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Cytopathology of cultured cells infected with herpes simplex virus Haines, Patricia Jean 1972

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. 1 CYTOPATHOLOGY OF CULTURED CELLS INFECTED WITH HERPES SIMPLEX VIRUS by PATRICIA JEAN HAINES B.Sc. University of British Columbia, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In the Department of Microbiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1972 In p resen t ing t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree tha t the L i b r a r y sha l l make i t f r e e l y a v a i l a b l e f o r reference and s tudy. I f u r t h e r agree t h a t permiss ion f o r ex tens ive copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood tha t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed w i t h o u t my w r i t t e n permiss ion . Department o f ^h^-A^M^Q L i o Io The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada ABSTRACT The cytopathology of herpes simplex virus (HSV) in H.Ep.2 and BHK-21 cells was studied using the techniques of light microscopy, immunofluorescence, electron microscopy, autoradiography and cytogenetics. Both c e l l types supported rapid growth cycles of HSV resulting i n the production of maximum ti t r e s after 22 - 24 hours of infection. Cultures treated with 10 yg/ml ara-C or 100 yg/ml IDU at the time of infection showed a 99% decrease i n infectious virus production. HSV-infected H.Ep.2 and BHK-21 cells revealed typical virus-induced inclusion bodies and a generalized disorganization of the nucleus and .cytoplasm. Syncytia formation was not observed but after 24 hours of infection, nearly 100% of the cells were rounded and often detached from the glass surface. Addition of 10 yg/ml ara-C or 100 yg/ml IDU failed to prevent virus cytopathology but did cause a characteristic cytoplasmic disruption and rounding of uninfected c e l l s . Virus-infected cells also revealed at least four separate immuno-fluorescent elements after exposure to hyperimmune serum prepared i n guinea pigs. These elements included small nuclear granules, amorphous nuclear masses, diffuse cytoplasmic antigens, and intense surface fluorescence. The nuclear antigens and cytoplasmic fluorescence appeared after treatment with ara-C or IDU but the surface fluorescence was not i i produced i n the presence of the a n t i - v i r a l agents. Herpes simplex v i r u s developed i n the nucleus of i n f e c t e d H.Ep.2 and BHK-21 c e l l s . The v i r i o n s were enveloped at the inner lamella of the nuclear membrane and a f t e r passing i n t o the cytoplasm, were released from the c e l l s by a process of reverse phagocytosis. Ara-C and IDU allowed the synthesis of c e r t a i n v i r a l antigens and the development of nuclear cytopathology but completely prevented the formation of i n f e c t -ious HSV p a r t i c l e s . Both drugs caused a marked d i s t o r t i o n of the mitochondria and endoplasmic reticulum i n uninfected c e l l s . 3 DNA synthesis i n HSV-infected c e l l s , as measured by H-thymidine incorporation, was almost completely i n h i b i t e d by 4 hours of i n f e c t i o n . This e a r l y i n h i b i t i o n of c e l l u l a r DNA synthesis was followed by an 3 immediate increase i n H-thymidine uptake corresponding to the synthesis of v i r a l DNA. Both c e l l types showed a b r i e f stimulation of mitosis p r i o r to the complete i n h i b i t i o n observed a f t e r 20 hours of i n f e c t i o n . C e l l u l a r and v i r a l DNA synthesis and mitosis appeared to be i n h i b i t e d i n v i r u s - i n f e c t e d and uninfected c e l l s treated with ara-C or IDU. I n f e c t i o n with HSV r e s u l t e d i n severe chromosomal damage to H.Ep.2 and BHK-21 c e l l s . Chromosomal abnormalities included chromatid gaps and breaks, enhanced secondary c o n s t r i c t i o n s , fragmentation, erosion, and endoreduplication, and were dependent on v i r u s dose and time of i n f e c t i o n . The capacity of the v i r u s to induce chromosomal aberrations i n cultured c e l l s was UV-inactivated approximately f i v e times l e s s r a p i d l y than the i n f e c t i o u s property. Ara-C acted s y n e r g i s t i c a l l y with the v i r u s to produce a large number of c e l l s with multiple chromosome breaks and also caused a s i g n i f i c a n t number of abnormalities i n uninfected c e l l s . In contrast, IDU treatment r e s u l t e d i n few aberrations over and above those produced by HSV and l i t t l e damage i n uninfected c e l l s . I t was concluded that HSV was capable of pro-ducing severe morphological and genetic a l t e r a t i o n s i n cultured human and hamster c e l l s . The a n t i v i r a l agents ara-C and IDU were able to completely i n h i b i t v i r u s m u l t i p l i c a t i o n but were unable to prevent any of the virus-induced cytopathic e f f e c t s i n v i t r o . i v ABBREVIATIONS ara-C Cytosine arabinoside (1-B-D arabinofuranosyl cytosine) BHK-21 Baby hamster kidney l i n e 21 (clone 13). Derived from a primary Syrian hamster kidney c u l t u r e . DNA Deoxyribonucleic a c i d HBSS Hanks' balanced s a l t s o l u t i o n H. Ep.2 Human epidermoid l i n e #2. Derived from a carcinoma of the larynx. IDU 5-iodo-2'-deoxyuridine I. U. i n t e r n a t i o n a l u n i t MEM Minimal e s s e n t i a l medium (Eagle) mg milligram nm nanometer pfu plaque forming u n i t pH logarithm of the r e c i p r o c a l of the hydrogen ion concentration rpm revolutions per minute TCID 5 Q Infectious dose destroying 50% of the t i s s u e cultures tested yg microgram V TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 3 Herpes simplex virus 3 1. History 3 2. Classification 4 3. Structure, Composition and Physical Properties 5 4. Growth i n Tissue Culture 7 5. Host C e l l Response 12 6. Pathogenesis 17 MATERIALS AND METHODS 24 Cells and Medium 24 Virus 25 1. Origin 25 2. Virus Preparation and Purification 25 Virus Assay 26 1. End-point Dilution Technique 26 2. Plaque Assay 26 Chemicals and Radioisotopes 27 UV Inactivation of HSV 28 In Vitro Infection Procedure 28 v i TABLE OF CONTENTS (continued) Page Light Microscopy 29 Indirect Fluorescent Antibody Technique 29 Electron Microscopy 30 1. Negative Staining 30 2. Thin Sectioning 31 Autoradiography 32 Metaphase Preparations 33 RESULTS 35 Growth Studies 35 Light Microscopy 35 1. Cytopathology of HSV 35 2. Effect of ara-C and IDU on HSV Cytopathology 37 3. Effect of ara-C and IDU on Uninfected Cells 40 Fluorescent Antibody Studies 40 1. HSV Antigen Production 40 2. Effect of ara-C and IDU on HSV Antigen Production 42 Electron Microscopy 42 1. Negative Staining 42 2- Thin Sectioning 45 v i i TABLE OF CONTENTS (continued) Page (a) Uninfected H.Ep.2 and BHK-21 Cells 45 (b) Uninfected BHK-21 Cells: Abnormal Particle Formation 45 (c) HSV Development i n H.Ep.2 and BHK-21 Cells 48 (d) Effect of ara-C and IDU on HSV Development 56 (e) Effect of ara-C and IDU on Uninfected Cells 58 Autoradiographic Studies 60 1. DNA Synthesis i n HSV-Infected Cells 60 2. DNA Synthesis i n Cells Treated with ara-C and IDU 63 Cytogenetic Studies 63 1. Mitotic Rates 63 2. Normal Cell Karyotypes 65 3. HSV-Induced Chromosome Abnormalities 65 4. Effect of Multiplicity of Infection on HSV-Induced Chromosome Abnormalities 69 5. Effect of UV Irradiation of HSV on Virus-Induced Chromosome Abnormalities 76 6. Effect of Arginine Excess on HSV-Induced Chromosome Abnormalities 76 7. Effect of ara-C on the Chromosomes of Uninfected and HSV-Infected Cells 78 TABLE OF CONTENTS (continued) v i i i Page 8. E f f e c t of IDU on the Chromosomes of Uninfected and HSV-Infected C e l l s 82 9. E f f e c t of ara-C and IDU on the Chromosomes of Uninfected and HSV-Infected C e l l s 84 DISCUSSION 86 BIBLIOGRAPHY 106 LIST OF FIGURES Page Figure 1. Representative growth curves of HSV i n H.Ep.2 and BHK-21 c e l l s 36 Figure 2. Uninfected culture of H.Ep.2 c e l l s (X1750) 38 Figure 3. H.Ep.2 culture 12 hours a f t e r HSV i n f e c t i o n (X4400) 38 Figure 4. Uninfected c u l t u r e of H.Ep.2 c e l l s (X440) 39 Figure 5. H.Ep.2 culture 24 hours a f t e r HSV i n f e c t i o n (X440) 39 Figure 6 . H.Ep.2 cul t u r e a f t e r 72 hours of ara-C t r e a t -ment (X440) 41 Figure 7. H.Ep.2 culture a f t e r 72 hours of ara-C treatment (X1750) 41 Figure 8. Fluorescent antibody study of H.Ep.2 c e l l s 4 hours a f t e r HSV i n f e c t i o n (X4400) 43 Figure 9. Fluorescent antibody study of H.Ep.2 c e l l s 7 hours a f t e r HSV i n f e c t i o n (X4400) 43 Figure 10. Fluorescent antibody study of H.Ep.3 c e l l s 24 hours a f t e r HSV i n f e c t i o n (X4400) 44 Figure 11. E l e c t r o n micrograph of a negative s t a i n prep-a r a t i o n of HSV (X90,700) 46 Figure 12. Ele c t r o n micrograph of a negative s t a i n prep-a r a t i o n of HSV (X90,700) 46 Figure 13. E l e c t r o n micrograph of normal H.Ep.2 c e l l s showing i n t a c t nuclear and cytoplasmic structure (X10,000) 47 Figure 14. E l e c t r o n micrograph of a normal BHK-21 c e l l showing i n t a c t nuclear and cytoplasmic s t r u c t -ure (X42,600) 47 X LIST OF FIGURES (continued) Page Figure 15. E l e c t r o n micrograph of an uninfected BHK-21 c e l l maintained i n serumless medium (X55,800) 49 Figure 16. An abnormal p a r t i c l e i n the cytoplasm of an uninfected BHK-21 c e l l maintained i n serumless medium (X160,000) 49 Figure 17. Ele c t r o n micrograph of a BHK-21 c e l l 4 hours a f t e r HSV i n f e c t i o n (X50,000) : 50 Figure 18. Reduplicated nuclear membranes (RNM) and immature v i r u s p a r t i c l e s i n a BHK-21 c e l l 7 hours a f t e r HSV i n f e c t i o n (X39,500) 51 Figure 19. Immature v i r u s p a r t i c l e s i n the nucleus of a BHK-21 c e l l 7 hours a f t e r HSV i n f e c t i o n (X112,000) 51 Figure 20. Immature v i r u s p a r t i c l e budding through the nuclear membrane of a BHK-21 c e l l 7 hours a f t e r HSV i n f e c t i o n (X91,000) 53 Figure 21. Mature v i r u s p a r t i c l e s near a branching tubule i n the cytoplasm of a H.Ep.2 c e l l 12 hours a f t e r HSV i n f e c t i o n (X123,000) 53 Figure 22. Release of a mature HSV p a r t i c l e from a H.Ep.2 c e l l 12 hours a f t e r HSV i n f e c t i o n (X112,000) 54 Figure 23. I n t r a c e l l u l a r and e x t r a c e l l u l a r v i r u s i n a H.Ep.2 c e l l 20 hours a f t e r i n f e c t i o n (X62,500) 55 Figure 24. Intranuclear v i r a l c r y s t a l i n a BHK-21 c e l l 20 hours a f t e r i n f e c t i o n (X82,200) 57 Figure 25. Cytoplasmic aggregate i n a BHK-21 c e l l 20 hours a f t e r i n f e c t i o n (X39,500) 57 Figure 26. Intranuclear granules i n a BHK-21 c e l l 20 hours a f t e r HSV i n f e c t i o n and ara-C treatment (X35,300). 59 LIST OF FIGURES (continued) Page Figure 27. Cytoplasmic p a r t i c l e i n a BHK-21 c e l l 20 hours a f t e r HSV i n f e c t i o n and IDU treatment (X28,000)... 59 Figure 28. Mitochondria of a BHK-21 c e l l a f t e r 24 hours of ara-C treatment (X69,200) 61 Figure 29. A BHK-21 c e l l a f t e r 48 hours of ara-C treatment (X44,500) 61 Figure 30. DNA synthesis i n v i r u s - i n f e c t e d and chemically treated BHK-21 c e l l s 62 Figure 31. M i t o t i c rates of BHK-21 c e l l s following v i r u s i n f e c t i o n and chemical treatment 64 Figure 32. Karyotype of a normal H.Ep.2 c e l l (X4400) 66 Figure 33. Karyotype of a normal BHK-21 c e l l (X4400) 66 Figure 34. E f f e c t of HSV i n f e c t i o n on the chromosomes of H.Ep.2 and BHK-21 c e l l s 68 Figure 35. Chromosome complement of a BHK-21 c e l l 4 hours a f t e r HSV i n f e c t i o n (X4400) 70 Figure 36. Chromosome complement of a BHK-21 c e l l 8 hours a f t e r HSV i n f e c t i o n (X4400) 70 Figure 37. Chromosome complement of a BHK-21 c e l l 4 hours a f t e r HSV i n f e c t i o n (X4400) 71 Figure 38. Chromosome complement of a BHK-21 c e l l showing complete fragmentation a f t e r 10 hours of HSV i n f e c t i o n (X4400) 71 Figure 39. Erosion of a BHK-21 complement a f t e r 10 hours of HSV i n f e c t i o n (X4400) 72 x i i LIST OF FIGURES (continued) Page Figure 40. Endoreduplication of BHK-21 chromosomes 8 hours a f t e r HSV i n f e c t i o n (X4400) 72 Figure 41. The r e l a t i o n s h i p between m u l t i p l i c i t y of i n f e c t i o n and HSV-induced chromosome abnormali-t i e s i n BHK-21 c e l l s 75 Figure 42. E f f e c t of UV i r r a d i a t i o n of HSV on v i r a l i n f e c t i -v i t y and capacity to induce chromosome abnormalities i n BHK-21 c e l l s 77 Figure 43. Chromatid gaps found i n a H.Ep.2 c e l l 4 hours a f t e r a d d i t i o n of ara-C (X4400) 81 Figure 44. Translocation found i n a BHK-21 c e l l 4 hours a f t e r a d d i t i o n of IDU (X4400) 81 LIST OF TABLES x i i i Page Table I. Frequency d i s t r i b u t i o n of various l e v e l s of pl o i d y i n H.Ep.2 and BHK-21 c e l l s 67 Table I I . An analysis of HSV-induced chromosome abnormalities i n H.Ep.2 c e l l s 73 Table I I I . An analysis of HSV-induced chromosome abnormalities i n BHK-21 c e l l s 74 Table IV. E f f e c t of excess arginine on HSV-induced chromo-some abnormalities i n BHK-21 c e l l s 79 Table V. Chromosome abnormalities i n HSV-infected and non-infected BHK-21 c e l l s treated with ara-C 80 Table VI. Chromosome abnormalities i n HSV-infected and non-infected BHK-21 c e l l s treated with IDU 83 Table VII. Chromosome abnormalities i n HSV-infected and non-infected BHK-21 c e l l s treated with ara-C and IDU 85 ACKNOWLEDGEMENTS The author wishes to thank Dr. J.J.R. Campbell, Head of the Department of Microbiology, f o r granting t h i s opportunity f o r study and research. The guidance of Dr. J.E. Bismanis who supervised t h i s work and the advice of Dr. D.M. McLean and Dr. J.B. Hudson on thesis preparation are gr e a t l y appreciated. The author i s also indebted to Mrs. T. Walters f o r assistance i n the f i e l d of electron microscopy and to Miss J . Bellamy f o r the typing of t h i s manuscript. 1 INTRODUCTION In the past, herpes simplex v i r u s has mainly been of i n t e r e s t as a common disease agent of man, capable of producing i n f e c t i o n s of varying s e v e r i t y and persistence. However, present studies appear to be d i r e c t e d toward biochemically and c y t o l o g i c a l l y de-f i n i n g the virus-host r e l a t i o n s h i p i n terms of p o t e n t i a l mutagenesis and oncogenesis. This concern arose a f t e r a number of morphologically s i m i l a r herpes-type viruses were implicated i n c e l l transformation and tumor production i n d i f f e r e n t animals. Shortly afterward, herpes simplex v i r u s type 2 was epidemiologically associated with the occur-rence of human c e r v i c a l carcinoma and further implicated i n mammalian c e l l transformation i n v i t r o . As a r e s u l t of these i n t r i g u i n g reports, present research has focused on v i r u s - c e l l i n t e r a c t i o n s at a l l l e v e l s of i n f e c t i o n and at the same time i s concerned with the development and examination of e f f e c t i v e a n t i - v i r a l agents. For these reasons, the purposes of t h i s study are t h r e e f o l d : 1. To inv e s t i g a t e the cytopathology of herpes simplex v i r u s type 1 i n cultured human and hamster c e l l s with the a i d of l i g h t and electron microscopy, immunofluorescence, auto-radiography, and cytogenetics. 2 . To examine the e f f e c t s of two chemotherapeutic agents, IDU and ara-C, on herpes simplex v i r u s r e p l i c a t i o n and cyto-pathology i n the same human and hamster c e l l l i n e s . To observe and measure the biochemical and c y t o l o g i c a l e f f e c t s of IDU and ara-C on normal, uninfected human and hamster c e l l s i n c u l t u r e . 3 LITERATURE REVIEW Herpes Simplex Virus 1. History Over two thousand years ago, Hippocrates used the word "herpes" to describe a host of different skin diseases including eczema, skin cancer, erysipelas and herpes zoster (shingles). As late as the nineteenth century, medical texts s t i l l referred to many spreading, ulcerative lesions as herpes, but the term soon became restricted to certain vesicular eruptions of the skin and mucous membranes. By 1900, f a c i a l herpes, labial herpes, ocular herpes and genital herpes were generally recognized as c l i n i c a l manifestations of the same human disease, herpes simplex (6). In 1912, Gruter successfully transmitted herpetic keratitis to the cornea of a healthy rabbit and later reversed his procedure by transmitting experimentally induced ocular herpes to the cornea of a blind man. Following Gruter's i n i t i a l work, Lowenstein reported that herpetic lesions of the eye, skin and mucous membranes yielded an infectious agent capable of producing a characteristic keratitis in rabbits. In 1921, examination of herpes-infected corneal c e l l s led to the discovery of typical large intranuclear inclusion bodies. Later experiments confirmed that a single washed inclusion could successfully i n i t i a t e a herpes infection. Interest in the herpes 4 simplex virus was subsequently spurred by the report of herpetic encephalitis i n rabbits and man, and by Burnet and Williams' de-scription of v i r a l persistence in humans in 1939 (reviewed i n 31). Early characterization of the virus showed i t to be 100-150 nm i n diameter as estimated by membrane f i l t r a t i o n (16). Infecti-vity was lost upon exposure to various detergents and electron microscopy in 1954 established the virus as a large enveloped particle with a dense inner nucleoid (52). At present, herpes simplex i s s t i l l of medical interest as a major cause of blindness in man but i s perhaps of even more import as a disease model of latency and persistence. Recently, the genital strains of herpes simplex virus have also been epid-emiologically associated with human cervical carcinoma and the virus i s now the object of intensive s c i e n t i f i c investigation (56,57,76). 2. Classification Herpesviruses are formally defined as large enveloped virions with an icosahedral capsid of 162 capsomeres arranged around a DNA core. Included in this major group are herpes simplex virus, pseudo-rabies virus, varicella-zoster virus, the cytomegaloviruses, B virus, marmoset virus and various equine, bovine, canine, avian and feline herpesviruses (36,49). Future members may include the Epstein-5 Barr v i r u s found i n B u r k i t t ' s lymphoma c e l l cultures, the Marek's disease agent of fowl, and the leopard frog v i r u s i s o l a t e d from Lucke's adenocarcinoma (24,59,85). At present, herpesviruses are ei t h e r designated by t h e i r o r i g i n a l d e s c r i p t i v e names ( i . e . herpes simplex v i r u s , pseudorabies virus) or are c l a s s i f i e d according to Andrew's binomial system ( i . e . Herpesvirus hominis, Herpesvirus s u i s ) . The herpes simplex viruses have also been regrouped i n t o two types or subtypes on the basis of immunological and biochemical dif f e r e n c e s recently detected i n o r a l and g e n i t a l s t r a i n s . Type 1 i s o l a t e s are generally from the mouth, eye and b r a i n while the type 2 s t r a i n s are derived from the perineum and g e n i t a l i a . Apart from antigenic d i f f e r e n c e s , type 1 viruses are le s s s e n s i t i v e to heat, produce higher t i t r e s i n r a b b i t kidney c e l l s , and possess a lower p a r t i c l e : plaque forming u n i t r a t i o than type 2 v i r u s e s . On the other hand, type 2 s t r a i n s are capable of producing plaques on chick embryo c e l l s , have a higher buoyant density and guanine plus cytosine content, and are more neurotropic i n mice (20,23,41,72). 3. Structure, Composition and P h y s i c a l Properties. The mature herpes simplex v i r i o n i s a large enveloped p a r t i c l e 150 - 170 ran i n diameter. I t appears to c o n s i s t of a dense core, 6 three concentric s h e l l s or capsids, and two outer envelopes (88). These three main a r c h i t e c t u r a l u n i t s have been concurrently studied with the e l e c t r o n microscope and the u l t r a c e n t r i f u g e . The core i s revealed as a 25 nm p a r t i c l e containing DNA; i t i s thought to be c l o s e l y associated with the e l e c t r o n opaque inner capsid (92). The middle and outer capsids appear as electron translucent and electron dense s h e l l s i n t h i n sections of i n f e c t e d c e l l s ; the outer capsid consists of 162 hollow p r o t e i n capsomeres arranged i n an icosahedral shape possessing 5:3:2 a x i a l symmetry. The outer envelopes are composed of various l i p i d s and glycoproteins, c e r t a i n of which are necessary f o r v i r a l i n f e c t i v i t y (84). Herpes simplex v i r u s contains double-stranded DNA (4,95) of 6 molecular weight 99 ± 5 x 10 daltons (2,41). Native DNA i s reported to contain single-strand breaks and i s l i n e a r and not c r o s s - l i n k e d (41). No methylated or unusual bases are known to be present. The guanine plus cytosine content i s 68% f o r type 1 and 70% f o r type 2 viruses (23). A f t e r acrylamide g e l electrophoresis, p a r t i a l l y p u r i -f i e d v i r i o n s y i e l d up to 12 separate p r o t e i n bands, at l e a s t 6 of which are glycosylated and associated with the v i r a l envelope (106). Infectious herpes simplex v i r i o n s are r e l a t i v e l y s e n s i t i v e to heat, r a d i a t i o n , and l i p i d solvents (36). They are r a p i d l y i n a c t i v a t e d at 37°C i n a f i r s t order r e a c t i o n and are best stored i n d i s t i l l e d 7 water at -70°C. Similarly, photosensitization, X-ray, and UV i r -radiation a l l destroy v i r a l activity in an exponential manner. The virus i s also sensitive to most l i p i d solvents such as sodium deoxycholate, chloroform and ethyl ether as well as enzymes such as trypsin and phospholipase C. Moreover, infectivity and structural integrity are rapidly lost after prolonged centrifugation in CsCl, where the virus generally bands at a density of 1.255 to 1.280 gm/cc (107). 4. Growth in Tissue Culture (a) Host Range Herpes simplex virus multiplies in a wide variety of primary and continuous cells cultivated i n v i t r o . The virus replicates i n most mammalian c e l l lines, chick embryo fibroblasts and tortoise kidney c e l l s (36). Rabbit kidney cells are perhaps the most sensi-tive of the c e l l types found to support herpes simplex virus multi-plication. It has been reported that the virus requires the amino acid arginine to complete assembly and maturation i n vitro (3) and i s best produced at temperatures ranging from 35°C to 37°C. (b) Adsorption, Penetration and Uncoating The rate of virus adsorption is unaffected by DNA and protein inhibitors and by changes in temperature, suggesting that the process does not require energy or active c e l l metabolism. Heparin 8 (30) and parathyroid hormone (80) i n h i b i t s v i r u s adsorption while thyroid hormone (81) stimulates the process. Using purely p h y s i c a l techniques, Huang and Wagner revealed that penetration of herpes simplex v i r u s was 90% complete within 10 minutes of adsorption. They found the process to be temperature dependent and energy r e q u i r i n g (33). Penetration has also been studied by e l e c t r o n microscopy, although r e s u l t s have been confusing at best. For example, while one major report concluded that pene-t r a t i o n was e f f e c t e d by pinocytosis of v i r a l p a r t i c l e s (32) , another equally extensive study found that v i r u s fusion with the c e l l membrane provided the only means of entry (50). Such c o n f l i c t i n g data probably a r i s e from the use of extremely high m u l t i p l i c i t i e s of i n f e c t i o n ( i . e . 1000 p a r t i c l e s per c e l l ) with p a r t i c l e : plaque forming u n i t r a t i o s of 10 or greater. As a r e s u l t , the p o s s i b i l i t y e x i s t s that over 90% of the viruses observed i n t h i n sections may not be going through an i n f e c t i o u s process during entry and uncoating. I t i s known, however, that uncoated DNA appears i n the nucleus within 30 minutes of v i r u s penetration and that e x i s t i n g c e l l enzymes apparently uncoat the v i r u s i n the cytoplasm (33). (c) V i r a l Synthesis (i) DNA, RNA and Protein Synthesis V i r u s - s p e c i f i c DNA i s detected as e a r l y as 3 hours post i n f e c t i o n i n the nucleus of i n f e c t e d H.Ep.2 c e l l s . DNA synthesis 9 reaches a maximum at about 7 hours and thereafter e i t h e r declines or remains constant throughout the r e p l i c a t i o n cycle (85, 89). Experiments with puromycin have revealed that p r o t e i n synthesis i s a d e f i n i t e requirement f o r i n i t i a t i o n of v i r a l DNA synthesis (86). V i r u s - s p e c i f i c RNA i s also detectable at 3-4 hours and reaches a maximum at 8 hours, p a r a l l e l i n g the course of DNA synthesis (21). The nuclear RNA i s heterogenous i n s i z e and contains at l e a s t one species with a sedimentation c o e f f i c i e n t greater than 80s that does not appear i n the cytoplasmic RNA p r o f i l e . This and other evidence suggests that nuclear RNA i s cleaved i n t o pieces of lower molecular weight within 10-15 minutes of i t s synthesis and transported i n t o the cytoplasm of the in f e c t e d c e l l (121). Moreover, soon a f t e r i n f e c t i o n , the cytoplasmic polyribosomes r a p i d l y disaggregate and v i r u s - s p e c i f i c polysomes containing RNA o r i g i n a l l y synthesized i n the nucleus reform i n t h e i r place (113). In a recent paper on herpes simplex v i r u s RNA synthesis, Wagner also reported sequence differences i n "early" and " l a t e " RNAs from both the nucleus and cytoplasm, thus providing the f i r s t evidence of t r a n s c r i p t i o n a l c o n t r o l of the v i r a l genome (120). The bulk of v i r u s - s p e c i f i c p r o t e i n synthesis occurs within 4-6 hours post i n f e c t i o n i n the cytoplasm of in f e c t e d c e l l s (90). At l e a s t 9-11 d i f f e r e n t polypeptides are synthesized and incorporated i n t o the v i r i o n s , although i t i s not yet known whether a l l of these are coded f o r by the v i r u s genome (66). Synthesis of these s t r u c t u r a l proteins i s asynchronous and t h e i r transport i n t o the nucleus slow and r e l a t i v e l y s e l e c t i v e (67). A f t e r i n f e c t i o n of H.Ep.2 c e l l s , Spear and Roizman found 25 v i r u s -s p e c i f i c proteins ranging i n molecular weight from 25,000 to 275,000. Of the proteins incorporated i n t o the v i r i o n s , two contained l i p i d and at l e a s t s i x were glycosylated and associated with the v i r a l envelopes and c e l l membranes. These s i x glycoproteins v a r i e d i n s i z e and structure with the p a r t i c u l a r v i r u s s t r a i n used, suggesting t h e i r synthesis was i n part v i r u s - d i r e c t e d (106). ( i i ) Enzyme Production Herpes simplex v i r u s causes a marked increase i n the a c t i v i t i e s of several c e l l enzymes with concomitant changes i n t h e i r immunological and biochemical pro p e r t i e s . Evidence supports the contention that v i r u s - s p e c i f i c thymidine kinase (44), DNA polymerase (42) and a l k a l i n e DNA exonuclease (54) are a l l produced i n i n f e c t e d c e l l s , although conclusive proof awaits more rigorous examination of the virus-host system. ( i i i ) V i r u s - S p e c i f i c Antigens Immunofluorescent studies have revealed the presence of f i v e separate antigenic elements i n herpes simplex v i r u s i n f e c t e d c e l l s (22,45,91,94,124). Using hyperimmune serum prepared i n rabbit s from unheated i n f e c t e d c e l l debris, Roizman et a l . detected the presence of small and large nuclear granules, amorphous nuclear patches, cytoplasmic granules and d i f f u s e cytoplasmic fluorescence i n H.Ep.2 c e l l s i n f e c t e d with the v i r u s . They concluded that the d i f f u s e s t a i n i n g represented early development of nucleocapsids and v i r i o n s while the aggregates or granules found l a t e i n i n f e c t i o n corresponded to large v i r u s " f a c t o r i e s " or c r y s t a l formations (91). Roane and Roizman also discovered that the type of antiserum determines the number and kind of v i r u s - s p e c i f i c antigenic elements found i n i n f e c t e d c e l l s . Thus, human convalescent serum produces only cytoplasmic and perinuclear s t a i n i n g , and hyperimmune serum prepared i n laboratory animals from b o i l e d i n f e c t e d c e l l debris reveals only s p e c i f i c nuclear fluorescence (78). In a l l cases, how-ever, antigen production can be detected as ea r l y as 3 hours a f t e r i n f e c t i o n and involves nearly the e n t i r e c e l l population by 24 hours. (d) Virus Assembly and Release The s i t e of herpes simplex v i r u s assembly i s the nucleus of the in f e c t e d c e l l . CsCl c e n t r i f u g a t i o n studies and electron microscopy have suggested that a small core p a r t i c l e containing DNA and p r o t e i n acts as the v i r a l precursor (88). In the nucleus, the capsid subunits and inner envelope are assembled about the dense cores i n a highly asynchronous manner, producing p a r t i c l e s i n various stages of assembly throughout the r e p l i c a t i o n c y c l e . Maturation i s considered complete when the v i r u s acquires i t s outer envelope by budding through the inner lamella of the nuclear membrane (13, 53). However, some p a r t i c l e s may reach the cytoplasm before t h i s f i n a l step, i n which case envelopment usually occurs i n the cyto-plasmic membrane system. Once i n the c e l l cytoplasm, s i n g l y and doubly enveloped v i r i o n s , which are probably both i n f e c t i v e (107), tend to aggregate near and around vacuoles and tubules (13,98). For many years, i t was thought that mature herpes simplex v i r u s was released by budding through the c e l l membrane i n a continuous process that d i d not lyse the i n f e c t e d c e l l (19). However, more recent studies have indicated that a network of cytoplasmic tubules transport the v i r i o n s out of the c e l l (98) . Controversy over the method of egress i s s t i l l very apparent and w i l l probably remain so u n t i l the inherent d i f f i c u l t i e s i n t h i n section electron microscopy are circumvented. 5. Host C e l l Response (a) Macromolecular Synthesis and M i t o s i s Upon i n f e c t i o n with herpes simplex v i r u s , the i n i t i a l rates of c e l l DNA, RNA and p r o t e i n synthesis d e c l i n e . C e l l DNA synthesis i s r a p i d l y i n h i b i t e d by 2-3 hours of i n f e c t i o n while RNA and p r o t e i n synthesis enjoy a gradual decline over the same period. T o t a l macro-molecular synthesis increases b r i e f l y f o r the peak 4-5 hours of v i r a l synthesis and then decreases i r r e v e r s i b l y u n t i l c e l l death (25). In a d d i t i o n , the v i r u s causes a rapid i n h i b i t i o n of the m i t o t i c process and c e l l growth (112). This early event corresponds i n time to the i n h i b i t i o n of c e l l DNA synthesis and. i s probably mediated by a s t r u c t u r a l component of the v i r i o n or a v i r u s product made soon a f t e r i n f e c t i o n . (b) Cytopathology C e l l s productively i n f e c t e d with herpes simplex v i r u s d i s p l a y severe a l t e r a t i o n s i n t h e i r morphology and s o c i a l behavior. The f i r s t evidence of these a l t e r a t i o n s i s usually seen i n the infe c t e d c e l l nucleus (55,62). As early as 3-4 hours a f t e r i n f e c t i o n , the network of normal c e l l chromatin becomes highly condensed and displaced to the periphery of the nucleus. N u c l e o l i also condense and gradually d i s i n t e g r a t e and s h o r t l y afterwards, a large Feulgen-p o s i t i v e v i r a l i n c l u s i o n body forms (36). With the development of the i n c l u s i o n , the c e l l nucleus becomes grossly enlarged and d i s t o r t e d while the cytoplasm vacuolates and shrinks i n volume. Infected c e l l s eventually round up completely and detach from the container surface. Apart from such morphological changes, the v i r u s also causes a l t e r a t i o n s i n the s o c i a l behaviour of c e l l s (84). In f e c t i o n with d i f f e r e n t s t r a i n s may r e s u l t i n (i) the formation of polykaryocytes, ( i i ) the production of clumped, rounded c e l l s , or ( i i i ) the forma-t i o n of round "balloon" c e l l s that do not aggregate. Polykaryocytes r e s u l t from the fu s i o n of neighbouring c e l l s to form syncytia containing up to 20-30 n u c l e i . Studies on t h e i r formation reveal that the fusion process can only take place a f t e r the i n i t i a t i o n of v i r a l DNA synthesis i n in f e c t e d c e l l s (79). In a se r i e s of papers dealing with the s o c i a l behaviour of herpes-infected c e l l s , Roizman concluded that f u s i o n and clumping were the d i r e c t r e s u l t of virus-induced c e l l membrane a l t e r a t i o n s . He observed that c e l l s acquire new surface antigens and that c e l l membrane composition i s changed a f t e r i n f e c t i o n with herpes simplex v i r u s (84,87,93). Presumably, these a l t e r a t i o n s which lead to the formation of aggregates and polykaryocytes a i d i n the c e l l - t o - c e l l spread of the v i r u s . The type and extent of a l l a l t e r a t i o n s i n c e l l morphology and behaviour are dependent on the genetic c o n s t i t u t i o n of the v i r u s s t r a i n . Variants that a r i s e frequently a f t e r prolonged passage i n v i t r o have been reported to d i f f e r markedly from the parent s t r a i n i n the type of cytopathic e f f e c t s produced i n the same host c u l t u r e (15). Moreover, the species of host c e l l and the age and character of the monolayer exert a profound e f f e c t on v i r u s cytopathology. Thus, c e l l responses are influenced not only by the genotype but also by the phenotypic expression of the v i r u s (118). (c) Chromosome A l t e r a t i o n s Herpes simplex v i r u s i s one of a large number of mammalian viruses that induce chromosomal and m i t o t i c i r r e g u l a r i t i e s i n c u l -tured c e l l s (60,109). The f i r s t evidence of i t s mutagenic p o t e n t i a l came i n 1961 when Hampar and E l l i s o n discovered that i n f e c t i o n of the MCH Chinese hamster c e l l l i n e with herpes simplex v i r u s r e s u l t e d i n an increased incidence of chromatid breaks, accented secondary c o n s t r i c t i o n s , and t r a n s l o c a t i o n s . Most of the abnormalities appeared a f t e r the f i r s t c e l l d i v i s i o n and were p r i m a r i l y located on chromo-somes 1 and 2 of the aneuploid complement (27,28). Since t h i s i n i t i a l work, many inv e s t i g a t o r s have reported s i m i l a r e f f e c t s of v i r u s i n f e c t i o n i n a v a r i e t y of mammalian c e l l s i ncluding d i p l o i d Chinese hamster c e l l s (110), human embryonic lung c e l l s (68,70), human pe r i p h e r a l leukocytes (68), r a b b i t kidney c e l l s (97), BHK-21 c e l l s (126), and monkey kidney c e l l s (7). In a l l cases, save f o r the Chinese hamster c e l l s , the chromosome aberrations appear to be random and non-specific. Virus e f f e c t s depend on the host c e l l species and include chromatid and chromosome breaks and gaps, severe fragmentation, accented secondary c o n s t r i c t i o n s , and C-mitosis. The lesi o n s are without exception prevented by prolonged u l t r a v i o l e t i r r a d i a t i o n of the v i r u s or n e u t r a l i z a t i o n with herpes simplex v i r u s antibody. In a d d i t i o n , damage i s detectable as early as 3 hours a f t e r 16 i n f e c t i o n and i s found to increase with time and i n i t i a l v i r a l dose (60, 109). E a r l y studies suggested that v i r a l r e p l i c a t i o n was necessary f o r the induction of chromosome abnormalities. However, zur Hausen found no evidence of v i r a l DNA synthesis i n BHK-21 c e l l s with severely damaged metaphase plates and thus concluded that e i t h e r a v i r i o n component or an early enzyme was responsible f o r the damage (126). At present, the s p e c i f i c cause of a l l virus-induced chromo-some aberrations i s s t i l l unknown. (d) C e l l Transformation and Oncogenesis Transformation at the c e l l u l a r l e v e l i s generally defined as a stable, i n h e r i t a b l e genetic a l t e r a t i o n i n a c e l l , u s u a l l y r e -s u l t i n g i n a changed morphology and growth pattern. C e l l s trans-formed by viruses are very often oncogenic and are therefore studied i n an e f f o r t to define the causes and conditions of carcinogenesis. To date, there are at l e a s t four p o s s i b l e herpes-type viruses implicated i n the oncogenic process - the human Epstein-Barr v i r u s found i n B u r k i t t ' s lymphoma c e l l c u l t u r e s , the Marek's disease agent of chickens, the leopard frog adenocarcinoma v i r u s , and Herpes- v i r u s s a i m i r i which induces lymphomas i n marmoset monkeys (48,59,116). With the discovery of these viruses and the recent epidemiological evidence suggesting a r e l a t i o n s h i p between the occurrence of herpes simplex type 2 i n f e c t i o n s and human c e r v i c a l carcinoma (56,57,76), the herpes simplex viruses have been re-examined f o r po s s i b l e oncogenic and transforming c a p a c i t i e s . So f a r , the v i r u s has only been i m p l i -cated as a co-carcinogen i n l i v e animal studies (114). However, early i n 1971, Duff and Rapp reported the production of a hamster embryo f i b r o b l a s t l i n e transformed a f t e r exposure to u l t r a v i o l e t -i r r a d i a t e d herpes simplex type 2. T h i r t y days a f t e r v i r u s i n f e c t i o n , the c e l l s developed an a l t e r e d morphology and u n r e s t r i c t e d growth pattern. The r e s u l t i n g c e l l l i n e produced herpes simplex v i r u s antigens and was highly oncogenic i n newborn and weaning hamsters (14). I f t h i s preliminary report i s extended and v e r i f i e d by others, herpes simplex v i r u s may j o i n the ranks of DNA tumor viruses capable of i n t e r a c t i n g with t h e i r hosts to produce c e l l transformation as well as c y t o c i d a l and abortive i n f e c t i o n s . 6. Pathogenesis (a) Animals The natural host f o r herpes simplex v i r u s i s man. However, the v i r u s can also be experimentally transmitted to r a b b i t s , guinea pigs, mice, r a t s , chick embryos, geese and hedgehogs (36) . Perhaps the most common experimental animals are the adult r a b b i t and newborn mouse. Corneal i n o c u l a t i o n of herpes simplex v i r u s i n rabbits has been found to produce a t y p i c a l k e r a t i t i s beginning with the appearance of pinpoint d e n d r i t i c lesions within 24 hours of i n f e c t i o n . The l e s i o n s r a p i d l y enlarge and invade the corneal stroma, producing a purulent exudate and u l t i m a t e l y , blindness. Viremias can be detected a f t e r 24 hours and v i r u s can be s u c c e s s f u l l y i s o l a t e d from brain, spleen, kidney and lung homogenates. I f the k e r a t i t i s i s l e f t untreated, the disease often extends to the b r a i n and causes a f a t a l e n c e p h a l i t i s (40). Using 5-day o l d mice, Wildy found that footpad i n o c u l a t i o n r e -s u l t e d i n the r a p i d development of p o s t e r i o r p a r a l y s i s without evidence of viremia. He assumed that the normal route of v i r u s invasion was e x c l u s i v e l y v i a the p e r i p h e r a l nerves to the c e n t r a l nervous system (127). However, i n 1964, extensive immunofluores-cent studies of murine enc e p h a l i t i s provided evidence of both a neural and hematogenous spread. Johnson found that herpes simplex v i r u s e i t h e r produced a viremia that allowed i n f e c t i o n of small cerebral vessels serving the c e n t r a l nervous system, or r e s u l t e d i n a c e n t r i p e t a l i n f e c t i o n of endoneural c e l l s (not axons) leading to the v i t a l c r a n i a l nerves. Intracerebral i n o c u l a t i o n of the v i r u s produced an exclusive d i r e c t neural spread while i n t r a n a s a l and i n t r a p e r i t o n e a l i n o c u l a t i o n r e s u l t e d i n m u l t i p l e pathways of hemato-genous and neural invasion (35). (b) Man In man, primary herpes simplex v i r u s i n f e c t i o n s u s u a l l y occur between the ages of 6 months and 5 years. I t i s estimated that l e s s than 10% of the i n f e c t e d i n d i v i d u a l s ever demonstrate any overt disease although the v i r u s i n f e c t i o n can oc c a s i o n a l l y be severe and even f a t a l (10). Primary i n f e c t i o n s can a f f e c t up to 60 - 100% of the population, producing a v a r i e t y of v e s i c u l a r l e s i o n s . The c l i n i c a l expressions depend to a large degree on the p o r t a l of entry and include gingivostomatitis, k e r a t i t i s , v u l v o v a g i n i t i s , eczema herpeticum and r a r e l y , meningo-encephalitis. In most cases, the i l l n e s s e s are r e l a t i v e l y mild and s e l f - l i m i t i n g but i n newborn and premature i n f a n t s , generalized herpes simplex can be r a p i d l y f a t a l (36). Once the i n i t i a l v i r u s i n f e c t i o n subsides, however, the majority of people s u f f e r from a recurrent form of herpes simplex that i n -v a r i a b l y occurs i n the presence of c i r c u l a t i n g antibody (10,40,83). Skin le s i o n s tend to recur at or near the same s i t e and appear following s p e c i f i c p h y s i c a l or emotional s t i m u l i that include exposure to sun-l i g h t , i l l n e s s , fever, menstruation, hormone treatment, administration of f o r e i g n proteins, s p e c i f i c types of neurosurgery and emotional s t r e s s . I t i s now generally accepted that the herpes simplex v i r u s i s harbored i n a l a t e n t or inapparent form at some p a r t i c u l a r s i t e i n the body. C e r t a i n host f a c t o r s r e a c t i v a t e the i n f e c t i o n at various times and cause the v i r u s to manifest i t s e l f i n t y p i c a l herpetic les i o n s (36). At present, there are two theories to explain the persistence of herpes simplex i n man. The f i r s t proposes that v i r u s m u l t i p l i c a -t i o n occurs at a slow but constant rate at the s i t e of the recurrent l e s i o n . According to t h i s dynamic state hypothesis, various s t i m u l i provide a temporary permissive state i n uninfected c e l l s and thus allow b r i e f recurrences of overt disease (83). The second theory envisions the existence i n unknown c e l l s of a noninfectious form of the v i r u s . In t h i s hypothesis, v i r u s m u l t i p l i c a t i o n i s r e v e r s i b l y interrupted at some stage and can be reactivated by c e r t a i n agents and conditions of the host (83). In an e f f o r t to d i s t i n g u i s h between these two theories, many attempts have been made to i s o l a t e i n f e c t i o u s v i r u s from the healed s i t e of le s i o n s i n the interim between recurrences. To date, no v i r u s or i n f e c t e d c e l l s have been recovered i n t h i s manner. However, a number of i n v e s t i g a t o r s have reported the i s o l a t i o n of i n f e c t i o u s v i r u s from the tears and s a l i v a of apparently normal i n d i v i d u a l s as well as from the secretory glands and tears of r a b b i t s with recurrent k e r a t i t i s . ( 1 0 ) . Thus, the persistence of the herpes simplex v i r u s may indeed be due to a low-grade, chronic i n f e c t i o n of the host as envisaged by the dynamic state hypothesis. On the other hand, an inapparent form of the v i r u s has a l s o been encountered i n r a b b i t s with recurrent ocular herpes. Examination of various neurons including the trigeminal ganglion revealed no apparent v i r u s i n f e c t i o n between recurrences. However, one to two weeks a f t e r organ cultures had been established, the trigeminal ganglion c e l l s produced f u l l y i n f e c t i o u s herpes simplex v i r u s (108). In man, surgery of the same ganglion frequently r e s u l t s i n postopera-t i v e herpetic le s i o n s (17). Thus, the experimental and c l i n i c a l evidence appears to suggest that the v i r u s e x i s t s i n a l a t e n t , nonin-f e c t i o u s form i n s p e c i f i c neural ganglia of the host. P e r s i s t e n t herpes simplex v i r u s i n f e c t i o n s are rather unusual i n that c i r c u l a t i n g antibody i s normally present during the course of the recurrences. Moreover, there appears to be no change i n the serum l e v e l s of t h i s antibody either before or a f t e r overt i n f e c t i o n (26). This apparent c o n t r a d i c t i o n i s probably a r e s u l t of the method of v i r u s spread. Herpes simplex v i r u s can spread by d i r e c t c e l l - t o - c e l l contact and thus remain unaffected by c i r c u l a t i n g antibody (84). How-ever, the s e l f - l i m i t i n g nature of the l e s i o n s s t i l l requires s a t i s -f a c tory explanation from an immunological point of view. A recent analysis of immunoglobulins i n patients with p e r s i s t e n t i n f e c t i o n s shows a s i g n i f i c a n t l y greater amount of IgA i n the blood during a l a t e n t period than during an a c t i v e recurrence (26). Further studies showed that patients with p e r s i s t e n t herpes a l s o have impaired macrophage i n h i b i t i o n and lowered lymphocyte t o x i c i t y (128). In summary, recurrent herpes simplex v i r u s i n f e c t i o n s appear to be established and maintained by a complex i n t e r a c t i o n of a v a r i e t y of host f a c t o r s including IgA serum l e v e l s , cell-mediated immunity, temperature, i n t e r f e r o n and body hormones (83). (c) Chemotherapy To date, IDU and ara-C have provided the best c l i n i c a l r e s u l t s i n treatment of herpes simplex v i r u s i n f e c t i o n s (74). In c l i n i c a l t r i a l s , Kaufman found that t o p i c a l a p p l i c a t i o n s of IDU r e s u l t e d i n complete e r a d i c a t i o n of herpetic k e r a t i t i s i n man and animals (39). Subsequent double-blind studies confirmed that IDU had a s i g n i f i c a n t e f f e c t on s u p e r f i c i a l l e s i o n s but f a i l e d to reduce the recurrence rate or prevent the development of deep stromal l e s i o n s . In most cases, the chemical had no obvious t o x i c e f f e c t s , although at l e a s t one r e -port of corneal "speckling" i s recorded (36) . However, i n the i n t a c t animal, IDU i s highly t o x i c and i s used only i n very severe diseases. Patients with acute necrotizing e n c e p h a l i t i s and generalized herpes simplex are o c c a s i o n a l l y treated with massive doses of IDU. Most medical communications report an increased chance of recovery with the drug but l i t t l e i f any decrease i n subsequent neurological damage (47). Moreover, t o x i c s i d e - e f f e c t s such as leukopenia and thrombocytopenia are l i k e l y to occur a f t e r prolonged treatment (74). In v i t r o , IDU i s a potent i n h i b i t o r of herpes simplex v i r u s . At l e a s t 99% i n h i b i t i o n of i n f e c t i o u s v i r u s production i s obtained i f 50 yg/ml IDU i s added to an i n f e c t e d c u l t u r e as l a t e as 4 hours a f t e r i n f e c t i o n (9,82,104,105). The halogenated pyrimidine appears to be incorporated i n t o the newly synthesized v i r a l DNA i n place of thymidine, thus causing defective assembly of the v i r i o n s (37). Although repeated exposure to IDU r e s u l t s i n the development of drug-resistant mutants, these viruses u s u a l l y remain s e n s i t i v e to the a c t i o n of ara-C. Ara-C i s equally e f f e c t i v e against herpetic k e r a t i t i s (117) and herpes simplex v i r u s r e p l i c a t i o n i n v i t r o (9,46), but i s hi g h l y t o x i c to a l l mammalian c e l l s . Both IDU and ara-C prevent mitosis i n c u l t u r e and i n h i b i t DNA synthesis (11,38,43,73,101) while causing concomitant chromosome abnormalities (8,61,69). As a r e s u l t of the adverse e f f e c t s of these drugs, chemotherapy of herpes simplex v i r u s i n f e c t i o n s i s generally r e s t r i c t e d to l o c a l i z e d l e s i o n s or severe cases of systemic disease. MATERIALS AND METHODS C e l l s and Medium The H.Ep.2 continuous l i n e of human epidermoid c e l l s (51) and the BHK-21 (clone 13) l i n e of Syrian hamster kidney c e l l s (111) were obtained from M i c r o b i o l o g i c a l Associates, Bethesda, Md. The c e l l s were maintained as monolayer cultures i n milk d i l u t i o n b o t t l e s or Leighton tubes on Eagles' minimal e s s e n t i a l medium (MEM), AutoPow (Flow Labs., R o c k v i l l e , Md.). The medium was r o u t i n e l y supplemented with 10% f e t a l c a l f serum, p e n i c i l l i n G (100 I.U./ml), streptomycin (100 yg/ml), and 0.025% glutamine. C e l l cultures were divided r e g u l a r l y every three days by the use of 0.25% t r y p s i n i n Hank's balanced s a l t s o l u t i o n (HBSS). Suspensions of approximately 10 6 c e l l s i n medium with 20% f e t a l c a l f serum and 10% dimethylsulfoxide were frozen and stored a t -70°C to serve as stock c u l t u r e s . Frozen c e l l s were r e g u l a r l y revived every three months to ensure genetic and morphologic s t a b i l i t y of the l i n e s . E l e c t r o n microscopy and t r i t i a t e d thymidine autoradiography of normal H.Ep.2 and BHK-21 c e l l s f a i l e d to reveal the p a r t i c l e s and abnormal cytoplasmic l a b e l i n g c h a r a c t e r i s t i c of mycoplasma i n f e c t i o n (29,58). Virus 1. O r i g i n The H4253 herpes simplex v i r u s (HSV) s t r a i n was i s o l a t e d by Dr. D.M. McLean from the throat of a patient with multiple l i p l e s i o n s . S e r o l o g i c a l i d e n t i f i c a t i o n was made on the basis of s p e c i f i c n e u t r a l i z a t i o n t e s t s i n mice. Subsequent to i t s i s o l a t i o n , the v i r u s was passaged f i v e times i n mice and a t o t a l of twelve times i n H.Ep.2 monolayers. During t h i s time, the v i r u s was a l s o plaque-p u r i f i e d three times by T. Mosmann. Ele c t r o n microscopy revealed p a r t i c l e morphology t y p i c a l of the herpes group of v i r u s e s . 2. V i r a l Preparation and P u r i f i c a t i o n Stock v i r u s was prepared from 24 hour H.Ep.2 monolayers grown Q i n d i l u t i o n b o t t l e s and inoculated with 1.0 x 10 pfu HSV. The cultures were incubated at 37°C f o r 20 hours i n MEM containing 2% f e t a l c a l f serum. At the end of t h i s time, the medium was replaced with an equal volume of water, the c e l l s scraped o f f the glass b o t t l e s , and the r e s u l t i n g suspensions frozen and thawed three times. The crude lysates were then centrifuged at 3500 rpm f o r 10 minutes to remove large c e l l debris and the supernatants recentrifuged at 4°C i n a Beckman Model L2-65B U l t r a c e n t r i f u g e f o r one hour a t 15,000 rpm. P e l l e t s containing the v i r u s were resuspended i n MEM with 2% f e t a l 26 c a l f serum and were stored i n serum b o t t l e s at -70°C. Stock 7 5 preparations gave t i t r e s of 7.2 x 10 pfu/ml or 5.0 x 10 TCID /ml. Virus Assays 1. End-point D i l u t i o n Technique For rough assays of v i r u s preparations, the end-point d i l u t i o n method was used to c a l c u l a t e v i r u s t i t r e s . The v i r u s samples to be tested were s e r i a l l y d i l u t e d t e n f o l d i n MEM plus 2% f e t a l c a l f serum. Twenty-four hour H.Ep.2 monolayers grown i n Leighton tubes were inoculated i n duplicate with 0.1 ml of each d i l u t i o n and incubated at 37°C f o r 48 hours. The monolayers were then examined microscopi-c a l l y f o r cytopathic e f f e c t s and graded on a scale of +1 to +4. Virus t i t r e s were ca l c u l a t e d by the Reed and Meunch method (77) and expressed as TCID,.,. u n i t s . 50 2. Plaque Assay Quantitative plaque assays were a l s o performed using confluent H.Ep.2 monolayers grown i n 35 x 10 mm Falcon p l a s t i c p e t r i dishes. The c e l l s were maintained under a 5% C0 2 atmosphere i n Dulbecco's medium. Washed H.Ep.2 monolayers were i n f e c t e d i n duplicate with 0.1 ml of each d i l u t i o n and rotated p e r i o d i c a l l y over 30 minutes to ensure even inoculation. An agarose overlay was prepared by melt-ing and mixing in equal parts 1% agarose i n d i s t i l l e d water and serumless double-strength Dulbecco's medium. Each monolayer received 2.0 ml of the overlay before incubation at 37°C i n a 5% CO^ atmos-phere for 3-4 days. The resulting plaques were stained for 3 hours with a 0.1% neutral red solution and observed under indirect light. The plaques appeared as small heavily stained spots surrounded by clear halos against a ligh t l y stained background. Chemicals and Radioisotopes 1. L-Arginine Hydrochloride was obtained from Sigma Chemicals, St. Louis, Missouri. A stock 5% solution in d i s t i l l e d water was stored at 4°C. 2. Colcemid was donated by the Ciba Co. Ltd., Dorval, Quebec. Stock 10 yg/ml solutions in HBSS were stored at 4°C i n a light-tight container and were renewed every three weeks. A fresh 1.0 yg/ml preparation was made daily from the stock. 3. Cytosine Arabinoside Hydrochloride was obtained from Nutritional Biochemicals Corp., Cleveland, Ohio. Stock solutions of 1000 yg/ml in HBSS were stored at 4°C and were renewed every three weeks. A fresh 10 yg/ml solution was prepared for each experiment. 4. 5-Iododeoxyuridine, purchased from Calbiochem, Los Angeles, C a l i f o r n i a , was ki n d l y supplied by Dr. D.M. McLean. Stock pre-parations i n HBSS were stored at 4°C and were renewed every three weeks. A f r e s h 500 Ug/ml s o l u t i o n was made d a i l y from the stock. . . 3 5. T n t i a t e d thymidine ( H-thymidine) , s p e c i f i c a c t i v i t y 49.2 Ci/mM, was obtained from New England Nuclear Corporation, Boston, Mass., and generously supplied by Dr. J.B. Hudson. Fresh preparations of the radioisotope were made i n HBSS and held at 4°C u n t i l use. UV I n a c t i v a t i o n of Virus The herpes simplex v i r u s was i r r a d i a t e d i n 60 mm Falcon p l a s t i c p e t r i dishes (2.0 ml/dish) at a distance of 20 cm from a Sylvania G15T8 UV bulb. I r r a d i a t i o n was c a r r i e d out at room temperature with constant a g i t a t i o n f o r periods of time ranging from 10 seconds to 240 seconds. In V i t r o I n f e c t i o n Procedure Day-old c e l l cultures were washed once with HBSS and inoculated with HSV at an input m u l t i p l i c i t y of about 1 pfu per c e l l . The input m u l t i p l i c i t y was determined by c a l c u l a t i n g the average number of c e l l s per c ulture i n a Levy counting chamber (Max Levy, Phila d e l p h i a , Penn.). A f t e r 30 minutes adsorption at room temperature, the mono-layers were r i n s e d with HBSS and incubated at 37°C i n MEM with 10% 29 f e t a l c a l f serum. Light Microscopy C e l l monolayers grown on Leighton tube c o v e r s l i p s were i n f e c t e d with 7.2 x 10 pfu HSV, treated with 10 yg/ml ara-C or 100 yg/ml IDU, or l e f t to serve as co n t r o l s . A f t e r 5, 12, 24 and 72 hours, the cov e r s l i p s were removed and rinsed b r i e f l y i n p h y s i o l o g i c a l s a l i n e . F i x a t i o n i n absolute methanol f o r 3 minutes preceded s t a i n i n g i n a Giemsa s o l u t i o n c o n s i s t i n g of 3.0 ml stock Giemsa i n 50 ml phosphate buffer pH 7.2 f o r 30 minutes. The stained c o v e r s l i p s were given a f i n a l r i n s e i n s a l i n e , a i r - d r i e d , and mounted on clean glass s l i d e s with Apochromount mounting f l u i d (Aloe S c i e n t i f i c , St. Louis, Mis-souri) . The c e l l s were examined and photographed using a Zeiss semi-automatic photomicroscope. In d i r e c t Fluorescent Antibody Technique Monolayer cultures grown on Leighton tube c o v e r s l i p s were in f e c t e d with 7.2 x 10 pfu HSV per tube (MOI = 1 ) . In some cases, 10 yg/ml ara-C or 100 yg/ml IDU were added to both i n f e c t e d and non-infected c e l l s . At 4, 7, 12 and 24 hours post i n f e c t i o n , the co v e r s l i p s were removed, rin s e d i n p h y s i o l o g i c a l s a l i n e , a i r - d r i e d , and f i x e d i n acetone f o r 10 minutes. Each c o v e r s l i p was exposed to 30 0. 1 ml of 1:2 dilution of guinea pig anti-HSV gamma globulin (Ti.tre 1:32 Microbiological Associates, Bethesda, Md.) for 30 minutes i n a moist chamber at 37°C. The coverslips were then rinsed i n saline and treated with 0.1 ml fluorescein-labeled goat anti-guinea pig gamma globulin (Microbiological Associates, Bethesda, Md.) for 30 minutes i n a moist chamber at 37°C. The cells were given a f i n a l rinse in saline and mounted on clean glass slides using phosphate-buffered glycerol pH 7.2 (Baltimore Biological Labs., Baltimore, Md.). Observation and photography was accomplished using a Reichert microscope equipped with an Osram HBO 200 high pressure mercury vapor lamp as a UV source. Electron Microscopy 1. Negative Staining 7 A frozen stock HSV preparation containing 7.2 x 10 pfu/ml was rapidly thawed in a room temperature water bath and a drop of the virus suspension placed on a wax staining tray. A clean carbon and formvar coated copper grid was lowered onto the drop and allowed to remain i n contact with i t for one minute. The grid was then removed to a drop of 2% phosphotungstic acid pH 6.5 and stained for one minute before examination i n a Phil l i p s EM 300 electron microscope. Grids were observed within 30 minutes of i n i t i a l preparation i n order to preserve v i r u s i n t e g r i t y which was r a p i d l y l o s t a t room temperature. 2. Thin-Sectioning Confluent monolayers of H.Ep.2 and BHK-21 c e l l s grown i n milk d i l u t i o n b o t t l e s were used i n a l l electron microscopy studies. Normal, i n f e c t e d and chemically treated c e l l s were scraped from the glass with a rubber policeman, p e l l e t e d by low-speed c e n t r i f u g a t i o n (1000 rpm f o r 5 minutes i n an International Centrifuge Model CS), and immediately placed i n t o 2.5% glutaraldehyde i n 0.1 M phosphate buffer pH 7.2 f o r 30 minutes at 4°C. The specimens were washed four times i n 0.2 M phosphate buffered sucrose and pos t - f i x e d i n 1% osmium tetrox i d e pH 7.2 for 30 minutes. Dehydration was accomplished by passing the samples through a graded s e r i e s of ethanol concen-t r a t i o n s followed by a step-wise trans f e r i n t o propylene oxide. Specimens were embedded by placing the c e l l s i n increasing concen-t r a t i o n s of Epon 812 r e s i n d i s s o l v e d i n propylene oxide. A f t e r over-night incubation i n p l a s t i c a t room temperature, the embedded samples were placed i n g e l a t i n capsules and cured at 60°C f o r 24 hours. Sections of a l l specimens embedded i n Epon 812 were cut on a LKB I I I microtome and c o l l e c t e d on carbon and formvar coated 150 mesh copper g r i d s . They were stained with a saturated a l c o h o l i c s o l u t i o n of uranyl acetate f o r one minute and post-stained with Reynold's lead c i t r a t e f o r 30 seconds. The g r i d s were examined on a P h i l l i p s EM 300 e l e c t r o n microscope a t 60 kv. Autoradiography Twenty-four hour monolayer cultures grown on Leighton tube co v e r s l i p s were in f e c t e d with 7.2 x 10 6 pfu HSV (MOI = 1) and/or treated with e i t h e r 10 yg/ml ara-C or 100 yg/ml IDU. At various 3 times afterward, the c e l l s were exposed to 2.0 yCi/ml H-thymidine f o r 30 minutes at 37°C. Immediately following exposure to the radioisotope, the c o v e r s l i p s were washed with HBSS and f i x e d i n a 3:1 mixture of methanol and a c e t i c a c i d f o r 15 minutes. Residual f i x a t i v e was removed by repeated washings i n d i s t i l l e d water over a period for 20 minutes. The f i x e d c e l l s were then a i r - d r i e d and mounted on clean glass s l i d e s with Apochromount mounting f l u i d . The s l i d e s were dip-coated with I l f o r d L-4 Nuclear Emulsion, drained, a i r - d r i e d and stored f o r 7 days at 4°C i n a l i g h t - t i g h t box containing a small amount of D r i e r i t e . The dipped s l i d e s were developed according to the following schedule: 12 minutes i n Microdol-X developer (2 parts water and 1 part developer); 5 minutes i n tap water; 5 minutes i n f i x e r ; 4 - 2 minute washes i n tap water. The developed slides were air-dried and examined with an Olympus light microscope for the presence of tritium label. To f a c i l i t a t e observation of the grains, coverslips were stained bri e f l y i n 1% eosin. Metaphase Preparations Cells growing logarithmically (i.e. 16 - 18 hour cultures) i n Leighton tubes were used in a l l chromosome studies. Infected cells generally received 7.2 x 10 pfu HSV per tube and chemically treated cells either 10 yg/ml ara-C or 100 lig/ml IDU. Two hours prior to harvesting, infected, treated and control cultures also received 0.1 yg/ml colcemid. Cells were trysinized off the tubes with 0.25% trypsin in HBSS and centrifuged i n an International Centrifuge Model CS for 5 minutes. The supernatant was decanted and the cells gently resuspended i n 5.0 ml of 0.5% KC1 solution for 15 minutes at room temperature. This suspension was recentrifuged and 4.0 ml of a 3:1 methanol-glacial acetic acid mixture added drop by drop over a period of 20 minutes. After two further changes of fixative, the cells were f i n a l l y suspended i n about 1.0 ml of the solution. Four or five drops from a Pasteur pipette were dropped on a precleaned glass slide from a height of 4 - 5 inches. After air-drying, the slides were stained i n a Giemsa solution for 5 minutes. The stained slides were 34 then b r i e f l y rinsed i n tap water, a i r - d r i e d and examined preliminar-i l y with an Olympus l i g h t microscope. Metaphase plates were scored for chromosomal aberrations under an o i l immersion lens and photographed on Kodak Plus X panchromatic f i l m on a Zeiss semi-automatic photo-microscope. RESULTS Growth Studies Figure 1 shows the growth of HSV i n H.Ep.2 and BHK-21 monolayers at 37°C. Both c e l l types supported v i r a l m u l t i p l i c a t i o n but H.Ep.2 c e l l s were c l e a r l y more susceptible to HSV i n f e c t i o n . The f i r s t de-tectable increase i n v i r u s production occurred 8-12 hours a f t e r i n f e c t i o n and maximum y i e l d s were obtained a f t e r 22-24 hours. Virus 5 0 3 8 t i t r e s a t 22 hours were 10 * TCTD^/ml i n H.Ep.2 c e l l s and 10 * TCID r./ml i n BHK-21 c e l l s . A d d i t i o n of 10 yg/ml ara-C or 100 yg/ml 50 IDU at the time of i n f e c t i o n reduced v i r u s t i t r e s by 99.9% i n both c e l l types. Further e f f e c t s of the a n t i - v i r a l drugs are documented i n the various sections on l i g h t and el e c t r o n microscopy, autoradio-graphy and cytogenetics. Light Microscopy 1. Cytopathology of HSV Leighton tube cultures of H.Ep.2 c e l l s were in f e c t e d with an input m u l t i p l i c i t y of 1 pfu HSV per c e l l and examined at 5, 12, 24 and 72 hours of i n f e c t i o n . HOURS Figure 1. Representative growth curves of HSV in H.Ep.2 and BHK-21 c e l l s . Total virus (extracellular and intracellular) was measured by the standard end-point dilution technique after i n i t i a l infection with an input multiplicity of 1 pfu per c e l l . 37 Normal, uninfected H.Ep.2 monolayers were composed of closely packed epitheloid cells with large ovoid nuclei containing 2-4 well-defined nucleoli (Figs. 2,4). The f i r s t evidence of virus-induced cytopathology was observed i n the nuclei of infected c e l l s . Five hours after infection, many cells showed evidence of early nucleolar disintegration and peripheral chromatin displacement. At 12 hours, over one-half of the ce l l s also demonstrated some degree of rounding and cytoplasmic vacuolization (Fig. 3). The empty-appearing nuclei of these c e l l s were invariably swollen and usually contained dense v i r a l inclusion bodies surrounded by clear halos (Fig. 3). After 24 hours of infection, large numbers of ce l l s had detached from the glass surface and those remaining showed severe nuclear damage coupled with extensive cytoplasmic shrinking and round-ing. The damaged cells had a slight tendency to aggregate i n loose clumps but did not at any time form syncytia (Fig. 5). Monolayers were completely destroyed at 72 hours. A similar virus cytopathology was observed in BHK-21 cells infected with HSV. 2. Effect of Ara-C and IDU ON HSV Cytopathology Cultures inoculated with HSV received either 10 yg/ml ara-C or 100 yg/ml IDU at the time of virus infection. Observations made at 5, 12, and 24 hours revealed no decrease i n the severity or time of appearance of the virus-induced cytopathic effect i n H.Ep.2 and BHK-21 ce l l s . 38 Figure 2. Uninfected culture of H.Ep.2 c e l l s . X1750. Giemsa s t a i n . » J H NI 0 Figure 3. H.Ep.2 culture 12 hours a f t e r HSV i n f e c t i o n . Giemsa s t a i n . Note intranuclear i n c l u s i o n (NI) and swollen degenerating n u c l e i of in f e c t e d c e l l s . X4400. Figure 5. H.Ep.2 culture 24 hours a f t e r HSV i n f e c t i o n . Note loose aggregates of rounded, shrunken c e l l s . X440. 3. E f f e c t of Ara-C and IDU on Uninfected C e l l s Healthy H.Ep.2 and BHK-21 monlayers were treated with 10 yg/ml ara-C or 100 yg/ml IDU f o r 24 and 72 hours. Exposure to ara-C r e s u l t e d i n moderately severe damage to cultures a f t e r 72 hours. The monolayers were p a r t i a l l y destroyed (Fig. 6) and i n d i v i d u a l c e l l s h ighly vacuolized and often rounded (Fig. 7). IDU appeared to cause s i m i l a r but l e s s pronounced c e l l u l a r a l t e r a t i o n s a f t e r 3 days of treatment. Fluorescent Antibody Studies 1. HSV Antigen Production Leighton tube cultures of H.Ep.2 and BHK-21 c e l l s were i n f e c t e d with an input m u l t i p l i c i t y of 1 pfu HSV per c e l l and observed f o r production of v i r u s - s p e c i f i c antigens at 4, 7, 12 and 24 hours by the i n d i r e c t fluorescent antibody method. The f i r s t signs of v i r a l i n f e c t i o n appeared a f t e r 4 hours i n the form of weak cytoplasmic and nuclear fluorescence (Fig. 8). The d i f f u s e cytoplasmic fluorescence reached a maximum i n t e n s i t y a t 7 hours and was p a r t i c u l a r l y evident i n the perinuclear regions of i n f e c t e d c e l l s . E a r l y nuclear antigens were characterized by small, irregularly-shaped granules (Fig. 8) that were l a t e r obscured 41 Figure 6. H.Ep.2 culture a f t e r 72 hours of ara-C treatment. X440. Figure 7. H.Ep.2 culture a f t e r 72 hours of ara-C treatment. Note cytoplasmic v a c u o l i z a t i o n and prevalence of rounded, shrunken c e l l s . X1750. by the development of large amorphous masses. Between 7 and 12 hours, the number of antigen-producing c e l l s rose to over 70% of the population. C e l l s at t h i s time commonly exhibited intense cytoplasmic s t a i n i n g together with patchy nuclear fluorescence (Fig. 9). By 24 hours, v i r u s - s p e c i f i c fluorescence was observed i n over 80-90% of the i n f e c t e d c e l l c u l t u r e . Most of these c e l l s fluoresced strongly i n the cytoplasm and at the c e l l surface (Fig. 10). 2. The E f f e c t of Ara-C and IDU ON HSV Antigen Production The a d d i t i o n of 10 yg/ml ara-C or 100 yg/ml IDU to i n f e c t e d cultures f a i l e d to delay the appearance of HSV antigens or decrease the t o t a l number of f l u o r e s c i n g c e l l s . Strong nuclear and perinuclear s t a i n i n g was evident at 4, 7 and 12 hours of i n f e c t i o n but surface fluorescence was generally lacking i n the chemically treated c e l l s . E l e c t r o n Microscopy 1. Negative Staining HSV morphology was studied by the negative s t a i n method. T y p i c a l v i r u s p a r t i c l e s of mean diameter 170 nm were frequently observed i n a d d i t i o n to the c e l l debris normally found i n semi-purified v i r u s preparations. The v i r i o n s consisted of large envelopes enclosing Figure 8. Fluorescent antibody study of H.Ep.2 c e l l s 4 hours a f t e r HSV i n f e c t i o n . Note weak perinuclear staining and fluorescent nuclear granules. X4400. Figure 9. Fluorescent antibody study of H.Ep.2 c e l l s 7 hours a f t e r HSV i n f e c t i o n . Note amorphous nuclear masses and d i f f u s e cytoplasmic fluorescence. X4400. 44 Figure 10. Fluorescent antibody study of H.Ep.2 c e l l s 24 hours a f t e r HSV i n f e c t i o n . X1750. 68-70 nm nucleocapsids. The capsids were c l e a r l y composed of hollow, polygonal capsomeres arranged i n a regular icosahedral symmetry (Fig. 11). In cases where the phosphotungstic a c i d pene-tra t e d the external capsid, a dense inner "core" was sometimes observed. The core, which measured 30 nm i n diameter, was encased i n an outer s h e l l composed of hollow subunits s i m i l a r to those seen on the external capsid. Other p a r t i c l e s appeared to contain no inner structure (Fig. 12). 2. Thin Sectioning (a) Uninfected H.Ep.2 and BHK-21 C e l l s . Uninfected H.Ep.2 and BHK-21 c e l l s maintained i n MEM plus 10% c a l f serum revealed well preserved microanatomy with i n t a c t mem-branes, undistorted mitochondria, and normal chromatin d i s t r i b u t i o n (Fig. 13,14). Apart from an increased incidence of l i p i d granules i n BHK-21 c e l l s , the human and hamster l i n e s appeared to possess very s i m i l a r u l t r a s t r u c t u r e . (b) Uninfected BHK-21 C e l l s : Abnormal P a r t i c l e Formation BHK-21 c e l l s maintained i n serumless MEM repeatedly gave r i s e to abnormal p a r t i c l e s i n the cytoplasm of af f e c t e d c e l l s . The 90 nm p a r t i c l e s consisted of an ele c t r o n dense core and a less-dense membrane-bound outer region (Fig. 15). Close examination revealed 46 Figure 11. El e c t r o n micrograph of a negative s t a i n preparation of HSV. X90,700. Figure 12. Electron micrograph of a negative s t a i n preparation of HSV. The inner structure of two p a r t i c l e s can be observed where the s t a i n has penetrated the outer capsid. X90,700. Figure 13. Electron micrograph of normal H.Ep.2 c e l l s showing i n t a c t nuclear and cytoplasmic structure. X10,000. fine radial structures extending from the core to the external membrane (Fig. 16). The particles were normally observed i n cyto-plasmic spaces or between the swollen membranes of the endoplasmic reticulum. Numerous distorted mitochondria, ragged vacuoles, and myelin figures were also found i n affected c e l l s . Since serum starvation appeared to be the sole cause of these structural ab-normalities, a l l electron microscope studies of BHK-21 cel l s were carried out on cultures provided with normal growth medium. No particles were observed i n uninfected c e l l s grown in the presence of 10% fet a l calf serum. (c) HSV Development i n H.Ep.2 and BHK-21 Cells H.Ep.2 and BHK-21 monolayers were inoculated with an input multiplicity of 20 pfu HSV per c e l l and examined at 4, 7, 12 and 20 hours after infection. Evidence of v i r a l replication was f i r s t detected as early as 4 hours after infection. At this time, infected cells developed irregularly condensed and marginated nuclear chromatin. In addition, dense granular aggregates were often observed scattered throughout the nuclei, although v i r a l particles were not yet evident (Fig. 17). By 7 hours, the f i r s t particles could be seen adjacent to re-duplicated nuclear membranes i n both H.Ep.2 and BHK-21 cells (Fig. 18). Membrane proliferation was usually extensive and fusion often resulted 49 Figure 16. An abnormal p a r t i c l e i n the cytoplasm of an uninfected BHK-21 c e l l maintained i n serumless medium. X160,000 50 Figure 17. Electron micrograph of a BHK-21 c e l l 4 hours a f t e r HSV i n f e c t i o n . Note the marginated chromatin and granular aggregate i n the nucleus. X50,000 Figure 18. Reduplicated nuclear membranes (RNM) and immature vi r u s p a r t i c l e s i n a BHK-21 c e l l 7 hours a f t e r HSV i n f e c t i o n . X39,500. Figure 19. Immature v i r u s p a r t i c l e s i n the nucleus of a BHK-21 c e l l 7 hours a f t e r i n f e c t i o n . X112,000. i n the formation of b i z a r r e concentric lamallae. V i r a l p a r t i c l e s found i n the nucleus were generally 90 nm i n diameter and consisted of naked capsids enclosing electron dense or electron lucent cores. In a d dition, i n f e c t e d n u c l e i a l s o contained a small number of en-veloped p a r t i c l e s 130 nm i n diameter (Fig. 19). Very oc c a s i o n a l l y these immature v i r i o n s could be seen acquiring a second envelope by budding through the inner lamella of the nuclear membrane (Fig. 20). A few mature p a r t i c l e s with double membranes and a number of naked and immature v i r i o n s were found i n the cytoplasm at 7 hours of i n -f e c t i o n but no surface v i r u s was detected at t h i s time. Extensive v i r a l r e p l i c a t i o n was evident at 12 hours a f t e r i n f e c t i o : Nuclear p a r t i c l e s i n various stages of assembly were observed i n great numbers and cytoplasmic v i r u s was frequently found associated with smooth membranous structures. The mature and immature viruses appeared to p r e f e r e n t i a l l y aggregate i n c e l l vacuoles and within f i n e , branch-ing tubules i n the cytoplasm (Fig. 21). Mature p a r t i c l e s measuring 170-175 nm were released by a process resembling reverse phagocytosis (Fig. 22). No evidence of budding v i r u s was encountered i n e i t h e r c e l l type. By 20 hours, widespread c e l l degeneration was evident with cor-responding high l e v e l s of e x t r a c e l l u l a r v i r u s (Fig. 23). Enveloped HE ' ; ,• , Figure 20. Immature v i r u s p a r t i c l e budding through the nuclear membrane of a BHK-21 c e l l 7 hours a f t e r HSV i n f e c t i o n . X91,000. Figure 21. Mature v i r u s p a r t i c l e s near a branching tubule i n the cytoplasm of a H.Ep.2 c e l l 12 hours a f t e r HSV i n f e c t i o n . X123,000. 54 • f l Figure 22. Release of a mature HSV p a r t i c l e from a H.Ep.2 c e l l 12 hours a f t e r i n f e c t i o n . X112,000. 55 Figure 23. I n t r a c e l l u l a r and e x t r a c e l l u l a r v i r u s i n a H.Ep.2 c e l l 20 hours a f t e r HSV i n f e c t i o n . X62,500. p a r t i c l e s measuring 170 nm were often found at the c e l l surface lodged between prominent cytoplasmic processes. The surface i t s e l f was jagged and discontinuous and a great deal of c e l l u l a r debris was found f r e e i n the medium. In add i t i o n , nuclear membranes were often broken and the nucleoplasm appeared granular and s t r u c t u r e l e s s . At t h i s time, b i z a r r e v i r a l forms were also found i n many i n f e c t e d c e l l s . Large c r y s t a l s composed of v i r a l capsids enclosing a v a r i e t y of e l ectron dense components were frequently observed i n the n u c l e i of BHK-21 c e l l s (Fig. 24). Groups of membranous p a r t i c l e s and mature v i r i o n s were a l s o found i n the cytoplasm of degenerating c e l l s . Cytoplasmic aggregates were enclosed i n i r r e g u l a r l y shaped vacuoles and existed adjacent to naked HSV p a r t i c l e s and mature v i r u s ( Fig. 25). In general, the development of HSV was very s i m i l a r i n both H.Ep.2 and BHK-21 c e l l s . However, the hamster cultures appeared to give r i s e to a higher percentage of defective and unenveloped v i r u s p a r t i c l e s i n the nucleus and cytoplasm of i n f e c t e d c e l l s . Moreover, although b i z a r r e v i r a l aggregates were very common i n BHK-21 c e l l s , no c r y s t a l formations were ever observed i n i n f e c t e d H.Ep.2 c e l l s . Beyond these d i f f e r e n c e s , v i r u s r e p l i c a t i o n appeared much the same i n terms of assembly, envelopment and relea s e . (d) E f f e c t of Ara-C and IDU on HSV Development H.Ep.2 and BHK-21 monolayers were i n f e c t e d with 20 pfu HSV per c e l l . A f t e r r e c e i v i n g 10 yg/ml ara-C or 100 yg/ml IDU, the c e l l s Figure 24. Intranuclear v i r a l c r y s t a l i n a BHK-21 c e l l 20 hours a f t e r HSV i n f e c t i o n . X82,200. Figure 25. Cytoplasmic aggregate i n a BHK-21 c e l l 20 hours a f t e r HSV i n f e c t i o n . X39,500. were incubated for 20 hours and examined under the electron microscope. Ara-C prevented the formation of complete virus particles, but did not prevent the early nuclear alterations characteristic of HSV infection. Although membrane reduplication was not evident, condensed and marginated chromatin was readily observed in the nuclei of many infected c e l l s . In addition, both c e l l types revealed a large number of dense intranuclear aggregates. The nuclear granules measured 30 nm i n diameter and resembled v i r a l cores i n form and density (Fig. 26). No other virus-like particles were found i n the nucleus or cytoplasm of cells treated with ara-C. IDU had much the same inhibitory effect on HSV replication i n cultured c e l l s . In most cases, marginated chromatin and dense nuclear granules were observed in the chemically treated and infected c e l l s . However, i n addition to such particulate structures, a number of enveloped viruses measuring 130-135 nm in diameter were also found free i n the cytoplasm. The visually defective particles were ragged in appearance and their cores were often of low density (Fig. 27) . They appeared to possess only one envelope and were never seen at the surface of the infected c e l l s . (e) Effect of Ara-C and IDU on Uninfected Cells Cells treated with 10 yg/ml ara-C were morphologically altered 59 Figure 27. Cytoplasmic particle i n a BHK-21 c e l l 20 hours after HSV infection and IDU treatment. X28,000. by 24 hours. E a r l y changes included marked d i s t e n t i o n of mito-chondria (Fig. 28) and endoplasmic reticulum. Further treatment with ara-C r e s u l t e d i n conspicuous cytoplasmic v a c u o l i z a t i o n with los s of mitochondrial structure and d i s t o r t i o n of the i n t e r n a l membrane systems (Fig. 29). Exposure to 100 yg/ml IDU f o r 24 and 48 hours produced the same type of s t r u c t u r a l a l t e r a t i o n s i n uninfected H.Ep.2 and BHK-21 c e l l s . However, cytopathic e f f e c t s were evident only a f t e r 48 hours and were r e s t r i c t e d to a minimal d i s t o r t i o n of c e l l organelles and v a c u o l i z a t i o n . Autoradiographic Studies 1. DNA Synthesis i n HSV Infected C e l l s DNA synthesis i n HSV i n f e c t e d c e l l s was studied by pulse l a b e l i n g 3 the c e l l s at various times a f t e r i n f e c t i o n with 2.0 yci/ml H-thymidine f o r 30 minutes. HSV i n f e c t i o n of H.Ep.2 and BHK-21 c e l l s appeared to cause an immediate and almost complete i n h i b i t i o n of DNA synthesis. A f t e r 4 hours, however, the number of c e l l s synthesizing DNA rose r a p i d l y and reached a maximum of 28.2% a f t e r 8 hours of i n f e c t i o n . Following t h i s peak of synthetic a c t i v i t y , t o t a l DNA synthesis gradu-a l l y declined to approximately one-sixth of the 0 time f i g u r e ( F i g . 30). The e a r l y decrease found i n in f e c t e d c e l l s probably represented a virus-induced i n h i b i t i o n of c e l l DNA synthesis while the secondary Figure 28. Mitochondria of a BHK-21 c e l l a f t e r 24 hours of ara-C treatment. X69,200. o — o HSV HOURS Figure 30. DNA synthesis i n v i r u s - i n f e c t e d and chemically treated BHK-21 c e l l s . C e l l s were i n f e c t e d with an input m u l t i p l i c i t y of 1 p f u / c e l l and treated with 10 yg/ml ara-C and 100 yg/ml IDU. increase r e f l e c t e d the bulk of v i r a l DNA synthesis and pos s i b l y r e p a i r of damaged host c e l l DNA (126). 2. DNA Synthesis i n C e l l s Treated with Ara-C and IDU Ara-C and IDU produced an immediate and complete i n h i b i t i o n of c e l l and v i r a l DNA synthesis i n HSV i n f e c t e d c e l l s (Fig. 30). The a n t i - v i r a l drugs also completely i n h i b i t e d c e l l u l a r DNA synthesis i n uninfected H.Ep-2 and BHK-21 c e l l s within 2 hours of t h e i r a d d i t i o n . Cytogenetic Studies 1. M i t o t i c Rates M i t o t i c rates were studied i n HSV-infected and chemically treated H.Ep.2 and BHK-21 c e l l s . HSV i n f e c t i o n produced a ra p i d increase i n the m i t o t i c index of both c e l l types. In one experiment, the number of BHK-21 c e l l s observed i n metaphase t r i p l e d i n the f i r s t s i x hours of i n f e c t i o n and thereafter declined u n t i l the study was terminated. By 24 hours, mitosis was completely i n h i b i t e d i n a l l HSV-infected c e l l s ( F ig. 31). The m i t o t i c rate of c e l l s exposed to ara-C or IDU at the time of HSV i n f e c t i o n was s i m i l a r to that of untreated v i r u s i n f e c t e d c e l l s . 64 H S V ( M O I = l ) HOURS Figure 31. M i t o t i c rates of BHK-21 c e l l s following v i r u s i n f e c t i o n and chemical treatment. Colcemid was omitted from the standard metaphase pre-paration technique. However, chemically treated uninfected c e l l s underwent a ra p i d and complete i n h i b i t i o n of mitosis within 6-8 hours of exposure to the drugs (Fig. 31). 2. Normal C e l l Karyotypes H.Ep.2 c e l l s possessed a d i p l o i d male karyotype of 74 chromosomes (Fig. 32). The human cancer l i n e had a r e l a t i v e l y high percentage of aneuploidy i n the d i p l o i d range and a low percentage of t e t r a p l o i d y (Table I ) . BHK-21 c e l l s displayed a normal male karyotype of 44 chromosomes with one metacentric marker chromosome (Fig. 33). A s i m i l a r high percentage of aneuploidy and low percentage of t r i p l o i d y and t e t r a p l o i d y was observed i n the hamster l i n e (Table I ) . 3. HSV-induced Chromosome Abnormalities A comparative study was made concerning the e f f e c t of HSV on the chromosomes of H.Ep.2 and BHK-21 c e l l s . Virus treated cultures showed a s i g n i f i c a n t increase i n the number of c e l l s with chromosome aberrations when compared to con t r o l cultures (Fig. 34). The per-centage of abnormal metaphases began too r i s e within 2 hours of i n f e c t i o n and reached a maximum of 100% at 14-20 hours. Generally, there were fewer abnormalities observed i n BHK-21 c e l l s during the f i r s t 8 hours of i n f e c t i o n . For example, a f t e r 4 hours, almost 60% of the H.Ep.2 metaphases were abnormal, while only 40% of the BHK metaphases revealed 66 Figure 32. Karyotype of a normal H.Ep.2 c e l l . X4400. Figure 33. Table I. Frequency D i s t r i b u t i o n of Various Levels of Ploidy i n H.Ep.2 and BHK-21 C e l l s . Approximate Chromosome Number T o t a l * Haploid Aneuploid D i p l o i d Aneuploid T r i p l o i d T e t r a p l o i d C e l l s n <2n 2n >2n 3n 4n H.Ep.2 8 28 137 55 - 42 250 BHK-21 5 34 145 48 15 3 250 * H.Ep.2 n = 37 BHK-21 n = 22 68 HOURS A F T E R I N F E C T I O N Figure 34. The effect of HSV infection on the chromosomes of H.Ep.2 and BHK-21 c e l l s . chromosome lesions. However, the total damage to the c e l l chromosomes after 20 hours was similar in both lines. Quantitative analysis of the damage also showed that the same type of lesions were produced i n the human and hamster c e l l s . The lesions consisted of chromatid gaps or achromatic regions (Fig. 35), chromatid breaks (Fig. 36), secondary constrictions (Fig. 37), frag-mentation (Fig. 38), erosion (Fig. 39), and endoreduplication (Fig. 40). Chromatid gaps, breaks and secondary constrictions were more frequent in the f i r s t 4 hours of infection while severe fragmentation and erosion appeared more often later i n infection. In addition, H.Ep.2 cells revealed more extensive damage at an earlier time than BHK-21 c e l l s (Table II, III). Chromosome abnormalities in both c e l l types seemed to be random and nonspecific, although extensive karyotyping was not performed i n a l l cases. Moreover, a l l chromosome aberrations were completely prevented by the neutralization of the virus inoculum with specific HSV antiserum. 4. Effect of Multiplicity of Infection on HSV-induced Chromosome  Abnormalities. Figure 41 demonstrates the relationship between i n i t i a l multi-p l i c i t y of infection and amount of chromosome damage found in BHK-21 ce l l s . The percentage of abnormal metaphases increased i n proportion to the virus inoculum. At an input multiplicity of 10 pfu per c e l l , 70 Figure 3 5 . Chromosome complement of a BHK-21 c e l l 4 hours a f t e r HSV i n f e c t i o n . Note prominent chromatid gap. X4400. Figure 36 . Chromosome complement of a BHK-21 c e l l 8 hours a f t e r HSV i n f e c t i o n . Note mul t i p l e chromatid breaks and chromosome d i s t o r t i o n . X4400. Chromosome complement of a BHK-21 c e l l 4 hours a f t e r HSV i n f e c t i o n . Note chromatid break and prominent secondary c o n s t r i c t i o n s . X4400. Chromosome complement of a BHK-21 c e l l showing complete fragmentation a f t e r 10 hours of HSV i n f e c t i o n . X4400. 72 « Figure 39. Erosion of BHK-21 complement a f t e r 10 hours of HSV i n f e c t i o n . X4400. Figure 40. Endoreduplication of BHK-21 chromosomes 8 hours a f t e r HSV i n f e c t i o n . X4400. Table I I . An Analysis of HSV-Induced Chromosome Abnormalities i n H.Ep.2 C e l l s Hours a f t e r i n f e c t i o n No. c e l l s scored Single gaps and breaks Multiple gaps Secondary con s t r i c t i o n s Fragmen-t a t i o n Erosion Endore-d u p l i c a t i o n Condensation 1 250 12 - 4 - 2 1 1 2 250 31 5 7 4 4 - 2 4 250 37 37 13 56 2 - -6 250 13 49 8 97 7 1 -8 250 10 55 9 110 8 - -10 250 7 51 5 128 14 - -12 250 8 29 2 164 10 1 2 14 200 4 14 - 169 - 1 20 100 - 4 - 94 2 - -M u l t i p l i c i t y of i n f e c t i o n = 1 pfu per c e l l Table I I I . An Analysis of HSV-Induced a Chromosome Abnormalities i n BHK-21 C e l l s Hours a f t e r i n f e c t i o n No. c e l l s scored Single gaps and breaks Multiple gaps Secondary Constrictions Fragmen-t a t i o n Erosion Endore-d u p l i c a t i o n Condensation 1 250 8 - 2 - 2 1 - -2 250 14 - 10 - 3 2 1 4 250 30 18 27 18 4 1 -6 250 30 41 12 51 5 2 2 8 250 16 58 4 94 7 1 -10 250 7 50 1 137 10 - -12 250 - 41 - 180 8 - 1 14 200 - 17 - 179 4 - -20 100 — 1 - 99 — - — M u l t i p l i c i t y of i n f e c t i o n = 1 pfu per c e l l . 75 O 2 4 6 8 10 1 2 14 2 0 HOURS AFTER INFECTION Figure 41. The r e l a t i o n s h i p between m u l t i p l i c i t y of i n f e c t i o n and HSV-induced chromosome abnormalities i n BHK-21 c e l l s . 100% of the metaphases observed at 8 hours were abnormal, whereas fo r 10- and 100-fold lower doses of v i r u s , 82% and 44% of the metaphases revealed chromosome l e s i o n s . The number of a l t e r e d chromosomes per metaphase also increased i n r e l a t i o n to the dose of v i r u s and the time of incubation i n both H.Ep.2 and BHK-21 c e l l s . 5. E f f e c t of UV I r r a d i a t i o n of HSV on Virus-Induced Chromosome  Abnormalities. The number of virus-induced chromosome abnormalities i n H.Ep.2 and BHK-21 c e l l s decreased l o g a r i t h m i c a l l y a f t e r UV i r r a d i a t i o n of HSV. In one experiment using BHK-21 c e l l s , the capacity of the v i r u s to induce metaphase a l t e r a t i o n s was i n a c t i v a t e d approximately f i v e times more slowly than the i n f e c t i v i t y (Fig. 42). 6. E f f e c t of Excess Arginine on HSV-induced Chromosome Abnormalities Previous studies have established that a minimal concentration of arginine i s necessary for HSV development i n cultured c e l l s (3). I t i s also known that mycoplasma i n f e c t i o n generally r e s u l t s i n arginine depletion and that such s t a r v a t i o n can cause chromosome ab-normalities i n mammalian c e l l s (60). Since mycoplasma i n f e c t i o n has been r u l e d out i n the H.Ep and BHK-21 l i n e s , i t was therefore of i n t e r e s t to determine i f the abnormalities produced by HSV i n f e c t i o n were i n f a c t , due to virus-induced arginine s t a r v a t i o n of the c e l l s . This 77 Figure 42. E f f e c t of UV i r r a d i a t i o n of HSV on v i r a l i n f e c t i v i t y and capacity to induce chromosome abnormalities i n BHK-21 c e l l s . The nonirradiated v i r u s (1 p f u / c e l l ) induced chromosomal damage at 12 hours i n 92% of the observed metaphases, which was taken as 100%. I n f e c t i v i t y was measured by the end-point d i l u t i o n technique. was done by increasing the concentration of arginine i n the growth medium and observing the e f f e c t on HSV-induced chromosome aberrations. Table IV shows the r e s u l t s of such an experiment c a r r i e d out i n BHK-21 c e l l s . Increases i n arginine varying from 5- to 15-fold did not s i g n i f i c a n t l y a l t e r the number or type of aberrations produced i n the c e l l chromosomes a f t e r HSV i n f e c t i o n . S imilar r e s u l t s were obtained i n HSV-infected H.Ep.2 c e l l s . 7. E f f e c t of Ara-C on the Chromosomes of Uninfected and HSV-Infected  C e l l s . Rapidly d i v i d i n g BHK-21 c e l l s were exposed to various concentrations of ara-C f o r 4 hours. Chromosome an a l y s i s at the end of t h i s time showed that ara-C caused a s i g n i f i c a n t increase i n the number of si n g l e chromatid gaps and breaks (Table V). The drug-induced damage was prop o r t i o n a l to the concentration of ara-C used i n the experiment ( i e . 10 Ug/ml ara-C induced abnormalities i n 10% of the BHK-21 metaphases and 20 yg/ml ara-C induced les i o n s i n 20% of the metaphases). HSV-infected BHK-21 c e l l s were also exposed to ara-C f o r 4 hours. Ara-C d i d not i n h i b i t any of the HSV-induced chromosome abnormalities (Table V). Moreover, the drug appeared to act s y n e r g i s t i c a l l y with the v i r u s to produce an overly large number of c e l l s with s i n g l e and multiple gaps and breaks (Fig. 43). I t was estimated that the number of af f e c t e d Table IV. The Effect of Excess Arginine on HSV-Induced Chromosome Abnormalities i n BHK-21 Cells Treatment % Abnormal Single gaps Multiple Secondary Fragment- Erosion Endore- Condensation Metaphases and breaks gaps Constrictions ation duplication 5 x Arg 4 6 - - - 2 - -10 x Arg 6 7 - - - 3 1 1 15 x Arg 6 8 - - - 3 1 -5 x Arg + HSVb 46 31 14 30 12 4 - 2 10 x Arg + HSV 44 31 15 26 11 3 - 1 15 x Arg + HSV 40 27 14 24 10 5 1 1 HSV 46 30 15 29 13 1 - -Control 4 7 _ _ _ _ _ — Arginine increases were based on normal medium content of 6.0 x 10 M Multiplicity of infections = 1 pfu per c e l l 200 cells were scored 4 hours after HSV infection and/or arginine treatment. Table V. Chromosome Abnormalities i n HSV-Infected and Non-Infected BHK-21 Cells Treated with ara-C Treatment % Abnormal^ Single Gaps Multiple Secondary Fragment-Metaphases and Breaks Gaps Constrictions ation Erosion Endoreduplication ara-C,5 yg/ml 2 ara-C,10 yg/ml 10 ara-C,20 yg/ml 20 ara-C, 5 yg/ml + HSV 45 ara-C,10 yg/ml + HSV 64 ara-C,20 yg/ml + HSV 76 HSV 40 Control 4 4 14 28 8 23 13 7 3 2 4 30 56 52 20 7 5 44 42 70 36 5 12 3 4 4 Multiplicity of infection = 1 pfu per c e l l 200 cells were scored 4 hours after HSV infection and/or ara-C treatment. CD o Figure 44. Translocation found i n a BHK-21 c e l l 4 hours a f t e r addition of IDU. X4400. cells i n the HSV and ara-C treated cultures was approximately 1.3 times greater than the additive effects induced separately by the virus and the antimetabolite. The synergistic effect on chromosome damage could be observed i f ara-C was added as late as 2 hours after virus adsorp-tion. Exposure to the drug in the last 2 hours on infection, however, produced no increase i n HSV-induced abnormalities. Similar results were obtained i n H.Ep.2 c e l l s . 8. Effect of IDU on the Chromosomes of Uninfected and HSV-Infected  Cells. Uninfected BHK-21 cells were exposed to concentrations of IDU ranging from 25 to 200 yg/ml. Low doses of IDU had l i t t l e , i f any, effect on the number of chromosome aberrations found in the c e l l s after 4 hours of treatment. However, higher concentrations of the drug induced damage in 7-10% of the metaphases as compared to 3% i n the control culture. IDU-induced abnormalities included single chromatid gaps, breaks, and translocations (Fig. 44). HSV-infected BHK-21 cel l s were also exposed to IDU for 4 hours. IDU did not inhibit any of the virus-induced chromosome abnormalities nor did i t act synergistically with the virus (Table VI). Infected cells treated with the lower concentrations (25 and 50 yg/ml) of the drug had the same number of aberrations as untreated infected c e l l s . Cells receiving higher concentrations had damage that was equal to the Table VI. Chromosome Abnormalities in HSV-Infected and Non-Infected BHK-21 Cells Treated With IDU. Treatment % Abnormal. b Metaphases Single Gaps and Breaks Multiple Gaps Secondary Constrictions Fragment-ation Erosion Endore- Translocations duplication IDU, 25 yg/ml 2 1 - - - - -IDU, 50 yg/ml 4 5 - - - 2 1 IDU, 100 yg/ml 7 9 - - - 2 2 1 IDU, 200 yg/ml 10 15 - - - 1 2 2 IDU, 25 yg/ml + HSV 40 14 16 8 35 5 2 IDU, 50 yg/ml + HSV 44 18 17 9 39 3 2 IDU, 100 yg/ml + HSV 47 21 19 8 41 3 1 1 IDU, 200 yg/ml + HSV 53 17 28 6 53 2 -HSV .42 11 18 8 41 6 -Control 3 3 - — - 2 1 Multiplicity of infection = 1 pfu per c e l l 200 cells were scored 4 hours after HSV infection and/or IDU treatment additive effects induced separately by IDU and HSV. Similar results were obtained i n H.Ep.2 c e l l s . 9. Effect of Ara-C and IDU on the Chromosomes on Uninfected and HSV- Infected Cells. Infected and uninfected BHK-21 and H.Ep.2 cells were exposed to a combination of 10 yg/ml ara-C and 100 yg/ml IDU for 4 hours. The amount of chromosome damage incurred i n uninfected cells was equal to the additive effects of ara-C and IDU. Damage i n HSV-infected cells equalled the sum of the abnormalities induced by IDU alone and those induced by the synergistic action of ara-C and HSV. Aberrations were generally restricted to single and multiple gaps and fragmentation (Table VII). Table VII. Chromosome Abnormalities in HSV-Infected and Non-Infected HBK-21 Cells Treated With ara-C and IDU % Abnormal, Single Gaps Multiple Secondary Fragment- „ . _ , , .. ^ , d , „ , „ „ . . j_. . . Erosion Endoreduplication Treatment Metaphases and Breaks Gaps Constrictions ation ara-C IDUb ara-C + IDU HSV° + ara-C HSV + IDU HSV + ara-C + IDU HSV Control 11 8 20 62 49 72 38 3 5 4 15 17 11 21 13 2 1 6 4 7 8 8 7 11 7 29 25 32 10 4 4 4 2 1 ara-C was used at a concentration of 10 lig/ml IDU was used at a concentration of 100 yg/ml Multiplicity of infection = 1 pfu per c e l l 100 cells were scored 4 hours after HSV infection and/or chemical treatment DISCUSSION These r e s u l t s show that HSV i s capable of inducing severe morphological and biochemical a l t e r a t i o n s i n in f e c t e d human and hamster t i s s u e c u l t u r e c e l l s . The a n t i - v i r a l agents ara-C and IDU completely i n h i b i t HSV r e p l i c a t i o n i n the same c e l l s but are unable to prevent any of the cytopathic e f f e c t s of the v i r u s . The r a p i d growth cycles of HSV i n H.Ep.2 and BHK-21 c e l l s have been documented i n previous studies by Roizman et a l . (89) and Rus s e l l et a_l. (96) . Although both c e l l types supported a remarkably s i m i l a r course of v i r a l r e p l i c a t i o n , the BHK-21 l i n e was c l e a r l y l e s s s u i t a b l e f o r producing high t i t r e s of HSV. The i n h i b i t i o n of HSV r e p l i c a t i o n following a d d i t i o n of 10 yg/ml ara-C or 100 yg/ml IDU at the time of i n f e c t i o n confirms e a r l i e r reports on the a n t i - v i r a l nature of the drugs. Buthala (9) was the f i r s t to show that s i m i l a r concentrations of ara-C and IDU were a c t i v e i n v i t r o against a s e l e c t group of DNA viruses including HSV, pseudo-ra b i e s , B-virus, swine pox, fowl pox and v a c c i n i a v i r u s . P r i o r to t h i s work, Kaufman had reported that IDU was c l i n i c a l l y e f f e c t i v e against herpetic k e r a t i t i s i n ra b b i t s (39) and subsequent studies showed that ara-C was equally a c t i v e i n curing ocular herpes i n man and animals (36). 87 More recent l y , e f f o r t s have been focused on determining the mechanism of a c t i o n of the two a n t i - v i r a l agents. L e v i t t and Becker (46) found that ara-C appeared to prevent HSV r e p l i c a t i o n i n v i t r o by blocking the de novo synthesis of deoxycytidylate. Thus, i n f e c t e d c e l l s treated with the drug showed no evidence of v i r a l DNA synthesis or v i r i o n assembly. S i m i l a r l y , Roizman e_t a_l. (89) reported that IDU completely i n h i b i t e d HSV r e p l i c a t i o n i n human c e l l s at a concentration of 5 Ug/ml and appeared to prevent v i r a l DNA synthesis by blocking the u t i l i z a t i o n of thymidine. On the other hand, l a t e r i n v e s t i g a t o r s found evidence of IDU s u b s t i t u t i o n i n v i r a l DNA, suggesting that the drug a f f e c t e d production of defective l a t e proteins involved i n v i r u s assembly rather than DNA synthesis i t s e l f (87,73). C y t o l o g i c a l examination of HSV-infected c e l l s revealed the large i n c l u s i o n bodies and nuclear disorganization t y p i c a l of HSV i n f e c t i o n (62). The HSV s t r a i n used i n the present study produced marked rounding of both c e l l types with l i t t l e aggregation of i n f e c t e d c e l l s and no syncytia formation. A s i m i l a r e f f e c t on the s o c i a l behaviour of c e l l s was described by Roizman (84) with the VR-3 s t r a i n of HSV. Ara-C and IDU d i d not appear to reduce HSV cytopathology i n ei t h e r c e l l l i n e . Previous reports have also described persistence of v i r u s cytopathology i n treated c e l l s (9,43) , i n d i c a t i n g that the host damage i s an e a r l y v i r u s function independent of v i r a l DNA synthesis or assembly of i n f e c t i o u s p a r t i c l e s . Drug-induced c y t o t o x i c i t y was observed i n both H.Ep.2 and BHK-21 c e l l s . The generalized degeneration of c e l l s appeared to occur with increasing time of treatment and was more obvious i n ara-C treated c u l t u r e s . These r e s u l t s confirm Buthala's e a r l i e r work (9) on i n v i t r o t o x i c i t y of ara-C and IDU. He observed marked v a c u o l i z a t i o n and mitochondrial d i s i n t e g r a t i o n i n c e l l s exposed to e i t h e r drug f o r a prolonged period of time. In addition, ara-C appeared to be the more toxic of the two a n t i - v i r a l agents although both were more acti v e i n r a p i d l y growing c e l l s than i n stationary c u l t u r e s . The l a t t e r observation i s probably r e l a t e d to the f a c t that ara-C and IDU are known to i n h i b i t DNA synthesis and mitosis i n mammalian c e l l s (43,73). C l i n i c a l l y , the two drugs are al s o capable of causing a marked d i s r u p t i o n of the hematopoietic system of man (74). Thus, the present c y t o l o g i c a l and c l i n i c a l evidence strongly suggests that ara-C and IDU act not as s p e c i f i c a n t i - v i r a l agents but rather as mammalian antimetabolites. As a r e s u l t , t h e i r usefulness as therapeutic agents i n human disease i s severely l i m i t e d . There are many reports i n the l i t e r a t u r e concerning the large number of antigens formed i n t i s s u e culture c e l l s i n f e c t e d with HSV. Using the agar g e l p r e c i p i t a t i o n t e s t , Watson et al_. (124) have de-tected 12 d i f f e r e n t p r e c i p i t a t i o n bands with immune antiserum prepared against HSV-infected c e l l extracts. Several other i n v e s t i g a t o r s have demonstrated at l e a s t f i v e separate immunofluorescent elements i n HSV-in f e c t e d c e l l s (45,78,91,94). In the present study, four d i f f e r e n t fluorescent antigens were detected i n v i r u s - i n f e c t e d H.Ep.2 and BHK-21 c e l l s with a commercial HSV antiserum prepared i n guinea p i g s . Within 4 hours of i n f e c t i o n , fluorescence was observed i n the nucleus, perinuclear region, and cytoplasm of both c e l l types. The early nuclear antigens appeared as small, i r r e g u l a r l y shaped granules and the cytoplasmic antigens as a d i f f u s e fluorescence. Geder and Vaczi (22) described s i m i l a r immunofluorescent elements i n HSV-infected BSC-1 c e l l s with antiserum prepared against v i r u s - i n f e c t e d t i s s u e c u l t u r e e x t r a c t s . A f t e r 4 hours, large fluorescent masses appeared i n the nucleus of i n f e c t e d c e l l s and l a t e i n i n f e c t i o n , intense surface fluorescence was observed i n over 90% of the c e l l population. These l a t e r antigens were previously described by Ross et al^. (94) and Roane and Roizman (78), who also detected a second granular antigen i n the cytoplasm a f t e r 5-6 hours of i n f e c t i o n . The absence of cytoplasmic granules i n the present study may be r e l a t e d to the use of d i f f e r e n t v i r a l s t r a i n s or d i f f e r e n t HSV antiserums. Ara-C and IDU f a i l e d to prevent the formation of the nuclear or perinuclear antigens i n in f e c t e d c e l l s but d i d prevent the appearance of surface fluorescence. Geder and Vaczi (22) reported s i m i l a r r e s u l t s i n BSC-1 c e l l s a f t e r treatment with 10 U.g/ml ara-C. This evidence 90 i n d i c a t e s that the nuclear granules and perinuclear elements are due to early antigenic components or products of HSV i n f e c t i o n while the surface fluorescence i s dependent on v i r a l DNA synthesis and assembly. I t i s apparent from t h i s and other studies that the number and type of v i r u s - s p e c i f i c antigens found i n HSV-infected c e l l s i s depen-dent on the immunofluorescent technique, the v i r u s s t r a i n , and the preparation of the antiserum. Moreover, most of the virus-induced antigenic components are not synthesized a f t e r in_ v i t r o exposure of the c e l l s to the a n t i v i r a l agents ara-C and IDU. In the past/ e l e c t r o n microscopy has provided much us e f u l information concerning the morphology and development of HSV i n cultured c e l l s . However, various t e c h n i c a l problems have also im-posed c e r t a i n r e s t r i c t i o n s on the technique. For example, the use of high t i t r e samples containing large numbers of defective viruses does not permit the microscopic d i f f e r e n t i a t i o n of i n f e c t i o u s and noninfectious p a r t i c l e s . Moreover, asynchrony of v i r u s development makes i t d i f f i c u l t to deduce the k i n e t i c sequence of events from a s e r i e s of s t a t i c micrographs. As a r e s u l t of these disadvantages, any evaluation of e l e c t r o n microscope studies must be tempered with the knowledge that the r e s u l t s c o n s t i t u t e a biased view of v i r u s structure and r e p l i c a t i o n . The HSV p a r t i c l e s observed i n t h i s study a f t e r negative s t a i n preparation were s i m i l a r i n s i z e and morphology to the v i r i o n s described by Watson et a l . (125) , Spring et al_. (107) and Darlington and Moss (13). Present e l e c t r o n microscopy revealed four s t r u c t u r a l elements of the v i r u s - the outer envelope, the outer capsid, the inner capsid and the dense core. Other studies have also described a second inner envelope and a middle capsid (85). The majority of the HSV p a r t i c l e s appeared to be f u l l y enveloped, i n d i c a t i n g that the preparation used i n t h i s research contained a large number of morpho-l o g i c a l l y mature v i r i o n s . In a d d i t i o n , the r a p i d d i s i n t e g r a t i o n of p a r t i c l e structure observed at room temperature confirmed e a r l i e r reports of HSV thermosensitivity (122). Under normal growth conditions, thin-sectioned H.Ep.2 and BHK-21 c e l l s appeared quite healthy and i n t a c t . At the same time, severe morphological changes were commonly observed i n BHK-21 c e l l s maintained i n serumless growth medium. These changes included the development of abnormal p a r t i c l e s and marked cytoplasmic d i s r u p t i o n i n a f f e c t e d c e l l s . Such a l t e r a t i o n s have been previously described i n many continuous and oncogenic c e l l c u l t u r e s . Bernhard and Tournier (5) were the f i r s t to detect v i r u s - l i k e p a r t i c l e s i n an apparently normal BHK-21 (clone 13) l i n e . The s p h e r i c a l 85 nm structures were observed s i n g l y or i n groups within cytoplasmic vacuoles or the swollen c i s -ternae of the endoplasmic reticulum. The p a r t i c l e s were further distinguished by the presence of c h a r a c t e r i s t i c e l e c t r o n dense r a d i a l structures that appeared to emanate from the nucleoid. Sub-sequent studies have since confirmed the presence of s i m i l a r "R" (radial) p a r t i c l e s i n BHK-21/13s, BHK-21/4, BHK-21/13/TC6/A, and BHK-21/F c e l l s (71). P a r t i c l e s have also been found i n BHK-21 c e l l s transformed by S V ^ Q and polyoma v i r u s e s , i n hamster tumors induced by polyoma-transformed BHK-21 c e l l s , and i n a continuous c a l f kidney c e l l l i n e (6). Thus, abnormal p a r t i c l e development appears to be a widespread phenomenon. Previously, however, the "R" p a r t i c l e s were normally detected i n a c t i v e l y growing c e l l s while i n the present study, they were observed only a f t e r serum s t a r v a t i o n . Thus, i t can be concluded that the conditions of serum d e f i c i e n c y induced or a t l e a s t stimulated the formation of the abnormal v i r u s - l i k e structures i n the BHK-21 system. A s i m i l a r stimulatory e f f e c t has been described i n BHK-21 c e l l s i n f e c t e d with r u b e l l a v i r u s (71). Current theories tend to regard the "R" p a r t i c l e s and other abnormal structures as manifestations of l a t e n t v i r u s i n f e c t i o n s . However, to date, there i s l i t t l e evidence to support t h i s hypothesis other than the obvious morphological s i m i l a r i t y of the p a r t i c l e s to known v i r u s e s . The structures may equally w e l l a r i s e i n response to some medium de f i c i e n c y or may represent another unknown i n f e c t i o u s agent. Thin-section e l e c t r o n microscopy of productively i n f e c t e d H.Ep.2 and BHK -21 c e l l s revealed that HSV was assembled i n the nucleus of i n f e c t e d c e l l s and enveloped at the nuclear membrane. Previous in v e s t i g a t i o n s have demonstrated a s i m i l a r course of development i n a number of mammalian c e l l l i n e s ( 1 3 , 5 3 , 6 3 , 9 9 , 1 0 2 , 1 2 5 ) . Present observations showed that a c t u a l v i r u s assembly was preceded by a l t e r a t i o n s i n the c e l l chromatin and n u c l e o l i that were probably r e l a t e d to the ea r l y virus-induced i n h i b i t i o n of host DNA and RNA synthesis ( 9 9 ) . The a l t e r e d n u c l e i also exhibited a number of dense, granular aggregates p e c u l i a r to HSV-infected c e l l s . S i m ilar aggregates i n KB c e l l s tagged s p e c i f i c a l l y with f e r r i t i n -conjugated HSV antibodies and were thought to be some form of early v i r a l antigen or precursor p a r t i c l e ( 6 4 ) . In t h i s study, naked v i r a l p a r t i c l e s were f i r s t observed i n the nucleus of in f e c t e d c e l l s a f t e r 7 hours of i n f e c t i o n . These scattered p a r t i c l e s were soon supplanted by v i r i o n s with a s i n g l e envelope and large aggregations of v i r a l capsids. In general, the v i r a l assembly process appeared to be highly asynchronous and i n e f f i c i e n t , e s p e c i a l l y during the l a t e r stages of i n f e c t i o n ; The s i t e of HSV envelopment has been previously reported to be the nuclear membrane of i n f e c t e d c e l l s ( 1 3 , 5 3 ) . In the present study, v i r u s p a r t i c l e s were occasionally seen budding through the inner lamella of the nuclear membrane of i n f e c t e d H.Ep.2 and BHK-21 c e l l s . However, t h i s event was r e l a t i v e l y r a r e . More common was the f i n d i n g of extensively p r o l i f e r a t e d membranes extending i n t o the cytoplasm or back i n t o the nuclear matrix. Whether t h i s redup-l i c a t i o n was necessary f o r HSV envelopment or was merely the product of virus-induced a l t e r a t i o n s i n c e l l membranes i s not known. Further-more, the p o s s i b i l i t y of other s i t e s of v i r u s envelopment i n the cytoplasm could not be e n t i r e l y r u l e d out. Epstein (19) demonstrated HSV budding i n t o cytoplasmic vacuoles i n i n f e c t e d HeLa c e l l s while Siminoff and Menefee (102) observed HSV envelopment i n the v i c i n i t y of the Golgi apparatus. Virus envelopment may thus occur randomly at a number of d i f f e r e n t c e l l membranes, incl u d i n g those of the smooth endoplasmic reticulum and the Golgi system, although recent evidence strongly implicates the nuclear membrane as the primary s i t e . Mature v i r u s p a r t i c l e s 170 nm i n diameter were commonly observed i n membrane-bound vacuoles and tubules i n the cytoplasm of i n f e c t e d c e l l s . These tubules resembled structures described by Schwartz and Roizman (98) i n HSV-infected H.Ep.2 c e l l s . In the previous study, mature HSV p a r t i c l e s appeared to move out of the c e l l v i a a network of f i n e , branching tubules extending from the nuclear membrane to the c e l l surface. However, no evidence of a transport system could be seen i n the present study. V i r i o n s were released from the c e l l s by a form of reverse phagocytosis s i m i l a r to the method of egress described by Darlington and Moss (13). Budding v i r u s was not observed at the c e l l s u r f a c e despite repeated attempts to confirm Epstein's early report of HSV mode of release from HeLa c e l l s (19). The vast arrays of c r y s t a l s and membranous aggregates found i n BHK-21 c e l l s a f t e r 20 hours of i n f e c t i o n were very s i m i l a r to the structures observed by N i i e t a l . (63) i n in f e c t e d FL c e l l s . In both cases, b i z a r r e v i r a l forms were-restricted to c e l l s demonstrating extensive v i r a l r e p l i c a t i o n and degeneration. Moreover, they appeared to be composed to numerous v i r a l components not incorporated i n t o mature p a r t i c l e s . BHK-21 c e l l s c o n s i s t e n t l y gave r i s e to a larger number of morphologically defective viruses and aberrant v i r a l forms than d i d H.Ep.2 c e l l s . Previous growth studies also showed that the hamster c e l l s produced lower t i t r e s of i n f e c t i o u s v i r u s . Thus, i t would appear that i n e f f i c i e n c i e s i n assembly and production of v i r a l com-ponents give r i s e to the reduced y i e l d s of HSV found i n BHK-21 c e l l s . The present study also revealed that the addi t i o n of 10 yg/ml ara-C at the time of i n f e c t i o n prevented the formation of i n f e c t i o u s p a r t i c l e s i n H.Ep.2 and BHK-21 c e l l s . However, the drug d i d not prevent the ea r l y chromatin displacement or the production of granular aggregates i n the nucleus of c e l l s i n f e c t e d with HSV. These r e s u l t s confirm previous biochemical studies concerning the e f f e c t of ara-C on HSV r e p l i c a t i o n (9,46). In v i r u s - i n f e c t e d c e l l s , ara-C appears to permit formation of HSV s t r u c t u r a l units and antigens but completely i n h i b i t s p a r t i c l e assembly i n v i t r o . IDU s i m i l a r l y f a i l e d to prevent nuclear disorganization and precursor synthesis i n HSV-infected H.Ep.2 and BHK-21 c e l l s . How-ever, ragged, morphologically abnormal p a r t i c l e s were oc c a s i o n a l l y detected i n the cytoplasm of treated c e l l s . Smith (104) reported the presence of s i m i l a r d efective p a r t i c l e s i n HSV-infected c e l l s treated with IDU. Biochemical studies have also shown that IDU i s d i r e c t l y incorporated i n t o HSV DNA and that defective l a t e proteins are subsequently produced (37). As a r e s u l t , v i r u s assembly i s severely impaired, and although a few abnormal p a r t i c l e s are produced, no i n f e c t i o u s v i r u s can be detected. In contrast, HSV-infected c e l l s treated with another halogenated pyrimidine, BudR, produced v i r u s - s p e c i f i c antigens but no p a r t i c l e s of any d e s c r i p t i o n (102). The c y t o t o x i c i t y of ara-C and IDU observed previously i n t h i s study under the l i g h t microscope was c l o s e l y p a r a l l e l e d i n the t h i n sections of H.Ep.2 and BHK-21 c e l l s . A f t e r 48 hours of drug exposure, c e l l s exhibited marked a l t e r a t i o n s including d i s t o r t i o n of the mito-chondria, swelling of the endoplasmic reticulum and v a c u o l i z a t i o n of the cytoplasm. Thus, the present e l e c t r o n microscope work confirmed and extended previous observations of ara-C and IDU-induced cyto-pathology. HSV i n f e c t i o n r e s u l t e d i n an immediate reduction of thymidine uptake i n t o the DNA of H.Ep.2 and BHK-21 c e l l s . This early i n h i b i t i o n was followed by a sharp increase i n DNA synthesis at 5 hours of i n f e c t -i o n . Similar r e s u l t s have been obtained by Roizman et a l . (86,89) i n H.Ep.2 c e l l s and Russ e l l et a l . (96) i n BHK-21 c e l l s . Previous work has also shown that HSV i n h i b i t s host c e l l DNA synthesis within 2-3 hours and that v i r a l DNA i s f i r s t detected a f t e r 4-5 hours of i n f e c t i o n . V i r a l DNA synthesis i s v i r t u a l l y completed by 14 hours i n most c e l l systems. In the present study, the ea r l y i n h i b i t i o n of host DNA appeared chr o n o l o g i c a l l y r e l a t e d to the nuclear d i s r u p t i o n and chromatin d i s -placement observed i n in f e c t e d c e l l s with the l i g h t and ele c t r o n microscope. Furthermore, the onset of v i r a l DNA synthesis c l o s e l y preceded the appearance of immunofluorescent antigens and naked v i r a l capsids i n the nucleus. Maximum cytopathic e f f e c t s were usually seen a f t e r the bulk of v i r a l DNA had been synthesized i n H.Ep.2 and BHK-21 c e l l s . Ara-C and IDU completely i n h i b i t e d DNA synthesis as measured by 3 H-thymidine uptake i n uninfected and HSV-infected c e l l s (Fig. 30). These r e s u l t s correspond with e a r l i e r i n v i t r o studies by Smith (104) , Smith and Dukes (105), L e v i t t and Becker (46), and Cohen (11). The m i t o t i c index of H.Ep.2 and BHK-21 c e l l s increased sharply a f t e r i n f e c t i o n with HSV, reaching i t s peak at 6 hours and then de-c l i n i n g to a l e v e l of complete i n h i b i t i o n at 24 hours of i n f e c t i o n . 98 In the f i r s t hours of i n f e c t i o n following i n h i b i t i o n of host DNA synthesis, c e l l s already i n mitosis appeared to be prevented from completing the d i v i s i o n cycle and were thus detected i n increasing numbers at metaphase. However, a f t e r i n i t i a t i o n of v i r u s r e p l i c a t i o n and development of cytopathic e f f e c t s , the number of c e l l s i n mitosis decreased u n t i l c e l l d i v i s i o n ceased altogether i n in f e c t e d c u l t u r e s . In contrast, Stoker and Newton (112) reported that mitosis was r a p i d l y i n h i b i t e d i n parasynchronous HeLa cultures i n f e c t e d with HSV 2 hours p r i o r to the cal c u l a t e d time of c e l l d i v i s i o n . Ara-C and IDU d i d not appear to a l t e r the number of HSV-infected c e l l s i n m i t o s i s . M i t o t i c e f f e c t s were therefore an ea r l y v i r a l function independent of ensuing v i r a l DNA synthesis. On the other hand, ara-C and IDU completely i n h i b i t e d mitosis i n uninfected H.Ep.2 and BHK-21 c e l l s within 6-8 hours (Fig. 31). Similar drug-induced e f f e c t s have been observed i n ra b b i t kidney (9) and HeLa c e l l s (43). A number of mammalian viruses are capable of inducing chromosome i r r e g u l a r i t i e s i n cultured c e l l s . The b i o l o g i c a l s i g n i f i c a n c e of these changes i s not yet c l e a r but i s of p o t e n t i a l importance i n c e l l death, carcinogenesis, aging, teratogenesis, and somatic and germ c e l l mutation (60). HSV has been previously reported to cause chromo-some abnormalities i n a v a r i e t y of mammalian c e l l s (7,27,28,34,68, 97,110). In the present study, HSV i n f e c t i o n of H.Ep.2 and BHK-21 c e l l s r e s u l t e d i n v i s i b l e damage to c e l l chromosomes. The v i r u s -induced aberrations included chromatid gaps, breaks, secondary con-s t r i c t i o n s , fragmentation, erosion and endoreduplication. These cytogenetic terms commonly r e f e r to various lesions observed i n stained metaphase preparations of c e l l chromosomes. Thus, chromatid gaps appear as unstained, loosely c o i l e d chromosome segments while breaks co n s t i t u t e actual i n t e r r u p t i o n s of the chromosome that lead to d i s l o c a t e d a c e n t r i c fragments. Secondary c o n s t r i c t i o n s represent p a r t i a l chromosome le s i o n s and fragmentation, a s e r i e s of multiple breakages. The blurred o u t l i n e s of eroded chromosomes are apparently caused by tr a n s i e n t c o i l i n g anomalies and endoreduplication by chromosome r e p l i c a t i o n i n the absence of a spindle. The production of these virus-induced l e s i o n s has been v a r i o u s l y a t t r i b u t e d to i n h i b i t i o n of c e l l DNA synthesis, interference with c e l l p r o t e i n synthesis, enzyme e f f e c t s mediated by disrupted lysozomes, and a d i r e c t combination of v i r a l and c e l l u l a r n u c l e i c a c i d . As yet, however, no one mechanism has been established on the s i n g l e cause of chromosome abnormalities. Any or a l l of the aforementioned les i o n s may occur i n one or both chromatids of a given c e l l chromosome. Chromosomes behave as s i n g l e structures i n the Gl phase of the c e l l c ycle before DNA synthesis has taken place. Therefore, i f a defect i s produced at t h i s time, the l e s i o n i s r e p l i c a t e d along with the second chromatid during the S or DNA synthesis period and the r e s u l t i s a f u l l chromosome abnorm-a l i t y . However, i f the i n j u r y occurs a f t e r the chromosome has synthesized i t s DNA i n the l a t e S or G2 period when the chromosome i s already a dual structure, a s i n g l e chromatid l e s i o n i s the usual r e s u l t . Most of the aberrations observed to date i n v i r u s - i n f e c t e d c e l l s are of the chromatid v a r i e t y , i n d i c a t i n g that the chromosomes are a f f e c t e d i n the l a t e S or G2 phase of the c e l l c y c l e (60). In a d d i t i o n , v i r u s - i n f e c t e d cultures r a r e l y give r i s e to large numbers of exchanges or t r a n s l o c a t i o n s . I f Taylor (115) i s c o r r e c t i n as-suming that host DNA synthesis i s necessary to a reunion of broken chromosomes, many of the v i r u s e s prevent exchanges by i n h i b i t i n g c e l l DNA synthesis involved i n r e p a i r . In the present study, previous observations concerning the e f f e c t of v i r u s dose, type of host c e l l and time of i n f e c t i o n on the amount of HSV-induced damage i n i n f e c t e d c e l l s were e s s e n t i a l l y confirmed. The number of chromosome aberrations i n i n f e c t e d H.Ep.2 and BHK-21 c e l l s increased with time and v i r u s m u l t i p l i c i t y of i n f e c t -i o n . Defects were detected as e a r l y as 2 hours a f t e r i n f e c t i o n and by 20 hours, 100% of the c e l l s observed at metaphase exhibited some type of chromosome abnormality. Similar damage was observed i n human and hamster cultures although the v i r u s apparently produced more ab-errations at an e a r l i e r time i n H.Ep.2 c e l l s . No evidence of s p e c i f i c chromosome a l t e r a t i o n s was seen i n e i t h e r c e l l l i n e despite the f a c t that a number of i n v e s t i g a t o r s have reported non-random lesi o n s i n HSV-infected Chinese hamster c e l l s (110) and mastomys (34) c u l t u r e s . 101 Prolonged UV i r r a d i a t i o n of HSV prevented the induction of a l l aberrations i n infe c t e d c e l l s . Moreover, the capacity of the v i r u s to damage host chromosomes was s i g n i f i c a n t l y more r e s i s t a n t to UV i n a c t i v a t i o n than was the i n f e c t i o u s property. Waubke et al_. (126) reported s i m i l a r r e s u l t s a f t e r UV i r r a d i a t i o n of labeled HSV. Upon furthe r i n v e s t i g a t i o n , they found evidence of impaired v i r u s ad-sorption a f t e r i r r a d i a t i o n although they were not able to conclude that the absence of chromosome damage was e n t i r e l y due to t h i s e f f e c t . Waubke's group also showed that the induction of chromosome lesions preceded and was thus independent of v i r a l DNA r e p l i c a t i o n . Evidence f o r the interference of normal p r o t e i n synthesis as a mechanism f o r induction of chromosome breaks has been obtained from various systems employing d e f i c i e n t media and mycoplasma (60). Mycoplasma-induced breaks i n cultured c e l l s were e f f e c t i v e l y prevented by the ad d i t i o n of excess arginine which i s known to be depleted from in f e c t e d c e l l media. Moreover, a r g i n i n e - d e f i c i e n t media i t s e l f caused chromosome damage i n a number of a c t i v e l y growing c e l l s . Thus, although no evidence of mycoplasma i n f e c t i o n was found i n the H.Ep.2 or BHK-21 c e l l s used i n t h i s research, i t was of i n t e r e s t to determine i f the HSV-induced chromosome abnormalities r e s u l t e d from arginine depletion, since i t i s known that the amino a c i d i s required f o r i n  v i t r o v i r u s maturation (3). However, i n the present study, large increases i n arginine f a i l e d to prevent aberrations i n HSV-infected c e l l s . Thus, i t must be concluded that the virus does not injure c e l l chromosomes by interfering with the metabolism of arginine and the protein synthesis necessary for repair. Ara-C has been previously reported to induce chromosome breakage i n human leukocytes (8) and WI-38 cells (61) as early as 1-3 hours after inoculation. The present study confirms the production of chromatid breaks and secondary constrictions after exposure of H.Ep.2 and BHK-21 c e l l s to various concentrations of ara-C. The amount of chromosome damage was significantly greater than the controls and appeared to have developed i n the late S or G2 phase of the c e l l cycle when the chromosomes behave as dual structures. Since ara-C also acts as an inhibitor of DNA synthesis, the aberrations may have been related to the drug-induced inhibition of repair DNA synthesis i n mammalian cells (60). In HSV-infected cells treated with ara-C, the number of chromo-some abnormalities was far greater than the combined effects of the two potential mutagens. O'Neill and Rapp (69) reported a similar synergism i n HSV-infected human embryonic lung cultures. The drug and virus apparently act together to produce cells containing many multiple breaks. Simultaneous autoradiography performed in this study on uninfected and HSV-infected cells showed that ara-C complete-ly blocked DNA synthesis. Therefore, since ara-C did not prevent the chromosome abnormalities induced by HSV, i t can be concluded that the l e s i o n s occurred i n the absence of v i r a l DNA synthesis. Previous studies have suggested that the antimetabolite and v i r u s act to prevent the r e p a i r of c e l l DNA and thus induce v i s i b l e chromosome damage. In contrast to ara-C treated c u l t u r e s , c e l l s exposed to various concentrations of IDU d i d not generally e x h i b i t a s i g n i f i c a n t increase i n the number of chromosome aberrations except at very high drug l e v e l s . Thus, despite i t s capacity to i n h i b i t c e l l DNA synthesis as 3 measured by H-thymidine uptake, the a n t i - v i r a l drug i s not an ac t i v e agent of chromosome damage. S i m i l a r l y , i n the present study, HSV-infected c e l l s exposed to IDU showed no evidence of synergism. The number of chromosome ab-normalities i n such c e l l s equalled the sum of the lesi o n s produced by HSV and IDU alone. As with ara-C, the virus-induced defects occurred i n the absence of c e l l and v i r u s DNA synthesis. Similar r e -s u l t s were obtained by O ' N e i l l and Rapp (70) i n human embryonic lung c e l l s . The present work has revealed that uninfected and HSV-infected c e l l s treated with a combination of ara-C and IDU showed a simple a d d i t i v e type of chromosome damage. I t thus appeared that the mole-cular i n t e r a c t i o n s responsible f o r the synergism of ara-C and HSV were not present i n the ara-C/IDU system. Although the s i g n i f i c a n c e of aberrations induced by viruses and chemicals i s not known with c e r t a i n t y , there are at l e a s t four areas of actual or p o t e n t i a l importance i n mammalian c e l l s . These are c e l l death, genetic damage that prevents further c e l l d i v i s i o n , changes i n chromosome number, and somatic and germline mutations. Virus-induced c e l l death and m i t o t i c i n h i b i t i o n may be of greatest s i g n i f i c a n c e i n the f i e l d of teratogenesis where i t could lead to f e t a l abnormali-t i e s and spontaneous abortion. Most of the c e l l s i n f e c t e d with HSV are normally expected to cease d i v i d i n g and d i e . These include the small percentage of c e l l s that e x h i b i t v i s i b l e chromosome defects a t a metaphase and the large majority of c e l l s that do not undergo m i t o s i s . Thus, i n l i g h t of the capacity of the v i r u s to cause human g e n i t a l i n f e c t i o n s (57), the mutagenic a c t i o n of HSV could conceiv-ably be involved i n c e l l damage during embryogenesis. Virus-induced aberrations t h e o r e t i c a l l y could also r e s u l t i n changes i n chromosome number or somatic mutations that subsequently might lead to malignancy. HSV has already been implicated i n c e l l transformation (14) and p o s s i b l y i n the production of human c e r v i c a l carcinoma (56,76). Thus, the v i s i b l e and s u b v i s i b l e metaphase ab-normalities induced by the v i r u s may indeed be r e l a t e d to the process of oncogenesis and p o s s i b l y i n t e g r a t i o n and latency. Of even more i n t e r e s t at present, i s the secondary f i n d i n g that the chemotherapeutic agents used to combat HSV i n f e c t i o n induce s i m i l a r chromosome damage and thus also e x h i b i t a d e f i n i t e p o t e n t i a l f o r d i s r u p t i o n of normal c e l l growth and d i v i s i o n . In summary, HSV was found to cause profound metabolic and mor-pho l o g i c a l a l t e r a t i o n s i n i n f e c t e d H.Ep.2 and BHK-21 c e l l s . These a l t e r a t i o n s included an i n h i b i t i o n of host DNA synthesis and mitosis followed by c h a r a c t e r i s t i c nuclear d i s r u p t i o n , formation of i n t r a -nuclear i n c l u s i o n bodies, and production of various antigens and chromosome abnormalities. Ara-C and IDU f a i l e d to prevent the v i r u s -induced cytopathic e f f e c t s i n v i t r o but d i d i n h i b i t v i r a l DNA syn-thesis and assembly of i n f e c t i o u s p a r t i c l e s . Moreover, exposure of the c e l l s to the a n t i - v i r a l agents themselves r e s u l t e d i n severe cyto-pathic a l t e r a t i o n s i n v o l v i n g cytoplasmic disorganization, i n h i b i t i o n of DNA synthesis and mi t o s i s , and induction of various l e v e l s of chromosome damage. BIBLIOGRAPHY Baserga, R. 1965. 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