-SAGARPA biosafety level 3 facility in Palo Alto, Mexico City. On May 16, 2003, cytopathic effects were detected in Veto cells injected with brain suspension. Viral RNA from the isolate was genetically characterized at the National Institute for Epidemiology and Diagnostics (InDre) in Mexico City and at the University of Texas Medical Branch in Galveston. A 2,004-nt genome portion, including the prM-E protein region, was amplified by using a reverse transcription-polymerase chain reaction as described previously (5); the resulting amplicons were sequenced directly with the Big Dye sequencing kit and model 3100 sequencer (Applied Biosystems, Foster City, CA). The sequence of this WNV isolate (TM171-03, submitted to GenBank under accession no. AY371271) was aligned with all homologous WNV sequences of the same length available from the GenBank library (homologous to nt 4662,469 in the Flamingo382 strain, GenBank accession no. AF196835), and phylogenetic trees were constructed by using maximum parsimony, maximum likelihood (incorporating empirical base frequencies, a general time-reversible substitution model with the following frequencies: A [right arrow] C 1.34263; A [right arrow] G 4.18575; A [right arrow] U 1.55497; C [right arrow] G 0.044980; C [right arrow] T 15.08737; G [right arrow] U 1.00000, a g shape parameter of 0.228), and neighbor joining programs implemented in the PAUP 4.0 software package (6). All trees had nearly identical branching orders; the maximum parsimony tree is presented in Figure 2. All trees placed the Mexican raven isolate as a sister to a clade that comprises most WNV strains isolated in Texas during 2002. Two other WNV strains from the Bolivar Peninsula, Texas (362,476), were positioned basally to a 1998 Israeli stork isolate that grouped with all other North American isolates, suggesting that the North American strains may not all have originated from a point source introduction into the New York area in 1999. This topologic finding was the result of a single synonymous, third codon position (genomic position 969 by using the numbering of the Flamingo382 isolate, GenBank accession no. AF196835) U nucleotide (synapomorphy) shared by the 1998 Israeli and all North American strains except the Bolivar Peninsula isolates. However, the relatively poor support, represented by bootstrap values (1,000 replicates) of only 56-59 by using the three different phylogenetic methods, suggests that this topologic finding is not necessarily correct. More robust phylogenetic analyses using complete genomic sequences are needed to clarify these relationships.

[FIGURE 2 OMITTED]

Comparison of nucleotide sequences indicated mutations at 9 nt (0.45%) of the Mexican TMI71-03 sequence compared to the prototype NY99 strain (Flamingo382, GenBank accession no. AF196835). Some of these nucleotide positions vary among other strains sampled worldwide, suggesting that they are not under strong purifying selection. Comparison with sequences of Year 2002 Texas strains showed only one shared mutation (genomic position 2466 C [right arrow] U). WNV has remained genetically conserved in the New World; however, based on our limited sequencing, the Mexican strain appears to be the most divergent WNV isolate identified to date in the Americas (Table 2). Two of the mutations resulted in amino acid changes at prM141 (Ile [right arrow] Thr) and E156 (Ser [right arrow] Pro), which have not been reported to date in North American isolates. The Ser [right arrow] Pro amino acid substitution at residue E156 is of interest, as it abolishes a potential glycosylation site that is a putative WNV virulence determinant (7-9). This report is the first of a New World WNV isolate with probable altered E protein glycosylation. Further studies are in progress to assess the possible phenotypic effects of this mutation.

Conclusions

Two recent publications reported serologic evidence of WNV infection among equines in the states of Yucatan and Coahuila from serum samples collected beginning in July and December 2002, respectively (10,11). We obtained serologic evidence of more widespread circulation of WNV in five other Mexican states, also dating back to July 2002. We also report the first isolation of WNV from Mexico from a dead Common Raven that resided in a wildlife preserve in Tabasco.

Genetic studies indicated that the Mexican WNV strain was likely introduced from the central United States. The level of genetic divergence (9 nt) of the Mexican isolate and the unique amino acid substitutions in the prM and E proteins when compared to all other North American WNV isolates suggest that the Mexican strain has been evolving independently for some time and did not simply enter Mexico recently from Texas. We speculate that this strain descended from a WNV strain introduced into the Yucatan peninsula by migrating birds. Nucleotide sequences of viruses isolated from Mexican states close to the U.S. border, once obtained, may more closely resemble strains isolated in Texas during 2002.

Of particular interest is the overlap in distribution of WNV and VEEV (serologic data for VEEV not shown) in the southern Mexican states of Veracruz and Yucatan; the presence of other flaviviruses like SLEV in these states is also likely. Both WNV and VEEV produce clinically similar neurologic disease in horses, and past, presumptive diagnoses of VEEV may have been inaccurate. Steps are now in place at the Mexico City headquarters of the Animal Health Division of SAGARPA to implement appropriate laboratory diagnosis for flaviviruses and alphaviruses. Additionally, field personnel are instructed to investigate epidemiologic signs of possible WNV infection including avian death and unusual human neurologic syndromes.

The biologic and epidemiologic consequences of mosquito-borne encephalitis viruses (12) cocirculating in the same ecosystem should be examined. The impact of WNV on human health in regions (such as Mexico) where inhabitants may have extensive prior exposure to other flaviviruses such as dengue, SLEV, Ilheus, Bussuquara, Jutiapa, and Yellow fever viruses may differ from that in regions (e.g., the United States and Canada) where human exposure to flaviviruses is very limited. WNV infection in persons with previous flavivirus immunity, which could either attenuate disease because of cross-protective antibodies (13) or potentially worsen disease because of immune enhancement (14), should be studied. Our ongoing VEEV surveillance in southern Mexico may identify differences in transmission habitats for VEEV and WNV and assist with optimizing virus containment efforts.

Table 1. Serologic analysis (a) of 22 horse serum samples from five
Mexican states, positive for West Nile virus antibodies,
July 2002-March 2003 (b)

State, locality              Sample no.       Date       Age    Sex

Veracruz, Minatitlan          VER-015     Oct 28, 2002   7 y     M
Veracruz, Minatitlan          VER-011     Oct 28, 2002   18 m    M
Veracruz, Hidalgotitlan       VER-017     Oct 29, 2002   8 m     M
Veracruz, Texistepec           VER-26     Oct 29, 2002   2 y     M
Veracruz, Texistepec          VER-025     Nov 7, 2002    18 m    M
Veracruz, J. Carranza         VER-036     Nov 11, 2002   7 m     M
Veracruz, Angel R. Cabada       406       Mar 20, 2003   13 m    F
Veracruz, Cosamaloapan          187       Aug 28, 2002   5 y     F
Yucatan, Merida                 389       Mar 7, 2003    4 y     F
Yucatan, Tizimin                391       Mar 1, 2003    5 y     M
Chihuahua, Ojinaga              210       Oct 20, 2002   2 y     M
Coahuila, Cd Acuna              112       Oct 14, 2002   3 y     M
Coahuila, Hidalgo                4        Sep 1, 2002     5      F
Coahuila, Villa Union            26       Nov 2, 2002    4 y     M
Coahuila, Nava                   67       Nov 11, 2002   7 y     M
Coahuila, P. Negras              56       Nov 5, 2002    2 y     M
Coahuila, Zaragoza               39       Oct 10, 2002   5 y     F
Coahuila, Morelos                51       Oct 5, 2002    7 y     M
Tamaulipas, Diaz Ordaz          268       Nov 7, 2002    8 y     F
Tamaulipas, Camargo             279       Nov 11, 2002   4 y     F
Tamaulipas, Rio Bravo           349       Nov 5, 2002    8 y     M
Tamaulipas, Victoria            344       Nov 7, 2002    2 y     M

                                WNV serologic findings

State, locality            IgG-E   IgM     IgG   PRNT   HI   HI SLEV

Veracruz, Minatitlan        pos   1,600    400   >320   20     20
Veracruz, Minatitlan        pos    neg     400   >320  160     neg
Veracruz, Hidalgotitlan     pos   12,800   neg   >320  >640    160
Veracruz, Texistepec        pos    neg     100    40   160     neg
Veracruz, Texistepec        pos   6,400    neg   >320  >640    160
Veracruz, J. Carranza       pos    neg    6,400  >320  >640    40
Veracruz, Angel R. Cabada   pos    neg     400   >320   80     neg
Veracruz, Cosamaloapan      pos    neg    1,600  >320  160     20
Yucatan, Merida             pos    neg    1,600   nt    80     20
Yucatan, Tizimin            pos    neg    1,600  160    40     20
Chihuahua, Ojinaga          neg   1,600    neg    40   320     40
Coahuila, Cd Acuna          pos   1,600    neg    80    20     20
Coahuila, Hidalgo           pos    400     100   >320  >640    20
Coahuila, Villa Union       pos    400     100   >320  320     80
Coahuila, Nava              pos    400     100   >320  320     20
Coahuila, P. Negras         pos   6,400    100   >320  >640    40
Coahuila, Zaragoza          pos    neg     400   >160  320     20
Coahuila, Morelos           pos    neg     100   >160  160     40
Tamaulipas, Diaz Ordaz      pos   12,800  1,600   40   320     40
Tamaulipas, Camargo         pos   12,800  3,200  >320  320     80
Tamaulipas, Rio Bravo       pos    neg     400    80    8      neg
Tamaulipas, Victoria        pos    neg    6,400  >320  160     20

(a) Titers expressed as reciprocal of dilution; all tests were
enzyme-linked immunosorbent assays unless otherwise noted;
all tests were against WNV (West Nile virus) unless otherwise
noted.

(b) SLEV, St. Louis encephalitis virus; Ig, immunoglobin; G-E,
recombinant domain III of E protein; PRNT, plaque reduction
neutralization test; HI, hemagglutination inhibition test.

Table 2. Nucleotide and deduced amino acid difference in the
prM-E gene region between the prototype New York (382-99)
and Mexican (TM171-03) West Nile virus strains

Nucleotide (amino acid) (a)    Strain 382-99   Strain TM171-03

483                                  C                U
858                                  C                U
887 (prM141)                      U (Ile)          C (Thr)
1137                                 C                U
1432 (E156)                       U (Ser)          C (Pro)
1626                                 C                U
2328                                 C                U
2388                                 C                U
2466                                 C                U

(a) Nucleotide numbering used for the New York flamingo 382-99 sequence
(Genbank accession no. AF196835); locations of encoded amino acid
difference are shown in parentheses.

Table. Predicted epidemic cessation date and maximum number of cases
severe acute respiratory syndrome

              Parameter estimation (a)

Locality      [t.sub.m]    r     [alpha]

Beijing         8.94      0.16    1.00
Hong Kong       6.11      0.09    2.94
Singapore       14.50     0.12    1.51

Locality      Maximum no. of cases (95% CI) (b)

Beijing             2,595 (2,541 to 2,649)
Hong Kong           1,748 (1,619 to 1,777)
Singapore              207 (191 to 223)

Locality       Epidemic cessation date (95% CI)

Beijing       June 27, 2003 (June 14-July 10)
Hong Kong     June 29, 2003 (June 14-July 14)
Singapore     May 28, 2003 (May 20-June 5)

(a) [t.sub.m], the inflection point of the growth model; r, the
intrinsic growth rate; [alpha], the measurement of the extent of
deviation of S-shaped dynamics from the classic logistic growth
curve.

(b) CI, confidence interval.

Acknowledgments

We thank Igor Romero Sosa and many CPA-SAGARPA veterinarians for their contributions to this project, and Wenli Kang for technical assistance.

This research was supported by grant NO1-AI-25489 from the National Institutes of Health and grant U90 CCU 618754 from the Centers for Disease Control and Prevention.

References

(1.) Beaty BJ, Calisher CH, Shope RE. Arboviruses. In: Schmidt NJ, RW Emmons, editors. Diagnostic procedures for viral, rickettsial and chlamydial infections, 6th edition. Washington: American Public Health Association; 1989. p. 79-855.

(2.) Rossi CA, Drabick JJ, Gambel JM, Sun W, Lewis TE, Henchal EA. Laboratory diagnosis of acute dengue fever during the United Nations Mission in Haiti, 1995-1996. Am J Trop Med Hyg 1998;59:275-8.

(3.) Oberste MS, Schmura SM, Weaver SC, Smith JF. Geographic distribution of Venezuelan equine encephalitis virus subtype IE genotypes in Central America and Mexico. Am J Trop Med Hyg 1999;60:630-4.

(4.) Scherer WF, Dickerman RW, Chia CW, Ventura A, Moorhouse A, Geiger R, et al. Venezuelan equine encephalitis virus in Veracruz, Mexico, and the use of hamsters as sentinels. Science 1963;145:274-5.

(5.) Beasley DW, Davis CT, Guzman H, Vanlandingham DL, Travassos da Rosa AP, Parsons RE, et al. Limited evolution of West Nile virus has occurred during its southwesterly spread in the United States. Virology 2003;309:190-5.

(6). Swofford DL. PAUP *. Phylogenetic Analysis Using Parsimony (* and other methods). Version 4. Sunderland (MA): Sinauer Associates; 1998.

(7.) Scherret JH, Mackenzie JS, Khromykh AA, Hall RA. Biological significance of glycosylation of the envelope protein of Kunjin virus. Ann NY Acad Sci 2001;951:361-3.

(8.) Chambers TJ, Halevy M, Nestorowicz A, Rice CM, Lustig S. West Nile virus envelope proteins: nucleotide sequence analysis of strains differing in mouse neuroinvasiveness. J Gen Virol 1998;79(Pt 10):2375-80.

(9.) Halevy M, Akov Y, Ben-Nathan D, Kobiler D, Lachmi B, Lustig S. Loss of active neuroinvasiveness in attenuated strains of West Nile virus: pathogenicity in immunocompetent and SCID mice. Arch Virol 1994;137:355-70.

(10.) Lorono-Pino MA, Blitvich BJ, Farfan-Ale JA, Puerto FI, Blanco JM. Marlenee NL, et al. Serologic evidence of West Nile virus infection in horses, Yucatan State, Mexico. Emerg Infect Dis 2003;9:857-9.

(11.) Blitvich BJ, Fernandez-Salas I, Contreras-Cordero JF, Marlenee NL, Gonzalez-Rojas JI, Komar N, et al. Serologic evidence of West Nile virus infection in horses, Coahuila State, Mexico. Emerg Infect Dis 2003;9:853-6.

(12.) Monath TP. The arboviruses: epidemiology and ecology, Vol. I-V. Boca Raton (FL): CRC Press; 1988.

(13.) Tesh RB, Travassos da Rosa AP, Guzman H, Araujo TP, Xiao SY. Immunization with heterologous flaviviruses protective against fatal West Nile encephalitis. Emerg Infect Dis 2002;8:245-51.

(14.) Kurane I, Ennis FE. Immunity and immunopathology in dengue virus infections. Semin Immunol 1992;4:121-7.

Jose G. Estrada-Franco, * Roberto Navarro-Lopez, ([dagger]) David W.C. Beasley, * Lark Coffey, * Anne-Sophie Carrara, * Amelia Travassos da Rosa, * Tamara Clements, ([double dagger]) Eryu Wang, * George V. Ludwig, ([double dagger]) Arturo Campomanes Cortes, ([dagger]) Pedro Paz Ramirez, ([dagger]) Robert B. Tesh, * Alan D.T. Barrett, * and Scott C. Weaver *

* University of Texas Medical Branch, Galveston, Texas, USA; ([dagger]) Comision Mexico-Estados Unidos para la Prevencion de la Fiebre Aftosa y Otras Enfermedades Exoticas de los Animales, Mexico City, Mexico; and ([double dagger]) U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA

Dr. Estrada-Franco is an assistant professor at the University of Texas Medical Branch. His research interests include the ecology and epidemiology of vector-borne diseases, their human impact, vector genetics, and vector-host-pathogen interactions of arboviruses and parasitic diseases.

Address for correspondence: Scott Weaver, Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609, USA; fax [phone]; email: [e-mail]