Carl M. Eklund

Virologic and neurohistologic findings in dairy goats affected with natural scrapie.

Virologic and neurohistologic findings in three dairy goats that became affected with scrapie while living with naturally infected Suffolk sheep were essentially like those in affected sheep. Virus, detected by mouse inoculation, was widespread in non-neural sites, particularly in lymphatic tissues and intestine. In most sites, titers of virus ranged from 3.0 to 3.5 log10 mouse intracerebral LD50/30 mg of tissue. Virus was in nervous tissue in much higher titer. Ranging from 5.1 to 5.8 log10, the highest mean titers were in the diencephalon, midbrain, medulla oblongata and cerebellar cortex—sites of the most severe histologic changes. Although these changes were like those in naturally affected Suffolk sheep, they differed somewhat from those in goats affected with the experimental disease. Spongiform alteration of neuropil was minimal, and the more rostral parts of the brain, such as corpus striatum, globus pallidus and septal area, had few changes. Concentrations and distribution of virus in non-neural tissues were consistent with the conclusion that scrapie virus no doubt can be maintained by contagion in a herd of goats living apart from infected sheep.

Some Properties of the Scrapie Agent and Its Behavior in Mice

Discussion and summary The agent isolated by Chandler in mice inoculated intracerebrally with brain tissue from goats affected with experimental scrapie can be passed serially in mice by this route. In the Rocky Mountain Laboratory stock of Swiss mice it causes a relentlessly progressive, and invariably fatal, disease of the central nervous system, usually after an incubation period of 4 to 5 months. The disease is characterized clinically by incoordination of gait, muscular atrophy, and drowsiness and neuropathologically by prominent astrocytosis, degeneration of neurons, and spongy alteration of the neuroparenchyma. That the agent causing this disease in mice is the transmissible agent of scrapie may be questioned. At present, identification of the scrapie agent can be made only by observing its pathogenic capacity in susceptible sheep and goats. In these hosts it induces a distinctive clinicopathologic picture whose essential features also characterize the infection in mice. Thus, the long incubation period, the slowly progressive course of the disease, and the degenerative changes in the central nervous system all are consistent with the view that the agent under study is the transmissible agent of scrapie. Moreover, first passage mouse brain has induced scrapie in goats inoculated intracerebrally(4). At this laboratory, goats inoculated intracerebrally or subcutaneously with a 10-1 suspension of fourth passage mouse brain have been observed for too brief a period (5 months) to permit any conclusion about the ability of this inoculum to cause typical caprine scrapie. The high concentrations of the agent in the brain, spinal cord, spleen, and thymus of affected mice make it appear unlikely that a toxic substance is being passed. That the agent passes through a gradacol membrane of 100 mμ average pore diameter, but not one of 30–100 mμ, suggests it is a medium-sized virus.

Overwintering of Western Equine Encephalomyelitis Virus in Experimentally Infected Garter Snakes and Transmission to Mosquitoes

Summary Garter snakes were inoculated in September and November with Western equine encephalomyelitis (WEE) virus and were caused to hibernate under simulated natural conditions. They emerged during March, April, May and June. After varying periods when no virus was detectable in their blood, virus was detected in concentrations as high as 106, and for a period up to 70 days following emergence of snakes. Normal mosquitoes became infected by feeding on these snakes and after an extrinsic incubation period of approximately 3 weeks transmitted WEE virus to 1-day-old chicks. These data demonstrate that snakes may serve as a natural overwintering mechanism for WEE virus. The technical assistance of Jack Cory and Edward Patzer is gratefully acknowledged.

Experimental Scrapie in the Mouse: Electrophoretic and Sedimentation Properties of the Partially Purified Agent

: Some biochemical and biophysical properties of the scrapie agent in a partially purified fraction P5 from murine spleen are described in this communication. The agent was stable in the nonionic detergents Triton X‐100 and Nonidet P40 and stable in the nondenaturing, anionic detergents sodium cholate and sodium N‐lauroyl sarcosinate. In contrast, sodium dodecyl sulfate (SDS) inactivated the agent at high concentrations (1% or >) when the detergent‐to‐protein ratio approached 1.5 g SDS/g protein. The agent was resistant to inactivation by nucleases and proteases, even in the presence of 0.1% SDS. A broad peak of infectivity was exhibited in modified colloidal silica (Percoll) density gradients. Maximal titers were found at a Percoll density of 1.10 g/cm3 in the presence and absence of 0.05% SDS. Gel electrophoresis of the agent in the presence of 0.1% SDS resulted in inactivation of >95% of the agent loaded onto the gel. Free‐flow electrophoresis showed that >99% of the agent in fraction P5 migrated toward the anode, but not as a discrete species. Sedimentation analysis of the agent in fraction P5 in the presence of 1% lysolecithin showed that the agent has a sedimentation coefficient of <300S but >30S. Heating P5 preparations caused the agent to associate with cellular elements and form aggregates with sedimentation coefficients >10,000S. Removal by differential centrifugation of the large forms of the agent produced upon heating permitted characterization of a discrete subpopulation of scrapie agent particles. Rate‐zonal sucrose gradient studies showed that >>95% of the infectivity in this subpopulation sedimented as uniform particles with a sedimentation coefficient of 240S.

Overwintering of western equine encephalomyelitis virus in garter snakes experimentally infected by Culex tarsalis.

Summary Garter snakes were infected with western equine encephalomyelitis (WEE) virus by the bites of experimentally infected Culex tarsalis. The snakes hibernated overwinter under simulated natural conditions and viremia was detected up to 69 days after emergence the following spring. Snakes which were maintained at room temperature after emergence developed viremia within a few days, while those maintained under simulated natural conditions required up to approximately 5 weeks. As shown in previous studies, circulating virus was detected in concentrations sufficient to infect C. tarsalis. The use of fish tags proved satisfactory in assuring positive identification of individual snakes.

Susceptibility of Garter Snakes (Thamnophis Spp.) to Western Equine Encephalomyelitis Virus

Summary 1) It has been demonstrated that garter snakes are susceptible to infection with WEE virus. This is the first evidence, to the best of our knowledge, that a virus which is an important parasite of avian and mammalian hosts can infect a cold-blooded vertebrate and cause viremia of high titer and long duration. 2) The possibility that snakes may play a role in overwintering of the virus is being investigated.

Isolation of a virus closely related to Powassan virus from Dermacentor andersoni collected along North Cache la Poudre River, Colo.

Conclusions A virus closely related, if not identical, to Powassan virus has been isolated from Dermacentor andersoni from northern Colorado. A nonreciprocal relationship between RSSE virus and 791A-52 virus is indicated by the cross neutralization tests. The importance of Dermacentor andersoni in maintenance of this virus is unknown. Although thousands of this tick species collected in many areas in the western United States have been examined for viruses in this laboratory, this is the only isolation of this agent. Since these prior studies of ticks at the Rocky Mountain Laboratory were directed specifically toward study of the ecology of Coloradotick fever virus, the ecology of the 791A-52 virus is unknown. Field and laboratory studies on this subject are in progress.

Mosquito-transmitted encephalitis viruses: A review of their insect and vertebrate hosts and the mechanisms for survival and dispersion

Abstract The present review deals primarily with vectors, vertebrate hosts, and mechanisms for maintenance and dispersion of the mosquito-borne encephalitis viruses (western equine, eastern equine, St. Louis, Japanese, Murray Valley, and Venezuelan). The Venezuelan virus is restricted to tropical areas, western and St. Louis viruses to temperate zones, and the eastern, Japanese, and Murray Valley viruses occur in both tropical and temperate zones, although the latter two agents are found primarily in the temperate zone. The five agents important in the temperate zones are ecologically associated with birds and, except for eastern equine virus, with Culex mosquitoes. The common factors in the maintenance and dispersion of these viruses may be sought in the association of virus, birds, and Culex mosquitoes. The eastern equine virus has been isolated from birds, and there is abundant evidence to indicate that they play some part in the circulation of this virus. There is no evidence of its occurrence in Culex mosquitoes, but epidemiological observations indicate that Aedes sollicitans may be a vector. Culex tarsalis has been definitely shown to be infected with virus throughout the area in which western equine encephalitis is found. This agent has been isolated repeatedly from birds. The bird- Culex tarsalis cycle appears to be adequately established in the case of this virus. Although the St. Louis virus has not been obtained directly from birds, there is sufficient evidence to implicate birds as being important in the ecological picture. The agent has been isolated several times from C. tarsalis and once from Aedes dorsalis and C. pipiens . On an epidemiological basis, the latter has been suggested as a vector. Japanese virus has not been isolated from birds, but serological evidence of previous infection of birds has been obtained. The majority of isolations of Japanese virus have been from Culex tritaeniorhynchus . Antibodies against Murray Valley virus have been detected in the sera of birds. On epidemiological grounds, Culex annulirostris is suspected of being important in the transmission of this virus. The evidence at hand, although it is not conclusive for the last named virus, indicates that there is an ecological relationship between the encephalitis viruses of temperate zones and mosquitoes and birds which accounts for the ability of these viruses to survive in cold climates. The single encephalitis virus (Venezuelan) restricted to tropical zones is thought to be transmitted by Mansonia tittilans , and no evidence has been obtained which would indicate that Culex species are involved. No evidence regarding the role of birds in the maintenance of this agent is available. Such mosquito-transmitted viruses as those of dengue and yellow fever, which occur primarily in tropical areas, are known to be transmitted by Aedes species and by Aedes and Haemagogus species, respectively. In general, Culex mosquitoes do not appear to be concerned with transmission of viral agents in the tropics. Much more field study is required before the ecological associations governing the presence of encephalitis viruses in the temperate zones can be considered to be firmly established. The available data suggest that there is a basic difference in the ecology of viruses of temperate and tropical zones and that further studies of ecological associations are justified.

Tissue Culture Studies of the Virus of Progressive Pneumonia, A Slow Infectious Disease of Sheep

Summary Properties of progressive pneumonia virus (PPV) were studied in sheep lung cell monolayers. Growth studies revealed that, with a multiplicity of infection of 2 TCID50 virus/cell, virus replicated to a titer of 106.6 TCID50 virus/ml in 4 days. Typical polykaryocytes were first seen at 1 day post inoculation (PI) and became larger and more numerous as the virus titer increased. Inoculation of high concentrations of virus produced cell fusion within 3 hr PI. Indirect immunofluorescence revealed homogeneous cytoplasmic fluorescence in infected cells as early as 1 day PI. The number of cells showing fluorescence and the intensity of the fluorescence increased steadily with increase of virus titer. The genetic material for PPV is probably single-stranded RNA. The various properties indicate that PPV is closely related to maedi, zwoegerziekte, and visna viruses.

Mosquito-transmitted encephalitis viruses

Abstract The present review deals primarily with vectors, vertebrate hosts, and mechanisms for maintenance and dispersion of the mosquito-borne encephalitis viruses (western equine, eastern equine, St. Louis, Japanese, Murray Valley, and Venezuelan). The Venezuelan virus is restricted to tropical areas, western and St. Louis viruses to temperate zones, and the eastern, Japanese, and Murray Valley viruses occur in both tropical and temperate zones, although the latter two agents are found primarily in the temperate zone. The five agents important in the temperate zones are ecologically associated with birds and, except for eastern equine virus, with Culex mosquitoes. The common factors in the maintenance and dispersion of these viruses may be sought in the association of virus, birds, and Culex mosquitoes. The eastern equine virus has been isolated from birds, and there is abundant evidence to indicate that they play some part in the circulation of this virus. There is no evidence of its occurrence in Culex mosquitoes, but epidemiological observations indicate that Aedes sollicitans may be a vector. Culex tarsalis has been definitely shown to be infected with virus throughout the area in which western equine encephalitis is found. This agent has been isolated repeatedly from birds. The bird- Culex tarsalis cycle appears to be adequately established in the case of this virus. Although the St. Louis virus has not been obtained directly from birds, there is sufficient evidence to implicate birds as being important in the ecological picture. The agent has been isolated several times from C. tarsalis and once from Aedes dorsalis and C. pipiens . On an epidemiological basis, the latter has been suggested as a vector. Japanese virus has not been isolated from birds, but serological evidence of previous infection of birds has been obtained. The majority of isolations of Japanese virus have been from Culex tritaeniorhynchus . Antibodies against Murray Valley virus have been detected in the sera of birds. On epidemiological grounds, Culex annulirostris is suspected of being important in the transmission of this virus. The evidence at hand, although it is not conclusive for the last named virus, indicates that there is an ecological relationship between the encephalitis viruses of temperate zones and mosquitoes and birds which accounts for the ability of these viruses to survive in cold climates. The single encephalitis virus (Venezuelan) restricted to tropical zones is thought to be transmitted by Mansonia tittilans , and no evidence has been obtained which would indicate that Culex species are involved. No evidence regarding the role of birds in the maintenance of this agent is available. Such mosquito-transmitted viruses as those of dengue and yellow fever, which occur primarily in tropical areas, are known to be transmitted by Aedes species and by Aedes and Haemagogus species, respectively. In general, Culex mosquitoes do not appear to be concerned with transmission of viral agents in the tropics. Much more field study is required before the ecological associations governing the presence of encephalitis viruses in the temperate zones can be considered to be firmly established. The available data suggest that there is a basic difference in the ecology of viruses of temperate and tropical zones and that further studies of ecological associations are justified.