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The housefly virus, isolation, purification and structure
Micro,. 1980. Vol.: I I. pp. 43 I-a,32 Pergamon Press Ltd. Printed in Great Britain
THE HOUSEFLY VIRUS, ISOLATION, PURIFICATION AND STRUCTURE
A. Y. MOUSSA School of Zoology, University of New South Wales, P.O. Box 1, Kensington. N.S.W. 2033, Australia
The virus was isolated from laboratory colonies of adult houseflies Musca domestica during routine investigations of the effect of insecticides on midguts of adult flies. The flies developed symptoms typical of insect virus diseases together with the presence of dark undulating threads in their mid~ut cells (Fig. A). These threads cannot be distinguished in suspensions before dialyzingJ. The surface structure (subunits) of the threads (after dialyzing) is similar to that of the virus capsomeres and the diameter of the threads is comparable to the diameter of the virions (Fig. B). Despite feeding adult flies on virus suspension, virions were never found within sections of the midgut epithelial cells. However virus particles were detected and found in infected flies' crops (by negative staining) and within the peritrophic membrane (in sections) (Fig. C). Flight muscles, Fat tissue, Malpiqhian tubules and tracheoles were also checked for possible virus multiplication. New techniques were developed to detect the presence of virus within hemocytes, one of which has proved useful in screening infected blood cells alongside the flies' midgut ] (Fig. D). The housefly virus (HFV) particles have some morphological structure similar to those of reoviruses (20 capsomeres on the periphery of the outer shell; an outer shell and an inner shell) but were found to have structures not found in reoviruses. This was demonstrated during attempts to purify the virus. Virus particles extracted and suspended in distilled water (D.W., pH 5.7 to 6.0) measured 57 to 76 nm, while they measured 64 to 80 nm when extracted and suspended in D.W. of pH 7.0 or in phosphate buffer (PR, pH 7.0 - 7.4) (Fig. E). Carbontetrachloride (CCI 4) tends to damage some virus particles when used during virus extraction, particularly after the second CCI 4 treatment 2. The internal structure (inner components) of the broken particles could be shown if virus particles were first mounted on the grids, then negatively stained (Fig. G,H,I,J,K). Virus particles were rendered undetectable by electron microscopic examination when virus suspended in D.W. (pH 5.7 to 6.0) was purified on sucrose/D.W, gradients, while virus particles fragmented when virus suspended in PB was purified on sucrose/PB gradients 2. When Ficoll 400/PB gradients were used instead of sucrose, a visible opalascent (pale green) virus band was formed. This band was found to contain: (i) a few complete particles with diameters of 80 - 85 nm (2) many empty particles (70 - 80 nm) some of which still contained parts of the inner components (middle layer - suggested name), (3) a few empty ring-like particles (40 - 50 nm), possibly the empty inner shells, (4) numerous small particles of 30 - 35 nm in diameter (the nucleoprotein cores) many of which were empty, (5) unidentified materials. Virus antisera was raised in rabbits and virus particles were agglutinated with viral antibody, though the inner components of broken particles could be seen. From the above mentioned information a suggested model of the virus structure was drawn; it consists of (I) Capsomeres (20) on the periphery of, (2) the outer shell, (3) the inner shell, (4) the middle layer (appearing as threads), (5) the nucleoprotein core, (6) viral nucleoid. REFERENCES i.
A.Y. Moussa, A new virus disease in the houseflv, Musca domestica (Diptera), J. Invertebr pathol. 3 1 - 2 0 4 : 2 1 6 (]978).
431
432
A . Y . Moussa
2.
A.Y. Moussa, The breakdown of the housefly vlrus durlng purlfi~'atlon and th,.. structure of the virions. Sumbitted to v~rolocy (]979).
W
m
"
i
FIGURES A. T h r e a d s within a midgut cell's section surrounded by rough e n d o p l a s m i c reticulum. B. A thread, negatively stained with PTA after dialyzing. C. Three virions within the p e r i t r o p h i c membrane. D. Virus particles w i t h i n infected blood cell. E. HFV particles negatively stained w i t h PTA. F. Purified HFV on Ficoll 400 note the presence of the n u c l e o p r o t e i n cores. G , H , I , J , - a n d K. Damaged HFV particles after the second treatment with CCI 4, show their inner components and exposed to viral antisera in (K). Scale A , B , C , E , F = 100 nm, D = 500 nm, G,H,I,J,K = 50 nm.
THE HOUSEFLY VIRUS, ISOLATION, PURIFICATION AND STRUCTURE
A. Y. MOUSSA School of Zoology, University of New South Wales, P.O. Box 1, Kensington. N.S.W. 2033, Australia
The virus was isolated from laboratory colonies of adult houseflies Musca domestica during routine investigations of the effect of insecticides on midguts of adult flies. The flies developed symptoms typical of insect virus diseases together with the presence of dark undulating threads in their mid~ut cells (Fig. A). These threads cannot be distinguished in suspensions before dialyzingJ. The surface structure (subunits) of the threads (after dialyzing) is similar to that of the virus capsomeres and the diameter of the threads is comparable to the diameter of the virions (Fig. B). Despite feeding adult flies on virus suspension, virions were never found within sections of the midgut epithelial cells. However virus particles were detected and found in infected flies' crops (by negative staining) and within the peritrophic membrane (in sections) (Fig. C). Flight muscles, Fat tissue, Malpiqhian tubules and tracheoles were also checked for possible virus multiplication. New techniques were developed to detect the presence of virus within hemocytes, one of which has proved useful in screening infected blood cells alongside the flies' midgut ] (Fig. D). The housefly virus (HFV) particles have some morphological structure similar to those of reoviruses (20 capsomeres on the periphery of the outer shell; an outer shell and an inner shell) but were found to have structures not found in reoviruses. This was demonstrated during attempts to purify the virus. Virus particles extracted and suspended in distilled water (D.W., pH 5.7 to 6.0) measured 57 to 76 nm, while they measured 64 to 80 nm when extracted and suspended in D.W. of pH 7.0 or in phosphate buffer (PR, pH 7.0 - 7.4) (Fig. E). Carbontetrachloride (CCI 4) tends to damage some virus particles when used during virus extraction, particularly after the second CCI 4 treatment 2. The internal structure (inner components) of the broken particles could be shown if virus particles were first mounted on the grids, then negatively stained (Fig. G,H,I,J,K). Virus particles were rendered undetectable by electron microscopic examination when virus suspended in D.W. (pH 5.7 to 6.0) was purified on sucrose/D.W, gradients, while virus particles fragmented when virus suspended in PB was purified on sucrose/PB gradients 2. When Ficoll 400/PB gradients were used instead of sucrose, a visible opalascent (pale green) virus band was formed. This band was found to contain: (i) a few complete particles with diameters of 80 - 85 nm (2) many empty particles (70 - 80 nm) some of which still contained parts of the inner components (middle layer - suggested name), (3) a few empty ring-like particles (40 - 50 nm), possibly the empty inner shells, (4) numerous small particles of 30 - 35 nm in diameter (the nucleoprotein cores) many of which were empty, (5) unidentified materials. Virus antisera was raised in rabbits and virus particles were agglutinated with viral antibody, though the inner components of broken particles could be seen. From the above mentioned information a suggested model of the virus structure was drawn; it consists of (I) Capsomeres (20) on the periphery of, (2) the outer shell, (3) the inner shell, (4) the middle layer (appearing as threads), (5) the nucleoprotein core, (6) viral nucleoid. REFERENCES i.
A.Y. Moussa, A new virus disease in the houseflv, Musca domestica (Diptera), J. Invertebr pathol. 3 1 - 2 0 4 : 2 1 6 (]978).
431
432
A . Y . Moussa
2.
A.Y. Moussa, The breakdown of the housefly vlrus durlng purlfi~'atlon and th,.. structure of the virions. Sumbitted to v~rolocy (]979).
W
m
"
i
FIGURES A. T h r e a d s within a midgut cell's section surrounded by rough e n d o p l a s m i c reticulum. B. A thread, negatively stained with PTA after dialyzing. C. Three virions within the p e r i t r o p h i c membrane. D. Virus particles w i t h i n infected blood cell. E. HFV particles negatively stained w i t h PTA. F. Purified HFV on Ficoll 400 note the presence of the n u c l e o p r o t e i n cores. G , H , I , J , - a n d K. Damaged HFV particles after the second treatment with CCI 4, show their inner components and exposed to viral antisera in (K). Scale A , B , C , E , F = 100 nm, D = 500 nm, G,H,I,J,K = 50 nm.