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Bunyamwera virus replication in arthropod and vertebrate tissue. Peers, Robert Ross


Bunyamwera (BUN) virus multiplied readily in mosquitoes following intrathoracic injection and also after imbibing'-an infective blood meal. This agent also multiplied following inoculation of human and avian cells in tissue cultures, with production of cytopathic effects. Both in arthropod and vertebrate tissues, enveloped virions 84 nm diameter were visualized by electron microscopic observation of tissues collected after maximum viral proliferation was attained. Following intrathroacic injection of 10 ²•² mouse LD₅₀ of BUN virus into groups of wild-caught mosquitoes comprising both Aedes canadensis and A. vexans, increments of infectivity were first detected in salivary glands and gut at 3 days, and maximum titres of 10 ⁵•² mouse LD₅₀ per organ were attained in salivary glands at 10 days. However, 50 the virus content of legs, which provided a convenient means of sampling hemocelic fluid, increased at 2 days. Virus transmission by biting mice was demonstrated with mosquitoes injected 10 days previously, but not after shorter intervals. No virus replication was demonstrated following ingestion of 10⁴•⁰ mouse LD₅₀ of BUN virus in a blood meal. Aedes aegypti mosquitoes readily supported the replication of BUN virus following injection with 10 ³•³ mouse LD₅₀ or imbibing of 10 ⁴•⁶ mouse LD₅₀. After injection, virus titres of whole mosquitoes declined to 10 ¹•⁷ mouse LD₅₀ at 12 hours, followed by an increase to a peak amount of 10 ⁵•⁰ mouse LD₅₀ at 2 days. After feeding, virus was first detected in legs and salivary glands at 4 days, and attained maximum titres of 10 ⁵•⁰ mouse LD₅₀ in salivary glands at 10 days. Transmission of virus to mice was effected by A. aegypti following feeding and injection 10 days previously, but not at earlier intervals. Following exposure of whole gut cultures of adult A. aegypti mosquitoes to 10 ³•⁷mouse LD₅₀ maximum yields of 10 ⁶•⁰ mouse LD₅₀ per ml. were observed after k days incubation at 29°C, after an initial decline of infectivity to 10 ¹•⁸ mouse LD₅₀ at 12 hours. Enveloped virions with cores 45 nm diameter and total diameters 80 to 100 nm were observed within vacuoles and lining vacuolar membranes of salivary glands and gut cells of A. aegypti mosquitoes 10 days or more after infection with BUN virus. No particles were observed earlier, despite high virus titres 4 days or more after injection. After inoculation of continuous live tissue cultures of human epidermoid corcinoma cells (H.Ep. 2) with 10 ⁶•⁵ mouse LD₅₀, the highest amount of virus produced was 10 ⁷•⁰ mouse LD₅₀ per ml. cell suspension after 24 hours incubation at 37°C. Maximum yields of BUN virus (10 ⁶•² mouse LD₅₀ per ml. cell suspension) were attained 24 hours after inoculation of primary chick embryo fibroblast monolayers with 10 ⁵•² mouse LD₅₀ following incubation at 37 C . However, a peak titre of 10 ⁵•⁰ mouse LD₅₀ was attained 3 days after inoculation with 10 ³•⁷mouse LD₅₀ in cultures incubated at 29°C. Before an increment of virus titre was observed infectivity declined to zero during the initial 4 hours after inoculation of cultures incubated at 37°C, and a tenfold decline of infectivity was noted in cultures incubated at 29°C. Enveloped virions with total diameter 84 nm which contained electron-dense nucleoids 44 nm diameter were observed extracellularly in thin sections of chick embryo fibroblasts infected 12 hours previously with BUN virus. These particles were released by budding. Precursor particles 41 nm diameter were associated with intracellular membranes in occasional cells sectioned at 4 hours. Extracellular virions released one day after inoculation of H.Ep. 2 cultures were tagged by ferritin-labelled anti-BUN antibody. Enveloped virions with mean diameters 100 nm were observed in suspensions of suckling mouse brain infected with BUN virus and stained negatively with phosphotungstic acid. These results show clearly that BUN virus exhibits the essential biological and morphological characteristics of a mosquito-borne arbovirus.

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