pathogenic microorganism, although filtrates of the material were found to be

Transcription

1 DEMONSTRATION OF A NEW INSECT VIRUS NOT ASSOCIATED WITH INCLUSION BODIES' HARRIETTE BLOCK WASSER University of California, Berkeley, California Received for publication April 7, 1952 For the past several years four distinct groups of insect viruses have been recognized by some authorities. These virus infections of insects include groups generally characterized by the presence of inclusion bodies (polyhedra, refringent polymorphic bodies, and granules) and one group in which no inclusion body appears to be produced. Except for the possible demonstration of the latter in sacbrood diseased honeybees (Steinhaus, 1949b), no virus of this latter type has been reported. The present paper describes what is believed to be the first demonstration of a noninclusion type virus which has been verified by means of infectivity tests. Research was initiated in December, 1949, following receipt of material sent to E. A. Steinhaus of our laboratory by G. T. Boettger, U. S. D. A. Entomologist at Anaheim, California. Insectary reared stock of the cosmopolitan armyworm, Cirphis unipuncta (Haworth) [= Leucania unipuncta (Haworth)], is dying from an infection the symptoms of which appeared to be quite unlike those described in the polyhedrosis virus disease reported to infect the same insect (Chapman and Glaser, 1915). Diseased specimens were sent to us for diagnosis, but upon examination of two separate lots of larvae no evidence could be found of a recognizable microorganism. However, when healthy Cirphis larvae were fed alfalfa that had been contaminated by dipping into a water suspension made from ground-up bodies of insects that had died of the disease, the healthy larvae soon exhibited typical symptoms of the disease and died. Filtrates made of the triturated bodies of dead larvae and fed to healthy insects proved to be equally infectious. Since light microscope examinations of the diseased larvae revealed no inclusion bodies characteristic of known insect viruses and no recognizable pathogenic microorganism, although filtrates of the material were found to be infectious, Steinhaus (1951) reported that the infection was probably caused by a filterable virus of a type different from any previously encountered. Further experiments were initiated to clarify the problem and to attempt to determine with certainty the nature of the etiological agent. CHARACTERISTICS OF THE DISEASE Larvae of Cirphis unipuncta infected in late third-instar soon appear swollen and somewvhat darker than normal insects. The cuticula of the diseased larvae have a waxy appearance, and in some cases the midportion is slightly enlarged. The liquefaction and disintegration of tissue characteristic of the polyhedrosis I Contribution from the Laboratory of Insect Pathology, Division of Biological Control, College of Agriculture, University of California, Berkeley. 787

2 788 HARRIETTE BLOCK WASSER [VOL. 64 in this insect are absent. Deaths from the disease may occur in the larval or pupal stage. In Boettger's colony, the greatest percentage of mortality occurred in the pupal stage when mortalities of 90 to 100 per cent were common. As the disease progresses, the Cirphis larvae become sluggish, are soon eating little, and gradually succumb, usually within 6 to 14 days following infection. Upon dissection and light microscope examination no evidence can be seen of the inclusion bodies usually associated with insect virus diseases. In addition, there was no indication that the larvae were dying of nutritional or metabolic disease. Although saprophytic bacteria were present in the bodies of the dead and dying larvae, no infectious agent (bacterial, protozoan, or fungus) was isolated that could be considered responsible for the disease. EXPERIMENTATION AND RESULTS To establish the possible viral nature of the disease a series of infectivity tests was initiated. Experiment I was designed to determine the filterability of the disease agent. The concentrated infectious material, which had been prepared by triturating bodies of larvae that had died of the disease 9 months previous to the present experiment, was passed through a series of fine filters and the filtrates fed to healthy third instar Cirphis larvae. Filtrates tested included: (1) a cheesecloth filtrate of the triturated material; (2) an 8 pound Mandler filtrate of (1); (3) a 15 pound Mandler filtrate of (1); (4) a Seitz filtrate of (1); and (5) a sintered glass filtrate of (1). The cheesecloth filtrate was passed through Whatman no. 2 filter paper prior to its passage through the finer filters. Examination of concentrations of these filtrates with the electron microscope showed the presence of very minute, fairly regular, spherical to slightly ovoid bodies. One hundred and fifty third-instar larvae were used to test the infectivity of the various filtrates. Of these, twenty-five served as the control group and were fed uncontaminated alfalfa while five test groups of twenty-five larvae each were fed alfalfa that had been contaminated with the various filtrates of the infectious material. The larvae in each group were handled individually in sterile units which consisted of a cardboard carton covered with a petri dish top. Light microscope examination was made of the tissues and fluids of each larva following its death. All larvae of the test groups died within 6 to 14 days following infection, showing typical disease symptoms as described above. In the control group, although two larvae did not pupate normally, the remaining twentythree emerged as healthy adults. The above results indicated that we were dealing with a highly virulent, filterable entity. In experiment II, the infectious material was fractionated by means of high speed centrifugation and the various sediments and supernatants fed to healthy larvae (in groups of 25 larvae each) using the infectivity testing procedure described above. A portion of the cheesecloth and filter paper filtrate (11 months old) was used as the stock test suspension and centrifuged for 30 minutes in the Sorvall SS-1 centrifuge at 5,000 rpm (5,900 g) to remove the insect fragments. The supernatant of this centrifugation then was spun in the Spinco ultracentrifuge (Model L, no. 40) for 1 hour at 10,000 rpm (9,000 g), and the resulting

3 1952] NEW INSECT VIRUS NOT ASSOCIATED WITH INCLUSION BODIES 789 supernatant and sediment used for infectivity feeding tests. The 10,000 rpm supernatant now was recentrifuged in the Spinco for 1 hour at 40,000 rpm (144,000 g), and the resulting translucent, slightly bluish sediment resuspended in distilled water, then centrifuged for another hour in the Spinco at 40,000 rpm. This final sediment was also examined for infectivity. All fractions tested were highly infectious and lethal while the control group again remained healthy. Electron micrographs made from the fractions showed a heavy concentration of small, regular, spherical to slightly ovoid bodies in the 40,000 rpm sediment and a lighter concentration of similar appearing material in the other fractions. For experiment III, the larvae infected and killed following the feeding of each of the three fractions (the 10,000 rpm supernatant, the 10,000 rpm sediment, and the 40,000 rpm sediment) tested in experiment II were pooled into three corresponding lots. These dead larvae had been held in the refrigerator following the completion of experiment II and were approximately a month old by the time they were used in experiment III. The dead larvae were triturated, filtered through a Seitz filter to remove bacterial contamination, then subjected to several centrifuge cycles as previously outlined, and the supernatants and sediments of each group fed to healthy test larvae in the usual manner (see experiment I) to determine infectivity of the various fractions. The only change in procedure from that described above was the inadvertent use of 35,000 rpm (106,000 g) rather than 40,000 rpm for the high-speed cycle of centrifugation. The infection showed itself to be easily transmissible, as indicated by the infection of virtually all the test insects (25 larvae being used to test each supernatant and sediment). Again, electron micrographs of the infectious material showed the minute, spherical to slightly ovoid bodies previously observed. They were particularly densely packed in the 35,000 rpm sediment made from larvae that had been previously fed the 40,000 rpm sediment described in the preceding paragraph. The particles also occurred in the other fractions but in much lighter concentrations. A cursory dilution experiment (experiment IV) in which healthy larvae were fed a concentrated suspension of the infectious material made from ground-up pupae that had died of the disease, as well as 1-10, 1-100, 1-1,000 dilutions of this concentrated suspension, showed the infectious material to be virulent at dilutions up to and including 1-1,000. In all probability it would have been infectious at considerably higher dilutions as well. The test material was derived from a group of insects that had died as pupae following contamination in the larval stage with sick larvae. The diseased larvae represented the third successful passage, during a 16 month period, of the infectious material first used in experiment I. The pupae were triturated in a blender, filtered through gauze, and made up in a final concentration equal to 4.88 g per ml. Five ml of this suspension was cleaned and concentrated by means of a cycle of high- and lowspeed centrifugation as recorded in experiment II, and the final sediment and dilutions made from it were tested to determine potency using the infectivity testing procedure described under experiment I. In the last experiment of this series, experiment V, the infectious material

4 790 HARRIETTE BLOCK WASSER [VOL. 64 used in experiment IV was retested, a fresh stock of insects being used to determine infectivity. Four months had passed since the previous experiment had been initiated, a total of 20 months having elapsed since the first passage of the infectious material through insects was made. It was found that the prolonged refrigeration of the infectious material had caused sedimentation. The liquid portion was carefully decanted, and after passage through a Seitz filter to remove possible bacterial contamination, the supernatant and sediment were tested individually for infectivity. The experiment showed both fractions to be infective, killing all of the 25 test insects in each instance. In all infectious fractions, electron micrographs revealed the presence of minute, regularly shaped spherical to slightly ovoid bodies (figure 1) approxi- Figure 1. Morator nudus, nov. spec. Palladium shadowed, magnification 20,OOX. mately 25 mma in size. Normal insects which were fractionated by means of high speed centrifugation in the same manner as the infected larvae did not reveal the clumped masses of particles found in the infectious material. Large, irregular particles, in all probability representing a normal component, were present. However, these could be distinguished readily from the virus particles seen in preparations from diseased insects. DISCUSSION The results of the infectivity experiments described ini the foregoing paragraphs strongly indicate the presence of a submicroscopic infectious agent in the tissues of the diseased Cirphis larvae. Electroni micrographs of the several centrifugation fractions and the filtrates revealed the presence of characteristic particles apparently associated with infectivity. In no case could particles of a

5 1952] NEW INSECT VIRUS NOT ASSOCIATED WITH INCLUSION BODIES 791 like nature and morphology be found in similar preparations from healthy Cirphis larvae. Accordingly, the conclusion appears warranted that the characteristic minute particles found in such large numbers and only in the diseased insects represent a new virus agent. This constitutes the first definite isolation and electron microscope demonstration of a virus pathogenic for insects and yet not associated with a demonstrable inclusion body. There apparently exist several viruses pathogenic for insects which do not appear to be associated with an inclusion body of any kind (Steinhaus, 1949a). These may include such agents as those causing sacbrood of the honeybee, paralysis in the honeybee, and (in association with certain bacteria) flacherie and gattine in the silkworm. With the possible exception of sacbrood, in none of these infections has the virus been demonstrated with the electron microscope. In the case of sacbrood, Steinhaus and the writer (Steinhaus, 1949b) obtained electron micrographs of small particulate bodies which may possibly represent the virus. Definite proof of the identity of these bodies, however, remains to be obtained. Holmes, in 1948, proposed the genus Morator to include the single species Morator aetatutae Holmes, the cause of sacbrood of the honeybee. In 1949, Steinhaus (1949a,b) tentatively accepted the name and characterized the genus as including "viruses causing insect disease in which no visible pathological inclusion body of any kind is produced." Subsequent to this Steinhaus (1952) suggested that the generic name Morator be dropped or withheld from usage until the sacbrood virus (or presumably another virus belonging to the genus as described in the statement just quoted) is identified and conclusively demonstrated with the electron microscope. The virus described in the present paper would come under the general description of the genus as given provisionally by Steinhaus. Accordingly, it is proposed that the virus here described be considered a new species, that it be placed tentatively in the genus Morator, and that it be given the name Morator nudus, nov. spec. (from Latin nudus, nude or naked). SUMMARY A new insect virus found in diseased larvae of the cosmopolitan armyworm, Cirphis unipuncta (Haworth), is described. Unlike other insect viruses that have been demonstrated with the electron microscope, the virus described in this paper is not associated with any recognizable inclusion body. It is filterable, readily transmissible per os, and may be highly virulent. On the basis of electron microscope observations, the virus appears to be a minute, more-or-less regular, spherical to slightly ovoid body, and approximately 25 m, in size. The name Mlorator nudus, nov. spec., is proposed for this virus in accordance with present procedures in classifying and naming insect viruses. REFERENCES CHAPMAN, J. W., AND GLASER, R. W A preliminary list of insects which have wilt, with a comparative study of their polyhedra. J. Econ. Entomol., 8,

6 792 HARRIETTE BLOCK WASSER [VOL. 64 HOLMES, F Order Virales, the filterable viruses, pp In Bergey's manual of determinative bacteriology, 6th ed. The Williams & Wilkins Co., Baltimore. STEINHAUS, E. A. 1949a Principles of insect pathology. McGraw-Hill, New York. STEINHAUS, E. A. 1949b Nomenclature and classification of insect viruses. Bact. Revs., 13, STEINHAUS, E. A Report on diagnoses of diseased insects Hilgardia, 20, STEINHAUS, E. A Taxonomy of insect viruses. Paper presented at Conference on Virus and Rickettsial Classification and Nomenclature by the New York Academy of Sciences, New York, on January 11 and 12, 1952.