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Am. J. Trop. Med. Hyg., 61(4), 1999, pp. 587–597 Copyright q 1999 by The American Society of Tropical Medicine and Hygiene

MOLECULAR CHARACTERIZATION OF RABIES VIRUS ISOLATES FROM MEXICO: IMPLICATIONS FOR TRANSMISSION DYNAMICS AND HUMAN RISK ´ N, CECILIA C. DE MATTOS, CARLOS A. DE MATTOS, ELIZABETH LOZA-RUBIO, ALVARO AGUILAR-SETIE LILLIAN A. ORCIARI, AND JEAN S. SMITH Rabies Section, Viral and Rickettsial Zoonosis Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; Centro Nacional de Investigaciones Disciplinarias en Microbiologıá, Instituto Nacional de Investigaciones Forestales Agrıćolas y Pecuarias, Secretaria de Agricultura, Ganaderia y Desarrollo Rural, Me´xico City, Me´xico; Unidad de Investigacioń Me´dica en Inmunologıá, Instituto Mexicano del Seguro Social, Me´xico City, Me´xico

Abstract. Twenty-eight samples from humans and domestic and wild animals collected in Mexico between 1990 and 1995 were characterized by using anti-nucleoprotein monoclonal antibodies and limited sequence analysis of the nucleoprotein gene. The variants of rabies viruses identified in these samples were compared with other isolates from Mexico and the rest of the Americas to establish epidemiologic links between cases and outbreaks and to increase the understanding of rabies epidemiology in the Western Hemisphere. Antigenic and genetic diversity was found in all samples from dogs and dog-related cases, suggesting a long-term endemic situation with multiple, independent cycles of virus transmission. Two isolates from bobcats were antigenically and genetically homologous to the rabies variant circulating in the Arizona gray fox population, indicating a wider distribution of this variant than previously reported. Rabies isolates from skunks were unrelated to any variant analyzed in this study and represent a previously unrecognized cycle of rabies transmission in skunks in Baja California Sur. Two antigenic and genetic variants cocirculating in southern and eastern Mexico were found in viruses obtained from cases epidemiologically related to vampire bats. These results serve as a baseline for the better understanding of the molecular epidemiology of rabies in Mexico. Urban and sylvatic rabies is an important public health and economic problem in Latin America. During the period 1990–1996, there were 1,372 human rabies cases in this region, but the animal responsible for the exposure was known for only 1,117 cases. The domestic dog and various Chiroptera species, mainly the vampire bat (Desmodus rotundus), were represented in 80.6% and 11.3% of these cases, respectively.1 Urban rabies constitutes a dog-to-dog transmission cycle that is maintained in cities. Dogs are the primary source of infection for humans and other domestic animals. Rabies epidemiology is highly influenced by both human and dog population density and the cultural and socioeconomic factors that govern the interaction between those 2 populations.2,3 These factors are particularly relevant in the case of Mexico. This country has one-fifth of the human population (approximately 93 million people) and one-fourth of the canine population (approximately 13.5 million dogs) of Latin America.1,4 As a consequence of the implementation of an aggressive public health policy in Mexico for rabies control in the urban centers, there has been a substantial decrease in the number of canine rabies cases from 2,077 in 1992 to 521 in 1997.5 Although the annual canine vaccine coverage in Mexico is approximate 78%, ultimate rabies control has been difficult to achieve.4 Strategies for the application of control programs should involve careful planning based on the knowledge of the local epidemiologic situation.6 The availability of detailed surveillance data, the identification of different transmission cycles, and the geographic distribution in urban settings are essential for the successful implementation and evaluation of control programs. In contrast, sylvatic rabies is characterized by the involvement of wildlife that maintain stable cycles of transmission over time in particular geographic areas. Although cases in non-hematophagous bats 7 and terrestrial wildlife 8 have been reported in Mexico, no sylvatic reservoirs for rabies virus, other than D. rotundus, have been definitively identified.

In Latin America, approximately 74,8% of the population lives in urban areas. During 1997, 50.5% of the human rabies cases in Latin America occurred in rural areas.5 Here the proximity between human habitations and the local wildlife increases the opportunity of encounters between people and their domestic animals with sylvatic species. In this setting, the urban and sylvatic cycles can easily overlap, complicating the understanding of rabies epidemiology and control of the disease. These circumstances make it critical to identify the actual source of infection of human and domestic animal rabies cases, to determine the intraspecific and interspecific pathways of transmission, and to establish the epidemiologic relationships between cases and outbreaks. The molecular characterization of rabies virus isolates can assist in the elucidation of epidemiologic links that cannot easily be established by the application of other methods.9– 11 Little data exist on the molecular characteristics of rabies viruses currently maintained by the urban and sylvatic rabies cycles in Mexico.12 The objective of this study was the antigenic and genetic characterization of rabies virus isolates obtained from humans and domestic and wild animals in different regions of Mexico. These samples were compared with viral representatives throughout the Americas to reveal otherwise inconspicuous epidemiologic relationships in the apparent Mexican urban and sylvatic rabies cycles. MATERIALS AND METHODS

Virus samples. Twenty-eight rabies isolates from animal (n 5 27) and human (n 5 1) cases obtained in different regions of Mexico between 1990 and 1995 were analyzed in this study (Table 1). Isolates representing antigenic variants (AgV) known from the Americas8 were provided by the Centers for Disease Control and Prevention (CDC) (Atlanta, GA) (Table 2). Antigenic analysis. Antigenic characterization was con-

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DE MATTOS AND OTHERS

TABLE 1 Designations and origins of the 28 rabies isolates collected in Mexico and analyzed in this study Identification no.

Species

Place of isolation*

Year of isolation

Lineage†

Mxct3122 Mxfrr3142 Mxhm3121 Mxbv3123 Mxdg3124 Mxdg3125 Mxpg3126 Mxgt3127 Mxdg3128 Mxpg3134 Mxdk3138 Mxbv3143 Mxbv3144 Mxdg3147 Mxuk3151 Mxsk3141 Mxsk3136 Mxsk3145 Mxgm3148 Mxgm3140

Cat Ferret Human Cattle Dog Dog Pig Goat Dog Pig Donkey Cattle Cattle Dog No data Skunk Skunk Skunk Bobcat Bobcat

Huetamano, Michoacan Mexico F.D. Mexico F.D Tacambaro, Michoacan Puebla, Puebla Jamiltepec, Oaxaca Tlacotepec, Puebla Tlaxcala, Tlaxcala Gomez Palacios, Durango Mexico F.D. Uruapan, Michoacan Puebla, Puebla Chihuahua, Chihuahua Queretaro, Queretaro No data La Paz, B.C.S. Camardu, B.C.S. La Paz, B.C.S. Chihuahua, Chihuahua Hermosillo, Sonora

1990 1990 1991 1991 1991 1992 1993 1991 1991 1991 1991 1994 1994 1995 No data 1990 1992 1995 1990 1994

A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A2 A2 A2 A3 A3

Mxtbbt3150 Mxbv313O Mxhr3129 Mxbv3131 Mxbv3139 Mxbv3135 Mxbv3137 Mxbv3146

Free tail bat Cattle Horse Cattle Cattle Cattle Cattle Cattle

Mexico F.D. Escuintla, Chiapas Escuintla, Chiapas Tihuatlan, Veracruz Santiago Tuxtla, Veracruz Huimanguillo, Tabasco Tuxtla Gutierrez, Chiapas San Rafael, Veracruz

1995 1993 1993 1995 1991 1992 1994 1995

B1 B2 B2 B2 B2 B2 B2 B2

Antigenic variant‡

1 1 1 1 1 1 1 10 1 1 1 1 1 1 1 10 10 10 7 7 9, (Loza-Rubio E. and others, unpublished data) 3 3 3 11 11 11 11

* F.D. 5 Federal District; B.C.S. 5 Baja California Sur. † Significance of lineages are discussed in the Results and shown in Figure 2. ‡ Significance of antigenic variants are discussed in the Results.

ducted by using a panel of 8 monoclonal antibodies (MAbs) produced against the viral nucleoprotein by CDC (Table 3). The selection of the panel was based on a collaborative study conducted by the Instituto Panamericano de Proteccioń de Alimentos y Zoonosis (Buenos Aires, Argentina) and CDC.8 Characterization was performed by the indirect immunofluorescent technique on acetone-fixed touch impressions of brain material, as described previously.8 Genetic analysis. Genetic typing was based on nucleotide sequence differences in cDNA obtained by the reverse transcription/polymerase chain reaction (RT/PCR) amplification of viral RNA extracted from infected brain material. The RNA was extracted with TRIzolTM (GIBCO-BRL, Inc., Gaithersburg, MD) according to the directions of the manufacturer. The RT/PCR was achieved with primers 10g and 304, as described previously.11 The DNA sequencing was performed on a 377 DNA sequencer (Applied Biosystems, Foster City, CA) using the Taq DyeDeoxyTM Terminator System (Applied Biosystems) according to the manufacturer’s recommendations. A 320-basepair region of the nucleoprotein gene from position 1157 to position 1476, as compared with rabies virus SADB19,13 was aligned with the PileUp program in the Genetics Computer Group Software, version 9.114 as previously described.9 The genetic distance between any 2 nucleotide sequences was calculated by using the DNADIST program (Kimura two-parameter method) of the PHYLIP package, version 3.5.15 The phylogenetic analysis was conducted by using the

obtained distance matrix in the Neighbor-Joining method of the program NEIGHBOR. The phylogenetic analysis based in the parsimony method was performed using the DNAPARS program from the same package. The non-rabies Lyssaviruses European bat 1 (Eblvfr)16 and Duvenhage (Duv)16 were used as outgroups. The confidence limits of the branch pattern were assessed by bootstrap resampling (100 replicates) of the multiple-sequence alignments with the programs SEQBOOT, DNADIST, and NEIGHBOR for distancematrix methods, and SEQBOOT and DNAPARS for parsimony methods of PHYLIP. The program CONSENSE of this package was used to calculate the consensus tree. Bootstrap values of more than 70% were regarded as providing evidence for the phylogenetic grouping. 17 The program TREEVIEW was used to obtain the graphic output.18 RESULTS

Antigenic typing. Differential reaction with MAbs 1, 10, 12, 15, and 19 identified 5 AgV (Table 3) of rabies virus in the 28 samples from Mexico (Table 1). Four variants, AgV 1, 3, 7, and 9 had been identified previously.7,8 Antigenic variant 1, which is associated with rabies in domestic dogs in Latin America, was found in 14 samples from different species (1 human, 4 dogs, 3 cattle, 1 cat, 2 pigs, 1 ferret, 1 donkey, and 1 unidentified sample). Antigenic variant 3, which is associated with vampire bats in Latin America, was identified in 3 samples (2 bovines and 1 equine), and anti-

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RABIES IN MEXICO

TABLE 2 Rabies virus isolates used for comparison in the genetic analyses Identification no.

Place of isolation

Species

Year of isolation

Antigenic variant*

Mxmxdg555 Mxmxdg559 Mxsndg1274 Mxsndg672 Mxsndg671 Mxsndg673 Mxmxdg1278 Mxmxdg1282 Txushm835

Dog Dog Dog Dog Dog Dog Dog Dog Human

Mexico, F.D. Mexico, F.D. Hermosillo, Sonora Hermosillo, Sonora Hermosillo, Sonora Hermosillo, Sonora Mexico, F.D. Mexico, F.D. Texas

1978 1978 1988 1988 1988 1988 1991 1991 1976

1 1 1 1 1 1 1 1 1

Txushm834

Human

Texas

1979

1

Txushm2193

Human

Texas

1979

1

Txusdg216O Txusfx294 Txusfx3700 Txusdg2361 Txuscy2587 Txuscy2830 Caushm726 Causct674

Dog Fox Fox Dog Coyote Coyote Human Cat

Laredo, Texas Gillespie, Texas Texas Texas Texas West Texas U.S.-Mexico border California

1985 1983 1997 1994 1994 1995 1961 1987

1 1 1 1 1 1 1 1

Causcy2520 Caushm2184

Coyote Human

San Diego, California California

1990 1993

1 1

Cahum953

Human

California

1979

1

Caussk127 Caussk2521 Caussk732 Orushm848

Skunk Skunk Skunk Human

Colusa, California California Amador, California Oregon

1974 1994 1987 1989

1 1 1 1

Orusdg1303

Dog

Oregon

1991

1

Azushm947

Human

Arizona

1981

1

Arussk963 Wiussk3789 Azusfx398 Azusfx186 Arussk964 Ksussk3436 Brdrbt892 Vedrbt346 Vebv2785 Vebv2782 Artbbt1327 Citbbt232 Vebv2784 Vebv2797 Vebv2787 Caustbbt807 Txustbbt2075 Flustbbt874 Alustbbt2769 Causefbt804 Causefbt2320 Eblvfr Duv

Skunk Skunk Fox Fox Skunk Skunk Vampire bat Vampire bat Cattle Cattle Freetail bat Freetail bat Cattle Cattle Cattle Freetail bat Freetail bat Freetail bat Freetail bat Big brown bat Big brown bat European house bat Human

Arkansas Chippewa, Wisconsin Arizona Arizona Desha, Arkansas Kansas Brazil Miranda, Venezuela Falcon, Venezuela Carabobo, Venezuela Buenos Aires, Argentina Valpariso, Chile Cojedes, Venezuela Miranda, Venezuela Guarico, Venezuela Modesto, California Texas Florida Alabama California California France South Africa

1984 1 1998 1 1979 7 1986 7 1984 8 1997 8 Unknown 3 1976 3 1992 3 1993 3 1991 4 1985 4 1993 5 1994 5 1994 5 1976 9 1984 9 1989 9 1994 9 1987 Big brown AgV 1993 Big brown AgV 1989 NA 1986 NA

Comments (Ref.)

Urban dog endemic cycle (29) Urban dog endemic cycle (11) Urban dog endemic cycle (11) Urban dog endemic cycle (11) Urban dog endemic cycle (11) Urban dog endemic cycle (11) Urban dog endemic cycle (11) Urban dog endemic cycle (11) Urban dog endemic cycle; Mexican citizen bitten by a dog in Cohauila, Mexico Urban dog endemic cycle; Mexican citizen bitten by a dog in Cohauila, Mexico Urban dog endemic cycle; Mexican citizen bitten by a dog in Cohauila, Mexico Urban dog endemic cycle (11) Texas fox endemic cycle Texas fox endemic cycle Urban dog/coyote endemic cycle Urban dog/coyote endemic cycle Urban dog/coyote endemic cycle Urban dog endemic cycle (11) Urban dog endemic cycle; transported from Guerrero, Mexico to California (11) Urban dog endemic cycle Urban dog endemic cycle Mexican citizen bitten by a dog in Morelos, Mexico Urban dog endemic cycle; Mexican citizen bitten by dog in Chihuahua, Mexico California skunk endemic cycle California skunk endemic cycle California skunk endemic cycle Urban dog endemic cycle; Mexican citizen from Michoacan died in Oregon (11) Urban dog endemic cycle; transported from Chihuahua, Mexico to Oregon (11) Urban dog endemic cycle; U.S. citizen, bitten by dog in Sonora, Mexico (11) Northcentral skunk endemic cycle Northcentral skunk endemic cycle Arizona fox endemic cycle Arizona fox endemic cycle Southcentral skunk endemic cycle Southcentral skunk endemic cycle (21) (21) (9) (9) Insectivorous migratory bat, South America Insectivorous migratory bat, South America (9) (9) (9) Insectivorous migratory bat, North America Insectivorous migratory bat, North America Insectivorous migratory bat, North America Insectivorous migratory bat, North America Insectivorous non-migratory bat, U.S. Insectivorous non-migratory bat, U.S. (10) (10)

* NA 5 not applicable.

genic variant 7, which is associated with foxes in Arizona, was identified in 2 samples from bobcats (Felis rufus). Antigenic variant 9 was found in 1 sample from a freetail bat (Tadarida brasiliensis). Two previously unidentified antigenic reaction patterns were also recognized: AgV 10 was found

in 3 skunks (species not identified) and in 1 goat, and AgV11 was present in 4 cattle. Genetic analysis. These same samples collected in Mexico between 1990 and 1995 were characterized by partial genomic sequencing of the nucleoprotein gene (Figure 1).

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FIGURE 1. Comparison of the nucleotide sequences for the nucleoprotein gene of the 28 Mexican rabies isolates between positions 1157 and 1476 compared with rabies virus SADB19.14 Nucleotides identical to the consensus sequence are shown by a dash. The nucleotide differences are shown in lower cases.

TABLE 3 Monoclonal antibody reaction patterns of the rabies virus isolates obtained in Mexico between 1990 and 1995 Antigenic variant*

1 3 7 9 10 11

Reservoirs†

Doga Vampire batb Arizona gray foxc Freetail batd Baja California Sur skunke Vampire batf

C1‡

C4

C9

C10

C12

C15

C18

C19

1 2 1 1 1 2

1 1 1 1 1 1

1 1 1 1 1 1

1 1 2 1 1 1

1 1 1 1 2 2

1 2 1 2 1 2

2 2 2 2 2 2

1 1 1 2 1 1

* Antigenic variant designation used as previously described for the antigenic characterization of rabies virus isolates from Latin America.8,9 † Domestic and wildlife species identified (a to d)8,10 or proposed here (e and f) as the reservoirs of the corresponding rabies antigenic variant. ‡ Monoclonal antibody designation.

RABIES IN MEXICO

FIGURE 1.

The Mexican viruses were clustered by phylogenetic analysis into 2 main lineages identified as A and B in Figure 2. Lineage A included viruses isolated from a human and from domestic and wild terrestrial animals typed as AgV1, 7, and 10. Lineage B included samples from a non-hematophagous bat typed as AgV9 and livestock cases typed as AgV3 and 11. Although there was a marked tendency for correspondence between the phylogenetic lineages and the antigenic variant groups, this correspondence was not consistent for all samples. Lineage A was divided into 3 sublineages (A1, A2, and A3), with each presenting high values in the bootstrap analysis, supporting the concept that they were independent. Sublineage A1 was formed by isolates typed as AgV1 and the sample Mxgt3127, which was characterized with the

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Continued.

panel of 8 MAbs as AgV10. This sublineage contained the majority of samples, which segregated in 3 sample clusters (identified as A1a, A1b, and A1c in Figure 2). Clade partitioning was not determined by the geographic origin of a sample since rabies cases originating from the central region of the country were found in all 3 clusters. Sublineage A2 contained viruses isolated from skunks in Baja California Sur typed as AgV10. They showed an average genetic distance of 3.5%. Sublineage A3 was formed by the AgV7 isolates obtained from bobcats in Sonora and Chihuahua. These viruses were very closely related to each other, showing 99% nucleotide homology. Lineage B samples diverged as 2 sublineages. The single sample of AgV9 from a freetail bat obtained in Mexico City constituted sublineage B1. Samples of AgV3 and AgV11

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FIGURE 2. Consensus phylogenetic tree of the 28 rabies viruses isolated in Mexico between 1990 and 1995. Phylogenetic analysis was performed as described in the Materials and Methods. Bootstrap values obtained from 100 resamplings of the data using distance matrix and parsimony methods are shown at the top and at the bottom of the corresponding nodes, respectively. For clarity, only the bootstrap values that define the groups are shown. The significance of letter designations at nodes is discussed in the Results.

from livestock formed sublineage B2. Nucleotide differences at 6 positions separated samples of AgV3 and AgV11. Isolates of AgV11 formed a clade supported by a high bootstrap value. Isolates of AgV3 were more genetically diverse than AgV11 viruses and did not form a monophyletic group. Representation within a clade did not conform to the geographic origin of a sample. Rabies cases originating from Veracruz and Chiapas were found in both sample clusters. To help interpret the epidemiologic relationships between these 2 lineages with other genetic variants maintained in different endemic cycles in the Americas, these 2 lineages were compared with other rabies viruses of the urban and sylvatic cycles of the region as detailed in Table 2. Lineage A was compared with rabies isolates from dogs and dog-related cases from Mexico and the United StatesMexican border and with viruses obtained from terrestrial reservoirs of sylvatic rabies in areas of the United States adjoining Mexico (Figure 3). Lineage B was compared with viruses isolated from vampire bats and vampire bat-related cases in Venezuela and Brazil, with other viral sequences obtained from freetail bats from North and South America, and with 2 samples of a non-migratory bat species in the United States (Figure 4). In lineage A (Figure 3), all the AgV1 Mexican samples

and the isolate Mxgt3127 formed an independent monophyletic group (A1) clustering together with viruses epidemiologically related to the urban rabies cycle in Mexico and the United States-Mexico border. All but one (Causct674) of the members of Clade A1 segregated in 4 groups: A1a, A1b, and A1c (they included the samples from the corresponding branches from Figure 2) and group A1d that is a new cluster formed by samples from northern Mexico and Texas. Group A1a consisted of isolates from geographically and temporally dispersed cases. It included viruses from the States of Michoacan, Durango, Sonora, Chihuahua, and the western United States-Mexican border region. They were isolated between 1961 and 1995. Although the death of some human and domestic animal cases from this clade occurred in the United States, their epidemiologic history indicated that the infection was acquired in those Mexican States. All isolates were closely related, with an average genetic distance of 1.86%. Sample Causct674 showed a weak support for its inclusion in this group, and presented an average genetic distance of 4.5% with the members of this clade. Group A1b was formed by samples from the central region of the country and 1 isolate from California. Although it was not supported by a high bootstrap value, all its members showed a high degree of nucleotide homology, showing an average genetic distance of 1.99%. Within Group A1b, 2 subgroups, A1b1 and A1b2, were highly supported in the sampling analyzes. Subgroup A1b1 viruses were all obtained from Mexico City, a restricted area characterized by a high human population density with different socioeconomic levels. These viruses formed a highly homologous group presenting a low average genetic distance (0.37%). Subgroup A1b2 was formed by 2 viruses circulating in the central region of Mexico. They shared 99.7% nucleotide homology. One virus was isolated from a donkey in Michoacań and the other was from a human who had been bitten by a dog in Morelos, but the patient died in California.19 Group A1c was composed of rabies viruses isolated in Mexico City, the adjacent area of the central region of the country, and 1 isolate from Oregon. This monophyletic group was well supported by the bootstrap analysis. This genetic variant was co-circulating in Mexico City and the central region with viruses from Groups A1a and A1b. The sequence variation within this cluster was limited to an average genetic distance of 1.7%. None of the 28 isolates under study was placed in Group A1d. This clade was formed by samples previously obtained from humans bitten by dogs in the northeastern part of Mexico, and from dog and coyote (Canis latrans) samples isolated in the southeastern part of Texas between 1976 and 1994. The calculated mean genetic distance for this group was 0.90%. Bootstrapping by both the neighbor-joining and the parsimony methods suggested that the 2 clearly different lineages represented by Groups A1d and A1c were related. The analysis of the viruses obtained from wildlife in lineage A showed that the isolates from skunks in Baja California Sur formed the independent monophyletic group A2 and differed an average of 13%, 29.25%, and 13.8% from representatives of the northcentral, southcentral and Califor-

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RABIES IN MEXICO

FIGURE 3. Consensus phylogenetic tree of the comparison of the samples from lineage A in Figure 2 with isolates representing major sylvatic reservoirs for rabies in areas of the United States bordering Mexico and rabies cases associated with urban dog rabies in which the infection occurred in Mexico and the death occurred in the United States. Phylogenetic analysis was performed as described in the Materials and Methods. Bootstrap values obtained from 100 resamplings of the data using distance matrix and parsimony methods are shown at the top and at the bottom of the corresponding nodes, respectively. For clarity, only the bootstrap values that define the groups are shown. 1 5 U.S. Texas fox endemic cycle viruses; 2 5 U.S. Arizona fox endemic cycle viruses; 3 5 U.S. northcentral skunk endemic cycle viruses; 4 5 U.S. California skunk endemic cycle viruses; 5 5 Mexican Baja California Sur skunk viruses; 6 5 U.S. southcentral skunk endemic cycle viruses.

nia skunk rabies virus variants, respectively, from the United States. The bobcat isolates collected in Chihuahua and Sonora formed a monophyletic group (A3) with viruses from Arizona gray fox (Urocyon cineroargenteus) rabies virus variants (average genetic distance 5 1.54%). The bobcat viruses showed no close relationship with representatives of Texas gray fox or any of the United States skunk (Mephitis mephitis) rabies virus variants. In lineage B (Figure 4), all Mexican viruses obtained from cases epidemiologically related to vampire bat rabies, characterized as AgV3 and AgV11, segregated in the monophyletic Group B2 with other rabies isolates collected from vampire bats and domestic animals infected by this species.9,11 This close phylogenetic relationship and the epidemiologic data indicated that the reservoir and transmitter of these Mexican viruses was D. rotundus. The AgV11 samples remained as the highly supported Subgroup B2a.

The sample from freetail bat isolated in Mexico City in 1995 segregated with samples obtained from the same species in different parts of the United States and formed Group B1. This group was well supported by the bootstrap resampling analysis. These viruses were highly homologous, showing a divergence of only 1.26%. Viruses obtained from freetail bats in the southern cone of South America were grouped in an independent branch (Group C). The rabiesendemic viruses circulating in T. brasiliensis in the Northern and Southern Hemisphere presented an average genetic distance of 12.88% between each other. DISCUSSION

Monoclonal antibodies and partial sequencing were used to study the molecular epidemiology and patterns of geographic distribution of rabies virus variants circulating in Mexico. The panel of 8 MAbs used in this study proved to

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FIGURE 4. Consensus phylogenetic tree of the comparison between the Mexican bat rabies isolates and rabies viruses from hematophagous bats from Brazil and Venezuela and non-hematophagous bats from Chile, Argentina, and the United States. Phylogenetic analysis was performed as described in the Materials and Methods. Bootstrap values obtained from 100 resamplings of the data using distance matrix and parsimony methods are shown at the top and at the bottom of the corresponding nodes, respectively. For clarity, only the bootstrap values that define the groups are shown. A 5 rabies genetic variant circulating in big brown bat (Eptesicus fuscus) in North America; B2 5 rabies genetic variant circulating in vampires in Latin America; B1 5 rabies genetic variant circulating in freetail bats in North America; C 5 rabies genetic variant circulating in freetail bats in South America.

be a valuable screening tool in epidemiologic studies and was able to detect the presence of 3 antigenic variants previously identified in Latin America, AgV1, AgV3 and AgV7, and 2 antigenic variants, AgV10 and AgV11, described for the first time in this investigation. With the exception of sample Mxgt3127, all rabies viruses from the urban cycle in Mexico were characterized as AgV1. Sample Mxgt3127 showed 99% nucleotide sequence homology with rabies viruses isolated from dog and dog-related cases, and segregated in Group A1c. These results established a strong epidemiologic link between this sample and other viruses of canine origin, indicating the domestic dog as the reservoir for this particular isolate. This sample was typed as AgV10 and was antigenically indistinguishable from the AgV10 viruses isolated from skunks in Baja California Sur. Viral variants presenting the same pattern of reaction to a panel of MAbs can be differentiated by limited sequence analyzes.20 Virus variants from dogs, Texas foxes, and skunks from the northcentral United States, all typed as

AgV1, were clearly distinguished by genetic characterization.10,20 The use of a panel of 20 MAbs8,10,21 allowed the differentiation of Mxgt3127 from the Baja California Sur skunk viruses and its identification as a new antigenic variant. However, no conclusive epidemiologic information about the probable reservoir of this virus could be obtained with this methodology because this virus was isolated from a non-reservoir species. In these instances, methods of higher resolution should be applied. Surveillance data, together with typing methods using MAbs, offer the important advantages of rapidity, ease of use, and economy. To confirm the antigenic data and obtain a higher degree of resolution for the discrimination of the variants, the genetic typing of selected samples should be carried out. The phylogenetic analysis and surveillance data helped to establish epidemiologic relationships between rabies viruses isolated in Mexico over a period of 34 years. At least 4 rabies genetic variants (Groups A1a, A1b, A1c, and A1d) co-circulated in the urban rabies cycle.

RABIES IN MEXICO

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FIGURE 5. Geographic distribution of the samples from dogs and dog-related cases analyzed in this study. With the exception of the Oregon case (Orushm848), in which no history of exposure to a rabid animal was available,26 all symbols indicate the geographic location where each exposure occurred.

Groups A1a, A1b, and A1c were present in the central region of Mexico. The distribution of endemic rabies viruses in the urban cycle is highly influenced by human population density and by the mobility of people and dogs.22 The central region of the country has the highest population density and active intra-regional migration, and with its educational and economic opportunities, is a national and international center of attraction for migrants.23 These circumstances favor the introduction and movement of animals that contribute to the dispersion of extant rabies variants or could serve as the source of introduction of new ones in a region. Group A1a covered the most extensive territory, from the central region (Mxbv3123, Mxct3122) to Sonora (e.g., Mxsndg1274), Durango (Mxdg3128), Chihuahua (Mxbv3144), and the United States (e.g., Orusdg1303, Caushm953) following the western and eastern slopes of the Sierra Madre Occidental. This distribution coincides with the highest interregional and intra-regional migration routes in northern Mexico (Figure 5).23–25 The increment of international commerce, the development of the industrial sector, and the high demand for agricultural labor in the northern territory of Mexico transformed this region into an important center of population migration.23 Important destinations in this area

are Tijuana, Mexicali, Ciudad Juarez, Monterrey, and the United States (Figure 5).24 Migrants reach their destinations directly or indirectly after living for variable periods in different places along their route.25,26 For example, Orushm848 from group A1c was an agricultural laborer working in northern Oregon who had traveled for 1 month from his place of origin (Michoacań) by car through California to Oregon.27 Such migration flow may have contributed to the dispersion of Clade A1a viruses over this extensive geographic area. Dog ecology is a product of the cultural and socioeconomic conditions of the humans with whom these animals share their environment. The epidemiology of urban rabies is greatly influenced by these complex relationships. In Mexico, people moving from the outlying provinces to the new suburbs of the metropolitan areas often possess a few farm animals and use dogs, which are mostly unsupervised, to protect their belongings.2 The presence of livestock affected with the dog rabies variant (e.g., Mxpg3134, Mxbv3143, and Mxgt3127) in the most densely populated and highly industrialized areas of the central region of Mexico is a direct consequence of this epidemiologic situation. The analysis of lineage A provided evidence for the pres-

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ence of 2 cycles of transmission in wildlife species represented by the AgV7 and AgV10 isolates. Antigenic variant 7 has been isolated only from wild felids in Mexico.8 Feline species have not been identified as rabies reservoirs anywhere in the world. Comparative genetic studies strongly suggested the gray fox as the most probable reservoir of this rabies variant in the northern semi-desert plateau of Mexico and Sonora. In these regions, F. rufus is sympatric with several potential rabies reservoirs of the family Canidae, such as the gray fox, the coyote, and the desert fox (Vulpes macrotis).28 Surveillance data and the molecular characterization of rabies isolates from wildlife species would definitely identify the reservoir and determine the inter-species transmission pathway for this variant in this part of the Mexican territory. The high degree of nucleotide difference between the Baja California Sur AgV10 samples with isolates from the rabies viruses maintained endemically in skunk populations of the United States and other wildlife reservoirs suggested the presence of an independent cycle of rabies transmission in the skunk population of Baja California Sur. Although the taxa of the rabid skunks was not reported, the only species present in this area is the lloriguïn28 skunk (Spilogales putorious).28,29 The future characterization of this particular cycle will depend in part upon enhanced surveillance data and the taxonomic identification of the affected skunk species. The analysis of the samples from cases related with vampire bats showed that there are at least 2 rabies variants, AgV3 and AgV11, in the vampire bat population in Mexico. It is not known how changes in vampire bat populations precipitate new outbreaks, how one outbreak is related to others in a region, or how a new variant appears in an area. The systematic collection and molecular characterization of isolates from outbreaks, in conjunction with surveillance data, may allow the mapping of the actual distribution of AgV3 and AgV11 in Mexico and help in the understanding of their epidemiology. The application of the technology presented in this study for the laboratory surveillance for the antigenic and genetic variants of rabies associated with vampire bats may permit the earlier recognition of the problem and a more rapid institution of control methods. The analysis of the freetail bat sample obtained in Mexico City showed the close relationship of this virus with other samples obtained from the same species in the United States. Previous studies have shown the presence of a single rabies variant in geographically distant isolates obtained from this migratory bat species.30 Rabies cases in humans from exposures to infected bats become specially evident in countries where dog rabies has been controlled or successful vaccination programs have considerably reduced the number of dog cases such as in Mexico. In the United States, bats represent only a low percentage of the total number of rabid animals reported every year. However the rabies variants associated with bats were found in 17 of 32 humans cases reported between 1980 and 1996.31 In these epidemiologic situations, often there are no clear history of animal bite and frequently the disease is not clinically suspected with the consequent delay in the formulation of the right diagnosis.31,32 Under these circumstances, the molecular characterization of the virus is the only tool available to determine the actual source of infection.32

This study established epidemiologic relationships among rabies samples collected in different geographic and socioeconomic regions of Mexico and described their general distribution in the urban and sylvatic cycles. As consequence of the successful dog rabies vaccination program in Mexico, dog rabies cases have dramatically diminished during the last 2 years.5 In this new epidemiologic situation, the transport and importation of dogs from rural to metropolitan areas within the Mexican territory and internationally constitutes an important risk for reintroduction of rabies in regions where the disease is being controlled. The incorporation of rabies virus molecular typing in the surveillance programs will greatly contribute to monitor the occurrence of animal translocation and to evaluate the appropriate control measures to be applied at the national and international level. The results presented here revealed the presence of several rabies transmission cycles in wildlife species in urban and rural areas of the country and provided evidence for the identification of their most probable reservoirs. Active surveillance on wildlife species will confirm the proposed species and possibly recognize others as rabies reservoirs, determine the geographic distribution of their endemic cycles, and through the antigenic and genetic characterization of the obtained isolates, define the pathways of intraspecies and interspecies rabies transmission. This information is essential for evaluating the risk that these wild species represent for humans and their animals and to assess the need for the implementation of rabies control programs in wildlife using oral vaccination. Although only a relatively small number of samples was analyzed here and they could not provide a more comprehensive description of the molecular epidemiology of rabies in Mexico overall, this contribution serves in the establishment of important baseline data that are necessary to elucidate the patterns of urban and sylvatic rabies transmission in Mexico.

Acknowledgments: We thank Dr. Rina Pedroza Requenes (Direccioń General de Medicina Preventiva de la Secretarıá de Salud) and Dr. Carlos Gonzales and Dr. Marcela Mercado (Centro del Diagno´stico en Salud Animal–Secretaria de Agricultura, Ganaderia y Desarrollo Rural for providing some of the isolates analyzed in this study. We also thank Pamela A. Yager for help in the antigenic characterization of the isolates, and Dr. Charles E. Rupprecht for valuable discussions during the preparation of the manuscript. Authors’ addresses: Cecilia C. de Mattos, Carlos A. de Mattos, Lillian A. Orciari, and Jean S. Smith, Rabies Section, Viral and Rickettsial Zoonosis Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Mailstop G-33, 1600 Clifton Road NE, Atlanta, GA 30333. Elizabeth Loza-Rubio, Centro Nacional de Investigaciones Disciplinarias en Microbiologıá, Instituto Nacional de Investigaciones Forestales Agrıćolas y Pecuarias, Secretaria de Agricultura, Ganaderia, y Desarrollo Rural, Km 15.5 Carretera Me´xico-Toluca Cuajimalpa, 05110 Apartado Postal 41-682 Me´xico City 11001 D.F., Mexico. Alvaro Aguilar-Setień, Unidad de Investigacioń Me´dica en Inmunologıá, Instituto Mexicano del Seguro Social. Av. Cuauhte´moc 330, Col. Doctores, Me´xico City D.F., Mexico. REFERENCES

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