Venezuelan Encephalitis Virus Research Articles

Experimental infection of North American mammals with epidemic Venezuelan encephalitis virus.

Eighty-three wild mammals of ten species were inoculated with a first mouse passage level Venezuelan encephalitis (VE) virus isolated from south Texas in 1971 during the VE outbreak. Rodents were highly susceptible to VE infection and circulated VE virus at levels ranging from 10(7.7) to 10(11.5) suckling mouse intracranial lethal doses per ml for 2 to 4 days. These levels of virus could easily infect vector mosquitoes. Mortality was high in all species of rodents except Sigmodon hispidus, adult Neotoma micropus, and adult Peromyscus leucopus. Lagomorphs were susceptible to VE infection but circulated VE virus at or near mosquito threshold levels for 1 day only. Raccoons and opossums were relatively resistant to VE infection or circulated low levels of virus in the blood. Juvenile animals of four species were more susceptible and experienced higher viremias than did adults. In two of the four, mortality was higher in the juveniles than in adults. A consideration of results obtained during this study and those of other investigators led to the conclusion that equines were of major importance, that canines, lagomorphs and rodents were of minor importance, and that other species tested were of no importance as virus amplifiers in VE epizootics.

Search for persistent epizootic Venezuelan encephalitis virus in Guatemala, El Salvador and Nicaragua during 1970-1975.

Evidence was sought during 1970-1975 of persistence of equine-virulent Venezuelan encephalitis (VE) virus in regions of Central America that were heavily involved in the epidemic-equine epizootic of 1969. (a) Four sentinel horses were exposed in an arid, upland region of the Atlantic drainage of Guatemala during August-October 1970, but no horse became infected. (b) The epicenter region of the 1969 outbreak, in southwestern Guatemala and southwestern El Salvador, was studied during July 1970-February 1974; no antibody developed in sentinel horses, sentinel hamsters did not die, mosquitoes yielded no virus, wild rats had no detectable VE virus HI antibody. Unexplained decreases in populations of wild terrestrial mammals possibly limited maintenance of VE virus. However, mosquitoes were plentiful and present in the same species composition found at a focus of enzootic VE virus about 35 km northwest of the epicenter region. (c) In studies at two Guatemalan ranches near the epicenter, where horses died in 1969, VE viruse infected sentinel horses along one of three lakes on one ranch during the wet season of 1972 but not during the dry or wet seasons of 1973; the titers of neutralizing antibodies in these four horses were higher against an enzootic strain of VE virus than against an epizootic strain. During 1970 and 1971, VE virus was isolated from sentinel hamsters exposed at a marsh on the other ranch, but Vero plaque characteristics were those of enzootic VE virus. (d) The only epizootic activity of VE virus discovered in Central America in 1970-1975 occurred in Nicaragua between April and June 1972. Several hundred horses died, and N antibody, like that engendered by epizootic virus, was found in two young, unvaccinated horses. Whether this represented persistence of epizootic VE virus or reintroduction of virus is unknown.

Virus-induced pancreatic disease by Venezuelan encephalitis virus. Alterations in glucose tolerance and insulin release

Virus-induced pancreatic disease by Venezuelan encephalitis virus. Alterations in glucose tolerance and insulin release

Virus-induced pancreatic disease by Venezuelan encephalitis virus. Alterations in glucose tolerance and insulin release.

Viral infections have been implicated in the induction of diabetes mellitus in man and laboratory animals. Since virus-specific immunofluorescence (FA) is detectable in hamster pancreas during the acute phase of Venezuelan encephalitis (VE), experiments were designed to correlate pathologic and virologic events with metabolic studies in VE-infected hamsters. Golden Syrian hamsters were inoculated s.c. in groups of four to 12 with 100,000 plaque-forming units (PFU) of the vaccine strain (TC-83) of VE or 1,000 PFU of the virulent Trinidad strain of VE. Ultrastructurally, during Trinidad infection, mature virions were associated with the cell surfaces and within pancreatic beta cells in contrast to absence of virus-related changes in TC-83-infected hamsters. Virus-specific-FA was noted in islet cells and acinar cells of Trinidad-infected hamsters. VE growth curves demonstrated viral replication in pancreas with both strains. Although ultrastructural and FA changes were much more prominent in Trinidad-infected hamsters in contrast to TC-83-infected hamsters during the first few days of illness, the rapid lethality of the Trinidad-infected group necessitated performing all metabolic studies in TC-83-strain-infected hamsters. Accordingly, for the metabolic studies, glucose tolerance tests (GTT) using 2 mg. or 5 gm./kg. glucose i.p. were performed in groups of hamsters acutely infected two days earlier with the TC-83 vaccine strain and in 24-day and 90-day convalescent hamsters after TC-83 vaccine strain. Samples were obtained for glucose and immunoreactive insulin (IRI) determinations. Glucose intolerance occurred in hamsters in each of the infected groups given 5 gm./kg. glucose except for the 90-day convalescent TC-83 group. Severely decreased IRI responses occurred in the 24-day and 90-day convalescent TC-83 hamsters following both 2- and 5-gm./kg. glucose. Pancreatic IRI content in 24-day convalescent TC-83 hamsters was within normal limits, suggesting a defect in IRI release from the beta cells at this stage of convalescence.

Search for epizootic-like Venezuelan encephalitis virus at enzootic habitats in Guatemala during 1969-1971.

Seventy-four strains of Venezuelan encephalitis (VE) virus recovered from sentinel hamsters or mosquitoes at enzootic habitats in Guatemala in the two years following the 1969 epidemic-equine epizootic were examined for ability to produce small plaques in Vero African green monkey kidney cell cultures, like isolates obtained during the epizootic. (a) One strain recovered from a sentinel hamster in late October 1969 at an enzootic habitat near the epicenter of the hemagglutination-inhibition (HI) and equine-virulence properties like epizootic virus; this strain retained its small plaque characteristic after inoculation and recovery from bloods of three horses. (b) None of the other 73 strains produced uniformly small plaques, but 31 formed a few small plaques among large ones. Virions from small plaques of five strains were cloned twice in Vero cell cultures. Four clones produced uniformly small plaques after one more passage in Vero cells; three had hemagglutination-pH properties compatible with epizootic virus or intermediate between epizootic and enzootic virus, but HI tests with these three hemagglutinins or with antibody to the fourth cloned strain showed them to be like Central American enzootic virus. One of three cloned strains tested in horses produced encephalitis and death in one of four horses; another strain produced encephalitis with recovery in one of two horses. (c) Thus these small Vero plaque clones resembled Central American enzootic strains of VE virus in HI and equine-virulence tests, and the small Vero plaque characteristic was not a satisfactory marker for consistently isolating equine-virulent, epizootic VE virions. Nevertheless, this technic led to recognition of one epizootic strain isolated at an enzootic habitat in Guatemala at the end of 1969 outbreak. Whether this strain was there before the outbreak or subsequently penetrated the habitat is uncertain. During the next two years, this strain did not become dominant in that enzootic focus.

Equine viral encephalitis.

The most important neurotropic viral infections of the horse are the arthropod-borne encephalitides. These include Venezuelan encephalitis (VE), eastern encephalitis (EE) and western encephalitis (WE), which are found in the Americas, and Japanese B encephalitis which occurs in the Far East. All the viruses cause encephalitis in man. Between 1969 and 1972 an epidemic of VE occurred in Central America. In 1971 the disease was reported in Texas, where it was brought under control by the vaccination of susceptible horses with an attenuated live virus vaccine and by the reduction of the mosquito population with insecticides sprayed from aircraft. A high titre viraemia occurs with VE virus in the horse and epidemics are maintained by a mosquito/horse cycle; infection of man and other species is incidental. EE and WE have been recognised as separate diseases since 1933 and in the U.S.A. horses are protected by routine vaccination. Epidemics of these diseases are routine vaccination. Epidemics of these diseases are now uncommon. In contrast with VE, both EE and WE viruses are maintained by a bird/mosquito cycle. The viraemia in the horse is generally considered insufficient to infect mosquito vectors; the horse is a "dead end host". Several species of mosquito can act as vectors of VE, WE and EE. The extension of other arthropod-borne diseases to areas originally outside their geographical distribution (e.g. bluetongue in sheep) serves to illustrate the potential of VE, WE and EE to cause disease on other continents.

Vector competence of mosquitoes as a marker to distinguish Central American and Mexican epizootic from enzootic strains of Venezuelan encephalitis virus.

Two epizootic strains of Venezuelan encephalitis (VE) virus from Central America and Mexico were transmitted by a colonized epizootic vector mosquito, Aedes taeniorhynchus, at higher rates than were two enzootic strains when the mosquitoes were infected by intrathroacic inoculation or feeding of virus. Differences in transmission rates also occurred with colonized Aedes aegypti, but were less marked. Following intrathoracic inoculation of A. taeniorhynchus or A. aegypti, epizootic strains grew to slightly higher concentrations in the mosquitoes than did enzootic strains. Intestinal thresholds of infection for A. taeniorhynchus were slightly lower for epizootic than for enzootic virus strains, but were essentially equal for A. aegypti. Only a small percentage of individual Culex pipiens quinquefasciatus mosquitoes supported growth of epizootic VE virus, and only 1 of 6 tested C. p. quinquefasciatus transmitted virus by bite. Thus, transmission and growth of virus in these Aedes mosquitoes distinguished between these epizootic and enzootic strains of VE virus.

Studies of possible movement of Venezuelan encephalitis virus from an enzootic focus in Guatemala during 1971-1974.

During the wet seasons of 1972 and possibly 1971, sentinel horses became infected by Venezuelan encephalitis (VE) virus in a temporally and geographically progressive manner inland from an enzootic marsh focus of virus on the Pacific couast of southeastern Guatemala. During the wet seasons of 1972 and 1973, VE virus was detected by sentinel horses (and a sentinel hamster in 1972) in a small woods 10 km north of the marsh, but virus was undetectable there during the dry seasons of 1973 and 1974 and the wet season of 1974. Culex (Melanoconion) mosquitoes were found in this woods and at the marsh during August 1973. These observations are compatible with movement of VE virus from the marsh habitat during some wet seasons. However, virus activity in this region adjacent to the marsh was quantitatively unpredictable on a yearly basis and occurred in only very focal habitats during 1971 to 1974. Mechanisms of VE virus movement from the marsh are currently unknown, but bats are under study as a likely possibility.

Ecologic studies of Venezuelan encephalitis virus and isolations of Nepuyo and Patois viruses during 1968-1973 at a marsh habitat near the epicenter of the 1969 outbreak in Guatemala.

Ecologic studies of Venezuelan encephalitis (VE) virus at a marsh habitat near the epicenter of the 1969 outbreak in Guatemala revealed that the virus was enzootic there. VE virus was isolated yearly during 1968-1973 from sentinel hamsters exposed during the rainy seasons and from mosquitoes collected during July and August 1970. Hamsters yielded 41 strains of VE virus and virus was detected within 2 km of the edge of the marsh, in its interior, and at its western extreme 18 km from the central study site at La Avellana. One strain of virus came from a hamster that died in the dry season of January 1970. Culex mosquitoes yielded 20 strains of VE virus and Mansonia and Aedes one each. Culex (Melanoconion) and Aedes taeniorhynchus were most prevalent near the marsh. Hemagglutination-inhitibion (HI) and neutralization antibody tests of sera showed that wild terrestrial mammals (opossums and rodents), humans, and dogs, but not wild birds, were frequently infected. Seven of 16 susceptible residents of villages at the edge of the marsh developed antibodies without symptoms during an 18-month period between September 1971 and February 1973. Only 1 of 5 sentinel rabbits, and none of 30 sentinel chickens developed VE HI antibody during August-September 1971, a period when virus activity was readily detected by the use of sentinel hamsters. Five strains of group C arbovirus (one identified as Nepuyo) were recovered from sentinel hamsters during 1968 to 1970, and one strain of Nepuyo virus was isolated from the blood of a person with a febrile illness during 1972. Two strains of Patois group arboviruses were isolated from Culex mosquitoes during 1970.

Interference between virulent and vaccine strains of Venezuelan encephalitis virus in mixed infections of hamsters.

SUMMARY Hamsters inoculated with the TC-83 vaccine strain of Venezuelan encephalitis (VEE) virus were protected against a normally lethal challenge by virulent VEE virus, strain 68U201, inoculated 30 h after vaccination. Protection was correlated with significantly decreased levels of challenge virus in target tissues (spleen and bone marrow). Inhibition of challenge virus replication was correlated more closely with the interferon concentrations in spleen and bone marrow than with the vaccine virus infectivities in these tissues at the time of challenge. The induction of early defence mechanisms involves the efficient interaction of virus with the spleen; although this interaction is more efficient for TC-83 vaccine, virulent VEE viruses are sensitive to the induced protective mechanism. Interferon appears to be a mediator of this mechanism.

An attenuated variant of Eastern encephalitis virus: biological properties and protection induced in mice.

Wild type Eastern equine encephalitis virus (E) was compared with a mutant (Em) derived from it. The latter was tested as an attenuated vaccine in mice. They differed in the following properties: Em formed smaller plaques on chick embryo (CE) cell monolayers and, unlike E, did not plaque on mouse embryo (ME) monolayers. Futher, Em had a longer latent period and attained a lower peak titer than E after infection of CE cells, was more senssitive than E to chick interferon, and was less virulent for mice (SC and IP routes) and hamsters (IP route) than E. Both viruses were similar in several other properties tested. The mutant was found to induce a gradient in the specificity of protection in mice against challenge by selected viruses after a single subcutaneous injection of living virus. The protection was best against autologous (Em) challenge, was next best against challenge by the virulent parent (E) virus, but was not demonstrable against cross challenge by Venezuelan encephalitis (V) virus. Conventional hemagglutination-inhibiting (HI), complement-fixing (F), and neutralizing (N) antibodies could not be detected in Em-immunized mice even when fresh monkey or guinea pig serum was included in Ntests to provide complement and/or accessory factor(s). However, N antibodies were detected in protected mice by an indirect antiglobulin test. Passive protection by serum or ascites fluids (a.f.) was characterized by a lower but otherwise similar protection gradient like that found after active immunization with virus as described above. Interferon was not detected in the a.f. used for passive protection, nor was heterologous interference evident in Em immunized mice challenged 18 days later with vaccinia or vesicular stomatitis virus. Immunized mice that survived autologous (Em) challenge showed broadened protection against a second challenge by parent E virus, and cross protection against V virus. This typical protection was associated with the presence of HI and conventional N antibodies, except for V which showed no detectable neutralizing antibodies by either a standard or antiglobulin technique.

ECOLOGIC STUDIES OF VENEZUELAN ENCEPHALITIS VIRUS IN PERU DURING 1970–19711

Venezuelan encephalitis (VE) virus has intermittently produced epidemics and equine epizootics on the dry Pacific coastal plain of Peru since at least the 1930's. However, evidence that the virus exists in the Amazon region of Peru to the east of the Andes mountains was not obtained until antibodies were found in human sera collected in 1965, and 10 strains of the virus were isolated in a forest near the city of Iquitos, Peru during February and March 1971. Eight strains came from mosquitoes and two from dead sentinel hamsters. Three hamsters exposed in forests near Iquitos developed VE virus antibodies suggesting that hamster-benign strains also exist there. Antibody tests of equine sera revealed no evidence that VE virus was actively cycling during the late 1950's or 1960's in southern coastal Peru, where equine epizootics had occurred in the 1930's and 1940's. In northern coastal Peru bordering Ecuador, antibodies were present in equine sera, presumably residual from the 1969 outbreak caused by subtype I virus, since neutralizing antibody titers were higher to subtype I virus than to subtypes III or IV. No VE virus was detected in this northern region during the dry season of 1970 by use of sentinel hamsters. The possibility is considered that VE epidemics and equine epizootics on the Pacific coast of Peru are caused by movements of virus in infected vertebrates traversing Andean passes or in infected vertebrates or mosquitoes carried in airplanes from the Amazon region.

ANTIGENIC AND BIOLOGIC CHARACTERISTICS OF VENEZUELAN ENCEPHALITIS VIRUS STRAINS INCLUDING A POSSIBLE NEW SUBTYPE, ISOLATED FROM THE AMAZON REGION OF PERU IN 19711

Nine strains of Venezuelan encephalitis (VE) virus isolated from the Amazon region of Peru in 1971 were identified as antigenic subtype I based on plaque-reduction neutralization tests with four and 20 units of antibody. A tenth strain, 71D1252, was possibly a new subtype, but was related to subtypes I and III. Hemagglutinins of each strain made from infected mouse brains had optimals pHs of 6.2 and 6.4. Nine strains were pathogenic for adult hamsters and adult mice, but strain 71D1252 inapparently infected some adult hamsters and mice inoculated peripherally. Plaques of nine strains in Vero African green monkey kidney cell cultures were intermediate in size between representative epizootic and enzootic strains, but plaques of strain 71D1252 were small like epizootic strains.

Growth of Venezuelan encephalitis virus and disapperance of coxsackie A, Rous sarcoma, herpes simplex and vaccinia viruses following inoculation of Aedes aegypti and other mosquitoes.

Venezuelan encephalitis virus, an RNA-containing virus, uniformly grew to high titers in Aedes aegypti and A. triseriatus mosquitoes following intrathoracic inoculation. The minimal intrathoracic infectious dose per A. triseriatus was about 10 chicken embryonic cell culture pfu whereas the minimal oral dose was about 10,000 pfu. In contrast, only some inoculated C. tarsalis and very few C. pipiens quinquefasciatus supported viral growth. Failure of VE virus to grow in C. tarsalis and C. p. quinquefasciatus was probably not caused by virus inhibitors in mosquito tissues since no virus inhibitors were found in triturated mosquitoes of the 2 Culex or 2 Aedes species. Three RNA-containing non-arboviruses, Coxsackie A5 and A7 and Rous sarcoma, and 2 containing DNA, herpes simplex and vaccinia, did not multiply in Aedes aegypti following intrathoracic inoculation, nor did Rous sarcoma virus grow in Culex p. quinquefasciatus . Persistence of infectious Coxsackie A7 and vaccinia viruses for at least a week in mosquitoes indicated that isolations of these and similar viruses from mosquitoes collected in nature might represent virus survival without multiplication.

Correlates to increased lethality of attenuated Venezuelan encephalitis virus vaccine for immunosuppressed hamsters.

Splenectomy or pretreatment of adult hamsters with cyclophosphamide (Cytoxan) increased the lethality of the TC-83 vaccine strain of Venezuelan encephalitis virus (VEE), inoculated subcutaneously, from 12% for normal hamsters to 75 and 76%, respectively. Neither splenectomy nor cyclophosphamide treatment significantly increased the lethality of Pixuna virus. Cytoxantreated (Cy) hamsters developed and maintained levels of TC-83 virus higher than normal infected controls in blood, brain, spleen, and femoral bone marrow; splenectomy had a similar but less intense effect. A severe myeloid necrosis of femoral bone marrow developed 4 to 9 days after TC-83 virus inoculation in 78% of the Cy hamsters and in 48% of the splenectomized (Sx) hamsters. In contrast, only 13% of normal TC-83-infected hamsters developed this lesion. Extensive hemorrhagic lesions in the olfactory lobes and adjacent areas of the brain also developed more frequently in Cy or Sx hamsters than in normal infected controls. Lethally infected hamsters developed and maintained a severe thrombocytopenia, which may be related to the bone marrow lesion and to the hemorrhagic manifestations of lethal VEE infections.

The involvement of dogs in endemic cycles of Venezuelan encephalitis virus.

The involvement of dogs in endemic cycles of Venezuelan encephalitis virus.

Growth curves and clearance rates of virulent and benign Venezuelan encephalitis viruses in hamsters.

Four virulent strains of Venezuelan encephalitis virus attained higher concentrations of infectious virus in bloods of adult hamsters than two benign strains when given subcutaneously. One benign virus, the attenuated TC83 vaccine strain, reached higher concentrations in bone marrow and Peyer's patches than virulent subtype I strains, and another benign virus, subtype IV, grew to lower levels. When inoculated intracranially, the two benign viruses attained high concentrations in brain, but did not significantly alter in lethality although morbidity was increased. Intracardiac inoculation failed to increase virus concentrations of two benign viruses in blood or hematopoietic tissues above those found after subcutaneous inoculation, nor did it increase lethalities to those of virulent virus. Three benign virus strains were cleared more rapidly from hamster plasmas than six virulent strains. Differences in clearance rates were apparently not due to destruction of benign viruses by blood or tissue components. Thus viral concentrations in blood correlated directly and clearance rates inversely with hamster virulence, whereas the rate and extent of growth of a VE virus in hematopoietic or brain tissues did not correlate with ability to kill hamsters.

Homogeneity of Venezuelan Encephalitis Virion Populations of Hamster-Virulent and Benign Strains, Including the Attenuated TC83 Vaccine

A hamster-virulent strain of Venezuelan encephalitis virus (BeAn8, subtype III) and a benign strain (BeAr35645, subtype IV) each yielded 100 clones with virulence properties like those of the parental virion population. Another hamster-virulent strain (63U2, subtype I, variety E) was heterogeneous since 2 out of 100 clones were less virulent for hamsters than the other 98 clones or the parental virus population. The attenuated TC83 vaccine strain, (a derivative of subtype I, variety A), although over 99% homogeneous with respect to hamster virulence, was unstable since virulent virions could be selected from the parental population by passage through hamsters.

Ecological Profile of Culex (Melanoconion) aikenii (Diptera: Culicidae), Vector of Endemic Venezuelan Encephalitis in Panama1

Preliminary notes are presented on the ecology of Culex aikenii (Aiken & Rowland), vector of Venezuelan encephalitis (VE) in the middle Chagres river basin of Panama. C. aikenii probably represents a complex of at least 2 closely related species with similar habits and both are involved in the natural transmission of VE virus. However, until taxonomic work now in progress is completed the name in its present broad sense will be used. Immature stages of the species are almost always found closely associated with the aquatic floating plant water-lettuce, Pistia stratiotes . Eggs arc laid singly on the leaves. Larvae and pupae remain motionless for hours attached to airpockets under the Pistia leaves. When Pistia plants are dislodged from their anchorage by flooding, larvae and pupae move down with them and may seed new areas where the plants become tangled and proliferate. These movements of Pistia seem to serve the double effect of restricting growth of local populations of C. aikenii and of serving as one method for dispersal of the species. Females have a broad spectrum of hosts both homeothermic and poikilothermic. They have a slight predilection for mammals over birds. Among the latter, herons are the preferred hosts. The species bites a variety of mammals, but seems to prefer rodents, except where there are large concentrations of domestic mammals, such as cattle. They feed on blood between dusk and dawn with peaks of activity occurring irregularly throughout the night. Females feed preferably on the ground, seldom going up to the canopy of trees in search of hosts. Man is usually bitten very low on the legs, so that people wearing shoes and long pants arc seldom fed on.

Growth of Venezuelan encephalitis virus in embryonic cell cultures of wild birds.

Primary embryonic cell cultures made from nine species of wild and domestic birds were infected with Venezuelan encephalitis virus and reproduced each strain to moderately high levels.