Proc. Nati. Acad. Sc. USA Vol. 80, pp. 7664-7668, December 1983 Microbiology

Enveloped double-stranded DNA virus with novel structure and cytopathology (viral vesicles/cell cleavage/mitochondria/membrane synthesis/virion assembly) BRIAN A. FEDERICI Division of Biological Control, Department of Entomology, University of California, Riverside, CA 92521 Communicated by George A. Zentmyer, September 8, 1983 ABSTRACT An unusual type of virus has been isolated from tion of diseased on a regular basis and enabled the de- larvae of the cabbage looper, Trichoplusia ni (; Noc- velopment of techniques for the routine isolation and trans- tuidae). The virus infects a variety of tissues, including fat body, mission of the virus in the laboratory. epidermis, and tracheal matrix, causing a chronic, fatal disease. In the present paper, the results of more detailed studies on Viral replication begins in the nucleus and is accompanied by in- the development of the disease, structure of the virions, and vagination of the nuclear envelope and extensive nuclear and cel- type of nucleic acid of the virus isolated from T. ni are reported. lular hypertrophy. The nuclear envelope eventually ruptures and By using restriction enzyme analysis of the viral genome and fragments, after which viral-induced membranes are assembled the cel- along planes through the cell and around its periphery. Subse- electron microscopy to study disease development at quently, these membranes coalesce, partitioning most of the cell, lular level, it is shown that the virions contain double-stranded including viroplasms and virions in various stages of assembly, (ds) DNA and that the virus replicates initially in the nucleus. among a cluster of vesicles. The vesicles dissociate and are lib- The most novel aspects of the disease occur later in develop- erated into the hemolymph where they accumulate in large num- ment, however, when the nuclear envelope disrupts and the bers (>108 vesicles per ml), causing the blood to become opaque cell is partitioned into virion-containing vesicles. A comparison white. The virus has been isolated from T. ni and transmitted per of the structural and developmental properties of this virus with os and by injection to T. ni and several other species of the family other types of enveloped ds DNA viruses indicate it is a mem- . The virions produced by this virus are large (ca. 130 ber of a unique group of previously unknown invertebrate vi- X 400 nm), enveloped, and allantoid in shape with complex sym- ruses. metry and contain apparently linear, double-stranded DNA of M, of 1.00 x 108. The envelope contains subunits arranged in a MATERIALS AND METHODS hexagonal pattern that impart a distinctive reticular appearance to virions in negatively stained preparations. The unique struc- Purification of Virions. Larvae of T. ni were infected as de- tural and developmental properties of this virus indicate that it scribed below, and the virions were isolated from the hemo- is a member of a group of enveloped, double-stranded DNA vi- lymph 10-14 days after infection. Hemolymph was obtained ruses not observed previously. from diseased fifth-instar T. ni by puncturing the body with a pin. After collection of 3 ml, the hemolymph was centrifuged An unusual type of virus not observed previously has been found for 5 min at 2,000 X g to pellet the vesicles. The supernatant causing a chronic but ultimately fatal disease in larvae of the was discarded, and the pellet was resuspended in 15 ml of 1% noctuid Scotogramma trifolii (1), Trichoplusia ni (2), and Triton X-100. The suspension was then agitated vigorously for Heliothis zea (3) (order, Lepidoptera; family, Noctuidae). The ca. 30 sec by repeated pipetting through a Pasteur pipette, then most unusual feature of this virus is that it induces the host cell clarified by centrifugation for 10 min at 2,000 X g. Subse- to cleave into a cluster of virion-containing vesicles. These ves- quently, the supernatant was layered onto 0.1 M phosphate- icles are highly refractile, typically range in diameter from 2 to buffered 20-55% (wt/wt) sucrose gradients and centrifuged for 12 gm and, after cleavage of the host cell, dissociate and are 1 hr at 60,000 X g in a Beckman SW-27 rotor. The band con- liberated into the hemolymph where they accumulate in large taining the particles was collected, diluted five times with buff- numbers (>108 vesicles per ml) as the disease progresses. An er, and centrifuged at 143,000 X g in a type 35 fixed-angle rotor additional unusual feature of this virus is the unique structure to pellet the particles. The pelleted virions were used directly of the virion, which is a large enveloped particle, essentially for various studies if sufficiently pure or were resuspended in allantoid in shape, with an envelope that exhibits a distinctive 1 ml of buffer, purified further in a second sucrose gradient, hexagonal pattern in negatively stained preparations. and repelleted by centrifugation as above. In two initial reports on the disease caused by this virus, the Virus Transmission. The virus was transmitted to larvae of identity of the pathogen was not confirmed (1, 2). It was sug- T. ni either by injection of purified virions or per os by feeding gested that the causative agent was either a new type of virus larvae on an artificial diet (4) contaminated with hemolymph or, because of its large size, shape, and occurrence in mem- from diseased larvae. In the first case, larvae that had moulted brane-bound vesicles, a peculiar type of Rickettsia. A more de- to the third instar within the previous 24 hr were inoculated finitive characterization of the disease in S. trifolii was not un- with ca. 2 x 104 virions in 10 1.l of 0.1 M phosphate buffer. dertaken because attempts to transmit the virus in the laboratory The large size of the particles enabled them to be counted readily were unsuccessful. However, the occurrence of the virus in with scanning electron microscopy, and the approximate con- populations of T. ni throughout the year permitted the collec- centration of particles in the inoculum was determined by counting them in diluted replicated samples of 1 ul. For trans- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: ds DNA, double-stranded DNA; EtdBr, ethidium bro- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. mide. 7664 Downloaded by guest on October 1, 2021 Microbiology: Federici Proc. Natl. Acad. Sci. USA 80 (1983) 7665 mission by feeding, 1-day-old first-instar larvae were placed on the artificial diet, the surface of which was contaminated with infectious hemolymph diluted to yield a concentration of 3,600 viral vesicles per cm2 of diet surface. The hemolymph used for these trials was collected freshly from diseased larvae infected in the laboratory and diluted with 0.1 M phosphate buffer. The concentration of vesicles was determined with a hemocytom- eter. Control larvae were injected with 0.1 M phosphate buffer or fed a diet covered with dilute hemolymph from healthy lar- vae. Treated and control larvae were reared at 25 ± 20C. To test the host range of the virus, third-instar larvae of the fol- lowing species were inoculated with viral particles as described above: family Noctuidae, H. zea, Heliothis virescens, Spodop- tera exigua, Peridroma saucia; family Arctiidae, Estigmnene acrea; family Geometridae, Sabulodes aegrotata. Electron Microscopy. For transmission electron microscopy, tissues were fixed with 3% glutaraldehyde and 1% OS04 and embedded in Epon-Araldite by methods described previously (1). To determine the sequence of cytopathology, tissues were obtained from larvae injected with particles 3, 7, and 10 days after inoculation. For negatively stained preparations, purified virions or samples of hemolymph containing virions were stained with 1% neutral phosphotungstic acid. The size of the particles was determined from electron micrographs of negatively stained preparations. Isolation and Analysis of Viral DNA. The type, approximate size, and physical conformation of viral nucleic acid was de- termined by using centrifugation techniques, restriction en- donucleases, and electron microscopy. The DNA was isolated from purified particles by phenol extraction (5) or more typi- cally by centrifugation in CsCl/ethidium bromide (EtdBr) gra- dients after disruption of virions in 5% sodium lauryl sarcosi- nate (6). In the latter case, the particle pellet was suspended in 200 A.l of 0.15 M NaCl/0.015 M Na citrate/10 mM EDTA buff- er, pH 7, and transferred to an ultraclear centrifuge tube. Ap- proximately 200 ,u of 10% Sarcosyl was added, and the mixture was heated at 60°C in a water bath for 1 hr. The CsCl gradient (1.7 g/ml under 1.3 g/ml) was layered under the mixture of disrupted particles, and the gradient was centrifuged for 16 hr in a Ti 50 rotor at 183,000 X g. Digestion of purified DNA with the enzymes HindIII and Xho I was by standard procedures (5, 6), and the fragments were separated on 0.7% agarose gels. Mo- lecular weights of DNA fragments were calculated by using a regression line generated from EcoRI fragments of phage A DNA run simultaneously in the same gel. The physical configuration of the purified DNA was exam- ined by the technique of Kleinschmidt (7). Viral DNA was spread immediately after being collected from CsCl/EtdBr gradients. FIG. 1. Isolation and purification of virions. (a and b) Light (wet mount, phase contrast) (a) and electron (b) micrographs of virion-con- RESULTS tainingvesicles from hemolymph. (Bar ina = 5 pm; Bar in b = 400 nm.) Virus Transmission. The virus was successfully (c) Band of virions obtained by purification on sucrose gradients. (d) transmitted Negatively stained virions from the band inc. (Bar = 200 nm.) (e) Band to T. ni by using both the inoculation and per os methods, al- ofviral DNA obtained by centrifugation ofSarcosyl-disrupted particles though the former method was the most successful. The disease on CsCl/EtdBr gradients. (f) Portion of a single overlapping molecule developed in 88% (22 out of 25) of the larvae inoculated with of linear DNA. (Bar = 1 pn.) (g) Restriction digests of phage A and T. virions. In these larvae, virion-containing vesicles (Fig. 1 a and ni viral DNAs. Lanes: 1 and 2, HindI and Xho I digests, respectively, b) typical of the disease were present in the hemolymph within of T. ni viral DNA; 3,EcoRI digest of phage A DNA. Molecular weights 72 hr, and most larvae died 10-21 days after inoculation, dur- are shown x 106. ing the fifth instar. In samples of hemolymph taken from lar- vae, the number of virus vesicles averaged 2.8 X 108 per ml of came lethargic 2-3 days after feeding on virus-contaminated hemolymph. None of the control larvae died, and all pupated diet, and their development was highly retarded in comparison within 9 ± 1 days of inoculation. The rate of transmission for with that of control larvae. Typically, they did not progress be- the per os method was 12% (9 out of 25), and, though not nearly yond the third instar, although they survived for an average of as successful as inoculation, disease development in infected 34 days. Control larvae pupated in 21 ± 2 days. These results larvae was characteristic of that observed in diseased larvae confirm the chronic nature of the disease caused by this type collected from natural habitats (2). In general, these larvae be- of virus. Downloaded by guest on October 1, 2021 7666 Microbiology: Federici Proc. Nati. Acad. Sci. USA 80 (1983) Except for S. aegrotata, all test species inoculated with the larged, the nuclear envelope eventually disrupted (Fig. 2d). virus contracted the disease, and virion-containing vesicles in Virions first became apparent at about this stage, scattered most larvae were found in the hemolymph 4-6 days after in- sparsely throughout the cell in regions that could still be dis- oculation. The results indicate that the virus has a relatively tinguished as cytoplasm or nucleoplasm. Two other significant wide host range among the Lepidoptera, particularly among changes became apparent as the nuclear envelope fragmented. members of the family Noctuidae. First, the mitochondria, which had been pushed to the pe- Virus Purification and Preliminary Characterization of Viral riphery of the cell by the enlarging nucleus, changed in shape DNA. The treatment of the vesicles with Triton X-100 liberated from being basically tubular to spherical and stained more in- many virions, enabling them to be purified on sucrose gradient tensely than those in healthy cells. Second, membrane thatwould where they formed a diffuse band after centrifugation for 1 hr become part of bounding membrane of the virion-containing (Fig. 1c). Measurements made on 50 particles purified by this vesicles was assembled along planes through the cell, often in method (Fig. id) had an average length of 440 ± 36 nm and the vicinity of mitochondria (Fig. 2 e and f). The membrane width of 163 ± 13 nm at the widest dimension. However, ul- first assembled by forming small vesicles and then sheets, fre- trathin sections of purified virions indicated that the method quently in piles of several layers as well as long, narrow tu- of purification caused some particle hypertrophy and loss of bules. The layers of newly synthesized membrane then co- integrity, and thus the approximate size of the virions (130 x alesced and joined the plasma membrane partitioning the cell, 400 nm) from hemolymph preparations is probably closer to including fragments of nuclear envelope, nucleoli, virions, ri- their actual size. bosomes and mitochondria, among a cluster of vesicles. The The viral DNA obtained from Sarcosyl-disrupted viral par- number of virions apparent in the cell increased as vesicle for- ticles formed a single sharp band at a density of 1.59g/cm in mation proceeded, and many virions aggregated, forming re- CsCI/EtdBr gradients (Fig. le). Values obtained for the mo- fractile inclusions in the vesicles that could be observed with lecular weight of the viral DNA based on the number and size phase microscopy. As cleavage proceeded, the vesicles disso- of HindIII and Xho I digests were 1.01 x 108 and 1.03 x 108, ciated and eventually were liberated into the hemolymph, respectively (Fig. 3c). The preparations of DNA spread by us- probably through ruptures in the basement membrane. ing the Kleinschmidt technique indicated that the DNA was Assembly and Structure of Virions. Although typically the linear (Fig. if). No evidence was obtained with this technique assembly of virions did not appear to be directly associated with or banding in CsCI/EtdBr gradients for the existence of su- viroplasmic centers, two distinct types of nucleoprotein inclu- percoiled circular DNA. sions occurred in infected cells and vesicles. The first type was Histopathology/Cytopathology. Developing and mature vi- essentially spherical (Fig. 3a), resembled nucleoli, and actually rions were found in the tracheal matrix, epidermis, and fat body appeared to be involved in ribosome production in that dense cells; although other tissues were not examined for virus, this clusters of ribosomes were common adjacent to the inclusion range of tissues indicates that the virus is capable of infecting surface. The second type of inclusion was pleomorphic and a broad range of tissues. contained regions of distinctly different staining densities (Fig. The cytopathology caused by viral infection was examined in 3a). This type of inclusion may be the site of viral DNA syn- fat body and epidermal cells in late fourth- or fifth-instar T. ni. thesis. Because fat body cells in the fifth instar undergo significant Most virions assembled after the nuclear envelope dis- changes in ultrastructure due to the accumulation of lipid, gly- rupted. Assembly occurred throughout the cell or in devel- cogen, and protein in preparation for pupation (8), most ob- oping vesicles. The basic assembly process as observed in ul- servations on this cell type were made on tissue from fourth trathin sections consisted of the simultaneous formation of a instar larvae. In diseased larvae, infected cells could be found dense nucleoprotein core (ca. 80 X 300 nm) and surrounding in most thin-sections of fat body, but even in advanced stages matrix (ca. 25 nm thick) to form an allantoid inner particle (ca. of disease, the majority of the cells appeared to be uninfected. 130 X 350 nm) that was subsequently enveloped, forming the Although the cytopathology differed to some extent between mature virion (Fig. 3 b-g). The inner particle (Fig. 3b) con- fat body and epidermal cells, in general it could be divided into densed as it formed, revealing the presence of a possible inner four phases: (i) hypertrophy of the nucleus accompanied by in- membrane (indicated by an electron-translucent layer) and a vagination of the nuclear envelope, (ii) cell hypertrophy and surrounding layer of protein subunits, both of which were most disruption of the nuclear envelope, (iii) assembly of virions and clearly resolved after envelopment (Fig. 3g). The virion en- formation of virion-containing vesicles, and (iv) dissociation of velope contained subunits arranged in a hexagonal pattern, and, the viral vesicles, followed by their liberation into the hemo- although these subunits were only observed occasionally in ul- lymph. The gross aspects of this cytopathology could be ob- trathin sections (Fig. 3c), the hexagonal pattern was very dis- served with light microscopy. For example, Fig. 2a illustrates tinct in negatively stained preparations, apparently as a result a greatly hypertrophied fat body cell prior to cleavage and ves- of the superimposition of subunit layers upon one another (Fig. icle formation, whereas in Fig. 2b two epidermal cells are shown 3d). Similar hexagonal patterns were observed in large sections in which the formation of the virion-containing vesicles is nearly of membrane prepared by lysing virion-containing vesicles with complete. Triton X-100 directly on Formvar-coated grids. In these sec- At the ultrastructural level, the most prominent early signs tions, which may be fragments of the limiting vesicle mem- of infection were narrow and deep infoldings of the nuclear en- brane, the hexagonal pattern and moire patterns were most dis- velope accompanied by nuclear hypertrophy (Fig. 2c). Other tinct in areas where the membrane had folded upon itself (Fig. changes observed at this stage included disappearance of chro- 3e). The inner particle acquired the envelope by envelopment matin characteristic of healthy cells, except for the nucleoli, and in membranes in the cytoplasm of the host cells or vesicles (Fig. the development in some nuclei of what appeared to be a vi- 3f) or by budding through a membrane, either into intracy- rogenic stroma. However, neither mature virions nor recog- toplasmic spaces or out of vesicles. A completely formed virion nizable viral structural components were observed at this stage. in cross section is illustrated in Fig. 3g. Increased levels of glycogen in the cytoplasm also occurred at this stage. The hypertrophy of the nucleus caused extensive DISCUSSION cellular hypertrophy, with infected cells enlarging as much as The virus described above has several structural and devel- five times the diameter of healthy cells. As the nucleus en- opmental properties that, when considered together, readily Downloaded by guest on October 1, 2021 Microbiology: Federici Proc. Natl. Acad. 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FIG. 2. Light and electron micrographs showing viral cytopathology in fat body and epidermal cells. (a) Epon-Araldite section of diseased fat body with uninfected nuclei (small arrowheads) and a greatly hypertrophied infected cell (large arrowheads) in which the nucleus has disrupted. (Bar = 8 ,um.) (b) Two epidermal cells in which the formation of virion-containing vesicles is nearly complete. (Bar = 7 pm.) (c) Infected fat body cell with slightly hypertrophied nucleus and invaginated nuclear envelope (arrowheads). Compare with uninfected nucleus at lower left. (Bar = 2 an.) (d) Hypertrophied fat body cell in which the nuclear envelope (arrowheads) has disrupted. Vesicle membranes have begun to form along the cell periphery. (Bar = 4 pm.) (e and f) Spherical mitochondria in close proximity to small membrane vesicles (e) that will coalesce to form vesicle membrane (arrowheads in f). (Bars = 800 nm.) (g) Virion-containing vesicle from a completely cleaved epidermal cell such as that illustrated in b. (Bar = 2 gm.) distinguish it from all other known viruses. Structurally, the in this matrix suggests the possibility of an inner membrane virion is very large with a symmetry that is unique and is best surrounding the nucleoprotein core, a structural relationship characterized as being of the complex type (9). The shape of the similar to that found in the iridoviruses (10-12) and poxviruses virion, which is determined by the inner particle, is essentially (13). Clearly, however, on the basis of.structure and symmetry allantoid, although in thin sections the inner particle or mature alone, this novel virus from T. ni is not a member of the families virion can appear oval, reniform, elliptical, or naviculate, de- Poxviridae or Iridoviridae. There is a superficial resemblance pending on the plane of section through the inner particle or between this virus. and nonoccluded baculoviruses (type C) in enveloped virion. The substructure of the 25-nm inner mul- that virions of the former are enveloped and occasionally ap- tilaminar matrix that surrounds the DNA core was not clearly pear slightly bacilliform in thin sections. Yet baculoviruses typ- resolved, although the existence of an electron-translucent layer ically have rod-shaped nucleocapsids or nucleocapsids that ta- Downloaded by guest on October 1, 2021 7668 Microbiology: Federici Proc. Natl. Acad. Sci. USA 80 (1983) a~~~~~-: y0 Aim

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FRG. 3. Electron micrographs illustrating virion assembly and structure. (a) Site ofvirion assembly in an epidermal cell illustrating inclusions associated with assembly. (Bar = 500 nm.) (b) Developing inner particles prior to envelopment. (Bar = 100 nm.) (c) Ultrathin section through virion along longitudinal axis. Note repetitive subunits (arrowheads) in outer envelope. (Bar = 100 nm.) (d) Virion from hemolymph illustrating char- acteristic hexagonal pattern observed in negatively stained preparations. (Bar = 100 nm.) (e) Portion ofa large, negatively stained membrane frag- ment from a virion-containing vesicle exhibiting a pattern similar to that observed in virions. (Bar = 25 nm.) (f) Envelopment of inner particle. (Bar = 100 nm.) (g) Ultrathin cross section through virion. Large arrows indicate electron translucent layer of possible inner membrane, and the small arrows indicate the probable location of the subunits on the surface of the inner particle. (Bar = 100 nm.) per only slightly and a single lipid bilayer that exhibits little Autographa precationis, and Spodopterafrugiperda (J. J. Hamm, substructure in negatively stained preparations (14, 15)-struc- personal communication). Comparative studies must be un- tural properties that are considerably different from the new T. dertaken to determine whether these viruses are different from ni virus. Moreover, all baculoviruses contain circular DNA. the virus that occurs in T. ni and whether this new group war- The formation of virion-containing vesicles by cleavage of rants family status. the host cell is another characteristic that differentiates this vi- In summary, evidence is presented for the existence of an- rus from other known viruses and is certainly one of its most other type of enveloped ds DNA virus. The virus has virions unique features. The limiting membrane of the vesicles ap- with complex symmetry and structure that contain high mo- pears to be produced de novo in the case of the membranes lecular weight (>1.00 x 108) DNA. The virus undergoes a novel formed along cleavage planes through the cell and by modifi- developmental sequence that results in the formation of virion- cation and invagination, of the plasma membrane. An intriguing containing vesicles that form from membranes synthesized de aspect of membrane assembly is the apparent modification and novo as a result of virus infection. direct participation of host cell mitochondria. Most membrane assembly and vesicle formation occurs after the disruption of The author thanks J. J. Johnson, C. J. Lucarotti, andJ. Sudduth-Klinger the nuclear envelope, suggesting that these processes, includ- for their assistance in these studies and J. J. Hamm and G. R. Carner ing the involvement of mitochondria, may be viral-directed. for samples of similar viruses from other noctuids. In the initial description of the disease caused by a similar 1. Federici, B. A. (1982) J..Invertebr. Pathol. 40, 41-54. virus in S. trifolii, it was suggested that most viral reproduction 2. Browning, H. W., Federici, B. A. & Oatman, E. R. (1982) En- occurred in the vesicles as they circulated in the hemolymph viron. Entomol. 11, 550-554. because most circulating vesicles were considerably larger than 3. Carner, G. R. & Hudson, J. S. (1983)J Invertebr. Pathol. 41, 238- those observed in host tissues. based on 249. (1). However, the pres- 4. Shorey, H. H. -& Hale, R. L. (1965) J. Econ. Entomol. 58, 522- ent study it would appear that, at least in epidermal cells, large 524. numbers of virions are assembled prior to release of the ves- 5. Miller, L. K. & Dawes, K. P. (1978) Environ. Microbiol. 35, 1206- icles from host cells. Further studies are needed to determine 1210. whether viral replication and assembly continue after the ves- 6. McCarthy, W. J., Murphy, T. F. & Langridge, W. (1979) Virology icles are liberated into the hemolymph. 95, 593-597. 7. Kleinschmidt, A.. L. (1968) Methods Enzymol. 12B, 361-377. The significant differences between this virus type and known 8. Locke, M. (1980) in Insect Biology in the Future, VBW 80, eds. enveloped ds DNA viruses-i.e., Poxviridae, Herpesviridae, Locke, M. & Smith, D. S. (Academic, New York), pp, 227-252. Baculoviridae, and Plasmaviridae (9)-justify the establish- 9. Matthews, R. E. F. (1982) Intervirology 17, 1-200. ment of a new virus group. The Ascovirus is tentatively 10. Stoltz, D. B. (1973)J. Ultrastruct. Res. 43, 58-74. proposed to accommodate this virus and others with similar 11. Wrigley, N. G. (1969)J. Gen. Virol. 5, 123-134. properties. The prefix asco- (= sac, bladder) refers to the viral- 12. Kelley, D. C. & Vance, D. F. (1973)J.-Gen. Virol. 21, 417-423. 13. Granados, R. R. (1973) Misc. Publicat. Entomol. Soc. Am. 9, 73- induced virion-containing vesicles that characterize the disease 94. caused by this virus and differentiate it from known members 14. Summers, M. D. (1977) in TheAtlas of Insect and Plant Viruses, of the above families. Other likely members of this new group ed. Maramorosch, K. (Academic, New York), pp. 3-27. include viruses recently reported in S. trifolii (1), H. zea (3), 15. Harrap, K. A. (1972) Virology 50, 124-132. Downloaded by guest on October 1, 2021