Strains Of Venezuelan Encephalitis Virus Research Articles

Markers of Venezuelan encephalitis virus which distinguish enzootic strains of subtype I-D from those of I-E.

Strains of Venezuelan encephalitis virus isolated from enzootic habitats during interepizootic periods in Middle America and northern South America can be distinguished from each other antigenically by hemagglutination inhibition. This test has provided the basis for the classification of these virus strains into subtypes I-E and I-D, respectively. Virus strains of these two subtypes have been found to differ profoundly with respect to virulence for English short hair guinea pigs. Studies are described which confirm that virus strains of the I-D subtype are guinea pig virulent, and that virulence is not the result of cocycling subpopulations of epizootic subtype I-AB or I-C virions. Two additional markers were found which distinguish subtype I-D and I-E Venezuelan encephalitis virus strains. Firstly, hydroxylapatite chromatography of intact virions at pH 6.5 showed differential elution of I-D and I-E prototype strains. Virions of subtype I-D strains eluted at 0.08 to 0.11 M phosphate, while those of subtype I-E strains eluted at 0.15 to 0.20 M phosphate. Secondly, the isoelectric points of the E1 envelope glycoproteins of the I-D and I-E prototype strains were significantly different; pH 6.85 to 7.00 and pH 7.25 to 7.30, respectively. There was no significant difference in the isoelectric points of the E2 envelope glycoproteins. These distinguishing characteristics most likely reflect a fundamental difference in virion surface structure.

Ecologic observations of Venezuelan encephalitis virus in vertebrates and isolations of Nepuyo and Patois viruses from sentinel hamsters at Pacific and Atlantic habitats in Guatemala, 1968-1980.

La Avellana and Puerto Barrios, two enzootic foci of Venezuelan encephalitis (VE) virus on the Pacific and Caribbean lowlands (respectively) of Guatemala have been studied over a 13-year period. Data from sentinel hamsters and guinea pigs and wild and domestic vertebrates are reported. VE virus strains were isolated from hamsters each period they were exposed during the rainy seasons 1968-1980 and at the end of the dry season 1974. Rates of isolation of VE virus ranged from 0.2%-5.7% hamster/days/exposure. All strains tested were free of epizootic virions. Although virus was isolated from sentinel guinea pigs, their deaths were not attributable to infection with VE virus. Antibody titers in 26 of 28 terrestrial mammals bled at La Avellana in 1971 were higher to enzootic than to epizootic VE strains. Thirty-seven percent of 109 residents of Puerto Barrios had antibody to VE virus. In 13 of 20 tested, antibodies were engendered by the enzootic strain. Nepuyo and Patois viruses were isolated from sentinel hamsters at both La Avellana and Puerto Barrios.

An unusual strain of Venezuelan encephalitis virus existing sympatrically with subtype I-D strains in a Peruvian rain forest.

In 1971, an unusual strain of Venezuelan encephalitis (VE) virus (71D1252) was recovered from the same small area of a rain forest in the western Amazon basin of South America near Iquitos, Loreto, Peru, from which strains of subtype I-D were recovered. The marker characteristics of this strain resembled most closely those of VE subtype III (Mucambo) and were distinctly different from coexisting I-D strains. Thus the concurrent presence of two different VE virus subtypes in one place was a striking exception to the usual geographic allopatry of VE virus subtypes. Strain 71D1252 also contained temperature sensitive (ts) (37 degrees C versus 39 degrees C) virions in the original mosquito suspension and first suckling mouse passage brain tissue suspensions. It thus represents one of the few so-far-reported ts strains of viruses found in nature, and the only natural ts strain of VE virus.

Marker characteristics of Venezuelan encephalitis virus strains isolated before and after epidemics and equine epizootics in Middle America.

Ninety-four strains of Venezuelan encephalitis (VE) virus isolated from sentinel hamsters exposed in the Middle American countries of Mexico, Guatemala, Belize, and Honduras were examined for the presence of virions with marker characteristics of strains that cause large epidemics and equine epizootics. Thirty-four strains came from before and 60 strains came from after the Middle American epidemics and equine epizootics of 1966 and 1969-1972. Twenty-three virion clones that resembled epizootic strains by hydroxylapatite chromatography and Vero monkey kidney cell plaque size determinations were characterized further. However, the predominant virions in these clones were like enzootic strains from Middle America north of the Panama Canal region, and not like Middle American epizootic VE strains, since they were in hemagglutination-inhibition antigenic subtype IE, usually had optimal pH of hemagglutination at 6.2, and were avirulent for English shorthair guinea pigs inoculated subcutaneously. These results provide evidence against the theory of origin of epidemic-equine epizootic VE virus strains that posits that epizootic virions emerge in Middle America from strains containing mixtures of enzootic and epizootic virions in enzootic habitats.

Mesenteronal infection threshold of an epizootic strain of Venezuelan encephalitis virus in Culex (Melanoconion) taeniopus mosquitoes and its implication to the apparent disappearance of this virus strain from an enzootic habitat in Guatemala.

Culex (Melanoconion) taeniopus is a vector of Venezuelan encephalitis (VE) virus at a marsh focus in Guatemala and has low mesenteronal thresholds for infection by and transmission of two enzootic strains of VE virus. In contrast, samples of natural populations and subsequent F2 and F4 generations of these mosquitoes have a high mesenteronal threshold for infection by an epizootic VE strain isolated at the same marsh during the end of the 1969 VE epidemic-equine epizootic. The resistance of Cu. (Mel). taeniopus to mesenteronal infection by this VE strain probably represents a key factor in the apparent disappearance of epizootic VE virus from the marsh focus following the 1969 outbreak.

Intestinal threshold of an enzootic strain of Venezuelan encephalitis virus in Culex (Melanoconion) taeniopus mosquitoes and its implications to vector competency and vertebrate amplifying hosts.

The minimal intestinal dose of an enzootic strain of Venezuelan encephalitis (VE) virus for Culex (Melanoconion) taeniopus mosquitoes caught at a marsh habitat of VE virus in Guatemala was less than five plaque forming units (pfu) of virus. Ingestion of this dose of virus in blood of viremic hamsters resulted in transmission of virus to other hamsters. This low intestinal threshold of an enzootic strain of VE virus indicates that the natural Guatemalan population of Cu. (Mel.) taeniopus can acquire VE virus from vertebrates that have viremia levels as low as 1,000-5,000 pfu/ml of blood, provided other factors do not limit virus interchange between mosquitoes and vertebrates.

Studies of Venezuelan encephalitis in migrating birds in relation to possible transport of virus from South to Central America.

All of 13 species of northward migrating shorebirds and 7 species of songbirds captured on the Pacific coast of Guatemala during April and May of 1974--1976 were susceptible to infection with small doses of either an epizootic or an enzootic strain of Venezuelan encephalitis (VE) virus. They produce moderate to high levels of viremia for 2--4 days post-inoculation; levels high enough to infect both epizootic and enzootic vector mosquitoes. Viremias were often sufficient even on the 3rd day after inoculation, a time that might represent the end of a migratory flight, assuming that the physiological state of the birds after capture reflected that during migratory flight. Birds of many taxa react similarly to infection with strains of VE virus, and have the potential for being moderately to highly effective amplifying hosts. However, whether northward migrating birds could have been the agents for the introduction of the epizootic Ecuadorian strain that initiated the middle-American epizootic of 1969--1971 is less clear. Data are not available for the extent, rates or routes of migration between the region of Ecuador and Central America, but the best information on the real speed of migration from elsewhere indicates that even warblers that fly more slowly than shorebirds could make the flight in 72 hours or less. Still, that there are geographically segregated subtypes of VE virus suggests that avian transport has been of minimal importance over long time spans. The role of inactivated vaccines in the middle-American epizootic remains an open question.

Further Observations on Infections of Guinea Pigs with Venezuelan Encephalitis Virus Strains *

Guinea pigs from a Guatemalan colony died after subcutaneous inoculating of moderately small doses of equine-benign strains of Venezuelan encephalitis (VE) virus of hemagglutination-inhibition subtype I-E from enzootic habitats in Mexico and Guatemala. Thus these guinea pigs were unlike English short hair and inbred 13 guinea pigs, which usually survive infections with equine-benign VE strains of subtype I-E. We therefore caution others that not all strains of guinea pigs can be used to evaluate the potential equine virulence of VE viruses.

Heterogeneity of envelope polypeptides among strains of Venezuelan encephalitis virus

Abstract Comparative virion polypeptide analysis of 29 strains of purified Venezuelan encephalitis virus by electrophoresis side by side on discontinuous, slab polyacrylamide gels revealed heterogeneity in electrophoretic mobility and apparent numbers of polypeptides. All strains had two common proteins: a capsid protein of 36,000 molecular weight, and an envelope glycoprotein of 50,000–51,000 molecular weight. Another glycoprotein ranged in molecular weight between 51,000 and 55,000. Eight strains yielded an additional glycoprotein peak in the range of 56,000–58,000; two others showed a small glycoprotein peak at 45,000–46,000. These polypeptide patterns were not influenced by the cell species (chick, hamster, monkey) in which the virus was cultured.

Infections of congenitally athymic (nude) and normal mice with avirulent and virulent strains of Venezuelan encephalitis virus.

Two strains of Venezuelan encephalitis virus that are avirulent for normal BALB/c mice inoculated subcutaneously were also avirulent for infected congenitally athymic (nude) mice of the same strain. Viremias were of similar magnitudes and durations in normal and nude mice. Brain concentrations were higher in nude mice with the one avirulent strain tested, although the periods of detectable virus in brains were similar. No lesions were found in brains, spleens, or lymph nodes by ordinary histopathological examination. Viral neutralizing antibody titers in plasmas at 1 to 3 weeks after infection were lower and more transient in nude than in normal mice, and implantations of thymic tissues into nude mice partially restored their neutralizing antibody responses. Concentrations of spleen cells producing antibodies that lysed sheep erythrocytes 4 days after inoculation of erythrocytes and avirulent virus into nude mice were above the levels of uninfected nude mice. These concentrations were similar in infected and uninfected normal mice. In contrast, two mouse-virulent strains of Venezuelan encephalitis virus killed nude mice faster than normal mice after subcutaneous inoculation. Yet concentrations and durations of virus in bloods and brains were not consistently different between nude and normal mice. There were perivascular monocytes in brains and slight architectural alterations of spleens and lymph nodes. Concentrations of spleen cells producing antibodies hemolytic for sheep erythrocytes 4 days after inoculation with erythrocytes were depressed in nude and normal mice by infection with virulent strains.

Responses of guinea pigs to infections with strains of Venezuelan encephalitis virus, and correlations with equine virulence.

Five equine-virulent strains of Venezuelan encephalitis (VE) virus consistently killed guinea pigs (strain English short hair) inoculated subcutaneously, whereas ten equine benign strains did not. Two enzootic eastern Panamanian and Colombian strains of hemagglutination-inhibition antigenic subtype I-D and of undetermined virulence for equine animals also killed guinea pigs. This subtype of VE virus may represent an excpetion to a positive correlation between equine virulence and guinea pig lethality since another strain of ts subtype has been reported to be benign in horses inoculated subcutaneously. Within the strains tested, viruses of subtypes I-ABC and I-D were lethal for guinea pigs, and viruses of other subtypes were benign. There was no correlation between guinea pig and human virulence of VE strains.

Search for subtype I strains of Venezuelan encephalitis virus among isolates from Trinidad, W.I., during 1960-1972.

Twenty-one strains of Venezuelan encephalitis (VE) virus isolated from three habitats in Trinidad, W.I. during 1960 to 1972, were subtype III (Mucambo) VE virus by plaque-reduction neutralization tests. Like prototype Mucambo virus, each strain killed 8- to 15-week-old mice inoculated intraperitoneally. If the subtype I strain of VE virus that caused a major outbreak in Trinidad during 1943 to 1944 persisted on the island into the 1960s and early 1970s, it did not become the dominant VE virus in these three enzootic foci.

VIREMIA IN YOUNG HERONS AND IBIS INFECTED WITH VENEZUELAN ENCEPHALITIS VIRUS

Fifty-seven of 61 nestling, 8- to 30-day-old herons of three species (Black-crowned Night Heron, Great Egret, and Snowy Egret), developed viremia lasting one to three days following subcutaneous inoculation with small doses of endemic or epidemic strains of Venezuelan encephalitis virus from Mexico, Guatemala or Venezuela. Two epidemic strains from Guatemala or Venezuela stimulated levels of viremia similar to those following infection with enzootic strains. Great Egrets, Striated and Boat-billed Herons and Scarlet Ibis older than 30 days of age developed viremias of lower levels and shorter durtions than did young birds. Marked differences in levles of viremia were not observed among Black-crowned Night Herons, Great Egrets, or Snowy Egrets. Over 50% of viremic blood samples from herons 8-30 days of age contained 1000 or more chick embryo cell culture plaque forming units of Venezuelan encephalitis per ml, levels sufficient to infect some vector species mosquitoes.

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.

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.

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.

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.