VENEZUELAN ENCEPHALITIS VIRUS ALONG THE CENTRAL AND NORTHERN GULF COAST OF AS OF JULY-SEPTEMBER 1969 1

Drs. Robert W. Dickerman,’ Maria L. Zarate, William F. Scherer,’ and Julio de Mucha-Macias 5

VE virus is endemic in certuin marshy and wet forested habitats in the coastal lowlands of southeastern and central , Mexico. At , , during 1969, there was residual antibody evidente of the 1966 equine outbreak though endemic virus activity was not found. Size- able populations of susceptible equines and humans existed near Tampico and Matamoros, Tamaulipas. Thus spread of the disease to these areas is a real public health threat.

Introduction Sontecomapan on the Gulf coast near Lake (4), and VE virus antibodies were Venezuelan (equine) encephalitis (VE) found as far north as , about virus exists in endemic cycles involving vec- 100 kilometers West-northwest of Sonte- tor mosquitoes and amplifying vertebrates comapan (Figure 1) (5). In 1966, an along the Atlantic coast of Central America equine outbreak, identified by antibody tests and the Pacific coast of Costa Rica and as a VE virus epizootic, occurred in the Guatemala (1) . On the Atlantic lowlands, vicinity of Tampico, Tamaulipas and adja- virus has been isolated by use of sentinel cent northern Veracruz (6, 7). Further hamsters or from mosquitoes or wild verte- studies found antibody in serum of one of brates in Almirante, Panama; northeastern six bovines bled in October 1966 shortly Nicaragua; Puerto Cortes, Honduras; Puerto after the epizootic as far north as Soto la Barrios, Guatemala; Belize, British Hon- Marina in central Tamaulipas (7). This duras; and severa1sites in the State of Vera- epizootic was the first extensive outbreak cruz, Mexico (2-4). During intensive studies of VE virus in animals or man north of of this virus in Veracruz from 1961 to Panama and at most, only the second rec- 1967, virus was isolated only as far north as ognized outbreak in Central America or Mexico, prior to 1969.6 1 These investigations were performed in collabora- tion with the Pan Ameritan Health Organization and The Tampico equine epizootic in 1966 the Government of the United States of Mexico; they were supported in part by USPHS training grant raised the possibility of movement of VE 5-Tl-AI-231 from NIAID, in part by USPHS re- virus along the Gulf coast of Mexico north search grant AI-06248 and in part by research con- tract number DA-49-MD-2295 from the U.S. Army from endemic habitats in southeastern Vera- Medical Research and Development Command, De- partment of the Army, under sponsorship of the Com- cruz (4). It therefore seemed important to mission on Vira1 Infections of the Armed Forces Epidemiological Board. determine and record for future referente, 2 Assistant Professor, Department of Microbiology, the geographic distribution of VE virus along Cornell University Medical College, New York, N.Y., U.S.A. 3Instituto de Salubridad y Enfermedades Tropi- BDuring AprilJuly 1962, a small human epidemic cales, México, D.F., México. of encephalitis occurred in Campeche, Mexico on the 4 Professor and Chairman, Department of Micro- Yucatan Peninsula. There were 12 cases, 3 with biology, Cornell University Medical College, New neurologic sequelae and 5 deaths in ages from 7 York, N.Y., U.S.A. months to 47 years. Convalescent serum from one 6 Instituto Nacional de Virología de la Secretaría de person with sequelae was tested and found to have Salubridad y Asistencia, México, D.F., México. VE virus HI antibody (8). 143 144 BOLETIN DE LA OFICINA SANITARIA PANAMERICANA . Agosto 1971 f this coast, and to learn whether virus was sites increased by lt meters in the latter still active, though clinically silent, in the part of August 1969. Hamsters were ex- Tampico region. Such a study was made posed in both fresh and brackish (non-tidal) during July-September 1969 using antibody marshes. 4) Near , hamsters were tests of serum and isolations of virus from placed in mangroves and in thorn-brush ad- sentinel hamsters to map the virus. jacent to a freshwater cattail marsh at the mouth of Rio Tuxpan. 5) On the South side Materials and methods of the Rio Tecolutla hamsters were placed in mangroves along an estuary of the river. 6) Study sites Near Miraflores (32 kilometers South of Sentinel hamsters were exposed at 11, Nautla) hamsters were exposed in a moist, * near sea-leve1 locations along the Gulf of moderately-grazed forest with tal1 canopy- Mexico, extending from Matamoros, State of forming trees adjacent to open pasture. 7) Tamaulipas on the USA-Mexico border to Laguna la Mancha (50 kilometers north of Minatitlan, State of Veracruz a distance of Veracruz City) was a tidal lagoon bordered about 1,000 kilometers (figure 1). Of this by mangroves. 8) About one kilometer in- distance, approximately the northern 400 land from Barra de ‘Chachalacas, hamsters kilometers are in an arid to semi-arid zone were placed in vegetation along a permanent (localities 1 and 2 described below), the freshwater marsh bordered by open, grazed next 400 kilometers are in a semi-humid swamp forest. 9) Tlacotalpan on the Rio region (localities 3-8) and the southern Papaloapan is an area of savannah and ex- 200 kilometers are in a humid zone (locali- tensive freshwater habitats. Hamster cages ties 9-l 1). 1) Matamoros, a major port of were hung in low willow trees and brush entry in Mexico, has about 100,000 inhabi- along the river. In September 1969 the en- tants, and is directly across the Rio Grande tire area was inundated with nearly two (Rio Bravo del Norte) from Brownsville, meters of water in the village center. 10) Texas. Sorghum and other irrlgated crops At Sontecomapan the study site was a are grown in the area. Hamsters were ex- tropical wet forest of canopy-forming, posed in mesquite trees bordering the lower largely second-growth trees. ll) Near reaches of the Rio Grande in pasture land. Minatitlan hamsters were exposed in swamp 2) La Pesca was a small coastal village forest bordering the extensive freshwater near the mouth of the Rio Soto la Marina in Coatzacoalcos marsh. The latter three study the arid zone of Tamaulipas. Commercial areas have been described and illustrated and sport fishing, some farming and raising elsewhere; Tlacotalpan (9)) Sontecomapan of cattle and goats are the principal occupa- and Minatitlan ( IO). tions. Mangroves reach their northern limit along the coast at La Pesca. Collection of sera Inland from this narrow zone, the land is Pig and goat sera were collected at covered with dense, low, thorny-scrub forest. slaughter houses from severed cervical ves- 3) Tampico and adjacent Ciudad Madero sels as described elsewhere (II). Horses, (about 200,000 total inhabitants) are lo- birds, humans and dogs were bled by veni- cated at the mouth of the confluente of the puncture. Sera were stored at -20°C until Rio Tamasi and the Rio Panuco. These tested. cities are bordered by extensive freshwater marshes and more restricted brackish water marshes which become far more extensive VE virus antibody tests in the latter part of the rainy season (May to Hemagglutination-inhibition (HI) and September). Water depth at some hamster neutralization (N) antibody tests were per- Dickerman et al. * VE VIRUS IN MEXICO 145

FIGURE 1 .-M op of study sites of VE virus in Golf lowland Mexico during July-September 1969. The region where VE virus is currently consídered to be endemic is the shaded orea of the inset.

100km

+-Locotions of VE virus isolated from sentinel hamsters. A-Presente of VE virus ontibodies. O-Study sites without detectable VE virus by use of sentinel homsters. A-Sita with no detectable antibody. *-Places shown for orientotion only. 146 BOLETIN DE LA OFICINA SANITARIA PANAMERICANA . Agosto 1971 formed with Mexican strains of VE virus an endemic region (Minatitlan, Veracruz) (63U2) (3), Nepuyo virus (63Ull) (10) were collected in July 1969 and tested for and Patois virus (63A49) (12) by micro- VE virus HI antibody; 8 of 24 pigs less technics described elsewhere (13, 14), than 2 years of age had detectable HI antibody with titers ranging from 10 to 40. Exposure oj sentinel hamsters and recovery At various locations in northern Veracruz and identification of viruses and southern Tamaulipas, VE virus HI and N antibodies were found in sera of pigs, The methods used have been described horses and humans (table 1) . However, in (10, 12, 15) except that liquid nitrogen the Tampico region, no antibody was found tanks with vapor phase canisters (- lOOoC) in humans less than 3 years of age (i.e., were used for field storage of sick or dead born since the 1966 outbreak), and in hamsters in locations remote from our field only one horse, 1-2 years of age and thus headquarters; otherwise dry ice boxes born since the outbreak. (The HI titer of ( - 60°C) were used. this horse serum was 20 and N titer 4). Horses less than 1 year of age had HI and Results N antibodies, but the prevalence of N anti- VE virus antibody tests of sera from animals body was considerably higher than HI anti- and humans body (table 1). This observation plus the In April 1969, no VE virus N antibody high prevalence of antibodies in horses over was found in sera of 9 pigs and 12 cows 2 years of age and the fact that horses from Matamoros, 16 pigs from Tampico, 3 nurse until at least 1 year of age, made it pigs from Chicontepec, Veracruz and 7 likely that these antibodies in young equines pigs from Magosal, Veracruz; one additional were maternal in origin. HI antibody titers pig serum from Matamoros had an LNI of in horses less than one year of age were 10 1.6 but the HI titer was

TABLE 1-Results of VE virus HI and N antibody tests of 579 vertebrate sera collected from northern Veracrw to northern Tamaulipas, Mexico during July-September 1969.

Fractions with detectable VE antibody in serum and numbers tested Host Age Northern Veraqruz Central & northern sou&t&ernTamauhpas n Tamaulipas 11 N HI N Pigs +-2 yrs. 49 11 Horses < 1 yr. .lP 22 .45 22 .oo 7 .oo 7 1-2 yrs. .09 11 nO9 ll .OO 6 .oo 6 .PO 78 .61 78 *oo 50 .oo 50 Humans ;3y:s* JOO 17 .oo 17 .oo 4 .oo 4 3-20 yrk .OO 81 .oo 38 .oo 17 > 20 ws. .lO 122 .20 73 *OO 4 .oo 4 Ardeidae -< 6 dáys .oo ll’ lo-30 days .OO 24d Icteridae immature .oo 7 adult .oo 17 Goats .oo 46 Dogs .OO 23 .OO 23

n Pigs were from Tuxpan, Veracruz, horses, Icteridae and humans from Tampico, Tamaulipas and vicinity, and Ardeidae from Laguna la Mancha, Veracruz. b Goats were from Cruillas and Soto la Marina, Tamaulipas. Others were from Matamoros, Tamaulipas and vicinity. C8 Cattle Egrets and 3 Louisiana herons. d 23 Cattle Egrets and 1 Common Egret. Dickerman et al. - VE VIRUS IN MEXICO 147

N titers from 4 to 100. The pattems of N tions were 2.0 and 1.7 which were similar antibody titers against three strains of VE to results at Mmatitlan during 1964-1967 virus suggested that these antibodies had (average 2.4), but lower than the previous been engendered by an endemic strain like experience at Sontecomapan during 1963- Mexican 63U2; titers were 2 20 in 8 of 8 1966 (average 4.0) (15). During July and horses with 63U2 virus, in none of 8 with a August 1969, VE virus was isolated for the 1969 Guatemalan epizootic-epidemic strain first time at Tlacotalpan, Veracruz, a loca- (6921) and in only 3 of 8 with the atten- tion where antibody had been found as early uated TC83 vaccine strain. Small samples as 1961 (5), but 216 hamster-days of expo- of wild birds (Ardeidae and Icteridae) from sure during 1964 and 1965 had failed to northern Veracruz and southem Tamaulipas yield virus (15). VE virus was also isolated revealed no antibody in plasmas. north of Veracruz City as Chachalacas near In central Tamaulipas suckling goats were Carde1 (table 2). This site seemed particu- without N antibody and in northern Tamau- larly active, yielding 5.0 VE virus isola- lipas near Matamoros, horses, humans and tions per 100 hamster-days. Four habitats dogs were entirely negative for VE virus between Chachalacas and Tampico, 5 near HI and N antibodies (table 1) . Tampico, one in low vegetation near the coast at La Pesca, Tamaulipas and sites in VE virus isolations from sentinel hamsters vegetation along the Rio Grande adjacent to open fields at Matamoros yielded no strains Again, as positive controls, adult hamsters were exposed during July-August 1969 at of VE virus during more than 1,900 hamster- previously documented endemic habitats of days (table 2). No VE virus HI antibody VE virus in southern Veracruz to reinforce was detected in serum of any of 3 surviving the validity of possible negative results in hamsters at Minatitlan, 6 at Sontecomapan, more northern and arid regions along the 15 at Tlacotalpan, 17 at Tampico or 14 Gulf of Mexico. Two strains of VE virus at Matamoros. were recovered from 15 hamsters exposed at Nepuyo virus in arbovirns group C was Minatitlan and 2 from the same munber of recovered from 5 additional hamsters dying hamsters at Sontecomapan (table 2). Iso- or becoming ill at Minatitlan, from 1 at lations per 100 hamster-days at these loca- Sontecomapan, 1 at Chachalacas and 1 at

TABLE 2-Obsewations of 184 senflnel hamsters (5-11 weeks of age) on the Gulf coast of Mexico during July-August 1969.

No. of State and location hamster- days Veracruz Southem Minatitlan 102 2/15 2.0 to TlacotalpanSontecomapan 504118 2/202/15 0.7 Central Chachalacas 60 3/10 5:o to Laguna la Mancha (30 km N of Cardel) 64 0/8 0 Miraflores(32 km S of Nautla) Tecolutla Northem Tuxpan 13: 0:;” 0 Near Tampico 435 0/26 0 Tamaulipas South 611 ti%ld ~;e~c;mpicoMatamoros 356143 ~;~~0/15 i0 148 BOLETIN DE LA OFICINA SANITARIA PANAMERICANA - Amsto 1971

Tampico. CF antibody to Nepuyo virus objective of these studies was therefore to was found in post-exposure serum of 1 ham- leam whether equine and human populations ster at Tampico and 1 at Matamoros. Patois on the northem Gulf coast of Mexico were group viruses were isolated or CF antibody susceptible or were partially immune to VE found in post-exposure serum from 1 and 3 virus as of 1969. additional hamsters respectively at Minati- The results showed that VE virus existed tlan, 1 and 3 at Sontecomapan, 1 and 1 at during July-September 1969 in marshy and Tlacotalpan, 2 and none at Chachalacas, wet, forested, lowland habitats in Veracruz none and none at Tampico and none and 2 at as far north as Chachalacas nortlí of Vera- Matamoros. Thus apparently arboviruses cruz City. The virus was not present or other than VE virus were also active during sufficiently active to be recovered by the July-August 1969 along the Gulf coastal sensitive sentinel hamster technic (17) north lowlands of Mexico north to the USA border of this point. Also by antibody tests of ani- at Matamoros. Nepuyo and Patois group mal and human sera, there was no conclu- arboviruses were not previously known in sive evidente of persistent VE virus activity Mexico north of southeastern Veracruz (10, near Tampico, and there were large popu- 12). lations of susceptible equines and humans without detectable neutralizing antibody in Discussion serum there and farther north at Mata- These studies were done to learn, as of moros. Thus these regions are open to July-September 1969, the geographic distri- future attack by this virus. bution of VE virus along the Gulf coast of Of course, the precise northern limit of Mexico north of its endemic locations near VE virus in the northern Veracruz region Minatitlan and Sontecomapan, Veracruz. was probably not determined by these Also the question was asked whether the studies since not al1 available marshy or wet virus was still present near Tampico, three forested habitats were tested with sentinel years after it had produced an equine epi- hamsters. Moreover, in the Tampico re- zootic there. Historically the outbreak in gion, though the usual large lakes were 1966 was the first and only equine outbreak present there during July and August 1969, recognized to be due to VE virus north of these months were relatively dry, and it was Panama, prior to 1969 when these studies not until the latter part of August that inten- were done. Thus it raised the question as sive rains began and many fields became to whether the virus recently moved into flooded to provide additional areas for vec- the Tampico region and, if so, did it estab- tor mosquito breeding. Nevertheless, it can lish endemic cycles and remain there, or be stated that the limit of VE virus activity was the outbreak merely due to a temporary during July-September 1969 was somewhere penetration of virus followed by retreat. In in the Chachalacas to Tampico region of 1941, there was an equine encephalitis epi- northern Veracruz State. With further, more zootic near Matamoros, Tamaulipas on the detailed studies of this region, it should be Mexico-U.S.A. border and in Texas, but the possible to map the exact northern limit of virus recovered from horse brain was identi- virus activity, and to learn whether it fied as eastern encephalitis (16). Thus, fluctuates during wet and dry seasons and although the region north from Tampico to whether it is progressively moving north- Matamoros is fairly dry and arid much of the ward. year, evidently arbovirus outbreaks can oc- cur during rainy seasonsand in the presente Summary of irrigation such as exists in the Rio During July-September 1969 VE virus Grande valley near Matamoros. A third was recovered from sentinel hamsters ex- Dickerman et al. * VE VIRUS IN MEXICO 149 posed in marshy or wet, forested habitats lations of equines and humans were suscep- along the Gulf coastal lowlands of Mexico tible there as well as near Tampico. q as far north as Chachalacas (latitude 19” 26’ and longitude 96” 26’) north of Vera- Acknowledgements cruz City. Hamsters north of this site were negative for VE virus although they yielded Dr. Leandro Gonzales Gamboa, Centro de Nepuyo virus in arbovirus group C at Tam- Salud, Tampico, Dr. Louis Sataya Salazar, Centro de Salud, Ciudad Madero and Dr. Jorge pico, Tamaulipas and Patois group viruses at Burguete R., Centro de Salud, Matamoros and Chachalacas. Antibody evidente of senti- their associates enthusiastically supported these nel hamster infections by Nepuyo and Pa- studies and generously made available their tois group arbovirnses also occurred as far facilities. Without their help these investiga- north as Matamoros on the Mexico-U.S.A. tions would not have been possible. Also participation of the following persons border. By antibody tests of sera from is very gratefully acknowledged: Marcus horses, other animals and humans, evidente Simpson, Jr., John P. Mitchell and Frederick of VE virus infection was detected as far W. Schueler, as USPHS-supported trainees, north as Tampico and vicinity, but the re- Drs. Salvador Romero and Mateo Velazquez sults did not conclusively indicate that VE P., as veterinarians and Christine M. Bona- corsa and Maria E. Eugenia Ochoa L., as virus cycled there since the equine epizootic technical assistants in the field and Bette A. in 1966. There was no evidente of VE virus Pancake, Evangeline 1. DuCasse and Karen activity at Matamoros, and thus large popu- Anderson in the laboratory.

REFERENCES (1) Scherer, W. F., Dickerman, R. W. and Or- equina de Venezuela ocurrida en Tamauli- doñez, J. V. “Discovery and geographic pas, México.” Rev Invest Salud Publ distribution of Venezuelan encephalitis vi-

alitis virus in Veracruz with description distribution of virus measured by sentinel of antibody responses.” Bol Ofic Sanit Pan- .hamsters and mice.” Amer J Trop Med amer 6.5: 238-2.52, 1968. (In press) . (14) Scherer, W. F., Campillo-Sainz, C., de (16) ‘Tellez Giron, A. and Valdez Ornelas, 0. “La Mucha-Macias, J., Dickerman, R. W., presencia del virus tipo Este de la encefalo- Wong Chia, C. and Zarate, M. L. “Eco- mielitis equina en la epizootia ocurrida en logic studies of Venezuelan encephalitis vi- el estado de Tamaulipas, México, durante rus in southeastern Mexico. VII. Tnfection el año de 1941.” Rev Soc Mex Hist Nat of man.” Amer J Trop Med (In press) . 2: 251-259, 1941. (15) Scherer, W. F., Dickerman, R. W., Jordan, (17) Srihongse, S., Scherer, W. F. and Galindo, P. E. K., Saidi, S., Zarate, M. L. and Ven- “Detection of arboviruses by sentinel ham- tura, A. K. “Ecologic studies of Venezue- sters during the low period of transmis- lan encephalitis in southeastern Mexico. II. sion.” Amer J Trop Med 16: 519-524, Prevalence and geographic and temporal 1967.

Virus de EV en la costa central y septentrional del Golfo de México, en julio-septiembre de 1969 (Resumen) Durante los meses de julio a septiembre de y Patois en cricetos centinelas, en puntos tan 1969 se recobró virus de encefalitis venezo- septentrionales como Matamoros en la fron- lana (EV) en cricetos centinelas expuestos a tera Mexicano-Estadounidense. Las pruebas habitat pantanosos o húmedos de las llanuras de anticuerpos de sueros de caballo, otros ani- costeras del Golfo de México, a un punto tan males y seres humanos demostraron la presencia septentrional como Chachalacas (19” 26’ de de infecciones de virus EV en Tampico y sus latitud y 96” 26’ de longitud) al norte de la inmediaciones, pero no se obtuvieron resulta- ciudad de Veracruz. Los cricetos que habita- dos concluyentes que indicaran manifestaciones ban al norte de este lugar resultaron negativos de ciclos del virus EV en ese lugar desde la con respecto al virus EV aunque mostraban epizootia equina de 1966. No se demostró virus de Nepuyo en el grupo de arbovirus C ninguna actividad de virus EV en Matamoros en Tampico, Tamaulipas, y virus del grupo y, por consiguiente, una gran población equina Patois en Chachalacas. También se obtuvie- y humana de este lugar, así como de las cerca- ron pruebas de la presencia de anticuerpos de nías de Tampico, era susceptible a la enferme- infecciones por arbovirus del grupo Nepuyo dad.

Virus da encefalite Venezuelana na costa central e do nordeste do Golfo do México, iulho-setembro de 1969 (Resumo)

Durante julho-setembro de 1969 o virus VE ao norte como Matamoros, na fronteira do foi isolado em cobaias expostas em habitat México-E.U.A. Os testes de anticorpos de florestais pantonosos ou úmidos ao longo das serum de cavalos, outros animais e seres hu- terras baixas litorâneas do Golfo do México, manos localizaram urna infeccáo do virus VE táo ao norte como Chachalacas (latitude 19” bem ao norte, como em Tampico e suas vizi- 26’ e longitude 96” 26’), ao norte de Veracruz. nhancas; mas os resultados náo indicaram em As cobaias ao norte dêsse lugar eram negativas caráter definitivo a exist&rcia de um ciclo de ao virus VE embora registrassem virus Nepuyo virus VE alí, desde a epizootia eqüina de 1966. no arbovirus do grupo C em Tampico e Ta- Náo se verificou atividade do virus VE em maulipas, e viroses do grupo Patois em Cha- Matamoros e, portanto, grandes popula@es chalacas. A evidência de anticorpos em co- eqüinas e humanas eram suscetíveis nessa baias infectadas por arboviroses do grupo localidade bem como nas proximidades de Nepuyo e Patois foi registrada, também, táo Tampico. Dickerman et al. * VE VIRUS IN MEXICO 151

Virus de I’encéphalite vénézuelienne le long des parties septentrionale et centrale de la côte du Golfe du Mexique, Juillet-Septembre 1969 (Résumé) Pendant la période Juillet-Septembre 1969, tains hamsters par les arbovirus des groupes on a constaté des cas de contagion par virus Nepuyo et Patois dans des régions aussi éloi- de l’encéphalite vénézuelienne (EV) chez des gnées vers le nord que Matamoros, située entre hamsterstémoins exposés dans les régions boi- la frontière Mexique-Etats-Unis. En testant sées, et humides ou marécageuses, des basses les sérums d’équidés, d’autres animaux divers, terres de la cote du Golf du Mexique, y com- et d’hommes pour la présence d’anticorps, on pris en des points aussi septentrionaux que a pu déceler des cas d’infection par virus EV Chachalacas (19” 26’ de latitude et 96” 26’ de jusque dans le nord-Tampico et ses environs longitude) au nord de Veracruz. Les hamsters -mais depuis l’épizootie de 1966, on n’a postés au nord de cette région n’ont pas réagi trouvé aucune preuve indiquant de manière au virus EV, alors qu’ils ont été touchés par le certaine la présence d’un cycle de virus EV virus Nepuyo du groupe arbovirus C à Tam- dans cette région. A Matamoros, on n’a cons- pico, et par les virus des groupes Tamaulipas taté aucune trace d’activité virale EV, ce qui et Patois à Chachalacas. La présence d’anti- signifie que de nombreux humains et équidés y corps a également révélé la contagion de cer- sont exposés à la contagion.