Susceptibility of human skeletal muscle culture to influenza virus infection

Journal of the Neurological Sciences, 1978, 36: 63-81 © Elsevier/North-Holland Biomedical Press

63

SUSCEPTIBILITY OF H U M A N S K E L E T A L MUSCLE C U L T U R E TO INF L U E N Z A VIRUS I N F E C T I O N Part 2. Ultrastructural Cytopathology

A. F. MIRANDA, E. T. GAMBOA, C. L. ARMSTRONG and K. C. HSU Departments of Neurology, Pathology and Microbiology, College of Physicians and Surgeons, Columbia' University, New York, N. Y. 10032 and The H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases (U.S.A.)

(Received 1 September, 1977) (Accepted 15 November, 1977)

SUMMARY Cultured post-fused human skeletal muscle monolayers exposed to WSN influenza A virus were analyzed by scanning and transmission electron microscopy. At 12-14 h post-inoculation (p.i.), affected mononuclear cells retracted from the cell surface, but remained anchored to the substrate by taut filar processes. Retraction was accompanied by shortening of microvilli, appearance of hemispherical cytoplasmic protrusions and corrugation of the surface proper. These changes were more pronounced at 24 and 48 h p.i. The rounded, moribund mononuclear ceils eventually detached from the substratum. Surface alterations were accompanied by the intracellular appearance of electron-dense nuclear inclusions (often associated with the nucleolus) and paracrystalline ribosomestudded cytoplasmic bodies, which increased in size and number with time. In myotubes, distinct surface alterations appeared later (24 h p.i.). Early myotube retraction was accompanied by accentuation of the longitudinally oriented surface pleats and appearance o f " b l e b s " followed by cell-rounding. At 48-72 h, many myotubes detached from the substratum. The surfaces of those still adhering appeared corrugated. Intranuclear and cytoplasmic inclusions accumulated, and budding virions, often filamentous, could be demonstrated at the plasmalemma of mononuclear cells and myotubes. Late (end-stage) cytopathic effects included clumping of chromatin, breakdown of the nuclear envelope, disappearance of cortical and endoplasmic cytofilaments, mitochondrial swelling, and vesiculation of surface membranes. The lesions This study was supported by N.I.H. Training Grant No. 5T01NS0506221,Parkinson Disease Foundation, NS-11766 of the National Institute of Neurological and Communicative Disorders and Stroke, and the Muscular Dystrophy Association, Inc.

64 leading to cell injury and cell death appeared to be due to massive accumulation o~ virus-induced products that altered cellular metabolism, with physical and functional abnormalities of surface membranes.

INTRODUCTION Experimentally-induced influenza virus infections have been studied by electron microscope in animal hosts and tissue culture, but there has been no ultrastructural analysis of influenza virus-induced cytopathology of cultured striated muscle. A study of this kind is timely because of the numerous reports of virus-like particles, including myxovirus, in biopsied muscle of patients with polymyositis or dermatomyositis (Chou 1967; Mastaglia and Walton 1970; Tang, Sedmak, Siegesmund and McCreadie 1975). The data reported here complement those of a preceding paper (Gamboa, Armstrong, Miranda and Hsu 1978) which demonstrated that postfused adult human skeletal muscle in culture is susceptible to WSN-influenza A virus infection as evidenced by virus growth curves, hemadsorption, and immunocytochemistry. O'Neill and Kendal (1975) also showed influenza A virus-induced cytopathology by light microscopy, as well as production of hemagglutinins in cultures of embryonic chick muscle. We now describe the ultrastructural cytopathology (by transmission electron microscopy (TEM) and scanning electron microscopy (SEM)) of cells in adult human muscle cultures infected with WSN influenza A virus. Preliminary findings have been communicated (Miranda, Armstrong, Hsu and Gamboa 1976). MATERIALS AND METHODS Muscle culture methods, preparation of the influenza virus, and inoculation procedures have been described previously (Gamboa et al. 1978).

Transmission Electron Microscopy (TEM) Cultures were fixed in 2 ~ phosphate-buffered glutaraldehyde (pH 7.3), scraped off the substrate with a rubber "policeman", rinsed, osmicated and stained in bulk with 0.25 ~ aqueous uranyl acetate for 1 h. The uranyl acetate block stain was carried out to enhance contrast of virus components (Acheson and Tamm 1967). This treatment however, also extracted glycogen and water-soluble components. The preparations were then pelleted in agar according to a modification of the method of Hirsch and Fedorko (1968), dehydrated and embedded in Epon 812 according to standard procedure (Hayat 1970). 0.5/~m sections were examined with phase optics. Selected areas of the block face were thin-sectioned and examined with a Siemens Elmiskop I electron microscope.

Scanning Electron Microscopy (SEM) All monolayers were fixed according to the method of Porter, Kelley and Andrews (1972), dried by the critical point method in a Denton DCP-1 apparatus, covered

65 with carbon and gold-palladium in a Denton DV-502 evaporator (Denton Vacuum Inc., Cherry Hill, N.J.) and examined with a JSM-U3 scanning electron microscope, operated at 25 kV.

A utoradiography Muscle monolayer cultures were grown on 22-mm coverslips and infected with virus as described previously (Gamboa et al. 1978). The cultures were rinsed in phosphate-buffered saline (PBS) and then exposed to complete muscle medium containing 10 #Ci/ml of tritiated thymidine ([SH]TdR) (specific activity 29 Ci/mmol) for 15 and 30 min at 0, 4, 18, 24 and 48 h p.i. The cultures were rinsed 3 times with PBS, then fixed overnight in methanol-formalin-acetic acid (85:10:5) and prepared for autoradiography by dipping in a K-5 emulsion (Ilford Ltd., Essex, Great Britain). After 2 weeks at 4°C, they were developed and stained with hematoxylin and eosin. RESULTS

Sham-inoculated cultures By SEM, the myotubes appeared as raised, elongated, often branched structures, measuring up to 11 mm in length and up to 1 mm in width. Most small myotubes exhibited straight or slightly curved projections (microvilli) and occasional knobby protrusions on the dorsal surfaces. The larger myotubes were generally smoother with surfaces thrown into longitudinally oriented folds or pleats (Fig. I a). They appeared to be anchored to the glass substratum by lateral stout, flattened, often triangular peripheral cytoplasmic extensions. Interspersed with myotubes were flattened mononuclear cells (presumably fibroblasts and unfused myogenic cells). These cells were polygonal or triangular in shape. Their smooth surfaces had occasional microvilli (Fig. 2a). There were fewer spindle-shaped cells with rounded contours, often aligned in parallel. Solitary spherical cells, attached to substrate by taut slender cytoplasmic extensions and covered with long microvilli and knobs ("blebs"), seemed to be cells preparing for mitosis. By TEM, myotube nuclei were centrally located and usually contained 1 to 2 electron-dense nucleoli. In longitudinal sections, some syncytia exhibited developing myofibrils, but sarcomeres with distinct cross-striations were present in fewer than 5 of the myotubes. The sarcoplasm of these cells contained oval or filamentous mitochondria, profiles of developing sarcoplasmic reticulum (sr) and aggregates or bundles of myofibrils. Subsurface microfilaments and tubular invaginations of the surface membrane were commonly present (Fig. lb). The basal lamina was either absent or rudimentary, represented by irregular patches of electron-dense material. Most mononuclear cells of post-fused cultures had a fibroblast-like morphology. In the cytoplasm there were numerous profiles of often distended sarcoplasmic reticulum, some mono- and polyribosomes and moderate numbers of ovoid or rod-like mitochondria (Fig. 2b). Glycogen extracted during processing for electron microscopy (EM) was not demonstrable.

66

67

Fig. 3. TEM of a mononuclear cell in a WSN influenza-infected culture of adult human muscle 2 h p.i. The culture was exposed to Earle's balanced salt solution (EBSS), fixed and prepared for electron microscopy as indicated in Materials and Methods. A virus particle of the inoculum (arrow) has become attached to the membrane at an invagination of the plasmalemma. The cortical cytoplasm is markedly vacuolated. × 36,000.

Fig. la. Scanning electron micrograph (SEM) of a normal myotube in a sham inoculated culture of adult human muscle, 24 h after optimal fusion. The myotube is anchored to the substratum by blunt cytoplasmic extension. The surface is thrown into shallow pleats parallel to the long axis. Occasional small knobs are present on the free surface. The peripheral areas of cytoplasmic sheets of mononuclear cells are visible above and below the myotube. × 3,700. Fig. 1b. Transmission electron micrograph (TEM) of a peripheral and neighboring area of a myotube as represented in Fig. la. Myofibrils (My), cut obliquely, are prominent in the subcortex and interspersed with them are elements of the sarcoplasmic reticulum (sr). Beneath the plasmalemma there is a felt of 5 nm actin-type microfilaments (mf). Arrows point to rudimentary elements of a developing transverse tubular system. No basement membrane material is apparent at this time (mi: mitochondria; db: lysosomal dense body). × 33,800. Fig. 2a. SEM of part of a mononuclear fibroblast-like cell in a control post-fused culture. Asterisk indicates the nuclear area. The free surface of the cell is sparsely beset with microvilli. On the left, the cell appears to adhere to substrate and to a neighboring cell by a broad sheet of cytoplasm at the margin of which there are thin filopodial processes. × 2,000. Fig. 2b. TEM of a section of a fibroblast as illustrated in Fig. 2a. The cytoplasm contains numerous profiles of distended rough endoplasmic reticulum (er) and some ribosomes. A mitochondrion (mi) and nucleus (N) are indicated. × 23,000.

Fig. 4a. SEM of 2 mononuclear cells in a WSN influenza-infected human muscle culture 12 h p.~. The moderately retracted cell (top) still remains adherent to the substratum by several broad cytoplasmic extensions. The dorsal surface exhibits some microvilli and some small, clustered protrusions. "l-he curved filar processes on the dorsal cell surfaces represent cytoplasmic processes which apFear to have snapped while the cell was retracting. The more severely contracted cell below is still anchored to the glass by numerous taut filar cell extensions (retraction fibers), which radiate from the central portion of the cell body. A constricted band of cytoplasm is indicated by arrows. 2,000. Fig. 4b. TEM of a nuclear (N) area o f a mononuclear cell exposed to a WSN influenza-infected culture 12 h p.i. The amorphous nucleolus (nu) is enlarged and contains many electron-dense inclusions (arrow heads). × 20,800. Fig. 4c. Electron microscope (EM) section of a mononuclear cell in a WSN influenza-infected muscle culture 12 h p.i., showing an accumulation of electron-dense ribosome-studded inclusions (arrows) (nucleus: N; rough endoplasmic retieulum: er; droplets: L). × 21,000.

69

Cultures infected with the WSN strain of influenza A virus Early events. Muscle cultures, when examined by SEM at 2-4 h p.i. showed minor surface changes in a few of the mononuclear cells. Their flattened surfaces exhibited small to moderate numbers of knobby protrusions ("blebs") and a decreased number of microvilli. Myotubes were still of normal morphology. Sections examined by TEM showed solitary, round (110-120 nm) viral particles of the inoculum adherent to the plasmalemma of three of 32 mononuclear cells examined (Fig. 3). Small infoldings of the plasmalemma and vacuole; beneath the plasma membrane were commonly observed in both mononuclear cells and myotubes. At 12-16 hr p.i., peripheral cytoplasm had been retracted centripetally, although the cells still adhered to the substrate and to neighboring cells by flattened cytoplasmic ribbons or taut filamentous processes (retraction fibers), thus assuming a spidery or star-shaped appearance (Fig. 4a). The dorsal surfaces of these ceils showed microvilli of varying length and occasional knobby protrusions. In more severely contracted cells, the cytoplasm overlying the nuclear area appeared corrugated (Fig. 4a). TEM of these retracted mononuclear cells 12-16 h p.i. revealed prominent, dense nucleoli occupying a third to half of the nucleoplasm. In about one-third of these cells examined 16 h p.i., the nucleoli contained electron-dense, somewhat elongate inclusions 100-200 nm long (Fig. 4b), most numerous at or near the periphery of the nucleoli. The nucleoplasm proper was mostly unremarkable, but on occasion some clumping of chromatin was evident. An even more common feature at this time was the appearance of elongated or multiform, paracrystalline cytoplasmic inclusions of high electron density. The paracrystalline nature of these inclusions was only visible at high magnification (see inset: Fig. 9c). These structures, ranging in size from 100 to 1000 nm, were studded with ribosome-like particles (Fig. 4c). They were frequently present in cells with hypertrophic nucleoli in which nucleolar inclusions were absent. Except for dense bodies, the cytoplasm and other organelles appeared unaltered. At 12-16 h p.i., most myotube surfaces were still of normal morphology. A few myotubes showed some retraction away from the substratum and accentuation of the longitudinally-oriented surface pleats. In TEM sections some myotubes had large hypertrophic nucleoli but with no nucleolar inclusions. Cytoplasmic inclusions similar to those in mononuclear cells were only seen in 2 of 12 myotubes examined. Solitary budding virions at the plasmalemma were seen in some mononuclear cells but not in the myotubes. The intermediate phase of infection. At 24 h p.i., 80 ~ or more of the mononuclear cells showed marked cytopathic changes, but less than half of the myotubes appeared to be affected. When examined by SEM, the surfaces of most mononuclear cells were corrugated; filamentous processes and microvilli were rare or absent because most peripheral cytoplasmic extensions (retraction fibers) of contracting cells appeared to have snapped and recoiled. Some spherical or filamentous cytoplasmic remnants remained firmly adherent to the substrate (Fig. 5a). In many preparations, there were spaces within the cellular debris where cells had been dislodged or sloughed. Some mononuclear cells, often in groups of four or more, showed no cytopathic changes.

70

71 By T E M , affected mononuclear cells exhibited the nuclear and cytoplasmic changes seen earlier, but the n u m b e r o f nuclear and cytoplasmic inclusions had increased. M a n y nucleoli were segregated into discrete masses of high electron density. Vacuoles, probably derived f r o m plasmalemma, 0.1-0.5 # m in diameter, were frequently seen in the peripheral cytoplasm o f mononuclear cells and less c o m m o n l y in myotubes showing moderate cytopathic changes. Within the nuclei of some mononuclear cells and occasional myotubes there were straight tubular structures of varying length, with an outer diameter o f 38-40 n m and 20 nm electron-lucent core (Fig. 5d). Most of these structures were not associated with nucleoli. At this stage of infection, a b o u t half of the myotubes showed cell surface changes. The pleated surfaces were beset with solitary or clustered k n o b b y projections 0.1-3.0 /~m long (Fig. 6a). A few myotubes appeared rippled, with numerous infoldings perpendicular to the long axis. Others had retracted from the substratum at one or both ends in club-like or halter-like appearances. Sections of myotubes examined by T E M revealed intranuclear electron-opaque inclusions, 80-100 nm in diameter. They were most frequently seen within or adjacent to nucleoli (Fig. 6c). In the sarcoplasm there were paracrystalline ribosome-studded bodies. The intranuclear and sarcoplasmic inclusion bodies were similar to those in mononuclear ceils at 12 h and 24 h p.i. In some syncytia the electron-dense sarcoplasmic inclusions appeared in groups (Fig. 6b) or were clustered or fused into irregular masses of up to 2000 nm (Fig. 6d). The peripheral sarcoplasm, beneath irregularly contoured surface membranes was generally filled with a continuous pad or mat o f microfilamentous (Fig. 6b) material of greater compaction and density than in controls. A few myotubes exhibited tortuous tubular profiles in the cortical cytoplasm, some continuous with the extracellular space (Fig. 6c, d). Such tubular structures, also seen occasionally in sham-inoculated cultures, probably represent proliferation o f the developing transverse tubular system. Myofibrils, mitochondria and developing sarcoplasmic reticulum showed no alterations at this time. A few lysosome-like bodies, some o f high electron density, were c o m m o n l y seen in both mononuclear cells and myotubes but they were not a prominent feature (Figs. 6b, d).

Fig. 5a. SEM of a retracted fibroblast-like cell in WSN influenza-infected culture 24 h p.i. Most cell processes appear to have snapped. Some cytoplasmic fragments adhering to the substratum or to the cell surface have rounded up into globules. The cell surface is irregular; small protrusions on the dorsal cell surface may represent microvillar remnants. × 2,000. Fig. 5b. Section ofa mononuclear cell treated as in Fig. 5a and examined by TEM. The nucleus contains two large nucleolar areas of high electron density. No cytoplasmic inclusior~s are discernible. The cortical cytoplasm has a fenestrated appearance due to numerous membrane-bound vacuoles. A budding virion is indicated (arrow). × 22,700. Fig. 5c. EM of a mononuclear cell in WSN influenza-infected culture 36 b p.i. The nucleolus (nu) is only moderately enlarged. The cytoplasm is not remarkable except for some v~cuoles in the cortical cytoplasm. Several virions (v), mostly elongate or ovoid, are present at the dorsal cell surface. A virion is also detectable at the ventral surface (endoplasmic reticulum: er; microfilaments: mf). x 22,350. Fig. 5d. Section of a nucleus in a mononuclear cell treated as in Fig. 5c. In the nucleolar area there are several circular profiles with electron-lucent cores. A similar structure cut longitudinally is also indicated (arrow heads), x 85,000.

72

73 At 24-36 h p.i., virions with spikes at their surfaces, mostly filamentous in form (up to 2000 nm long and about 100 nm in diameter) budded off the plasmalemma of many mononuclear cells (Fig. 5b, c) and some myotubes. The cells showing large numbers of budding virions were not necessarily those with large numbers of nucleolar or cytoplasmic inclusion bodies. In fact, cells with many inclusions appeared to produce less virus (when judged by the number of virions demonstrable at or near the cell surface). Many virus-producing mononuclear cells and myotubes had prominent nucleoli, but with little nucleolar or cytoplasmic inclusions or other cytopathologic changes. At 36-48 h, most mononuclear cells and almost all myotubes had undergone striking surface changes. More than half of the affected mononuclear cells had been sloughed off and were seen to float in the feeding medium; those still adhering had retracted but remained attached to the glass by numerous branched cytoplasmic extensions. The cell surface, devoid of microvillar processes, was pitted or wrinkled (Fig. 7b). Myotubes frequently maintained an elongate form, but the surfaces appeared corrugated or pitted and cell-to-substrate adhesion points were fewer than in affected mononuclear cells (Fig. 7a). By TEM, elongated and ovoid or spherical virions could be demonstrated at cell surfaces (Figs. 7c, 8). Cytoplasmic ribosome-studded densities were sometimes observed, solitary or in clusters. The surface membrane appeared wsiculated in some cells but was not interrupted in most myotubes studied. Myofilaments, mitochondria and sarcoplasmic reticulum appeared unaltered. Late effects. At 48 and 72 h, in sister cultures treated with trypan blue, more than 80 ~ of the mononuclear cells took up the dye (i.e. were leaky or moribund); 50 ~ of the myotubes accumulated dye at 48 h and more than 80 ~ at 72 h (see Phillips 1973). Most affected mononuclear cells had become spherical by 48 h (Fig. 9b), with many cytoplasmic remnants still adhering to the substrate. At 72 h, most syncytia had lost the characteristic elongated shape; instead, there were bizarrely shaped structures (Fig. 9a). Intracellularly, these end-stage myotubes were mostly devoid of fibrillar elements (i.e. microfilaments and myofibrils) and intact mitochondria (Fig. 9c). Internal membranes were swollen. Virus-induced cytoplasmic bodies were denuded of ribosomelike particles and virions were frequently demonstrable outside of the vesiculated cell

Fig. 6a. Part of a myotube examined by SEM in WSN influenza-infected culture 12 h p.i. The cell surface exhibits pleats parallel to the long axis. There are numerous knobby protrusions (arrows) sometimes aggregated in clusters, x 3,750. Fig. 6b. Section of a myotube treated as in Fig. 6a. The cell surface is lumpy. The cytoplasmic cortex is filled with a continuous pad of microfilaments (mr). The subcortex exhibits a cluster of electron-dense inclusions with ribosomes at their surfaces (arrows). Myoflbrils cut tangentially (My), sarcoplasmic reticulum (sr) and mitochondria (mi) are of normal morphology (vacuole: v). x 32,500. Fig. 6c. Section of a myotube in a culture treated as in Fig. 6b. The nucleoplasm contains inclusions of high electron density. Membrane-bound structures budding from Golgi membranes are probably coated ("basket") vesicles, unrelated to influenza A. x 29,400. Fig. 6d. Section of a cortical and subcortical area of a myotube in WSN influenza-infected culture, as indicated in Fig. 6c. The plasmalemma exhibits deep infoldings and tubular structures (t), apparently in contact with extracellular space. A lysosome-like body is seen between the tubules. In the cortex there are large aggregates of virus-induced ribosome-studded cytoplasmic inclusions (arrow). Lipid : L. × 24,100.

V

i¸77¸

,++,

+i +,

Fig. 7a. SEM of a sector of a myotube in W S N influenza-infected culture 48 h p.i. The nlyotube is loosely adherent to the substratum. The entire myotube surface is corrugated and pitted with craterlike indentations. : 3,700. Fig. 7b. SEM of a mononuclear cell of a W S N influenza-infected culture 36 h p.i. The cell surface is irregular. The retracted cell body is still adherent to the glass by slender cytoplasmic processes. ~s 2,000. Fig. 7c. Cortical and endoplasmic area of a tangentially cut myotube of a W S N influenza-infected culture. The myotube surface is irregular. Two coated ("basket") vesicles (arrow heads) and a virion (v) are indicated. Virus-induced dense bodies: arrows ; sarcoplasmic retieulum; sr; myofibrils : My : l i pid : L; developing t-system: t. ~; 29,300.

75

Fig. 8. TEM of a sectioned myotube of a WSN influenza-infected culture 48 h p.i. showing budding virions (v) and extracellular filamentous viruses (V1). (Myofibrils: My; sarcoplasmic reticulum: sr; microfilaments: mr.) × 59,000.

A

. . . . . . . . . .

Fig. 9a. SEM micrograph of a myotube of a WSN influenza-infected culture 72 h p,i. The retractcd, branched myotube has lost its elongated profile. The corrugated surface is devoid of distinct protrusions or peripheral cytoplasmic projections. ~ 3,700. Fig. 9b. SEM micrograph o f a mononuclear cell o f a WSN influenza-infected culture 48 h p.i. ]-he cell has rounded-up into an irregular spheroid. Detached cytoplasmic fragments, adhered to the glass, are visible near the cell periphery. Part of a smooth, flattened, unaltered mononuclear cell is discernible below. × 2,000. Fig. 9c. Section of an end-stage myotube of a WSN influenza-infected culture 72 h p,i, Cytoplasmic filaments are no longer discernible. The sarcoplasm contains many membrane-bound globules, of uncertain identity, filled with proteinoid granular material. Numerous electron-dense cytoplasmic inclusions (arrows) are indicated. Virions: v. ~, 32,400. Inset: high magnification of paracrystaltine cytoplasmic inclusions. ~ 72,900.

77 membranes (Fig. 9c). In most instances, the chromatin was clumped and nuclear envelopes were either ruptured or absent. Even at this final stage lysosome-like structures were observed only infrequently. Despite the marked virus-induced changes in the nuclei of myotubes, re-initiation of DNA synthesis was not detected autoradiographically in cultures that were pulselabeled with [3H]TdR at intervals p.i. We saw no budding virions on internal membranes or intact virus particles in the cytoplasm of mononuclear cells or myotubes. No nucleocapsid-like material was seen in the cytoplasm at any time. DISCUSSION In this study we examined WSN influenza A virus production and virus-induced cytopathology on cultured monolayers of adult human skeletal muscle by TEM and SEM. In this heterogeneous cell system, both fibroblast-like mononuclear cells and multinucleate myotubesproduced virus. Virus growth and maturation was accompanied, intracellularly, by the appearance of inclusion bodies similar to those described in other cell culture systems susceptible to influenza A virus infection (Morgan, Hsu, Rifkind, Knox and Rose 1961; Compans and Dimmock 1969; Archetti, Bereczky, Rosati-Valenti and Steve-Bocciarelli 1970; Wolf, Barden, Takahata, Benitez, Gamboa, Harter and Duffy 1974). Virus-induced changes in the nucleus and cytoplasm were accompanied by pronounced cell surface alterations visible in SEM.

Virus-induced changes in the nucleus The first noticeable virus-induced nuclear change observed in EM was an increase in size and prominence of nucleolar components. Nucleolar hypertrophy coincided with the appearance of viral antigen in nuclei of infected cells, as demonstrated previously (Gamboa et al. 1978) and preceded formation of virus-induced nuclear and cytoplasmic inclusion bodies. The functional significance of virus-induced inclusions, usually associated with nucleoli is not known, but they underscore the significance of a host nucleolar function in influenza A virus replication. The virus-induced nucleolar-associated inclusion bodies were described in WSN influenza A-infected chick embryo fibroblasts by Compans and Dimmock (1969) and may be identical to the structures in influenza A-infected cells (Morgan et al. 1961), which bind ferritinconjugated antibody to influenza A virus. The virus-induced tubular inclusions (Fig. 5d) were similar to those seen in monkey kidney cells infected with several strains of influenza virus, which consist of virus-induced ribonucleoprotein (Archetti et al. 1970). Virus-induced cytoplasmic changes In the interim period between virus ingress into cells and the appearance of nucleolar hypertrophy, the only observable cytoplasmic change was the appearance of deep plasmalemmal infoldings and subsurface membrane-bound vesicles which were rarely seen to contain intact virus particles. The first ribosome-studded, electron-

78 dense cytoplasmic inclusions were noted 12-16 h p.i. They were similar in size and form to those described by Compans and Dimmock (1969) at 5 h p.i. in WSN influenza A-infected chick embryo fibroblast cytoplasm and at 12 h p.i. by Kopp, Kempf and Kroeger (1968). These inclusions often appeared before discrete nucleolar inclusions were noticeable, lending credence to the hypothesis of Compans and Dimmock (1969) that the nucleolar inclusions are not precursors of the cytoplasmic bodies. The basophilic HCl-hydrolyzable inclusions and "basophilic streaks" in myotubes observed by light microscopy (Gamboa et al. 1978) appear to be identical with the aggregates of the ribosome-studded inclusions we observed here in EM. Neither nucleolar nor cytoplasmic bodies seem to be essential for virus production because they were frequently lacking in virus-producing ceils and in WSNinfluenza-induced encephalopathy; however, these inclusions may be absent even in cells producing large numbers of virions (Duffy, Wolf, Harter, Gamboa and Hsu 1973). Compans and Dimmock (1969) suggested that the large crystalloid aggregates could represent "virus-derived products which will not be incorporated into virus particles".

Virus-induced cell surface changes and cell death Microvilli are normally found on the dorsal surface of cells cultured in monolayer. Prior to the retraction and rounding of mononuclear cells and myotubes, these microvilli appeared to decrease in length and coincident with the shortening, hemispherical knobby protrusions ("blebs") were noted. Later, the cells began to retract and subsurface filaments (microfilaments) appeared compacted and denser than in controls. This "agonal contraction" (Bessis 1964) is not unique to influenza A-infected cells; it is commonly observed in lytic infections produced by viruses (Tamm 1975) and can be induced by chemicals, such as cytochalasin (Miranda, Godman, Deitch and Tannenbaum 1974). Prior to cell lysis, detachment and cell death, the most prominent change in the cytoplasm of infected cells was the massive accumulation of ribosome-studded dense bodies. It preceded or occurred simultaneously with pronounced cell surface corrugation as noted by SEM (Figs. 5a, 7a, b, 9a). Mitochondrial swelling and detachment of ribosomes from the endoplasmic membranes, indicative of lysis and cell death, were noted only later. Influenza A virus replication is not strictly related to cell injury (Gavrilov, Asher, Vialushkina, Ratushkina, Zmieva and Tumyam 1972). From our studies, the major factors contributing to cell injury seem to be the massive accumulation of virus-derived ribonucleoproteins, altering cellular metabolism and membrane function. Cell lysis is probably not related to lysosomal enzyme release because lysosomes were not prominent in infected cells at any time. However, these assumptions should be put to more rigid experimental test. Influenza A virus replication requires some poorly understood host-nuclear function. This function does not involve replication of host DNA because inhibitors of DNA synthesis do not affect virus production (Scholtissek and Rott 1961 ; Barry, Ives, Cruickshank 1962; Nayak and Rasmussen, Jr. 1966). Moreover, intact nuclei

79 of cells in which DNA replication has ceased, such as avian erythrocytes hybridized with permissive mammalian cells (Kelly and Dimmock 1975), differentiated neurons (Wolf et al. 1974), and post-fused muscle syncytia (O'Neill and Kendal 1975; Gamboa et al. 1978), can support influenza A virus replication. In addition, there appears to be no virus-induced re-initiation of DNA synthesis because Bell and Masaab (1969) reported only a mild, transient increase in incorporation of radiothymidine in DNA of conjunctival cells infected with NWS influenza A virus, and Nayak and Rasmussen (1966) failed to detect an increase of DNA synthesis in infected chick fibroblasts. In our study, also, re-initiation of DNA synthesis was not detected autoradiographically in post-fused muscle cells. Thus, it seems that a post-DNA replication step is involved. Evidence to support this notion is that agents (such as actinomycin D) which interfere with DNA-dependent RNA synthesis and ultraviolet light irradiation (Barry et al. 1962; Barry 1964; Compans and Choppin 1975) inhibit virus replication. An intact cell nucleus is essential for influenza A replication because cytoplasts of permissive cells, enucleated with the fungal metabolite cytochalasin failed to become infected when exposed to influenza A. Replication of other RNA viruses which did not require a nucleus, were unaffected (Follett, Pringle, Wunner and Skehel 1974; Kelly, Avery and Dimmock 1974; also see Scholtissek and Klenk 1975). Our study demonstrated that both myotubes and mononuclear cells contribute to production of virus in post-fused human muscle cultures. The virus-induced changes were similar to those in other host cell systems. Since DNA replication has ceased in post-fused muscle, this culture system seems ideal for investigating the precise postDNA replication step in the cell nucleus which is essential for influenza A virus synthesis. In vitro studies of this kind may also contribute to understanding the possible viral etiology of myalgia and acute myositis sometimes associated with human influenza infections (Mejlszenkier, Safran, Healy, Embree and Ouellette 1973; Dietzman, Schaller, Ray and Reed 1976). A variety of host factors play a role in susceptibility to influenza in vivo because skeletal muscle of infected adult animals are known to be insusceptible to viral infection (Wagner 1955). However, mouse skeletal muscle becomes susceptible to WSN influenza A virus after denervation (Hays and Gamboa 1977). Since mature muscle resists infection and cultured muscle is susceptible, developmental changes in the surface membrane probably alter virus receptor sites. Alternatively, a fully developed muscle basal lamina (which is rudimentary or absent in the culture system used here) could provide an efficient mechanical barrier to virus adsorption. Studies of innervated skeletal muscle in vitro might answer these questions. ACKNOWLEDGEMENTS The authors would like to express their thanks to Mrs. Viola Mahoney, Dr. Myrna Retino and Mrs. Shahnaz Khan for technical assistance and to Mrs. Carolyn Waysse for her advice and editorial assistance.

80 REFERENCES Acheson, N. H. and I. Tamm (1967) Replication of Semliki Forest virus - - An electron microscopic study, Virology, 32: 128-143. Archetti, I., E. Bereczky, F. Rosati-Valenti and D. Steve-Bocciarelli (1970) Elongated structures present in cells infected with influenza viruses, Arch. ges. Virusforsch., 29: 275-286. Barry, R. D. (1964) The effects of actinomycin D and ultraviolet irradiation on the production of fowl plague virus, Virology, 24: 563-569. Barry, R. D., D. R. lves and J. G. Cruickshank (1962) Participation of deoxyribonucleic acid in the multiplication of influenza virus, Nature (Lond.), 194: 113%1140. Bell, W. C. and H. F. Masaab (1969) Nucleo-cytoplasmic studies in the development of influenza virus in mammalian cells, Arch. ges. Virusforsch., 27: 128-137. Bessis, M. (1964) Studies on cell agony and death - - An attempt at classification. In: A. Renck and J. Knight (Eds.), Cellular Injury, Little, Brown and Co., Boston, Mass., pp. 287-328. Chou, S. M. (1976) Myxovirus-like structures in a case of human chronic polymyositis, Science, 158: 1453-1455. Compans, R. W. and P. W. Choppin (1975) Reproduction of myxoviruses. In: H. Fraenkel-Conrat and R. R. Wagner (Eds.), Comprehensive Virology, Vol. 4 (Reproduction of Large RNA Viruses), Plenum Press, New York and London, pp. 179-252. Compans, R. W. and N. J. Dimmock (1969) An electron microscope study of single-cycle infection of chick embryo fibroblasts by influenza virus, Virology, 39: 499-515. Dietzman, D. E., J. Schaller, C. G. Ray and H. E. Reed (1976) Myositis associated with influenza B infections, Pediatrics, 57: 255-258. Duffy, P. E., A. Wolf, D. Harter, E. Gamboa and K. C. Hsu (1973) Murine influenza virus encephalomyelitis, Part 2 (Electron microscopic observations), J. Neuropath. exp. Neurol., 32: 72-91. Follett, E. A. C., C. R. Pringle, W. H. Wunner and J. J. Skehel (1974) Virus replication in enucleate cells - - Vesicular stomatitis virus and influenza virus, J. Virol., 13: 394-399. Gamboa, E. T., C. L. Armstrong, A. F. Miranda and K. C. Hsu (1978) Susceptibility of human skeletal muscle culture to influenza virus infection, Part 1 (Cytopathology and immunofluorescence), J. neurol. Sci., 35: 43-57. Gavrilov, V. I., D. M. Asher, S. D. Vyalushkina, L. S. Ratushkina, R. G. Zmieva and G. B. Tumyam (1972) Persistent infection of continuous line of pig kidney cells with a variant of WSN strain of influenza A0 virus, Proc. Soc. exp. Biol. Med., 140: 109-117. Hayat, M. (1970) Principles and Techniques of Electron Microscopy, Vol. 1 (Biological Applications), Van Nostrand Reinhold Co., Div. of Litton Educational Publishing Inc., New York, pp. 155-159. Hays, A. P. and E. T. Gamboa (1977) Influenza infection in mouse-denervated muscle (Abstract), Presented at the 1 lth World Congress of Neurology, Amsterdam, 13 Sept., 1977. Hirsch, J. and Fedorko (1968) Ultrastructure of leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and "post-fixation" in uranyl acetate, J. CellBiol., 38 : 615-627. Kelly, D. C., R. J. Avery and N. J. Dimmock (1974) Failure of an influenza virus to initiate infection in enucleate BHK cells, J. Virol., 13: 1155-1161. Kelly, D. C. and N. J. Dimmock (1975) Cell fusion: A new approach to the study of influenza virus multiplication. In: R. D. Barry and W. J. Mahy (Eds.), Negative Strand Viruses, Academic Press, New York, pp. 469-473. Kopp, J. F., J. E. Kempf and A. V. Kroeger (1968) Cytoplasmic inclusions observed by electron microscopy late in influenza virus infection of chick embryo fibroblasts, Virology, 36: 681-4~83. Mastaglia, F. L. and J. N. Walton (1970) Coxsackie virus-like particles in skeletal muscle from a case of polymyositis, J. neurol. Sci., l l : 593-599. Mejlszenkier, J. D., A. P. Safran, J. J. Healy, L. Embree and E. M. Ouellette (1973) The myositis of influenza, Arch. Neurol. (Chic.), 29: 441~143. Miranda, A. F., C. L. Armstrong, K. C. Hsu and E. T. Gamboa (1976) Cytopathology and localization of influenza virus in cultured human muscle, J. Nel~ropath. exp. NeuroL, 35:339 (Abstract). Miranda, A. F., G. C. Godman, A. D. Deitch and S. Tanenbaum (1974) Action of cytochalasin D on cells of established lines, Part 1 (Early events), J. Cell Biol., 61 : 481-500. Morgan, C., K. C. Hsu, R. A. Ritkind, A. W. Knox and H. M. Rose (1961) The application of ferritinconjugated antibody to electron microscopic studies of influenza virus in infected cells, Part 2 (The interior of the cell), J. exp. Med., 114: 833-836. Nayak, D. P. and A. F. Rasmussen, Jr. (1966) Influence of Mitomycin C on the replication of influenza viruses, Virology, 30: 673-683.

81 O'Neill, M. C. and A. P. Kendal (1975) Infection of differentiating muscle cells with influenza and Newcastle disease viruses, Nature (Lond.), 253 : 195-198. Phillips, H. J. (1973) Dye exclusion test for cell viability. In: P. F. Kruse, Jr. and M. K. Patterson, Jr. (Eds.), Tissue Culture Methods and Applications, Academic Press, New York and London, pp. 406-408. Porter, K., D. Kelley and P. M. Andrews (1972) The preparations of cultured cells and soft tissues for scanning electron microscopy. In: O. Johari and I. Corvin (Eds.), Scanning Electron Microscopy, I.I.T. Research Institute, Chicago, I11., pp. 1-19. Scholtissek, C. and H.-D. Klenk (1975) Influenza virus replication. In: E. D. Kilbourne (Ed.), The Influenza Viruses andlnfluenza, Academic Press, New York, San Francisco, London, pp. 215-242. Scholtissek, C. and R. Rott (1961) Zusammenh/inge zwischen der Synthesen yon Ribonucleins~iure und Protein bei der Vermehrung eines Virus der Influenzagruppe (Virus der klassischen Geflfigelpest), Z. NaturJbrsch., 16b: 663-673. Tamm, I. (1975) Cell injury with viruses, Amer. J. Path., 81 : 163-177. Tang, T. T., G. V. Sedmak, K. A. Siegesmund and S. R. McCreadie (1975) Chronic myopathy associated with coxsackievirus type A9 - - A combined electron microscopical and viral isolation study, New EngL J. Med., 292:608-611. Wagner, R. R. (1955) A pantropic strain of influenza virus - - Generalized infection and viremia in the infant mouse, Virology, 1 : 497-515. Wolf, A., H. Barden, N. Takahata, H. H. Benitez, E. T. Gamboa, D. H. Harter and P. E. Duffy (1974) Cellular pathology in cultures of embryonic mouse hypothalamus inoculated with influenza Ao WSN virus, J. Neuropath. exp. NeuroL, 33 : 582-594.