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Summary Comparison of seven strains of WEE virus by neutralization, hemagglutination-inhibition and complement-fixation indicated considerable antigenic differences among these strains. Four strains (RI-Y, RI-C, A-42 and McM) maintained for several passages in the laboratory were indistinguishable by any of the serologic methods employed. Three other strains (Florida, Massachusetts, and HJ), representing relatively recent isolates, showed significant differences from the four laboratory-maintained strains in at least two or all three of the serological tests. In addition, significant one-way cross-differences were recognizable among these three strains by one or more of the serologic methods utilized. These antigenic variations were further reflected by differences in the prevalence of HI antibodies to WEE in approximately 1000 bloods screened with RI-Y and HJ strains. The number of positive HI reactors was twice as high with the HJ strain as with the RI-Y strain.

Summary A method for monitoring variations in antigenic constitution of arbovirus strains has been developed. The technique is based upon the magnitude of cross neutralization of virus strains by antisera prepared against virus fractions obtained from gradient sedimentation of a single strain. At a specific point in time, antigenically distinct Western encephalitis viral populations were found to coexist within various tissues of an infected mouse. The antigenic nature of such populations was found to be unstable and in a continuous state of flux. Eastern encephalitis virus strains had distinct antigenic properties when stocks were examined directly. The antigenic properties also varied when strains were carried from one passage to another in mice or in tissue culture, or when passaged in parallel in mice and tissue culture. The phenomenon of antigenic variation of strains was explained in part by the differential selection and ultimate accumulation of certain antigen subpopulations over others during virus replication in various tissue or cell types.

Summary Genetron-treated fluids of chick embryo cultures infected with Eastern and Western equine encephalomyelitis viruses were found, after freezing and thawing, to possess hemolytic activity over a wide temperature range (4 to 37°C). The virus hemolysin and hemagglutinin were inactivated by heat, whereas treatment with trypsin, ether and Ca++ tended to cause a selective suppression of the hemolysin activity of both viruses. Certain changes in the pH at which assays were performed resulted in a reduction of hemolytic activity which was greater than any change in hemagglutinating activity. Treatment of chick red blood cells with RDE and trypsin failed to remove the receptors for either of the activities. However, trypsin treatment significantly increased the hemagglutinating activity of both viruses. The hemolysins were inhibited by specific viral antibody and were sedimentable with the hemagglutinins.

Summary Examination of Navarro, Kwatta,and Mossuril viruses by electron microscopy revealed typical rhabdovirus morphology. They are longer than most other rhabdoviruses. All three viruses were observed in sections of infected mouse brain while Navarro and Kwatta viruses were also observed in infected Vero cells. The three viruses were unrelated antigenically to each other and to other rhabdoviruses of animals.

SummaryA total of 25 arboviruses representing 5 different serogroups (A, B, C, Bunyamwera and California) were studied and found to possess hemolytic activity. With the exception of Japanese B virus, special treatment of viral antigens was necessary to demonstrate hemolytic activity by the group B viruses. The hemolytic properties appeared to be a common property of the arboviruses.

SummaryNariva virus induced syncytium formation in BHK-21 and Vero cultures. Such infected cultures showed positive hemadsorption for guinea pig red blood cells (RBC). A hemagglutinin for guinea pi...

Summary A probing ecologic study was conducted in south-central Florida during May, June and July, 1960, and August, 1961, to determine the nature and distribution of arthropod-borne viruses in this area. Collections for virus and/or antibody examination included 1380 specimens composed of arthropod pools, birds, small mammals, reptiles and sentinels (mice and chicks). EEE virus was isolated from two pools of Culex mosquitoes (mixed species) and from a Common Grackle. WEE was recovered from a pool of Aedes taeniorhynchus, and the Highlands J strain of WEE from the blood of two Blue Jays and the brain of a sentinel mouse. Antibody rates in birds which were high for EEE (21%) and WEE (23%) in the 1960 collection and indicated recent infections, were markedly lower (2.7% and 3.4%, respectively) in 1961. Bird plasma reactors to SLE were similar (8% in 1960, 5% in 1961), except positives were found with blood from juvenile birds in 1961 but not in 1960, indicating probably recent activity of SLE or a related group B arthropodborne virus in the 1961 bird population sampled. HI antibody rates in wild-caught rodents in 1960 were: WEE, 10%; SLE, 30%; EEE, 0%. There were marked differences in two WEE strain antigens when used to measure antibody levels in birds. Forty-three or 10% of the samples were positive using the RI strain and 95 or 23% using the HJ strain of WEE. Moreover, a variance in the level of antibody was noted in bird plasmas examined by the HI and neutralization tests. These differences were attributed to the asynchronous development and/or disappearance of these antibody types.

Preparations of eastern encephalitis virus were disrupted by treatment with sodium deoxycholate. Tween 80 and ether, and sodium lauryl sulfate. The effect of these treatments on the viral specific activities of eastern encephalitis virus were studied. Untreated and disrupted preparations of eastern encephalitis virus were physically characterized by buoyant density centrifugation in cesium chloride density gradients and by rate zonal sedimentation in sucrose density gradients. Disruption by both sodium deoxycholate and Tween 80-ether resulted in the release of hemagglutinating subunits of higher density than the hemagglutinin associated with the intact virion. The hemagglutinating subunits released by both treatments were similar in density and size. A dense complement-fixing component, believed to represent released viral nucleoprotein core, was detected in cesium chloride gradients after sodium deoxycholate treatment of the virus. It was not detected in untreated preparations or in preparations treated with Tween 80-ether. Even at low concentrations, sodium lauryl sulfate was destructive of all viral activities save complement-fixation activity. The hemolytic activity of eastern encephalitis virus was physically closely associated with infectivity and hemagglutinating activity of the virus and was apparently dependent on the structural integrity of the virion.

Antigenic relations among viruses isolated from bats were studied by complement-fixation, hemagglutination-inhibition, and neutralization testing. This information would help to determine which of these viruses could be identified as strains and which could be recognized as antigenically distinct agents. The viruses were also characterized with respect to their comparative infectiousness for several host systems. Most of the viruses isolated from bats have been previously placed in serogroup B; two were considered ungrouped. The group B viruses included Rio Bravo, Burns, Dakar, BP 180, Ib An 8646, Bukalasa, and Entebbe virus. Antigenic studies indicated that BP 180 and Ib An 8646 were strains of Dakar virus. Bukalasa virus was shown to be closely related to the Dakar virus strains. Rio Bravo and Burns viruses were indistinguishable and were distantly related to MML virus, which, by certain tests, was also distantly related to the Dakar virus strains. The two ungrouped viruses, Kern Canyon and Lagos, were not related to one another nor to any of the other viruses. Plaque formation on LLC-MK2 or Vero cell cultures, or both, and the production of cytopathic effects (CPE) in BHK-21 cell cultures were also studied. Lagos bat and Bukalasa viruses did not produce plaques and Lagos bat virus did not produce CPE in BHK-21 cell cultures. A comparison of the in vitro assays with assays performed in suckling mice indicated that the intracerebral inoculation of suckling mice provided a slightly more sensitive assay system than any of the in vitro systems. Neutralization in LLC-MK2 cell cultures was significantly enhanced by the presence of “fresh” normal serum in the neutralization mixtures, although in certain instances, the enhancement of neutralization was accompanied by a lessening in the specificity of the neutralization reaction.

Two complement fixation (CF) components were detected in all of the various preparations (mouse liver, brain, serum, and BHK-21 cell culture) of Oriboca examined by equilibrium density centrifugation. The CF components possessed a buoyant density of 1.21 and 1.18 g/ml, and they were precipitated by treatment with protamine. Analysis of Oriboca virus by rate zonal sedimentation separated three components. The most rapidly sedimenting component was the infectivity which cosedimented with a peak of hemagglutinating (HA) activity designated VHA. A second, more slowly sedimenting, noninfectious HA component (HA-2) was followed by a peak of CF activity which barely moved from the top of the gradient. The single rate zonal CF peak and the two density gradient CF components were broadly cross-reactive serologically with Murutucu antiserum. They could not be identified as Oriboca on the basis of their CF reactions with Murutucu and Oriboca antisera. Oriboca-infective virions possessed an equilibrium density of 1.20 g/ml if derived from BHK-21 cell culture fluid concentrates or mouse serum. Infective virions prepared from mouse brain or liver banded at a buoyant density of 1.17 to 1.18 g/ml. These density differences, along with observed differences in reactivity with protamine and in rate of neutralization, are consistent with the interpretation that these are related to the problem of whether the source preparations of Oriboca represented primarily intracellular or extracellular particles.

Summary Of 129 different arboviruses (134 strains) inoculated into cultures of the baby-hamster kidney-cell line BHK-21 (clone 13), all but five viruses produced cytopathic effect. The agents tested comprised members of 18 serogroups and a large number of ungrouped arboviruses. Comparative titrations of 10 viruses indicated that BHK-21 tissue culture, although slightly less sensitive than infant mice, could be of value as a routine assay system. Results were satisfactory in a limited series of BHK-21 tissue-culture neutralization tests. A total of 31 arboviruses, 10 in the larger serogroups and 21 mostly ungrouped, was tested in tissue culture for hemadsorption of guinea-pig erythrocytes. Results were negative except for one ungrouped virus, both strains of which were shown to be a myxovirus. The applicability of the hemadsorption test as a screening method in field laboratories is discussed.