Transmission of western equine encephalitis virus by Saskatchewan mosquitoes and behavior of the virus in selected laboratory vertebrates in relation to epidemiological studies

Abstract

For laboratory experiments on transmission of Western equine encephalitis (WEE) virus by Saskatchewan mosquitoes, a local virus isolate of human origin, strain 1540-1544 Regina 66, was selected. The half-day old chick which was used as the principal vertebrate host in these experiments, showed a consistent response to subcutaneous inoculation with this strain of WEE virus. After infection with moderate doses, viremia was maximal at 18 to 24 hours, when virus concentration was within the range of 108 to 109 intracerebral three-week old mouse LD50 (ic mouse LD50 ) per 0.03 ml of blood. Most infected chicks showed inactivity followed by prostration before death; very few displayed overt clinical signs of neurologic disturbance. In comparative titrations, chicks were more sensitive than weanling mice for detection and titration of virus. An indirect fluorescent antibody test applied directly to brain sections from WEE infected mice, was satisfactory for diagnostic purposes. Sections were fixed in Carney's fluid and transferred through chloroform to paraffin. The use of Evans blue, of absorbed antisera to WEE virus, and the inclusion of negative and positive control preparations, ensured specificity of staining reactions in the tests. The technique could not be applied to brain sections from chicks infected subcutaneously, despite the presence of large amounts of virus in this tissue. Confirmation of WEE infection in chicks which were bitten by infected mosquitoes was achieved by passage of virus to mice, from which brain sections served for diagnosis by immunofluorescence. These initial investigations formed the basis for the design and interpretation of detailed quantitative transmission experiments with mosquitoes.

Nine species of local mosquitoes were examined for their ability to transmit WEE virus by bite. Culex tarsalis was a very efficient transmitter at 69 and 75°F. This mosquito transmitted from four days after infection and remained infective for up to 44 days, which was the longest period of observation. Throughout this period, infection rates remained at 100% or close to this level at both temperatures, but at 69°F, transmission rates declined after about two weeks. At the higher temperature, almost all infected mosquitoes transmitted after the first week of incubation, and this trend continued throughout. The minimum concentration of virus required to infect 50% of C. tarsalis was 102.5 ic mouse LD50 per 0.03 ml of donor blood, and for the same level of infection in Culiseta inornata, a concentration of 104.0 was necessary. The latter did not transmit WEE virus by bite. However, infection rates were maintained between 63 and 88% from the second to the sixth week of incubation, and the quantities of virus in individual mosquitoes were comparable to those in C. tarsalis. Anopheles earlei and Aedes vexans failed to support viral growth, while Aedes campestris, A. flavescens and A. spencerii maintained infection for close to two weeks at 69°F. Infected A. dorsalis did not survive beyond the first week of incubation, at which time, individual mosquitoes had minimal amounts of virus. Apart from c. tarsalis, A. fitchii was the only other mosquito which transmitted the infection, and it did so after an extrinsic incubation period of nine days. All the mosquitoes, except for C. tarsalis, were reluctant to feed on the chick. In an attempt to provide an alternative laboratory host for future transmission studies, a short investigation was undertaken to determine the susceptibility of the Mongolian gerbil (Meriones unguiculatus) to peripheral infection with WEE virus. Four-week old animals responded by exhibiting uniform viremia patterns to a standard dose of virus, and small doses regularly caused death. Resistance to infection increased with age but ten-week old gerbils were as susceptible as three-week old mice. Brains from gerbils which died within ten days after infection, contained large amounts of virus.