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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 B r i t i s h 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 t h i s thesis as conforming to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA August, 1972  In p r e s e n t i n g 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 r e q u i r e m e n t s  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, the L i b r a r y  s h a l l make i t  freely available  for  I agree  for  that  r e f e r e n c e and s t u d y .  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s  representatives.  It  of t h i s thesis f o r financial written  i s u n d e r s t o o d t h a t copying o r gain shall  permission.  Department o f  ^h^-A^M^Q  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  L i o Io  Columbia  publication  not be a l l o w e d w i t h o u t my  ABSTRACT  The cytopathology of herpes simplex v i r u s (HSV) i n H.Ep.2 and BHK-21 c e l l s was studied using the techniques of l i g h t microscopy, immunofluorescence, electron microscopy, autoradiography and cytogenetics.  Both c e l l types supported rapid growth cycles of  HSV r e s u l t i n g i n the production of maximum t i t r e s a f t e r 22 - 24 hours of i n f e c t i o n .  Cultures treated with 10 yg/ml ara-C or 100 yg/ml IDU  at the time of i n f e c t i o n showed a 99% decrease i n infectious v i r u s production. HSV-infected H.Ep.2 and BHK-21 c e l l s revealed t y p i c a l v i r u s induced i n c l u s i o n bodies and a generalized disorganization of the nucleus and .cytoplasm.  Syncytia formation was not observed but a f t e r  24 hours of i n f e c t i o n , nearly 100% of the c e l l s were rounded and often detached from the glass surface.  Addition of 10 yg/ml ara-C or  100 yg/ml IDU f a i l e d to prevent v i r u s cytopathology but did cause a c h a r a c t e r i s t i c cytoplasmic disruption and rounding of uninfected c e l l s . Virus-infected c e l l s also revealed a t least four separate immunofluorescent elements a f t e r exposure to hyperimmune serum prepared i n guinea pigs.  These elements included small nuclear granules, amorphous  nuclear masses, d i f f u s e cytoplasmic antigens, and intense fluorescence.  surface  The nuclear antigens and cytoplasmic fluorescence appeared  a f t e r treatment with ara-C or IDU but the surface fluorescence was not  ii  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 n u c l e u s o f 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 a t the i n n e r l a m e l l a o f  the n u c l e a r membrane and a f t e r p a s s i n g i n t o the cytoplasm, were r e l e a s e d from the c e l l s by a p r o c e s s o f r e v e r s e p h a g o c y t o s i s .  Ara-C and  IDU  a l l o w e d the s y n t h e s i s o f c e r t a i n v i r a l a n t i g e n s and the development o f n u c l e a r c y t o p a t h o l o g y b u t c o m p l e t e l y p r e v e n t e d the f o r m a t i o n o f i n f e c t i o u s HSV  particles.  Both drugs caused a marked d i s t o r t i o n of the  m i t o c h o n d r i a and endoplasmic  reticulum i n uninfected c e l l s . 3  DNA  s y n t h e s i s i n H S V - i n f e c t e d c e l l s , as measured by  i n c o r p o r a t i o n , was  H-thymidine  almost c o m p l e t e l y i n h i b i t e d by 4 hours o f i n f e c t i o n .  T h i s e a r l y i n h i b i t i o n o f c e l l u l a r DNA  s y n t h e s i s was  f o l l o w e d by  an  3  immediate i n c r e a s e i n o f v i r a l DNA.  H-thymidine uptake c o r r e s p o n d i n g t o t h e s y n t h e s i s  Both c e l l types showed a b r i e f s t i m u l a t i o n of m i t o s i s  p r i o r t o t h e complete  i n h i b i t i o n observed a f t e r 20 hours o f i n f e c t i o n .  C e l l u l a r and v i r a l DNA  s y n t h e s i s and m i t o s i s appeared  t o be  inhibited  i n v i r u s - i n f e c t e d and u n i n f e c t e d c e l l s t r e a t e d w i t h ara-C o r I n f e c t i o n w i t h HSV and BHK-21 c e l l s .  IDU.  r e s u l t e d i n severe chromosomal damage t o H.Ep.2  Chromosomal a b n o r m a l i t i e s i n c l u d e d chromatid gaps  and b r e a k s , enhanced secondary c o n s t r i c t i o n s , f r a g m e n t a t i o n , e r o s i o n , and e n d o r e d u p l i c a t i o n , and were dependent on v i r u s dose and time o f infection.  The c a p a c i t y of the v i r u s t o induce chromosomal a b e r r a t i o n s  i n c u l t u r e d c e l l s was  U V - i n a c t i v a t e d a p p r o x i m a t e l y f i v e times  less  r a p i d l y than t h e i n f e c t i o u s p r o p e r t y .  Ara-C a c t e d  synergistically  w i t h t h e v i r u s t o produce a l a r g e number o f c e l l s w i t h  multiple  chromosome breaks and a l s o caused a s i g n i f i c a n t number o f a b n o r m a l i t i e s i n uninfected c e l l s .  I n c o n t r a s t , IDU treatment r e s u l t e d i n few  a b e r r a t i o n s over and above those produced by HSV and l i t t l e damage i n uninfected c e l l s . ducing and  I t was c o n c l u d e d t h a t HSV was c a p a b l e o f p r o -  severe morphological  hamster c e l l s .  completely  and g e n e t i c a l t e r a t i o n s i n c u l t u r e d human  The a n t i v i r a l agents ara-C and IDU were a b l e t o  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 b u t were unable t o p r e v e n t any  of the v i r u s - i n d u c e d c y t o p a t h i c e f f e c t s i n v i t r o .  iv  ABBREVIATIONS  ara-C  Cytosine arabinoside  (1-B-D a r a b i n o f u r a n o s y l  cytosine) BHK-21  Baby hamster kidney  l i n e 21 (clone 1 3 ) .  Derived  from a p r i m a r y S y r i a n hamster k i d n e y c u l t u r e . DNA  Deoxyribonucleic  HBSS  Hanks' b a l a n c e d  H. Ep.2  Human epidermoid  acid salt solution l i n e #2.  D e r i v e d from a carcinoma  of the larynx. IDU  5-iodo-2'-deoxyuridine  I . U.  international unit  MEM  M i n i m a l e s s e n t i a l medium  mg  milligram  nm  nanometer  pfu  plaque forming  pH  l o g a r i t h m o f t h e r e c i p r o c a l o f t h e hydrogen i o n  (Eagle)  unit  concentration rpm TCID  r e v o l u t i o n s p e r minute 5 Q  I n f e c t i o u s dose d e s t r o y i n g 50% o f t h e t i s s u e cultures tested  yg  microgram  V  TABLE OF CONTENTS  Page  INTRODUCTION  1  LITERATURE REVIEW  3  Herpes simplex v i r u s  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  C e l l s and Medium  24  Virus  25  1.  25  Origin  2. Virus Preparation and P u r i f i c a t i o n  25  Virus Assay  26  1.  End-point D i l u t i o n Technique  26  2.  Plaque Assay  26  Chemicals and Radioisotopes  27  UV Inactivation of HSV  28  In V i t r o Infection Procedure  28  vi  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.  E f f e c t of ara-C and IDU on HSV Cytopathology  37  3.  E f f e c t of ara-C and IDU on Uninfected C e l l s  40  Fluorescent Antibody Studies  40  1.  HSV Antigen Production  40  2.  E f f e c t of ara-C and IDU on HSV Antigen Production  42  Electron Microscopy  42  1.  Negative Staining  42  2-  Thin Sectioning  45  vii  TABLE OF CONTENTS  (continued)  Page  (a)  Uninfected H.Ep.2 and BHK-21 C e l l s  (b)  Uninfected BHK-21 C e l l s : Abnormal P a r t i c l e  45  Formation  45  (c)  HSV Development i n H.Ep.2 and BHK-21 C e l l s  48  (d)  E f f e c t of ara-C and IDU on HSV Development  56  (e)  E f f e c t of ara-C and IDU on Uninfected C e l l s  58  Autoradiographic Studies  60  1.  DNA Synthesis i n HSV-Infected  Cells  2.  DNA Synthesis i n C e l l s Treated with ara-C and IDU  60 63  Cytogenetic Studies  63  1.  Mitotic Rates  63  2.  Normal C e l l Karyotypes  65  3.  HSV-Induced Chromosome Abnormalities  65  4.  E f f e c t of M u l t i p l i c i t y of Infection on HSV-Induced Chromosome Abnormalities  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  6.  76  E f f e c t of Arginine Excess on HSV-Induced Chromosome Abnormalities  7.  69  76  E f f e c t of ara-C on the Chromosomes of Uninfected and HSV-Infected C e l l s  78  viii  TABLE OF CONTENTS (continued)  Page  8.  E f f e c t o f IDU on t h e Chromosomes o f U n i n f e c t e d and HSV-Infected C e l l s  9.  82  E f f e c t o f ara-C and IDU on the Chromosomes o f U n i n f e c t e d and HSV-Infected C e l l s  DISCUSSION BIBLIOGRAPHY  84 86 106  LIST OF FIGURES  Page  Figure  1.  Representative  growth curves  o f HSV i n  H.Ep.2 and BHK-21 c e l l s c u l t u r e o f H.Ep.2 c e l l s  36  Figure  2.  Uninfected  (X1750)  Figure  3.  H.Ep.2 c u l t u r e 1 2 hours a f t e r HSV i n f e c t i o n  38  (X4400)  38  Figure  4.  Uninfected  Figure  5.  Figure  6.  H.Ep.2 c u l t u r e 24 hours a f t e r HSV i n f e c t i o n (X440) H.Ep.2 c u l t u r e a f t e r 72 hours o f ara-C t r e a t ment (X440)  Figure  Figure  Figure  7.  8.  9.  F i g u r e 10.  F i g u r e 11.  F i g u r e 12.  F i g u r e 13.  F i g u r e 14.  c u l t u r e o f H.Ep.2 c e l l s  (X440)  H.Ep.2 c u l t u r e a f t e r 72 hours o f ara-C (X1750)  39  39 41  treatment 41  F l u o r e s c e n t a n t i b o d y study o f 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) F l u o r e s c e n t a n t i b o d y study o f H.Ep.2 c e l l s a f t e r HSV i n f e c t i o n (X4400)  43  7 hours 43  F l u o r e s c e n t a n t i b o d y study o f 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  E l e c t r o n micrograph o f a negative a r a t i o n o f HSV (X90,700)  s t a i n prep46  E l e c t r o n micrograph of a negative a r a t i o n o f HSV (X90,700)  s t a i n prep46  E l e c t r o n m i c r o g r a p h o f normal H.Ep.2 c e l l s showing i n t a c t n u c l e a r and c y t o p l a s m i c s t r u c t u r e (X10,000)  47  E l e c t r o n m i c r o g r a p h o f a normal BHK-21 c e l l showing i n t a c t n u c l e a r and c y t o p l a s m i c s t r u c t u r e (X42,600)  47  X  LIST OF FIGURES  (continued)  Page  F i g u r e 15.  F i g u r e 16.  F i g u r e 17.  F i g u r e 18.  F i g u r e 19.  F i g u r e 20.  F i g u r e 21.  F i g u r e 22.  F i g u r e 23.  F i g u r e 24.  F i g u r e 25.  F i g u r e 26.  E l e c t r o n m i c r o g r a p h o f an u n i n f e c t e d BHK-21 c e l l m a i n t a i n e d i n serumless medium (X55,800)  49  An abnormal p a r t i c l e i n t h e cytoplasm o f an u n i n f e c t e d BHK-21 c e l l m a i n t a i n e d i n serumless medium (X160,000)  49  E l e c t r o n m i c r o g r a p h o f 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  R e d u p l i c a t e d n u c l e a r 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  Immature v i r u s p a r t i c l e s i n t h e n u c l e u s o f 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  Immature v i r u s p a r t i c l e budding through t h e n u c l e a r membrane o f 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  Mature v i r u s p a r t i c l e s near a b r a n c h i n g t u b u l e i n t h e c y t o p l a s m o f 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  R e l e a s e o f 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  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  I n t r a n u c l e a r 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  C y t o p l a s m i c 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  I n t r a n u c l e a r g r a n u l e s 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  F i g u r e 27.  C y t o p l a s m i c 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  F i g u r e 28.  M i t o c h o n d r i a o f a BHK-21 c e l l a f t e r 24 hours o f ara-C treatment (X69,200)  61  A BHK-21 c e l l a f t e r 48 hours o f ara-C treatment (X44,500)  61  DNA s y n t h e s i s i n v i r u s - i n f e c t e d t r e a t e d BHK-21 c e l l s  62  F i g u r e 29.  F i g u r e 30.  F i g u r e 31.  and c h e m i c a l l y  M i t o t i c r a t e s o f BHK-21 c e l l s f o l l o w i n g v i r u s i n f e c t i o n and c h e m i c a l treatment  64  F i g u r e 32.  Karyotype o f a normal H.Ep.2 c e l l  (X4400)  66  F i g u r e 33.  Karyotype o f a normal BHK-21 c e l l  (X4400)  66  F i g u r e 34.  E f f e c t o f HSV i n f e c t i o n on t h e chromosomes o f H.Ep.2 and BHK-21 c e l l s Chromosome complement o f 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)  F i g u r e 35.  F i g u r e 36.  F i g u r e 37.  F i g u r e 38.  F i g u r e 39.  68 70  Chromosome complement o f 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  Chromosome complement o f 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  Chromosome complement o f a BHK-21 c e l l showing complete f r a g m e n t a t i o n a f t e r 10 hours o f HSV i n f e c t i o n (X4400)  71  E r o s i o n o f a BHK-21 complement a f t e r 10 hours o f HSV i n f e c t i o n (X4400)  72  xii  LIST OF FIGURES (continued)  Page  F i g u r e 40.  F i g u r e 41.  F i g u r e 42.  F i g u r e 43.  F i g u r e 44.  E n d o r e d u p l i c a t i o n o f BHK-21 chromosomes 8 hours a f t e r HSV i n f e c t i o n (X4400)  72  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 o f i n f e c t i o n and HSV-induced chromosome a b n o r m a l i t i e s i n BHK-21 c e l l s  75  E f f e c t o f UV i r r a d i a t i o n o f HSV on v i r a l v i t y and c a p a c i t y t o i n d u c e chromosome a b n o r m a l i t i e s i n BHK-21 c e l l s  77  infecti-  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 o f ara-C (X4400)  81  T r a n s l o c a t i o n 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 o f IDU (X4400)  81  xiii  LIST OF TABLES  Page  Table I .  Table I I .  Table I I I .  T a b l e IV.  T a b l e V.  Table VI.  Table V I I .  Frequency d i s t r i b u t i o n o f v a r i o u s l e v e l s o f p l o i d y i n H.Ep.2 and BHK-21 c e l l s  67  An a n a l y s i s o f HSV-induced chromosome a b n o r m a l i t i e s i n H.Ep.2 c e l l s  73  An a n a l y s i s o f HSV-induced chromosome a b n o r m a l i t i e s i n BHK-21 c e l l s  74  E f f e c t o f excess a r g i n i n e on HSV-induced chromosome a b n o r m a l i t i e s i n BHK-21 c e l l s  79  Chromosome a b n o r m a l i t i e s i n H S V - i n f e c t e d and n o n - i n f e c t e d BHK-21 c e l l s t r e a t e d w i t h ara-C  80  Chromosome a b n o r m a l i t i e s i n H S V - i n f e c t e d and n o n - i n f e c t e d BHK-21 c e l l s t r e a t e d w i t h IDU  83  Chromosome a b n o r m a l i t i e s i n H S V - i n f e c t e d and n o n - i n f e c t e d BHK-21 c e l l s t r e a t e d w i t h ara-C and IDU  85  ACKNOWLEDGEMENTS  The a u t h o r wishes t o thank Dr. J.J.R. Campbell, Head o f t h e Department o f M i c r o b i o l o g y , f o r g r a n t i n g t h i s o p p o r t u n i t y f o r study and r e s e a r c h . The guidance o f Dr. J . E . Bismanis who s u p e r v i s e d t h i s work and t h e a d v i c e o f Dr. D.M. McLean and Dr. J.B. Hudson on t h e s i s preparation are greatly appreciated. The a u t h o r i s a l s o i n d e b t e d t o Mrs. T. W a l t e r s f o r a s s i s t a n c e i n t h e f i e l d o f e l e c t r o n m i c r o s c o p y and t o M i s s J . Bellamy f o r t h e typing of t h i s manuscript.  1  INTRODUCTION  In  the p a s t , herpes simplex v i r u s has m a i n l y been o f i n t e r e s t  as a common d i s e a s e agent o f man, of  capable of producing  v a r y i n g s e v e r i t y and p e r s i s t e n c e .  infections  However, p r e s e n t s t u d i e s  appear t o be d i r e c t e d toward b i o c h e m i c a l l y and c y t o l o g i c a l l y f i n i n g the v i r u s - h o s t r e l a t i o n s h i p i n terms o f p o t e n t i a l and o n c o g e n e s i s .  de-  mutagenesis  T h i s concern a r o s e a f t e r a number o f m o r p h o l o g i c a l l y  s i m i l a r h e r p e s - t y p e v i r u s e s were i m p l i c a t e d i n c e l l t r a n s f o r m a t i o n and tumor p r o d u c t i o n i n d i f f e r e n t a n i m a l s . simplex v i r u s type 2 was  S h o r t l y a f t e r w a r d , herpes  e p i d e m i o l o g i c a l l y a s s o c i a t e d w i t h the o c c u r -  rence o f human c e r v i c a l carcinoma and f u r t h e r i m p l i c a t e d i n mammalian c e l l transformation i n v i t r o .  As a r e s u l t o f these i n t r i g u i n g  reports,  p r e s e n t r e s e a r c h has f o c u s e d on v i r u s - c e l l i n t e r a c t i o n s a t a l l l e v e l s of  i n f e c t i o n and a t the same time i s concerned w i t h the development  and examination o f e f f e c t i v e a n t i - v i r a l a g e n t s .  For these reasons,  t h e purposes o f t h i s study a r e t h r e e f o l d : 1.  To i n v e s t i g a t e the c y t o p a t h o l o g y o f herpes simplex v i r u s type 1 i n c u l t u r e d human and hamster c e l l s w i t h the a i d o f l i g h t and e l e c t r o n m i c r o s c o p y , immunofluorescence,  auto-  r a d i o g r a p h y , and c y t o g e n e t i c s . 2.  To examine the e f f e c t s o f 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 c y t o -  pathology  i n t h e same human and hamster c e l l  lines.  To o b s e r v e and measure t h e b i o c h e m i c a l and c y t o l o g i c a l e f f e c t s o f IDU and ara-C on normal, u n i n f e c t e d 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 d i f f e r e n t skin diseases including eczema, skin cancer, erysipelas and herpes zoster  (shingles).  As l a t e as the nineteenth  century, medical texts s t i l l referred to many spreading, u l c e r a t i v e lesions as herpes, but the term soon became r e s t r i c t e d to c e r t a i n vesicular eruptions of the skin and mucous membranes.  By 1900, f a c i a l  herpes, l a b i a l herpes, ocular herpes and g e n i t a l 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 k e r a t i t i s to  the cornea of a healthy r a b b i t and l a t e r reversed h i s procedure by transmitting  experimentally induced ocular herpes to the cornea of  a b l i n d 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 c h a r a c t e r i s t i c k e r a t i t i s i n rabbits.  In 1921, examination of herpes-infected  to the discovery  corneal c e l l s led  of t y p i c a l large intranuclear i n c l u s i o n bodies.  Later experiments confirmed that a single washed i n c l u s i o n could successfully i n i t i a t e a herpes i n f e c t i o n .  Interest i n the herpes  4  simplex v i r u s was  subsequently spurred by the report of herpetic  encephalitis i n rabbits and man,  and by Burnet and Williams'  s c r i p t i o n of v i r a l persistence i n humans i n 1939  de-  (reviewed i n 31).  Early characterization of the v i r u s 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). v i t y was  Infecti-  l o s t upon exposure to various detergents and electron  microscopy i n 1954  established the v i r u s as a large enveloped  p a r t i c l e with a dense inner nucleoid  (52).  At present, herpes simplex i s s t i l l of medical i n t e r e s t as a major cause of blindness i n man  but i s perhaps of even more  import as a disease model of latency and persistence.  Recently,  the g e n i t a l s t r a i n s of herpes simplex v i r u s have also been epidemiologically associated with human c e r v i c a l carcinoma and the v i r u s i s now the object of intensive s c i e n t i f i c i n v e s t i g a t i o n (56,57,76).  2.  Classification Herpesviruses  are formally defined as large enveloped v i r i o n s  with an icosahedral capsid of 162 capsomeres arranged around a core.  DNA  Included i n t h i s major group are herpes simplex v i r u s , pseudo-  rabies v i r u s , v a r i c e l l a - z o s t e r v i r u s , the cytomegaloviruses,  B  v i r u s , marmoset v i r u s and various equine, bovine, canine, avian and f e l i n e herpesviruses  (36,49).  Future members may  include the Epstein-  5  B a r r 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 d i s e a s e agent o f f o w l , and the l e o p a r d f r o g v i r u s i s o l a t e d Lucke's  adenocarcinoma  (24,59,85).  from  At present, herpesviruses are  e i t h e r d e s i g n a t e d 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 , p s e u d o r a b i e s v i r u s ) o r a r e c l a s s i f i e d a c c o r d i n g t o Andrew's b i n o m i a l system  ( i . e . Herpesvirus hominis,  Herpesvirus  suis). The herpes simplex v i r u s e s have a l s o been regrouped t y p e s o r subtypes on the b a s i s o f immunological  into  two  and b i o c h e m i c a l  d i f f e r e n c e s r e c e n t l y d e t e c t e d 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 a r e g e n e r a l l y from the mouth, eye and b r a i n w h i l e t h e t y p e 2 s t r a i n s a r e d e r i v e d from the perineum and g e n i t a l i a .  Apart  from a n t i g e n i c d i f f e r e n c e s , type 1 v i r u s e s are l e s s s e n s i t i v e t o h e a t , produce h i g h e r t i t r e s i n r a b b i t k i d n e y c e l l s , and p o s s e s s a lower p l a q u e forming u n i t r a t i o t h a n type 2 v i r u s e s .  particle:  On the o t h e r hand,  t y p e 2 s t r a i n s a r e c a p a b l e o f p r o d u c i n g p l a q u e s on c h i c k embryo c e l l s , have a h i g h e r buoyant d e n s i t y and guanine p l u s c y t o s i n e c o n t e n t , and a r e more n e u r o t r o p i c i n mice  3.  (20,23,41,72).  S t r u c t u r e , C o m p o s i t i o n and P h y s i c a l P r o p e r t i e s . The mature herpes simplex v i r i o n i s a l a r g e enveloped  150  - 170 ran i n diameter.  I t appears  particle  t o c o n s i s t o f a dense c o r e ,  6  t h r e e c o n c e n t r i c s h e l l s o r c a p s i d s , and  two  o u t e r envelopes  These t h r e e main a r c h i t e c t u r a l u n i t s have been c o n c u r r e n t l y w i t h the e l e c t r o n m i c r o s c o p e and  the u l t r a c e n t r i f u g e .  r e v e a l e d as a 25 nm p a r t i c l e c o n t a i n i n g DNA;  The  core i s  dense s h e l l s i n t h i n s e c t i o n s o f i n f e c t e d c e l l s ;  the o u t e r  h o l l o w p r o t e i n capsomeres a r r a n g e d i n an  shape p o s s e s s i n g  5:3:2  a x i a l symmetry.  The  be  (92).  o u t e r c a p s i d s appear as e l e c t r o n t r a n s l u c e n t and  c o n s i s t s o f 162  studied  i t i s thought t o  c l o s e l y a s s o c i a t e d w i t h the e l e c t r o n opaque i n n e r c a p s i d m i d d l e and  (88).  The  electron capsid  icosahedral  o u t e r envelopes  are  composed o f v a r i o u s l i p i d s and g l y c o p r o t e i n s , c e r t a i n o f which a r e 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 c o n t a i n s d o u b l e - s t r a n d e d DNA  (4,95) o f  6 molecular  weight 99  ±5  x 10  daltons  t o c o n t a i n s i n g l e - s t r a n d breaks and (41).  (2,41).  N a t i v e DNA  i s l i n e a r and  i s reported  not c r o s s - l i n k e d  No m e t h y l a t e d or u n u s u a l bases are known t o be p r e s e n t .  guanine p l u s c y t o s i n e c o n t e n t viruses  (23).  i s 68%  A f t e r acrylamide  f o r t y p e 1 and  f o r type 2  gel electrophoresis, p a r t i a l l y  f i e d v i r i o n s y i e l d up t o 12 s e p a r a t e a r e g l y c o s y l a t e d and  70%  The  puri-  p r o t e i n bands, a t l e a s t 6 o f which  a s s o c i a t e d w i t h the v i r a l envelope  (106).  I n f e c t i o u s herpes simplex v i r i o n s a r e r e l a t i v e l y s e n s i t i v e t o h e a t , r a d i a t i o n , and  l i p i d solvents  (36).  a t 37°C i n a f i r s t o r d e r r e a c t i o n and  They are r a p i d l y i n a c t i v a t e d  are best stored i n d i s t i l l e d  7  water at -70°C.  S i m i l a r l y , photosensitization, X-ray, and UV i r -  r a d i a t i o n a l l destroy v i r a l a c t i v i t y i n 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, i n f e c t i v i t y and s t r u c t u r a l  i n t e g r i t y are r a p i d l y l o s t a f t e r prolonged centrifugation i n CsCl, where the v i r u s generally bands a t a density of 1.255  4.  to 1.280  gm/cc (107).  Growth i n Tissue Culture (a)  Host Range Herpes simplex virus m u l t i p l i e s i n a wide variety of primary  and continuous c e l l s c u l t i v a t e d i n v i t r o .  The virus r e p l i c a t e s i n  most mammalian c e l l l i n e s , chick embryo f i b r o b l a s t s and t o r t o i s e kidney c e l l s  (36).  Rabbit kidney c e l l s are perhaps the most sensi-  t i v e of the c e l l types found to support herpes simplex virus m u l t i plication.  I t has been reported that the virus requires the amino  acid arginine to complete assembly and maturation i n v i t r o (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 i s unaffected by DNA  and  protein i n h i b i t o r s and by changes i n temperature, suggesting that the process does not require energy or active c e l l metabolism.  Heparin  8  (30) and p a r a t h y r o i d hormone (80)  i n h i b i t s v i r u s adsorption while  t h y r o i d hormone (81) s t i m u l a t e s the  process.  Using p u r e l y p h y s i c a l techniques,  Huang and Wagner r e v e a l e d t h a t  p e n e t r a t i o n o f herpes simplex v i r u s was minutes o f a d s o r p t i o n . dependent and  They found  90%  the process  energy r e q u i r i n g (33).  temperature  r e s u l t s have been c o n f u s i n g  F o r example, w h i l e one major r e p o r t concluded  t r a t i o n was  e f f e c t e d by p i n o c y t o s i s o f v i r a l p a r t i c l e s  e q u a l l y e x t e n s i v e study found  t h a t pene(32) , another  t h a t v i r u s f u s i o n w i t h the c e l l membrane  p r o v i d e d the o n l y means o f e n t r y a r i s e from the use o f extremely 1000  t o be  10  P e n e t r a t i o n has a l s o been  s t u d i e d by e l e c t r o n m i c r o s c o p y , a l t h o u g h at best.  complete w i t h i n  (50).  Such c o n f l i c t i n g d a t a  probably  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 .  p a r t i c l e s p e r c e l l ) w i t h p a r t i c l e : plaque forming u n i t r a t i o s o f  10 o r g r e a t e r .  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 t h a t over 90%  the v i r u s e s observed  i n t h i n s e c t i o n s may  n o t be g o i n g  i n f e c t i o u s p r o c e s s d u r i n g e n t r y and u n c o a t i n g . t h a t uncoated DNA p e n e t r a t i o n and  Viral (i)  through an  I t i s known, however,  appears i n the n u c l e u s w i t h i n 30 minutes o f v i r u s  t h a t e x i s t i n g c e l l enzymes a p p a r e n t l y u n c o a t the v i r u s  i n the cytoplasm (c)  of  (33). Synthesis  DNA,  RNA  and P r o t e i n S y n t h e s i s  V i r u s - s p e c i f i c DNA  i s d e t e c t e d as e a r l y as 3 hours p o s t  i n f e c t i o n i n the nucleus o f i n f e c t e d H.Ep.2 c e l l s .  DNA  synthesis  9  reaches a maximum a t about 7 hours and t h e r e a f t e r e i t h e r d e c l i n e s or remains c o n s t a n t  throughout t h e r e p l i c a t i o n c y c l e (85, 8 9 ) .  Experiments w i t h puromycin have r e v e a l e d t h a t p r o t e i n s y n t h e s i s i s a d e f i n i t e r e q u i r e m e n t f o r i n i t i a t i o n o f v i r a l DNA s y n t h e s i s ( 8 6 ) . V i r u s - s p e c i f i c RNA i s a l s o d e t e c t a b l e a t 3-4 hours and r e a c h e s a maximum a t 8 hours, p a r a l l e l i n g t h e course o f DNA s y n t h e s i s ( 2 1 ) . The  n u c l e a r RNA i s heterogenous i n s i z e and c o n t a i n s a t l e a s t one  species with a sedimentation not appear i n t h e c y t o p l a s m i c  c o e f f i c i e n t g r e a t e r t h a n 80s t h a t does RNA p r o f i l e .  T h i s and o t h e r  suggests t h a t n u c l e a r RNA i s c l e a v e d i n t o p i e c e s o f lower  evidence molecular  weight w i t h i n 10-15 minutes o f i t s s y n t h e s i s and t r a n s p o r t e d i n t o the cytoplasm  of the i n 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 , t h e c y t o p l a s m i c p o l y r i b o s o m e s r a p i d l y d i s a g g r e g a t e and v i r u s - s p e c i f i c polysomes c o n t a i n i n g RNA o r i g i n a l l y s y n t h e s i z e d i n t h e nucleus  reform  i n their place  (113).  I n a r e c e n t paper on herpes  simplex  v i r u s RNA s y n t h e s i s , Wagner a l s o r e p o r t e d sequence d i f f e r e n c e s  i n " e a r l y " and " l a t e " RNAs from b o t h t h e nucleus thus p r o v i d i n g t h e f i r s t evidence v i r a l genome The  and cytoplasm,  o f t r a n s c r i p t i o n a l c o n t r o l of the  (120).  b u l k o f v i r u s - s p e c i f i c p r o t e i n s y n t h e s i s o c c u r s w i t h i n 4-6  hours p o s t i n f e c t i o n i n the cytoplasm l e a s t 9-11 d i f f e r e n t p o l y p e p t i d e s  of infected cells  (90).  At  a r e s y n t h e s i z e d and i n c o r p o r a t e d  i n t o the v i r i o n s , a l t h o u g h i t i s not y e t known whether a l l o f t h e s e a r e coded f o r by the v i r u s genome (66). p r o t e i n s i s asynchronous  structural  and t h e i r t r a n s p o r t i n t o the nucleus  and r e l a t i v e l y s e l e c t i v e After infection  Synthesis of these  slow  (67).  o f H.Ep.2 c e l l s , Spear and Roizman found 25  s p e c i f i c p r o t e i n s r a n g i n g i n m o l e c u l a r weight  virus-  from 25,000 t o 275,000.  Of the p r o t e i n s i n c o r p o r a t e d i n t o the v i r i o n s , two c o n t a i n e d  lipid  and a t l e a s t s i x were g l y c o s y l a t e d and a s s o c i a t e d w i t h the v i r a l envelopes and c e l l membranes.  These s i x g l y c o p r o t e i n s v a r i e d i n s i z e  and s t r u c t u r e w i t h the p a r t i c u l a r v i r u s s t r a i n used, s u g g e s t i n g t h e i r s y n t h e s i s was (ii)  i n part virus-directed  (106).  Enzyme P r o d u c t i o n Herpes simplex v i r u s causes a marked i n c r e a s e i n t h e  a c t i v i t i e s o f s e v e r a l c e l l enzymes w i t h concomitant changes i n t h e i r immunological  and b i o c h e m i c a l p r o p e r t i e s .  E v i d e n c e s u p p o r t s the  c o n t e n t i o n t h a t v i r u s - s p e c i f i c thymidine k i n a s e (44), DNA (42)  and a l k a l i n e DNA  exonuclease  (54)  a r e a l l produced  polymerase  i n infected  c e l l s , a l t h o u g h c o n c l u s i v e p r o o f awaits more r i g o r o u s examination o f the v i r u s - h o s t (iii)  system.  V i r u s - S p e c i f i c Antigens Immunofluorescent s t u d i e s have r e v e a l e d the  o f f i v e s e p a r a t e a n t i g e n i c elements cells  (22,45,91,94,124).  i n herpes  simplex v i r u s  presence infected  U s i n g hyperimmune serum p r e p a r e d i n r a b b i t s  from unheated i n f e c t e d c e l l d e b r i s , Roizman e t a l . d e t e c t e d t h e p r e s e n c e o f s m a l l and l a r g e n u c l e a r g r a n u l e s , amorphous n u c l e a r patches,  c y t o p l a s m i c g r a n u l e s and d i f f u s e c y t o p l a s m i c  i n H.Ep.2 c e l l s i n f e c t e d w i t h t h e v i r u s . d i f f u s e s t a i n i n g represented  They concluded  that the  e a r l y development o f n u c l e o c a p s i d s  and v i r i o n s w h i l e t h e aggregates o r g r a n u l e s found corresponded  fluorescence  late i n infection  t o l a r g e v i r u s " f a c t o r i e s " o r c r y s t a l formations (91).  Roane and Roizman a l s o d i s c o v e r e d t h a t t h e type o f a n t i s e r u m determines t h e number and k i n d o f v i r u s - s p e c i f i c a n t i g e n i c elements found  i n infected c e l l s .  Thus, human c o n v a l e s c e n t  serum produces  o n l y c y t o p l a s m i c and p e r i n u c l e a r s t a i n i n g , and hyperimmune serum prepared  i n l a b o r a t o r y animals  from b o i l e d i n f e c t e d c e l l  reveals only s p e c i f i c nuclear fluorescence  (78).  debris  I n a l l c a s e s , how-  ever, a n t i g e n p r o d u c t i o n c a n be d e t e c t e d as e a r l y as 3 hours a f t e r i n f e c t i o n and i n v o l v e s n e a r l y t h e e n t i r e c e l l p o p u l a t i o n by 24 hours. (d)  V i r u s Assembly and Release The  s i t e o f herpes simplex  of t h e i n f e c t e d c e l l .  v i r u s assembly i s t h e nucleus  C s C l c e n t r i f u g a t i o n s t u d i e s and e l e c t r o n  m i c r o s c o p y have suggested  t h a t a s m a l l c o r e p a r t i c l e c o n t a i n i n g DNA  and p r o t e i n a c t s as t h e v i r a l p r e c u r s o r  (88).  I n the n u c l e u s , t h e  c a p s i d s u b u n i t s and i n n e r envelope a r e assembled about t h e dense c o r e s i n a h i g h l y asynchronous manner, p r o d u c i n g  p a r t i c l e s i n various  s t a g e s o f assembly throughout i s c o n s i d e r e d complete by budding  the r e p l i c a t i o n c y c l e .  Maturation  when the v i r u s a c q u i r e s i t s o u t e r  envelope  through the i n n e r l a m e l l a o f the n u c l e a r membrane  (13,  53).  However, some p a r t i c l e s may  final  s t e p , i n which case envelopment u s u a l l y o c c u r s i n the c y t o -  p l a s m i c membrane system.  r e a c h the cytoplasm b e f o r e t h i s  Once i n the c e l l cytoplasm, s i n g l y  doubly enveloped v i r i o n s , which a r e p r o b a b l y both i n f e c t i v e t e n d t o aggregate  near and around v a c u o l e s and t u b u l e s  F o r many y e a r s , i t was was  r e l e a s e d by budding  and (107),  (13,98).  thought t h a t mature herpes simplex  through the c e l l membrane i n a  p r o c e s s t h a t d i d not l y s e the i n f e c t e d c e l l  (19).  virus  continuous  However, more  r e c e n t s t u d i e s have i n d i c a t e d t h a t a network o f c y t o p l a s m i c t u b u l e s t r a n s p o r t t h e v i r i o n s o u t o f the c e l l  (98) .  C o n t r o v e r s y over  the  method o f egress i s s t i l l v e r y apparent and w i l l p r o b a b l y remain until are  5.  the i n h e r e n t d i f f i c u l t i e s  i n thin section electron  so  microscopy  circumvented.  Host C e l l Response (a)  Macromolecular  S y n t h e s i s and  Mitosis  Upon i n f e c t i o n w i t h herpes simplex v i r u s , the i n i t i a l r a t e s of  c e l l DNA,  RNA  and p r o t e i n s y n t h e s i s d e c l i n e .  C e l l DNA  i s r a p i d l y i n h i b i t e d by 2-3 hours o f i n f e c t i o n w h i l e RNA  synthesis and  protein  synthesis  enjoy a g r a d u a l d e c l i n e over the same p e r i o d .  molecular synthesis synthesis  increases  b r i e f l y f o r the peak 4-5  T o t a l macrohours o f  and then d e c r e a s e s i r r e v e r s i b l y u n t i l c e l l death  viral  (25).  In a d d i t i o n , the v i r u s causes a r a p i d i n h i b i t i o n of the m i t o t i c p r o c e s s and c e l l growth  (112).  t o the i n h i b i t i o n o f c e l l DNA  T h i s e a r l y event corresponds i n time synthesis  and. i s p r o b a b l y mediated  by  a s t r u c t u r a l component o f the v i r i o n o r a v i r u s p r o d u c t made soon after infection. (b)  Cytopathology Cells productively  i n f e c t e d w i t h herpes simplex v i r u s  d i s p l a y s e v e r e 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 b e h a v i o r . The f i r s t e v i d e n c e o f t h e s e a l t e r a t i o n s i s u s u a l l y seen i n the i n f e c t e d c e l l nucleus  (55,62).  As e a r l y as 3-4  hours a f t e r i n f e c t i o n ,  the network o f normal c e l l chromatin becomes h i g h l y condensed displaced  t o the p e r i p h e r y o f t h e n u c l e u s .  and g r a d u a l l y positive v i r a l  and  N u c l e o l i a l s o condense  d i s i n t e g r a t e and s h o r t l y a f t e r w a r d s , a l a r g e F e u l g e n i n c l u s i o n body forms  (36).  With t h e development o f  the i n c l u s i o n , the c e l l n u c l e u s becomes g r o s s l y e n l a r g e d and d i s t o r t e d w h i l e the cytoplasm v a c u o l a t e s and s h r i n k s eventually  i n volume.  Infected  cells  round up c o m p l e t e l y and d e t a c h from the c o n t a i n e r s u r f a c e .  A p a r t from such m o r p h o l o g i c a l changes, a l t e r a t i o n s i n the s o c i a l b e h a v i o u r o f c e l l s d i f f e r e n t s t r a i n s may  the v i r u s a l s o (84).  causes  Infection with  r e s u l t i n ( i ) the f o r m a t i o n o f p o l y k a r y o c y t e s ,  (ii)  t h e p r o d u c t i o n o f clumped, rounded c e l l s , o r ( i i i )  t h e forma-  t i o n o f round " b a l l o o n " c e l l s t h a t do n o t aggregate. P o l y k a r y o c y t e s r e s u l t from the f u s i o n o f n e i g h b o u r i n g form s y n c y t i a c o n t a i n i n g up t o 20-30 n u c l e i .  c e l l s to  S t u d i e s on t h e i r  f o r m a t i o n r e v e a l t h a t t h e f u s i o n p r o c e s s can o n l y take p l a c e a f t e r the i n i t i a t i o n o f v i r a l DNA s y n t h e s i s i n i n f e c t e d c e l l s ( 7 9 ) . In a s e r i e s o f papers d e a l i n g w i t h t h e s o c i a l behaviour o f h e r p e s - i n f e c t e d c e l l s , Roizman concluded  t h a t f u s i o n and clumping  were t h e d i r e c t r e s u l t o f v i r u s - i n d u c e d c e l l membrane a l t e r a t i o n s . He observed  t h a t c e l l s a c q u i r e new s u r f a c e a n t i g e n s and t h a t c e l l  membrane c o m p o s i t i o n virus  (84,87,93).  i s changed a f t e r i n f e c t i o n w i t h herpes  simplex  Presumably, t h e s e a l t e r a t i o n s which l e a d t o the  f o r m a t i o n o f aggregates  and p o l y k a r y o c y t e s a i d i n t h e c e l l - t o - c e l l  spread o f t h e v i r u s . The behaviour strain.  type and e x t e n t o f a l l a l t e r a t i o n s i n c e l l morphology and a r e dependent on t h e g e n e t i c c o n s t i t u t i o n o f t h e v i r u s V a r i a n t s t h a t a r i s e f r e q u e n t l y a f t e r prolonged  passage  i n v i t r o have been r e p o r t e d t o d i f f e r markedly from t h e p a r e n t  strain  i n t h e type o f c y t o p a t h i c e f f e c t s produced i n t h e same h o s t c u l t u r e (15).  Moreover, t h e s p e c i e s o f h o s t c e l l and t h e age and c h a r a c t e r  o f t h e monolayer e x e r t a p r o f o u n d Thus, c e l l responses by the p h e n o t y p i c  e f f e c t on v i r u s  cytopathology.  a r e i n f l u e n c e d n o t o n l y by t h e genotype b u t a l s o  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 o f a l a r g e number o f mammalian  v i r u s e s t h a t i n d u c e 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).  came i n 1961 the MCH  The f i r s t e v i d e n c e o f i t s mutagenic p o t e n t i a l  when Hampar and E l l i s o n d i s c o v e r e d t h a t i n f e c t i o n o f  C h i n e s e hamster c e l l l i n e w i t h herpes  simplex v i r u s  i n an i n c r e a s e d i n c i d e n c e o f chromatid b r e a k s , a c c e n t e d 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 . a f t e r the f i r s t c e l l  resulted  secondary  Most o f the a b n o r m a l i t i e s appeared  d i v i s i o n and were p r i m a r i l y l o c a t e d on chromo-  somes 1 and 2 o f the a n e u p l o i d complement  (27,28).  S i n c e t h i s i n i t i a l work, many i n v e s t i g a t o r s have r e p o r t e d s i m i l a r e f f e c t s o f v i r u s i n f e c t i o n i n a v a r i e t y o f mammalian c e l l s i n c l u d i n g d i p l o i d Chinese hamster c e l l s  (110), human embryonic lung  cells  (68,70), human p e r i p h e r a l l e u k o c y t e s (68), r a b b i t k i d n e y  (97),  BHK-21 c e l l s  (126), and monkey k i d n e y c e l l s  save f o r the Chinese hamster c e l l s , to  be random and n o n - s p e c i f i c .  (7).  cells  In a l l cases,  the chromosome a b e r r a t i o n s appear  V i r u s e f f e c t s depend on the h o s t  cell  s p e c i e s and i n c l u d e chromatid and chromosome breaks and gaps, severe f r a g m e n t a t i o n , a c c e n t e d secondary  c o n s t r i c t i o n s , and C - m i t o s i s .  l e s i o n s a r e w i t h o u t e x c e p t i o n p r e v e n t e d by p r o l o n g e d  ultraviolet  i r r a d i a t i o n o f t h e v i r u s o r n e u t r a l i z a t i o n w i t h herpes antibody.  The  simplex  virus  I n a d d i t i o n , damage i s d e t e c t a b l e as e a r l y as 3 hours  after  16  i n f e c t i o n and dose (60,  i s found t o i n c r e a s e w i t h time and  initial  109).  E a r l y s t u d i e s suggested t h a t v i r a l r e p l i c a t i o n was f o r the i n d u c t i o n o f chromosome a b n o r m a l i t i e s . found no evidence  o f v i r a l DNA  thus concluded  v i r i o n component or an e a r l y enzyme was At present,  necessary  However, zur Hausen  s y n t h e s i s i n BHK-21 c e l l s  s e v e r e l y damaged metaphase p l a t e s and  (126).  viral  with  that e i t h e r a  r e s p o n s i b l e f o r the damage  the s p e c i f i c cause o f a l l v i r u s - i n d u c e d chromo-  some a b e r r a t i o n s i s s t i l l unknown. (d)  C e l l Transformation Transformation  and  Oncogenesis  a t the c e l l u l a r l e v e l i s g e n e r a l l y d e f i n e d  as a s t a b l e , i n h e r i t a b l e g e n e t i c 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 p a t t e r n .  Cells trans-  formed by v i r u s e s a r e v e r y o f t e n oncogenic and a r e t h e r e f o r e s t u d i e d i n an e f f o r t t o d e f i n e the causes and c o n d i t i o n s of  carcinogenesis.  To date, t h e r e a r e a t l e a s t f o u r p o s s i b l e h e r p e s - t y p e v i r u s e s i m p l i c a t e d i n the oncogenic p r o c e s s  - the human E p s t e i n - B a r r 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 d i s e a s e agent of c h i c k e n s ,  the l e o p a r d f r o g adenocarcinoma v i r u s , and  v i r u s s a i m i r i which induces  lymphomas i n marmoset monkeys (48,59,116).  With the d i s c o v e r y o f these v i r u s e s and evidence  suggesting  Herpes-  the r e c e n t  epidemiological  a r e l a t i o n s h i p between the o c c u r r e n c e  o f 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 the herpes simplex v i r u s e s have been re-examined and t r a n s f o r m i n g c a p a c i t i e s .  (56,57,76),  for possible  oncogenic  So f a r , the v i r u s has o n l y been i m p l i -  c a t e d as a c o - c a r c i n o g e n i n l i v e animal s t u d i e s  (114).  However,  e a r l y i n 1971, Duff and Rapp r e p o r t e d t h e p r o d u c t i o n o f a hamster embryo f i b r o b l a s t l i n e t r a n s f o r m e d a f t e r exposure i r r a d i a t e d herpes simplex type 2.  to ultraviolet-  T h i r t y days a f t e r v i r u s  infection,  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  a n t i g e n s and was h i g h l y oncogenic i n newborn and weaning (14).  hamsters  I f t h i s p r e l i m i n a r y r e p o r t i s extended and v e r i f i e d by o t h e r s ,  herpes simplex v i r u s may j o i n t h e ranks o f DNA tumor v i r u s e s c a p a b l e of  i n t e r a c t i n g w i t h t h e i r h o s t s t o produce  w e l l as c y t o c i d a l and a b o r t i v e  6.  c e l l t r a n s f o r m a t i o n as  infections.  Pathogenesis (a)  Animals The n a t u r a l h o s t f o r herpes simplex v i r u s i s man.  However,  the v i r u s c a n a l s o be e x p e r i m e n t a l l y t r a n s m i t t e d t o r a b b i t s , g u i n e a p i g s , mice, r a t s , c h i c k embryos, geese and hedgehogs (36) .  Perhaps  the most common e x p e r i m e n t a l animals a r e t h e a d u l t r a b b i t and newborn mouse. C o r n e a l i n o c u l a t i o n o f herpes simplex v i r u s i n r a b b i t s has been  found t o produce a t y p i c a l k e r a t i t i s b e g i n n i n g w i t h the of p i n p o i n t d e n d r i t i c l e s i o n s w i t h i n l e s i o n s r a p i d l y e n l a r g e and a p u r u l e n t exudate and  b r a i n , spleen,  24 hours o f i n f e c t i o n .  invade the c o r n e a l  ultimately, blindness.  d e t e c t e d a f t e r 24 hours and k i d n e y and  appearance  v i r u s can be  stroma, p r o d u c i n g V i r e m i a s can  be  s u c c e s s f u l l y i s o l a t e d from  lung homogenates.  I f the k e r a t i t i s i s  l e f t u n t r e a t e d , the d i s e a s e o f t e n extends to the b r a i n and f a t a l encephalitis  The  causes a  (40).  U s i n g 5-day o l d mice, Wildy found t h a t f o o t p a d i n o c u l a t i o n  re-  s u l t e d i n the r a p i d development o f p o s t e r i o r p a r a l y s i s w i t h o u t e v i d e n c e of v i r e m i a . i n v a s i o n was  He  assumed t h a t the normal r o u t e o f v i r u s  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 t o the  nervous system  (127).  However, i n 1964,  extensive  central  immunofluores-  c e n t s t u d i e s o f murine e n c e p h a l i t i s p r o v i d e d e v i d e n c e o f b o t h a neural  and  hematogenous s p r e a d .  v i r u s e i t h e r produced a v i r e m i a cerebral vessels  serving  the  Johnson found t h a t herpes simplex that allowed i n f e c t i o n of  c e n t r a l nervous system, o r r e s u l t e d  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 the v i t a l c r a n i a l n e r v e s . produced an e x c l u s i v e  small  Intracerebral  d i r e c t neural  (not axons) l e a d i n g  i n o c u l a t i o n o f the  spread w h i l e i n t r a n a s a l  in to  virus 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 o f hematogenous and  neural  invasion  (35).  (b)  Man I n man,  p r i m a r y herpes simplex v i r u s i n f e c t i o n s u s u a l l y  o c c u r between the ages o f 6 months and 5 y e a r s .  I t i s estimated  t h a t l e s s than 10% o f the i n f e c t e d i n d i v i d u a l s ever demonstrate o v e r t d i s e a s e a l t h o u g h the v i r u s i n f e c t i o n can o c c a s i o n a l l y s e v e r e and even f a t a l  (10).  any  be  Primary i n f e c t i o n s can a f f e c t up t o  60 - 100% o f the p o p u l a t i o n , p r o d u c i n g a v a r i e t y o f v e s i c u l a r  lesions.  The c l i n i c a l e x p r e s s i o n s depend t o a l a r g e degree on the p o r t a l o f e n t r y and i n c l u d e g i n g i v o s t o m a t i t i s , k e r a t i t i s ,  vulvovaginitis,  eczema herpeticum and r a r e l y , m e n i n g o - e n c e p h a l i t i s .  I n most c a s e s ,  the i l l n e s s e s a r e r e l a t i v e l y m i l d and s e l f - l i m i t i n g b u t i n newborn and premature i n f a n t s , g e n e r a l i z e d herpes simplex can be fatal  rapidly  (36). Once t h e i n i t i a l v i r u s i n f e c t i o n s u b s i d e s , however, the m a j o r i t y  o f p e o p l e s u f f e r from a r e c u r r e n t form o f herpes simplex t h a t i n v a r i a b l y o c c u r s i n the p r e s e n c e o f c i r c u l a t i n g a n t i b o d y  (10,40,83).  S k i n l e s i o n s t e n d t o r e c u r a t o r near the same s i t e and appear s p e c i f i c p h y s i c a l o r e m o t i o n a l s t i m u l i t h a t i n c l u d e exposure  following  t o sun-  l i g h t , i l l n e s s , f e v e r , m e n s t r u a t i o n , hormone treatment,  administration  o f f o r e i g n p r o t e i n s , s p e c i f i c t y p e s o f n e u r o s u r g e r y and  emotional  stress.  I t i s now  g e n e r a l l y a c c e p t e d t h a t the herpes simplex v i r u s i s  h a r b o r e d i n a l a t e n t o r i n a p p a r e n t form a t 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 h o s t 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 a t v a r i o u s  times and cause the v i r u s t o m a n i f e s t i t s e l f i n t y p i c a l h e r p e t i c lesions  (36).  A t p r e s e n t , t h e r e are two o f herpes simplex  i n man.  t h e o r i e s to e x p l a i n the p e r s i s t e n c e  The f i r s t proposes t h a t v i r u s m u l t i p l i c a -  t i o n o c c u r s a t a slow b u t c o n s t a n t r a t e a t the s i t e o f the r e c u r r e n t lesion.  A c c o r d i n g t o t h i s dynamic s t a t e h y p o t h e s i s , v a r i o u s s t i m u l i  p r o v i d e a temporary p e r m i s s i v e s t a t e i n u n i n f e c t e d c e l l s and b r i e f recurrences of overt disease  (83).  The  thus  allow  second t h e o r y e n v i s i o n s  the e x i s t e n c e i n unknown c e l l s o f a n o n i n f e c t i o u s form o f t h e v i r u s . In t h i s h y p o t h e s i s , 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 i n t e r r u p t e d a t some stage and can be r e a c t i v a t e d by c e r t a i n agents and c o n d i t i o n s of the host  (83).  I n an e f f o r t t o d i s t i n g u i s h between t h e s e two  t h e o r i e s , many  attempts have been made t o i s o l a t e i n f e c t i o u s v i r u s from the h e a l e d s i t e o f l e s i o n s i n the i n t e r i m between r e c u r r e n c e s .  To d a t e ,  v i r u s o r i n f e c t e d c e l l s have been r e c o v e r e d i n t h i s manner. a number o f i n v e s t i g a t o r s have r e p o r t e d the i s o l a t i o n o f v i r u s from the t e a r s and  no However,  infectious  s a l i v a o f a p p a r e n t l y normal i n d i v i d u a l s  as  w e l l as from the s e c r e t o r y g l a n d s and t e a r s o f r a b b i t s w i t h r e c u r r e n t keratitis.(10). may  indeed be due  Thus, the p e r s i s t e n c e o f the herpes simplex  virus  t o a low-grade, c h r o n i c i n f e c t i o n o f the h o s t  envisaged by the dynamic s t a t e h y p o t h e s i s .  as  On the o t h e r hand, an i n a p p a r e n t form o f the v i r u s has a l s o been encountered  i n r a b b i t s with r e c u r r e n t o c u l a r herpes.  Examination  of v a r i o u s neurons i n c l u d i n g the t r i g e m i n a l g a n g l i o n r e v e a l e d no apparent v i r u s i n f e c t i o n between r e c u r r e n c e s .  However, one  to  two  weeks a f t e r organ c u l t u r e s had been e s t a b l i s h e d , the t r i g e m i n a l g a n g l i o n c e l l s produced I n man,  f u l l y i n f e c t i o u s herpes  simplex v i r u s  (108).  s u r g e r y o f the same g a n g l i o n f r e q u e n t l y r e s u l t s i n p o s t o p e r a -  tive herpetic lesions evidence appears  (17).  Thus, the e x p e r i m e n t a l and  clinical  t o suggest t h a t 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 n e u r a l g a n g l i a o f the h o s t . 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 a r e r a t h e r u n u s u a l  i n t h a t c i r c u l a t i n g antibody i s normally present during the course of the r e c u r r e n c e s .  Moreover, t h e r e appears  t o be no change i n the serum  l e v e l s of t h i s antibody e i t h e r before or a f t e r overt i n f e c t i o n T h i s apparent v i r u s spread.  (26).  c o n t r a d i c t i o n i s p r o b a b l y a r e s u l t o f the method o f Herpes simplex v i r u s can spread by d i r e c t  cell-to-cell  c o n t a c t and thus remain u n a f f e c t e d by c i r c u l a t i n g a n t i b o d y  (84).  How-  e v e r , t h e s e l f - l i m i t i n g n a t u r e o f the l e s i o n s s t i l l r e q u i r e s s a t i s f a c t o r y e x p l a n a t i o n from an immunological  p o i n t o f view.  A r e c e n t a n a l y s i s of immunoglobulins i n p a t i e n t s w i t h 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 g r e a t e r amount of IgA i n the b l o o d d u r i n g a l a t e n t p e r i o d than d u r i n g an a c t i v e r e c u r r e n c e  (26).  Further impaired  s t u d i e s showed t h a t p a t i e n t s w i t h p e r s i s t e n t herpes a l s o have macrophage i n h i b i t i o n and  lowered lymphocyte t o x i c i t y  (128).  I n summary, r e c u r r e n t herpes simplex v i r u s i n f e c t i o n s appear t o be e s t a b l i s h e d and m a i n t a i n e d by a complex i n t e r a c t i o n of a v a r i e t y o f h o s t f a c t o r s i n c l u d i n g IgA temperature, i n t e r f e r o n and (c)  serum l e v e l s , c e l l - m e d i a t e d  body hormones  immunity,  (83).  Chemotherapy To date, IDU  and  ara-C have p r o v i d e d  the b e s t 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  t r i a l s , Kaufman found t h a t t o p i c a l a p p l i c a t i o n s of IDU  resulted i n  complete e r a d i c a t i o n of h e r p e t i c k e r a t i t i s i n man Subsequent d o u b l e - b l i n d  and  s t u d i e s c o n f i r m e d t h a t IDU  clinical  animals  had  a  (39).  significant  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 b u t f a i l e d t o reduce the r e c u r r e n c e o r p r e v e n t the development o f deep s t r o m a l the c h e m i c a l had p o r t of c o r n e a l a n i m a l , IDU  lesions.  I n most  i s h i g h l y t o x i c and  (36) .  i s used o n l y i n v e r y  severe  generalized  simplex are o c c a s i o n a l l y t r e a t e d w i t h massive doses o f IDU. m e d i c a l communications r e p o r t an i n c r e a s e d little  i f any  chance of r e c o v e r y  diseases. herpes Most with  d e c r e a s e i n subsequent n e u r o l o g i c a l damage  Moreover, t o x i c s i d e - e f f e c t s such as l e u k o p e n i a  re-  However, i n the i n t a c t  P a t i e n t s w i t h a c u t e n e c r o t i z i n g e n c e p h a l i t i s and  drug but  cases,  no o b v i o u s t o x i c e f f e c t s , a l t h o u g h a t l e a s t one "speckling" i s recorded  rate  and  the (47).  thrombocytopenia  a r e l i k e l y t o o c c u r a f t e r p r o l o n g e d treatment In v i t r o ,  (74).  IDU i s a p o t e n t i n h i b i t o r o f herpes  simplex  virus.  A t l e a s t 99% i n h i b i t i o n o f i n f e c t i o u s v i r u s p r o d u c t i o n i s o b t a i n e d i f 50 yg/ml IDU infection  i s added t o an i n f e c t e d c u l t u r e as l a t e as 4 hours  (9,82,104,105).  The h a l o g e n a t e d p y r i m i d i n e appears  i n c o r p o r a t e d i n t o the newly s y n t h e s i z e d v i r a l DNA thus c a u s i n g d e f e c t i v e assembly o f the v i r i o n s A l t h o u g h r e p e a t e d exposure  i n place of  (117) and herpes  thymidine,  t o IDU r e s u l t s i n the development o f s e n s i t i v e t o the  Ara-C i s e q u a l l y e f f e c t i v e a g a i n s t h e r p e t i c simplex v i r u s r e p l i c a t i o n i n v i t r o  h i g h l y t o x i c t o a l l mammalian c e l l s . i n c u l t u r e and i n h i b i t DNA  t o be  (37).  d r u g - r e s i s t a n t mutants, these v i r u s e s u s u a l l y remain a c t i o n o f ara-C.  after  keratitis  (9,46), b u t i s  Both IDU and ara-C p r e v e n t m i t o s i s  s y n t h e s i s (11,38,43,73,101) w h i l e c a u s i n g  concomitant chromosome a b n o r m a l i t i e s (8,61,69).  As a r e s u l t o f the  adverse e f f e c t s o f these drugs, chemotherapy o f herpes simplex v i r u s i n f e c t i o n s i s g e n e r a l l y 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 o f s y s t e m i c d i s e a s e .  MATERIALS AND METHODS  C e l l s and Medium The H.Ep.2 c o n t i n u o u s l i n e o f human epidermoid c e l l s the BHK-21 ( c l o n e 13) l i n e o f S y r i a n hamster k i d n e y c e l l s o b t a i n e d from M i c r o b i o l o g i c a l A s s o c i a t e s , Bethesda,  Md.  were m a i n t a i n e d as monolayer c u l t u r e s i n m i l k d i l u t i o n L e i g h t o n tubes on E a g l e s ' minimal (Flow Labs., R o c k v i l l e , Md.).  e s s e n t i a l medium  (51) and (111) were The c e l l s  bottles or  (MEM), AutoPow  The medium was r o u t i n e l y  supplemented  w i t h 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), s t r e p t o m y c i n (100 yg/ml), and 0.025% g l u t a m i n e .  C e l l c u l t u r e s were d i v i d e d  r e g u l a r l y every t h r e e days by t h e use o f 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 Suspensions  (HBSS).  o f approximately 1 0  6  c e l l s i n medium w i t h 20% f e t a l  c a l f serum and 10% d i m e t h y l s u l f o x i d e were f r o z e n and s t o r e d a t -70°C to  s e r v e as s t o c k c u l t u r e s .  F r o z e n c e l l s were r e g u l a r l y r e v i v e d  every t h r e e months t o ensure g e n e t i c and morphologic lines. of  E l e c t r o n microscopy  and t r i t i a t e d thymidine  s t a b i l i t y o f the autoradiography  normal H.Ep.2 and BHK-21 c e l l s f a i l e d t o r e v e a l t h e p a r t i c l e s and  abnormal c y t o p l a s m i c l a b e l i n g c h a r a c t e r i s t i c o f mycoplasma i n f e c t i o n (29,58).  Virus  1.  Origin The H4253 herpes simplex v i r u s  Dr. D.M. lesions.  s t r a i n was  i s o l a t e d by  McLean from the t h r o a t o f a p a t i e n t w i t h m u l t i p l e l i p 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 b a s i s o f 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. v i r u s was  (HSV)  passaged  Subsequent t o i t s i s o l a t i o n , the  f i v e times i n mice and a t o t a l o f twelve  i n H.Ep.2 monolayers.  D u r i n g t h i s time, the v i r u s was  p u r i f i e d t h r e e times by T. Mosmann.  times  a l s o plaque-  E l e c t r o n microscopy  revealed  p a r t i c l e morphology t y p i c a l o f the herpes group o f v i r u s e s .  2.  V i r a l Preparation Stock v i r u s was  and P u r i f i c a t i o n p r e p a r e d 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 i n o c u l a t e d  w i t h 1.0  x 10  p f u HSV.  c u l t u r e s were i n c u b a t e d a t 37°C f o r 20 hours i n MEM f e t a l c a l f serum.  The  containing  A t the end o f t h i s time, the medium was  2%  replaced  w i t h an e q u a l volume o f water, the c e l l s s c r a p e d o f f the g l a s s b o t t l e s , and the r e s u l t i n g suspensions f r o z e n and thawed t h r e e t i m e s . l y s a t e s were then c e n t r i f u g e d large c e l l debris  a t 3500 rpm  and the s u p e r n a t a n t s r e c e n t r i f u g e d  Beckman Model L2-65B U l t r a c e n t r i f u g e P e l l e t s containing  f o r 10 minutes  t o remove  a t 4°C i n a  f o r one hour a t 15,000  t h e v i r u s were resuspended  i n MEM  The  w i t h 2%  rpm. fetal  crude  26  c a l f serum and were s t o r e d i n serum b o t t l e s a t -70°C.  Stock  7 5 p r e p a r a t i o n s gave t i t r e s o f 7.2 x 10 pfu/ml o r 5.0 x 10 TCID  /ml.  V i r u s Assays  1.  End-point D i l u t i o n  Technique  F o r rough a s s a y s o f v i r u s p r e p a r a t i o n s , t h e e n d - p o i n t d i l u t i o n method was used t o 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  t o be  t e s t e d 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 p l u s 2% f e t a l c a l f serum. Twenty-four hour H.Ep.2 monolayers grown i n L e i g h t o n tubes were i n o c u l a t e d i n d u p l i c a t e w i t h 0.1 ml o f each d i l u t i o n and i n c u b a t e d a t 37°C f o r 48 h o u r s .  The monolayers were then examined m i c r o s c o p i -  c a l l y f o r c y t o p a t h i c e f f e c t s and graded on a s c a l e o f +1 t o +4. t i t r e s were c a l c u l a t e d by t h e Reed and Meunch method  Virus  (77) and expressed  as TCID,.,. u n i t s . 50  2.  Plaque Assay Q u a n t i t a t i v e p l a q u e a s s a y s were a l s o performed u s i n g  H.Ep.2 monolayers grown i n 35 x 10 mm F a l c o n p l a s t i c p e t r i The c e l l s were m a i n t a i n e d under a 5% C 0 medium.  2  atmosphere  confluent dishes.  i n Dulbecco's  Washed H.Ep.2 monolayers were i n f e c t e d i n d u p l i c a t e w i t h  0.1 ml o f each d i l u t i o n and r o t a t e d 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 i n 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 a t 37°C i n a 5% CO^ atmosphere f o r 3-4 days.  The r e s u l t i n g plaques were stained f o r 3 hours  with a 0.1% neutral red solution and observed under i n d i r e c t l i g h t . The plaques appeared as small heavily stained spots surrounded by clear halos against a l i g h t l y stained background.  Chemicals and Radioisotopes  1.  L-Arginine Hydrochloride was obtained from Sigma Chemicals,  St. Louis, Missouri.  A stock 5% solution i n d i s t i l l e d water was  stored a t 4°C. 2.  Colcemid was donated by the Ciba Co. Ltd., Dorval, Quebec.  Stock 10 yg/ml solutions i n HBSS were stored at 4°C i n a l i g h t t i g h t container and were renewed every three weeks.  A fresh 1.0  yg/ml preparation was made d a i l y from the stock. 3.  Cytosine Arabinoside Hydrochloride was obtained from N u t r i t i o n a l  Biochemicals Corp., Cleveland, Ohio.  Stock solutions of 1000 yg/ml  i n HBSS were stored a t 4°C and were renewed every three weeks. A fresh 10 yg/ml solution was prepared for each experiment.  4.  5 - I o d o d e o x y u r i d i n e , purchased from Calbiochem, Los A n g e l e s ,  C a l i f o r n i a , was k i n d l y s u p p l i e d by D r . D.M. McLean. Stock p r e p a r a t i o n s i n HBSS were s t o r e d a t 4°C and were renewed every t h r e e 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 t h e s t o c k . . .  5.  3  T n t i a t e d t h y m i d i n e ( H-thymidine) , s p e c i f i c a c t i v i t y 49.2  Ci/mM, was o b t a i n e d from New England N u c l e a r C o r p o r a t i o n , Boston, Mass., and g e n e r o u s l y s u p p l i e d by Dr. J.B. Hudson. of  Fresh preparations  the r a d i o i s o t o p e were made i n HBSS and h e l d a t 4°C u n t i l u s e .  UV I n a c t i v a t i o n o f V i r u s The herpes simplex v i r u s was i r r a d i a t e d i n 60 mm F a l c o n p l a s t i c p e t r i d i s h e s (2.0 m l / d i s h ) a t a d i s t a n c e o f 20 cm from a S y l v a n i a G15T8 UV b u l b .  I r r a d i a t i o n was c a r r i e d o u t a t room temperature  w i t h c o n s t a n t a g i t a t i o n f o r p e r i o d s o f time r a n g i n g 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  c u l t u r e s were washed once w i t h HBSS and i n o c u l a t e d  w i t h HSV a t a n i n p u t m u l t i p l i c i t y o f about 1 p f u p e r c e l l .  The i n p u t  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 t h e average number o f c e l l s per  c u l t u r e i n a Levy c o u n t i n g chamber (Max Levy, P h i l a d e l p h i a ,  Penn.).  A f t e r 30 minutes a d s o r p t i o n a t room temperature, t h e mono-  l a y e r s were r i n s e d w i t h HBSS and i n c u b a t e d a t 37°C i n MEM w i t h 10%  29  fetal calf  serum.  L i g h t Microscopy C e l l monolayers grown on L e i g h t o n tube c o v e r s l i p s were i n f e c t e d w i t h 7.2 x 10  p f u HSV,  t r e a t e d w i t h 10 yg/ml ara-C o r 100 yg/ml  or l e f t t o s e r v e as c o n t r o l s .  After  5, 12, 24 and 72 hours, the  c o v e r s l i p s were removed and r i n s e d b r i e f l y i n p h y s i o l o g i c a l F i x a t i o n i n a b s o l u t e methanol  f o r 30 m i n u t e s .  saline.  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 o f 3.0 ml s t o c k Giemsa i n 50 ml b u f f e r pH 7.2  The s t a i n e d  phosphate  c o v e r s l i p s were g i v e n 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 c l e a n g l a s s w i t h Apochromount mounting souri) .  fluid  slides  (Aloe S c i e n t i f i c , S t . L o u i s , M i s -  The c e l l s were examined and photographed u s i n g a  semi-automatic  IDU,  Zeiss  photomicroscope.  I n d i r e c t F l u o r e s c e n t A n t i b o d y Technique Monolayer c u l t u r e s grown on L e i g h t o n tube c o v e r s l i p s were i n f e c t e d w i t h 7.2 x 10  p f u HSV per tube  (MOI = 1 ) .  I n some c a s e s ,  10 yg/ml ara-C o r 100 yg/ml IDU were added t o b o t h i n f e c t e d non-infected c e l l s .  and  A t 4, 7, 12 and 24 hours p o s t i n f e c t i o n , the  c o v e r s l i p s were removed, r i n 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 m i n u t e s .  Each c o v e r s l i p was  exposed t o  30  0. 1 ml of 1:2 d i l u t i o n of guinea p i g anti-HSV gamma globulin (Ti.tre 1:32 Microbiological Associates, Bethesda, Md.) f o r 30 minutes i n a moist chamber a t 37°C.  The coverslips were then rinsed i n saline  and treated with 0.1 ml fluorescein-labeled goat anti-guinea p i g gamma g l o b u l i n (Microbiological Associates, Bethesda, Md.) f o r 30 minutes i n a moist chamber a t 37°C.  The c e l l s were given a f i n a l  rinse i n s a l i n e and mounted on clean glass s l i d e s using phosphatebuffered g l y c e r o l pH 7.2 (Baltimore B i o l o g i c a l 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 i n a room temperature water bath and a drop of the v i r u s suspension placed on a wax staining tray.  A clean carbon  and formvar coated copper g r i d was lowered onto the drop and allowed to remain i n contact with i t f o r one minute.  The g r i d was then  removed to a drop of 2% phosphotungstic acid pH 6.5 and stained for one minute before examination i n a P h i l l i p s EM 300 electron  microscope.  G r i d s were observed w i t h i n 30 minutes  of i n i t i a l  p r e p a r a t i o n i n o r d e r t o p r e s e r v e 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. T h i n - S e c t i o n i n g C o n f l u e n t monolayers  o f H.Ep.2 and BHK-21 c e l l s grown i n m i l k  d i l u t i o n b o t t l e s were used i n a l l e l e c t r o n microscopy  studies.  Normal, i n f e c t e d and c h e m i c a l l y t r e a t e d c e l l s were s c r a p e d from t h e g l a s s w i t h a rubber policeman, p e l l e t e d by low-speed (1000 rpm f o r 5 minutes  centrifugation  i n an I n t e r n a t i o n a l C e n t r i f u g e Model C S ) ,  and immediately p l a c e d i n t o 2.5% g l u t a r a l d e h y d e i n 0.1 M b u f f e r pH 7.2 f o r 30 minutes times i n 0.2 M phosphate  phosphate  a t 4°C. The specimens were washed f o u r  b u f f e r e d s u c r o s e and p o s t - f i x e d i n 1%  osmium t e t r o x i d e pH 7.2 f o r 30 minutes.  D e h y d r a t i o n was accomplished  by p a s s i n g t h e samples through a graded s e r i e s o f e t h a n o l concent r a t i o n s f o l l o w e d by a step-wise t r a n s f e r i n t o p r o p y l e n e o x i d e . Specimens were embedded by p l a c i n g t h e c e l l s i n i n c r e a s i n g t r a t i o n s o f Epon 812 r e s i n d i s s o l v e d i n p r o p y l e n e o x i d e . n i g h t i n c u b a t i o n i n p l a s t i c a t room temperature,  concenA f t e r over-  t h e embedded  samples  were p l a c e d i n g e l a t i n c a p s u l e s and c u r e d a t 60°C f o r 24 h o u r s . S e c t i o n s o f a l l specimens embedded i n Epon 812 were c u t on a LKB I I I microtome and c o l l e c t e d on carbon and formvar c o a t e d 150  mesh copper g r i d s .  They were s t a i n e d w i t h a s a t u r a t e d  alcoholic  s o l u t i o n o f u r a n y l a c e t a t e f o r one minute and p o s t - s t a i n e d w i t h Reynold's l e a d 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 m i c r o s c o p e a t 60 kv.  Autoradiography Twenty-four hour monolayer c u l t u r e s grown on L e i g h t o n tube c o v e r s l i p s were i n f e c t e d w i t h 7.2 x 1 0  6  p f u HSV  (MOI = 1) and/or  t r e a t e d w i t h e i t h e r 10 yg/ml ara-C o r 100 yg/ml IDU.  At various 3  times a f t e r w a r d , t h e c e l l s were exposed t o 2.0 y C i / m l f o r 30 minutes a t 37°C.  H-thymidine  Immediately f o l l o w i n g exposure t o the  r a d i o i s o t o p e , the c o v e r s l i p s were washed w i t h HBSS and f i x e d i n a m i x t u r e o f methanol and a c e t i c a c i d f o r 15 m i n u t e s .  Residual  3:1  fixative  was removed by r e p e a t e d washings i n d i s t i l l e d water o v e r a p e r i o d for 20 m i n u t e s .  The f i x e d c e l l s were t h e n a i r - d r i e d and mounted on  c l e a n g l a s s s l i d e s w i t h Apochromount  mounting  fluid.  The s l i d e s were d i p - c o a t e d w i t h I l f o r d L-4 N u c l e a r E m u l s i o n , d r a i n e d , a i r - d r i e d and s t o r e d f o r 7 days a t 4°C i n a l i g h t - t i g h t  box  c o n t a i n i n g a s m a l l amount o f D r i e r i t e . The d i p p e d s l i d e s were d e v e l o p e d a c c o r d i n g t o t h e f o l l o w i n g s c h e d u l e : 12 minutes i n M i c r o d o l - X d e v e l o p e r (2 p a r t s water and 1 p a r t d e v e l o p e r ) ; 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 s l i d e s were a i r - d r i e d and examined  with an Olympus l i g h t microscope f o r the presence of t r i t i u m l a b e l . To f a c i l i t a t e observation of the grains, coverslips were stained b r i e f l y i n 1% eosin.  Metaphase Preparations C e l l s growing logarithmically ( i . e . 16 - 18 hour cultures) i n Leighton tubes were used i n a l l chromosome studies. generally received 7.2 x 10  Infected c e l l s  pfu HSV per tube and chemically  c e l l s either 10 yg/ml ara-C or 100 lig/ml IDU.  treated  Two hours p r i o r to  harvesting, infected, treated and control cultures also received 0.1 yg/ml colcemid.  C e l l s were t r y s i n i z e d o f f the tubes with 0.25%  trypsin i n HBSS and centrifuged i n an International Centrifuge Model CS f o r 5 minutes.  The supernatant was decanted and the c e l l s gently  resuspended i n 5.0 ml of 0.5% KC1 solution f o r 15 minutes at room temperature.  This suspension was recentrifuged and 4.0 ml of a 3:1  methanol-glacial of 20 minutes.  acetic acid mixture added drop by drop over a period After two further changes of f i x a t i v e , the c e l l s were  f i n a l l y suspended i n about 1.0 ml of the s o l u t i o n .  Four or f i v e  drops from a Pasteur pipette were dropped on a precleaned from a height of 4 - 5 inches.  glass s l i d e  A f t e r a i r - d r y i n g , the s l i d e s were  stained i n a Giemsa s o l u t i o n f o r 5 minutes.  The stained s l i d e s were  34  then b r i e f l y r i n s e d i n t a p water, a i r - d r i e d i l y w i t h an Olympus l i g h t m i c r o s c o p e . f o r chromosomal a b e r r a t i o n s  and examined p r e l i m i n a r -  Metaphase p l a t e s were  under an o i l immersion l e n s and  scored photographed  on Kodak P l u s X panchromatic f i l m on a Z e i s s semi-automatic p h o t o microscope.  RESULTS  Growth S t u d i e s F i g u r e 1 shows t h e growth o f HSV i n H.Ep.2 and BHK-21 monolayers a t 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 b u t H.Ep.2  c e l l s were c l e a r l y more s u s c e p t i b l e t o HSV i n f e c t i o n .  The f i r s t de-  t e c t a b l e i n c r e a s e i n v i r u s p r o d u c t i o n o c c u r r e d 8-12 hours  after  i n f e c t i o n and maximum y i e l d s were o b t a i n e d a f t e r 22-24 h o u r s .  Virus  5 0 3 8 t i t r e s a t 22 hours were 10 * T C T D ^ / m l i n H.Ep.2 c e l l s and 10 * TCID ./ml i n BHK-21 c e l l s . 50 r  A d d i t i o n o f 10 yg/ml ara-C o r 100 yg/ml  IDU a t t h e time o f 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 b o t h c e l l types.  F u r t h e r e f f e c t s o f t h e a n t i - v i r a l drugs a r e documented  i n t h e v a r i o u s s e c t i o n s on l i g h t and e l e c t r o n microscopy, a u t o r a d i o graphy and c y t o g e n e t i c s .  L i g h t Microscopy  1.  C y t o p a t h o l o g y o f HSV L e i g h t o n tube c u l t u r e s o f H.Ep.2 c e l l s were i n f e c t e d w i t h an  i n p u t m u l t i p l i c i t y o f 1 p f u HSV p e r c e l l and examined a t 5, 12, 24 and 72 hours o f i n f e c t i o n .  HOURS  Figure 1.  Representative growth curves of HSV i n H.Ep.2 and BHK-21 c e l l s . Total virus (extracellular and i n t r a c e l l u l a r ) was measured by the standard end-point d i l u t i o n technique after i n i t i a l i n f e c t i o n with an input m u l t i p l i c i t y of 1 pfu per c e l l .  37  Normal, uninfected H.Ep.2 monolayers were composed of c l o s e l y packed e p i t h e l o i d c e l l s with large ovoid n u c l e i containing 2-4 well-defined n u c l e o l i (Figs. 2,4).  The f i r s t evidence of v i r u s -  induced cytopathology was observed i n the n u c l e i of infected c e l l s . Five hours a f t e r i n f e c t i o n , many c e l l s showed evidence of early nucleolar d i s i n t e g r a t i o n and peripheral chromatin displacement. At 12 hours, over one-half of the c e l l s also demonstrated  some degree  of rounding and cytoplasmic v a c u o l i z a t i o n ( F i g . 3 ) . The emptyappearing n u c l e i of these c e l l s were invariably swollen and usually contained dense v i r a l i n c l u s i o n bodies surrounded by clear halos (Fig. 3 ) . A f t e r 24 hours of i n f e c t i o n , large numbers of c e l l s had detached from the glass surface and those remaining showed severe nuclear damage coupled with extensive cytoplasmic shrinking and rounding.  The damaged c e l l s had a s l i g h t tendency to aggregate i n loose  clumps but d i d not a t any time form syncytia (Fig. 5 ) . were completely destroyed a t 72 hours.  Monolayers  A s i m i l a r v i r u s cytopathology  was observed i n BHK-21 c e l l s infected with HSV.  2.  E f f e c t of Ara-C and IDU ON HSV Cytopathology Cultures inoculated with HSV received either 10 yg/ml ara-C or  100 yg/ml IDU a t the time of v i r u s i n f e c t i o n .  Observations made a t  5, 12, and 24 hours revealed no decrease i n the severity or time of appearance of the virus-induced cytopathic e f f e c t i n H.Ep.2 and BHK-21 cells.  38  F i g u r e 2.  Uninfected culture X1750.  »  JH  o f H.Ep.2 c e l l s .  Giemsa s t a i n .  NI  0 F i g u r e 3.  H.Ep.2 c u l t u r e 12 hours a f t e r HSV i n f e c t i o n . Giemsa stain. Note i n t r a n u c l e a r i n c l u s i o n (NI) and s w o l l e n degenerating n u c l e i of i n f e c t e d c e l l s . X4400.  Figure  5.  H.Ep.2 c u l t u r e 24 hours a f t e r HSV i n f e c t i o n . l o o s e aggregates o f rounded, shrunken c e l l s . X440.  Note  3.  E f f e c t o f Ara-C and IDU on U n i n f e c t e d  Cells  H e a l t h y H.Ep.2 and BHK-21 monlayers were t r e a t e d w i t h 10 yg/ml ara-C o r 100 yg/ml IDU f o r 24 and 72 h o u r s .  Exposure t o ara-C  r e s u l t e d i n m o d e r a t e l y s e v e r e damage t o c u l t u r e s a f t e r 72 h o u r s . The monolayers were p a r t i a l l y d e s t r o y e d h i g h l y v a c u o l i z e d and o f t e n rounded  ( F i g . 6) and i n d i v i d u a l c e l l s  ( F i g . 7 ) . IDU appeared t o cause  s i m i l a r b u t 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 o f treatment.  Fluorescent  1.  Antibody  HSV A n t i g e n Leighton  Studies  Production  tube c u l t u r e s o f H.Ep.2 and BHK-21 c e l l s were i n f e c t e d  w i t h an i n p u t m u l t i p l i c i t y o f 1 p f u HSV p e r c e l l and observed f o r production  o f v i r u s - s p e c i f i c antigens  a t 4, 7, 12 and 24 hours by  the i n d i r e c t f l u o r e s c e n t a n t i b o d y method. The  f i r s t s i g n s o f 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 o f weak c y t o p l a s m i c The  d i f f u s e cytoplasmic  and n u c l e a r  fluorescence  i n the p e r i n u c l e a r  E a r l y nuclear antigens  small, irregularly-shaped granules  (Fig. 8).  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 e v i d e n t of i n f e c t e d c e l l s .  fluorescence  regions  were c h a r a c t e r i z e d by  ( F i g . 8) t h a t were l a t e r o b s c u r e d  41  Figure  6.  H.Ep.2 c u l t u r e a f t e r 72 hours o f ara-C t r e a t m e n t . X440.  Figure  7.  H.Ep.2 c u l t u r e a f t e r 72 hours o f ara-C t r e a t m e n t . Note c y t o p l a s m i c v a c u o l i z a t i o n and p r e v a l e n c e o f rounded, shrunken c e l l s . X1750.  by the development o f l a r g e amorphous masses.  Between 7 and  12  hours, the number o f a n t i g e n - p r o d u c i n g c e l l s r o s e t o over 70% population.  C e l l s a t t h i s time commonly e x h i b i t e d i n t e n s e  s t a i n i n g together  with patchy nuclear  fluorescence  24 hours, v i r u s - s p e c i f i c f l u o r e s c e n c e was o f the i n f e c t e d c e l l c u l t u r e . s t r o n g l y i n the cytoplasm and  2.  The The  E f f e c t o f Ara-C and  a t the c e l l s u r f a c e  ON  HSV  Antigen  a d d i t i o n o f 10 yg/ml ara-C or 100  cultures f a i l e d to delay  s t a i n i n g was  evident  fluorescence  was  a t 4, 7 and  cytoplasmic  (Fig. 9).  By  fluoresced ( F i g . 10).  Production  yg/ml IDU  the appearance of HSV  the t o t a l number o f f l u o r e s c i n g c e l l s .  the  observed i n over 80-90%  Most of t h e s e c e l l s  IDU  of  to infected  antigens  Strong n u c l e a r  or decrease and  12 hours of i n f e c t i o n but  g e n e r a l l y l a c k i n g i n the c h e m i c a l l y  perinuclear surface  treated  cells.  E l e c t r o n Microscopy  1.  Negative Staining HSV  morphology was  s t u d i e d by the n e g a t i v e  v i r u s p a r t i c l e s o f mean diameter 170  nm  s t a i n method.  Typical  were f r e q u e n t l y observed i n  a d d i t i o n t o the c e l l d e b r i s n o r m a l l y found i n s e m i - p u r i f i e d v i r u s preparations.  The  v i r i o n s c o n s i s t e d o f l a r g e envelopes  enclosing  F i g u r e 8.  F l u o r e s c e n t a n t i b o d y study o f 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 p e r i n u c l e a r s t a i n i n g and f l u o r e s c e n t n u c l e a r g r a n u l e s . X4400.  Figure  9.  F l u o r e s c e n t a n t i b o d y study o f 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 n u c l e a r masses and d i f f u s e c y t o p l a s m i c f l u o r e s c e n c e . X4400.  44  Figure  10.  F l u o r e s c e n t a n t i b o d y study o f 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 n u c l e o c a p s i d s .  The c a p s i d s were c l e a r l y composed o f  h o l l o w , p o l y g o n a l capsomeres arranged symmetry  i n a regular icosahedral  ( F i g . 1 1 ) . I n cases where the p h o s p h o t u n g s t i c  a c i d pene-  t r a t e d t h e e x t e r n a l c a p s i d , a dense i n n e r " c o r e " was sometimes observed.  The c o r e , which measured 30 nm i n diameter,  i n an o u t e r s h e l l composed o f h o l l o w on t h e e x t e r n a l c a p s i d .  was encased  s u b u n i t s s i m i l a r t o those  seen  Other p a r t i c l e s appeared t o c o n t a i n no  inner s t r u c t u r e ( F i g . 12).  2.  Thin Sectioning (a)  U n i n f e c t e d H.Ep.2 and BHK-21 C e l l s . U n i n f e c t e d H.Ep.2 and BHK-21 c e l l s m a i n t a i n e d  i n MEM p l u s  10% c a l f serum r e v e a l e d w e l l p r e s e r v e d microanatomy w i t h i n t a c t membranes,  undistorted mitochondria,  ( F i g . 13,14).  (b)  distribution  A p a r t from an i n c r e a s e d i n c i d e n c e o f l i p i d  i n BHK-21 c e l l s , very s i m i l a r  and normal chromatin  granules  t h e human and hamster l i n e s appeared t o p o s s e s s  ultrastructure.  U n i n f e c t e d BHK-21 C e l l s : Abnormal P a r t i c l e F o r m a t i o n BHK-21 c e l l s m a i n t a i n e d  i n serumless MEM r e p e a t e d l y gave  r i s e t o abnormal p a r t i c l e s i n t h e cytoplasm  of affected c e l l s .  The  90 nm p a r t i c l e s c o n s i s t e d o f an e l e c t r o n dense core and a l e s s - d e n s e membrane-bound o u t e r r e g i o n  ( F i g . 1 5 ) . C l o s e examination  revealed  46  F i g u r e 11.  E l e c t r o n micrograph o f a n e g a t i v e p r e p a r a t i o n o f HSV. X90,700.  stain  F i g u r e 12.  E l e c t r o n micrograph o f a n e g a t i v e s t a i n p r e p a r a t i o n o f HSV. The i n n e r s t r u c t u r e o f two p a r t i c l e s can be observed where t h e s t a i n has p e n e t r a t e d the o u t e r c a p s i d . X90,700.  Figure  13.  E l e c t r o n micrograph o f normal H.Ep.2 c e l l s showing i n t a c t n u c l e a r and c y t o p l a s m i c s t r u c t u r e . X10,000.  fine r a d i a l structures extending from the core to the external membrane (Fig. 16). The p a r t i c l e s were normally observed i n cytoplasmic 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 s t r u c t u r a l abnormalities, a l l electron microscope studies of BHK-21 c e l l s were carried out on cultures provided with normal growth medium. No p a r t i c l e s were observed i n uninfected c e l l s grown i n the presence of 10% f e t a l c a l f serum. (c)  HSV Development i n H.Ep.2 and BHK-21 C e l l s H.Ep.2 and BHK-21 monolayers were inoculated with an input  m u l t i p l i c i t y of 20 pfu HSV per c e l l and examined at 4, 7, 12 and 20 hours a f t e r i n f e c t i o n . Evidence of v i r a l r e p l i c a t i o n was f i r s t detected as early as 4 hours a f t e r i n f e c t i o n .  At t h i s time, infected c e l l s developed  i r r e g u l a r l y condensed and marginated nuclear chromatin.  In addition,  dense granular aggregates were often observed scattered throughout the n u c l e i , although v i r a l p a r t i c l e s were not yet evident (Fig. 17). By 7 hours, the f i r s t p a r t i c l e s could be seen adjacent to r e duplicated nuclear membranes i n both H.Ep.2 and BHK-21 c e l l s (Fig. 18). Membrane p r o l i f e r a t i o n was usually extensive and fusion often resulted  49  F i g u r e 16.  An abnormal p a r t i c l e i n t h e cytoplasm o f an u n i n f e c t e d BHK-21 c e l l m a i n t a i n e d i n serumless medium. X160,000  50  F i g u r e 17.  E l e c t r o n micrograph o f 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 g r a n u l a r aggregate i n the n u c l e u s . X50,000  F i g u r e 18.  R e d u p l i c a t e d n u c l e a r 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.  F i g u r e 19.  Immature v i r u s p a r t i c l e s i n the n u c l e u s o f 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 c o n c e n t r i c l a m a l l a e . found i n the n u c l e u s were g e n e r a l l y 90 nm  Viral  particles  i n diameter and c o n s i s t e d  of  naked c a p s i d s e n c l o s i n g e l e c t r o n dense o r e l e c t r o n l u c e n t c o r e s .  In  a d d i t i o n , i n f e c t e d n u c l e i a l s o c o n t a i n e d a s m a l l number o f  v e l o p e d p a r t i c l e s 130 nm  i n diameter  ( F i g . 19).  Very  occasionally  t h e s e immature v i r i o n s c o u l d be seen a c q u i r i n g a second budding  en-  envelope  by  through the i n n e r l a m e l l a o f the n u c l e a r membrane ( F i g . 20).  A few mature p a r t i c l e s w i t h double membranes and a number o f naked and immature v i r i o n s were found i n the cytoplasm a t 7 hours o f i n f e c t i o n b u t no s u r f a c e v i r u s was Extensive v i r a l  detected at t h i s  r e p l i c a t i o n was  time.  e v i d e n t a t 12 hours a f t e r  infectio:  N u c l e a r p a r t i c l e s i n v a r i o u s s t a g e s o f assembly were observed i n g r e a t numbers and c y t o p l a s m i c v i r u s was smooth membranous s t r u c t u r e s . to p r e f e r e n t i a l l y aggregate ing  t u b u l e s i n the cytoplasm  f r e q u e n t l y found a s s o c i a t e d w i t h  The mature and immature v i r u s e s  i n c e l l v a c u o l e s and w i t h i n f i n e , ( F i g . 21).  Mature p a r t i c l e s  appeared branch-  measuring  170-175 nm were r e l e a s e d by a p r o c e s s r e s e m b l i n g r e v e r s e p h a g o c y t o s i s (Fig.  22).  cell  type.  No e v i d e n c e o f budding v i r u s was  By 20 h o u r s , widespread  encountered  c e l l d e g e n e r a t i o n was  responding h i g h 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  i n either  evident with cor-  ( F i g . 23).  Enveloped  HE  '  ;  ,• ,  F i g u r e 20.  Immature v i r u s p a r t i c l e budding through t h e n u c l e a r membrane o f 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.  F i g u r e 21.  Mature v i r u s p a r t i c l e s near a b r a n c h i n g t u b u l e i n t h e cytoplasm o f 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  •fl  Figure  22.  R e l e a s e o f 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 o f t e n found a t t h e c e l l s u r f a c e  lodged between prominent  cytoplasmic processes.  The s u r f a c e i t s e l f  was jagged and d i s c o n t i n u o u s and a g r e a t d e a l o f c e l l u l a r d e b r i s was found f r e e i n the medium.  I n a d d i t i o n , n u c l e a r membranes were  o f t e n broken and t h e nucleoplasm appeared g r a n u l a r 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 a l s o found i n many i n f e c t e d  cells.  Large c r y s t a l s composed o f v i r a l c a p s i d s e n c l o s i n g a v a r i e t y  of  e l e c t r o n dense components were f r e q u e n t l y observed i n t h e n u c l e i  of  BHK-21 c e l l s  ( F i g . 2 4 ) . Groups o f 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 t h e cytoplasm o f d e g e n e r a t i n g  cells.  C y t o p l a s m i c aggregates were e n c l o s e d i n i r r e g u l a r l y shaped v a c u o l e s and e x i s t e d a d j a c e n t t o naked HSV p a r t i c l e s and mature v i r u s  ( F i g . 25).  In g e n e r a l , t h e development o f HSV was v e r y s i m i l a r i n b o t h H.Ep.2 and BHK-21 c e l l s . to  However, t h e hamster c u l t u r e s appeared t o g i v e r i s e  a h i g h e r p e r c e n t a g e o f d e f e c t i v e and unenveloped v i r u s  i n t h e n u c l e u s and cytoplasm o f i n f e c t e d c e l l s .  particles  Moreover, a l t h o u g h  b i z a r r e v i r a l aggregates were v e r y common i n BHK-21 c e l l s , f o r m a t i o n s were ever observed i n i n f e c t e d H.Ep.2 c e l l s .  no c r y s t a l  Beyond t h e s e  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 t h e same i n terms o f assembly, (d)  envelopment and r e l e a s e . E f f e c t o f Ara-C and IDU on HSV Development H.Ep.2 and BHK-21 monolayers  per c e l l .  were i n f e c t e d w i t h 20 p f u HSV  A f t e r r e c e i v i n g 10 yg/ml ara-C o r 100 yg/ml IDU, t h e c e l l s  F i g u r e 24.  I n t r a n u c l e a r 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.  F i g u r e 25.  C y t o p l a s m i c aggregate i n a BHK-21 c e l l a f t e r HSV i n f e c t i o n . X39,500.  20 hours  were incubated f o r 20 hours and examined under the electron microscope. Ara-C prevented the formation of complete virus p a r t i c l e s , but did not prevent the early nuclear a l t e r a t i o n s c h a r a c t e r i s t i c of HSV infection.  Although membrane reduplication was not evident, condensed  and marginated chromatin was r e a d i l y observed i n 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 v i r u s - l i k e p a r t i c l e s were found i n the nucleus or cytoplasm of c e l l s treated with ara-C. IDU had much the same i n h i b i t o r y e f f e c t on HSV r e p l i c a t i o n i n cultured c e l l s .  In most cases, marginated chromatin and dense nuclear  granules were observed i n the chemically treated and infected c e l l s . However, i n addition to such p a r t i c u l a t e structures, a number of enveloped viruses measuring 130-135 nm i n diameter were also found free i n the cytoplasm.  The v i s u a l l y defective p a r t i c l e s were ragged  i n appearance and t h e i r 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)  E f f e c t of Ara-C and IDU on Uninfected C e l l s C e l l s treated with 10 yg/ml ara-C were morphologically altered  59  Figure 27.  Cytoplasmic p a r t i c l e i n a BHK-21 c e l l 20 hours after HSV i n f e c t i o n and IDU treatment. X28,000.  by  24 h o u r s .  chondria  E a r l y changes i n c l u d e d marked d i s t e n t i o n o f m i t o -  ( F i g . 28) and endoplasmic r e t i c u l u m .  w i t h ara-C r e s u l t e d i n conspicuous c y t o p l a s m i c  Further  treatment  v a c u o l i z a t i o n with  l o s s o f m i t o c h o n d r i a l s t r u c t u r e and d i s t o r t i o n o f t h e i n t e r n a l membrane systems  ( F i g . 29).  Exposure t o 100 yg/ml IDU f o r 24 and 48 hours produced t h e same type o f s t r u c t u r a l a l t e r a t i o n s i n u n i n f e c t e d H.Ep.2 and BHK-21 c e l l s . However, c y t o p a t h i c e f f e c t s were e v i d e n t o n l y a f t e r 48 hours and were r e s t r i c t e d t o a m i n i m a l d i s t o r t i o n o f c e l l o r g a n e l l e s and v a c u o l i z a t i o n .  Autoradiographic  1.  DNA S y n t h e s i s  Studies  i n HSV I n f e c t e d C e l l s  DNA s y n t h e s i s i n HSV i n f e c t e d c e l l s was s t u d i e d by p u l s e  labeling  3 t h e c e l l s a t v a r i o u s times a f t e r i n f e c t i o n w i t h f o r 30 m i n u t e s .  2.0 y c i / m l  H-thymidine  HSV i n f e c t i o n o f H.Ep.2 and BHK-21 c e l l s appeared t o  cause an immediate and a l m o s t complete i n h i b i t i o n o f DNA s y n t h e s i s . A f t e r 4 h o u r s , however, t h e number o f c e l l s s y n t h e s i z i n g DNA r o s e r a p i d l y and reached a maximum o f 28.2% a f t e r 8 hours o f i n f e c t i o n . F o l l o w i n g t h i s peak o f s y n t h e t i c a c t i v i t y , t o t a l DNA s y n t h e s i s gradua l l y d e c l i n e d t o approximately The  o n e - s i x t h o f t h e 0 time f i g u r e ( F i g . 3 0 ) .  e a r l y d e c r e a s e found i n i n f e c t e d c e l l s p r o b a b l y  represented  a v i r u s - i n d u c e d i n h i b i t i o n o f c e l l DNA s y n t h e s i s w h i l e t h e secondary  Figure  28.  M i t o c h o n d r i a o f a BHK-21 c e l l a f t e r 24 hours of ara-C t r e a t m e n t . X69,200.  o—o  HSV  HOURS  F i g u r e 30.  DNA s y n t h e s i s i n v i r u s - i n f e c t e d and c h e m i c a l l y t r e a t e d BHK-21 c e l l s . C e l l s were i n f e c t e d w i t h an i n p u t m u l t i p l i c i t y o f 1 p f u / c e l l and t r e a t e d w i t h 10 yg/ml ara-C and 100 yg/ml IDU.  i n c r e a s e r e f l e c t e d t h e b u l k o f v i r a l DNA s y n t h e s i s and p o s s i b l y r e p a i r o f damaged h o s t c e l l DNA  2.  (126).  DNA S y n t h e s i s i n C e l l s T r e a t e d w i t h Ara-C and IDU Ara-C and IDU produced  an immediate and complete  c e l l and v i r a l DNA s y n t h e s i s i n HSV i n f e c t e d c e l l s anti-viral  drugs a l s o c o m p l e t e l y i n h i b i t e d c e l l u l a r  i n h i b i t i o n of  ( F i g . 3 0 ) . The DNA s y n t h e s i s  i n u n i n f e c t e d H.Ep-2 and BHK-21 c e l l s w i t h i n 2 hours o f t h e i r  addition.  Cytogenetic Studies  1.  Mitotic  Rates  M i t o t i c r a t e s were s t u d i e d i n H S V - i n f e c t e d and c h e m i c a l l y t r e a t e d H.Ep.2 and BHK-21 c e l l s .  HSV i n f e c t i o n produced  the m i t o t i c index o f b o t h c e l l t y p e s .  a rapid increase i n  I n one experiment,  of  BHK-21 c e l l s observed i n metaphase t r i p l e d  of  i n f e c t i o n and t h e r e a f t e r d e c l i n e d u n t i l  t h e number  i n t h e f i r s t s i x hours  t h e study was t e r m i n a t e d .  By 24 hours, m i t o s i s was c o m p l e t e l y i n h i b i t e d i n a l l H S V - i n f e c t e d cells  ( F i g . 31). The m i t o t i c r a t e o f c e l l s exposed t o ara-C o r IDU a t t h e time o f  HSV i n f e c t i o n was s i m i l a r t o t h a t o f u n t r e a t e d v i r u s i n f e c t e d  cells.  64  HSV(MOI = l )  HOURS  F i g u r e 31.  M i t o t i c r a t e s o f BHK-21 c e l l s f o l l o w i n g v i r u s i n f e c t i o n and c h e m i c a l treatment. Colcemid was o m i t t e d from the s t a n d a r d metaphase p r e p a r a t i o n technique.  However, c h e m i c a l l y t r e a t e d u n i n f e c t e d c e l l s underwent a r a p i d and complete drugs  2.  inhibition  o f m i t o s i s w i t h i n 6-8 hours o f exposure t o t h e  ( F i g . 31).  Normal C e l l  Karyotypes  H.Ep.2 c e l l s p o s s e s s e d a d i p l o i d  male k a r y o t y p e o f 74 chromosomes  ( F i g . 3 2 ) . The human c a n c e r l i n e had a r e l a t i v e l y of a n e u p l o i d y i n t h e d i p l o i d (Table I ) .  high percentage  range and a low p e r c e n t a g e o f t e t r a p l o i d y  BHK-21 c e l l s d i s p l a y e d a normal male k a r y o t y p e o f 44  chromosomes w i t h one m e t a c e n t r i c marker chromosome ( F i g . 3 3 ) . A s i m i l a r h i g h percentage o f a n e u p l o i d y and low percentage o f 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 t h e hamster l i n e  3.  HSV-induced  (Table I ) .  Chromosome A b n o r m a l i t i e s  A comparative study was made c o n c e r n i n g t h e e f f e c t the  chromosomes o f H.Ep.2 and BHK-21 c e l l s .  showed a s i g n i f i c a n t  o f HSV on  Virus treated  cultures  i n c r e a s e i n t h e number o f c e l l s w i t h chromosome  a b e r r a t i o n s when compared t o c o n t r o l c u l t u r e s  ( F i g . 3 4 ) . The p e r -  centage o f abnormal metaphases began too r i s e w i t h i n 2 hours o f and r e a c h e d a maximum o f 100% a t 14-20 h o u r s .  infection  G e n e r a l l y , t h e r e were  fewer a b n o r m a l i t i e s observed i n BHK-21 c e l l s d u r i n g t h e f i r s t 8 hours of  infection.  F o r example, a f t e r 4 hours, almost 60% o f t h e H.Ep.2  metaphases were abnormal, w h i l e o n l y 40% o f t h e BHK metaphases r e v e a l e d  66  Figure 32.  Figure 33.  Karyotype of a normal H.Ep.2 c e l l .  X4400.  Table I .  Frequency D i s t r i b u t i o n o f V a r i o u s L e v e l s o f P l o i d y i n H.Ep.2 and BHK-21 C e l l s .  Approximate Chromosome Number * Haploid n  Aneuploid <2n  Diploid 2n  Aneuploid >2n  Triploid 3n  H.Ep.2  8  28  137  55  -  BHK-21  5  34  145  48  15  *  H.Ep.2  n = 37  BHK-21  n = 22  Total Tetraploid 4n  Cells  42  250  3  250  68  HOURS A F T E R  Figure 34.  INFECTION  The 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 .  chromosome lesions.  However, the t o t a l damage to the c e l l chromosomes  after 20 hours was similar i n both l i n e s . 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), f r a g mentation (Fig. 38), erosion (Fig. 39), and endoreduplication (Fig. 40). Chromatid gaps, breaks and secondary constrictions were more frequent i n the f i r s t 4 hours of i n f e c t i o n while severe fragmentation and erosion appeared more often l a t e r i n i n f e c t i o n .  In addition, H.Ep.2 c e l l s  revealed more extensive damage at an e a r l i e r time than BHK-21 c e l l s (Table I I , I I I ) . Chromosome abnormalities i n 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 n e u t r a l i z a t i o n of the v i r u s inoculum with s p e c i f i c HSV antiserum.  4.  E f f e c t of M u l t i p l i c i t y of Infection on HSV-induced  Chromosome  Abnormalities. Figure 41 demonstrates the r e l a t i o n s h i p between i n i t i a l m u l t i p l i c i t y of i n f e c t i o n and amount of chromosome damage found i n BHK-21 cells.  The percentage of abnormal metaphases increased i n proportion  to the virus inoculum.  At an input m u l t i p l i c i t y of 10 pfu per c e l l ,  70  Figure 35.  Chromosome complement o f 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.  Figure  36.  X4400.  Chromosome complement o f 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 m u l t i p l e chromatid breaks and chromosome d i s t o r t i o n . X4400.  Chromosome complement o f 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 c h r o m a t i d break and prominent secondary c o n s t r i c t i o n s . X4400.  Chromosome complement o f a BHK-21 c e l l showing complete f r a g m e n t a t i o n a f t e r 10 hours o f HSV infection. X4400.  72  «  F i g u r e 39.  E r o s i o n o f BHK-21 complement a f t e r 10 hours o f HSV i n f e c t i o n . X4400.  F i g u r e 40.  E n d o r e d u p l i c a t i o n o f BHK-21 chromosomes 8 hours a f t e r HSV i n f e c t i o n . X4400.  T a b l e I I . An A n a l y s i s o f HSV-Induced  Hours a f t e r infection  No. c e l l s scored  S i n g l e gaps and breaks  Chromosome A b n o r m a l i t i e s  Multiple gaps  Secondary constrictions  i n H.Ep.2 C e l l s  Fragmentation  Erosion  Endoreduplication  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  -  -  M u l t i p l i c i t y of i n f e c t i o n = 1 p f u per c e l l  2  Table I I I .  An A n a l y s i s of HSV-Induced  Hours a f t e r No. c e l l s scored infection  Chromosome A b n o r m a l i t i e s i n BHK-21 C e l l s  a  S i n g l e gaps and breaks  Multiple gaps  Secondary Constrictions  Fragmentation  Erosion  Endoreduplication  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 o f i n f e c t i o n = 1 p f u per c e l l .  —  -  —  75  O  2  4  6  8  HOURS A F T E R  F i g u r e 41.  10  12  14  20  INFECTION  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 o f i n f e c t i o n and HSV-induced chromosome a b n o r m a l i t i e s i n BHK-21 cells.  100%  o f t h e metaphases observed a t 8 hours were abnormal, whereas  f o r 10- and 100-fold lower doses o f v i r u s , 82% and 44% o f t h e metaphases r e v e a l e d chromosome l e s i o n s . metaphase a l s o i n c r e a s e d  The number o f a l t e r e d chromosomes p e r  i n r e l a t i o n t o t h e dose o f v i r u s and the time  o f i n c u b a t i o n i n b o t h H.Ep.2 and BHK-21 c e l l s .  5.  E f f e c t o f UV I r r a d i a t i o n o f HSV on V i r u s - I n d u c e d Chromosome Abnormalities. The  and  number o f v i r u s - i n d u c e d  chromosome a b n o r m a l i t i e s  i n H.Ep.2  BHK-21 c e l l s d e c r e a s e d 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 o f HSV.  I n one experiment u s i n g BHK-21 c e l l s , t h e c a p a c i t y o f t h e v i r u s t o 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 a p p r o x i m a t e l y f i v e times more s l o w l y t h a n t h e i n f e c t i v i t y  6.  ( F i g . 42).  E f f e c t o f Excess A r g i n i n e on HSV-induced Chromosome Previous  s t u d i e s have e s t a b l i s h e d t h a t a minimal  Abnormalities  concentration  o f a r g i n i n e i s n e c e s s a r y f o r HSV development i n c u l t u r e d c e l l s (3). I t i s a l s o known t h a t mycoplasma i n f e c t i o n g e n e r a l l y r e s u l t s i n a r g i n i n e d e p l e t i o n and t h a t such s t a r v a t i o n can cause chromosome abn o r m a l i t i e s i n mammalian c e l l s  (60). S i n c e mycoplasma i n f e c t i o n has  been r u l e d o u t i n t h e H.Ep and BHK-21 l i n e s , i t was t h e r e f o r e o f i n t e r e s t t o determine i f t h e a b n o r m a l i t i e s f a c t , due t o v i r u s - i n d u c e d  produced by HSV i n f e c t i o n were i n  arginine starvation of the c e l l s .  This  77  F i g u r e 42.  E f f e c t o f UV i r r a d i a t i o n o f HSV on v i r a l i n f e c t i v i t y and c a p a c i t y t o induce chromosome a b n o r m a l i t i e s i n BHK-21 c e l l s . The n o n i r r a d i a t e d v i r u s (1 p f u / c e l l ) induced chromosomal damage a t 12 hours i n 92% o f the observed metaphases, which was taken as 100%. I n f e c t i v i t y was measured by t h e end-point d i l u t i o n technique.  was done by i n c r e a s i n g t h e c o n c e n t r a t i o n o f a r g i n i n e i n t h e growth medium and o b s e r v i n g t h e e f f e c t on HSV-induced  chromosome a b e r r a t i o n s .  T a b l e IV shows t h e r e s u l t s o f such an experiment c a r r i e d o u t i n BHK-21 cells.  I n c r e a s e s i n a r g i n i n e v a r y i n g from 5- t o 1 5 - f o l d d i d n o t  s i g n i f i c a n t l y a l t e r t h e number o r t y p e o f a b e r r a t i o n s produced i n t h e c e l l chromosomes a f t e r HSV  infection.  S i m i l a r r e s u l t s were o b t a i n e d  i n H S V - i n f e c t e d H.Ep.2 c e l l s .  7.  E f f e c t o f Ara-C on t h e Chromosomes o f U n i n f e c t e d and HSV-Infected Cells. R a p i d l y d i v i d i n g BHK-21 c e l l s were exposed t o v a r i o u s c o n c e n t r a t i o n s  o f ara-C f o r 4 h o u r s .  Chromosome a n a l y s i s a t t h e end o f t h i s  time  showed t h a t ara-C caused a s i g n i f i c a n t i n c r e a s e i n the number o f s i n g l e chromatid gaps and breaks  (Table V ) .  The d r u g - i n d u c e d damage was  p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n o f ara-C used i n t h e experiment ( i e . 10 Ug/ml ara-C i n d u c e d a b n o r m a l i t i e s i n 10% o f the BHK-21 metaphases and 20 yg/ml ara-C i n d u c e d l e s i o n s i n 20% o f the metaphases). H S V - i n f e c t e d BHK-21 c e l l s were a l s o exposed t o ara-C f o r 4 h o u r s . Ara-C d i d n o t i n h i b i t any o f t h e HSV-induced (Table V ) .  Moreover,  chromosome a b n o r m a l i t i e s  t h e drug appeared t o a c t s y n e r g i s t i c a l l y w i t h t h e  v i r u s t o produce an o v e r l y l a r g e number o f c e l l s w i t h s i n g l e and m u l t i p l e gaps and breaks  ( F i g . 43).  I t was e s t i m a t e d t h a t t h e number o f a f f e c t e d  Table IV.  The E f f e c t of Excess Arginine on HSV-Induced Chromosome Abnormalities  Treatment  % Abnormal Metaphases  i n BHK-21 C e l l s  Single gaps and breaks  Multiple gaps  Secondary Constrictions  Fragment- Erosion EndoreCondensation ation duplication  -  -  2  -  -  -  -  3  1  1  5 x Arg  4  6  10 x Arg  6  7  -  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  -  -  4  7  _  _  _  _  _  —  Control  Arginine increases were based on normal medium content of 6.0 x 10  M  M u l t i p l i c i t y of infections = 1 pfu per c e l l 200 c e l l s were scored 4 hours a f t e r HSV i n f e c t i o n and/or arginine treatment.  Table V.  Chromosome Abnormalities i n HSV-Infected and Non-Infected BHK-21 C e l l s Treated with ara-C  Treatment  ara-C,5 yg/ml  %  Abnormal^ Metaphases  Single Gaps and Breaks  Multiple Gaps  Secondary Constrictions  Fragmentation  Erosion  Endoreduplication  2  4  ara-C,10 yg/ml  10  14  2  4  ara-C,20 yg/ml  20  28  4  4  ara-C, 5 yg/ml + HSV  45  8  30  44  ara-C,10 yg/ml + HSV  64  23  56  42  13  52  7  70  5  7  20  5  36  12  ara-C,20 yg/ml + HSV HSV Control  76 40 4  3  3  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 200 c e l l s were scored 4 hours a f t e r HSV i n f e c t i o n and/or ara-C treatment.  CD  o  Figure 44.  T r a n s l o c a t i o n found i n a BHK-21 c e l l 4 hours a d d i t i o n o f IDU. X4400.  after  c e l l s i n the HSV and ara-C treated cultures was approximately  1.3 times  greater than the additive e f f e c t s induced separately by the v i r u s and the antimetabolite.  The s y n e r g i s t i c e f f e c t on chromosome damage could  be observed i f ara-C was added as l a t e as 2 hours a f t e r v i r u s adsorption.  Exposure to the drug i n the l a s t 2 hours on i n f e c t i o n , however,  produced no increase i n HSV-induced abnormalities.  Similar r e s u l t s  were obtained i n H.Ep.2 c e l l s .  8.  E f f e c t of IDU on the Chromosomes of Uninfected and HSV-Infected Cells. Uninfected BHK-21 c e l l s 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,  e f f e c t on the number of chromosome aberrations found i n the c e l l s a f t e r 4 hours of treatment. induced damage i n 7-10% control c u l t u r e .  However, higher concentrations of the drug of the metaphases as compared to 3% i n the  IDU-induced abnormalities included single chromatid  gaps, breaks, and translocations (Fig. 44). HSV-infected  BHK-21 c e l l s were also exposed to IDU for 4 hours.  IDU d i d not i n h i b i t any of the virus-induced chromosome abnormalities nor d i d i t act s y n e r g i s t i c a l l y with the v i r u s (Table VI). c e l l s treated with the lower concentrations  Infected  (25 and 50 yg/ml) of the  drug had the same number of aberrations as untreated infected c e l l s . C e l l s receiving higher concentrations had damage that was  equal to the  Table VI.  Chromosome Abnormalities i n HSV-Infected  and Non-Infected BHK-21 C e l l s Treated With IDU.  Secondary Multiple Constrictions Gaps  FragmentErosion ation  EndoreTranslocations duplication  % Abnormal. b Metaphases  Single Gaps and Breaks  IDU, 25 yg/ml  2  1  -  -  -  -  IDU, 50 yg/ml  4  5  -  -  -  2  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  IDU, 200 yg/ml + HSV  53  17  28  6  53  2  -  HSV  .42  11  18  8  41  6  -  3  3  -  -  2  1  Treatment  Control  —  M u l t i p l i c i t y of infection = 1 pfu per c e l l 200 c e l l s were scored 4 hours after HSV infection and/or IDU treatment  1  1  additive e f f e c t s induced separately by IDU and HSV.  Similar r e s u l t s  were obtained i n H.Ep.2 c e l l s .  9.  E f f e c t of Ara-C and IDU on the Chromosomes on Uninfected and HSVInfected C e l l s . Infected and uninfected BHK-21 and H.Ep.2 c e l l s were exposed to a  combination of 10 yg/ml ara-C and 100 yg/ml IDU f o r 4 hours.  The amount  of chromosome damage incurred i n uninfected c e l l s was equal to the additive e f f e c t s of ara-C and IDU. Damage i n HSV-infected c e l l s 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  r e s t r i c t e d to single and multiple gaps and fragmentation (Table V I I ) .  Table VII.  Chromosome Abnormalities i n HSV-Infected and Non-Infected HBK-21 C e l l s Treated With ara-C and IDU  ^ Treatment  % Abnormal, , d Metaphases  Single Gaps ,„ , and Breaks  Multiple „ Gaps  Secondary Fragment„ . . j_. .. Constrictions a t i o n  „ . Erosion  11  5  4  8  4  4  ara-C + IDU  20  15  1  HSV° + ara-C  62  17  6  8  29  2  HSV + IDU  49  11  4  7  25  1  HSV + ara-C + IDU  72  21  7  11  32  HSV  38  13  8  7  10  3  2  ara-C IDU  b  Control  4  ara-C was used at a concentration of 10 lig/ml IDU was used at a concentration of 100 yg/ml 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 100 c e l l s were scored 4 hours after HSV i n f e c t i o n and/or chemical treatment  _ , , .. Endoreduplication  DISCUSSION  These r e s u l t s show t h a t HSV i s c a p a b l e o f i n d u c i n g  severe  m o r p h o l o g i c a l and b i o c h e m 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 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  c o m p l e t e l y i n h i b i t HSV r e p l i c a t i o n i n t h e same c e l l s b u t a r e unable t o p r e v e n t any o f t h e c y t o p a t h i c The  effects of the v i r u s .  r a p i d growth c y c l e s o f HSV i n H.Ep.2 and BHK-21 c e l l s have  been documented i n p r e v i o u s s t u d i e s by Roizman e t a l . (89) and R u s s e l l e t a_l. (96) .  A l t h o u g h b o t h c e l l types s u p p o r t e d a remarkably  s i m i l a r c o u r s e o f v i r a l r e p l i c a t i o n , t h e 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 p r o d u c i n g h i g h t i t r e s o f HSV. The  i n h i b i t i o n o f HSV r e p l i c a t i o n f o l l o w i n g a d d i t i o n o f 10 yg/ml  ara-C o r 100 yg/ml IDU a t the time o f i n f e c t i o n c o n f i r m s e a r l i e r reports first  on t h e a n t i - v i r a l n a t u r e o f t h e d r u g s .  Buthala  (9) was t h e  t o show t h a t s i m i l a r c o n c e n t r a t i o n s o f 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 o f DNA v i r u s e s  rabies, B-virus,  swine pox, f o w l pox and v a c c i n i a v i r u s .  work, Kaufman had r e p o r t e d herpetic  t h a t IDU was c l i n i c a l l y  keratitis i n rabbits  ara-C was e q u a l l y  i n c l u d i n g HSV, pseudo-  effective  (39) and subsequent s t u d i e s  active i n curing  Prior to this against  showed t h a t  o c u l a r herpes i n man and animals ( 3 6 ) .  87  More r e c e n t l y , e f f o r t s have been f o c u s e d on d e t e r m i n i n g the mechanism o f a c t i o n o f the two a n t i - v i r a l a g e n t s . (46) found t h a t ara-C appeared  t o p r e v e n t HSV  L e v i t t and  replication i n vitro  by b l o c k i n g the de novo s y n t h e s i s o f d e o x y c y t i d y l a t e .  Thus, i n f e c t e d  c e l l s t r e a t e d w i t h the drug showed no e v i d e n c e o f v i r a l DNA o r v i r i o n assembly.  Becker  synthesis  S i m i l a r l y , Roizman e_t a_l. (89) r e p o r t e d t h a t  c o m p l e t e l y i n h i b i t e d HSV o f 5 Ug/ml and appeared  r e p l i c a t i o n i n human c e l l s a t a c o n c e n t r a t i o n t o p r e v e n t v i r a l DNA  the u t i l i z a t i o n of thymidine. found evidence o f IDU  IDU  s y n t h e s i s by b l o c k i n g  On the o t h e r hand, l a t e r  s u b s t i t u t i o n i n v i r a l DNA,  investigators  suggesting t h a t the  drug a f f e c t e d p r o d u c t i o n o f d e f e c t i v e l a t e p r o t e i n s i n v o l v e d i n v i r u s assembly r a t h e r than DNA  synthesis i t s e l f  (87,73).  C y t o l o g i c a l examination o f H S V - i n f e c t e d c e l l s r e v e a l e d t h e l a r g e i n c l u s i o n b o d i e s and n u c l e a r d i s o r g a n i z a t i o n t y p i c a l o f HSV (62).  The HSV  s t r a i n used i n the p r e s e n t study produced  infection  marked  r o u n d i n g o f both c e l l t y p e s w i t h l i t t l e a g g r e g a t i o n o f i n f e c t e d and no s y n c y t i a f o r m a t i o n . o f c e l l s was  A s i m i l a r e f f e c t on the s o c i a l  d e s c r i b e d by Roizman (84) w i t h the VR-3  Ara-C and IDU d i d n o t appear t o reduce HSV either c e l l line.  cells  behaviour  s t r a i n of  HSV.  cytopathology i n  P r e v i o u s r e p o r t s have a l s o d e s c r i b e d p e r s i s t e n c e  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 t h a t the  h o s t damage i s an e a r l y v i r u s f u n c t i o n independent s y n t h e s i s or assembly o f i n f e c t i o u s  particles.  of v i r a l  DNA  Drug-induced 21 c e l l s .  c y t o t o x i c i t y was observed i n b o t h H.Ep.2 and BHK-  The g e n e r a l i z e d d e g e n e r a t i o n o f c e l l s appeared  t o occur  w i t h i n c r e a s i n g time o f treatment and was more obvious i n ara-C cultures.  treated  These r e s u l t s c o n f i r m B u t h a l a ' 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 o f ara-C and IDU.  He observed marked v a c u o l i z a t i o n  and m i t o c h o n d r i a l d i s i n t e g r a t i o n i n c e l l s exposed t o e i t h e r drug f o r a prolonged p e r i o d of time.  I n a d d i t i o n , ara-C appeared  t o be  t h e more t o x i c o f t h e two a n t i - v i r a l agents a l t h o u g h b o t h were more a c t i v e i n r a p i d l y growing  c e l l s than i n s t a t i o n a r y c u l t u r e s .  The  l a t t e r o b s e r v a t i o n i s p r o b a b l y r e l a t e d t o t h e f a c t t h a t ara-C and IDU a r e known t o i n h i b i t DNA s y n t h e s i s and m i t o s i s i n mammalian (43,73).  cells  C l i n i c a l l y , t h e two drugs a r e a l s o c a p a b l e o f c a u s i n g a  marked d i s r u p t i o n o f t h e h e m a t o p o i e t i c system o f man  (74). Thus, t h e  p r e s e n t c y t o l o g i c a l and c l i n i c a l e v i d e n c e s t r o n g l y suggests t h a t  ara-C  and IDU a c t n o t as s p e c i f i c a n t i - v i r a l agents b u t r a t h e r as mammalian antimetabolites.  As a r e s u l t , t h e i r u s e f u l n e s s as t h e r a p e u t i c agents  i n human d i s e a s e i s s e v e r e l y  limited.  There a r e many r e p o r t s i n the l i t e r a t u r e c o n c e r n i n g t h e l a r g e number o f a n t i g e n s formed  i n t i s s u e c u l t u r e c e l l s i n f e c t e d w i t h HSV.  U s i n g the agar g e l p r e c i p i t a t i o n t e s t , Watson e t al_.  (124) have de-  t e c t e d 12 d i f f e r e n t p r e c i p i t a t i o n bands w i t h immune a n t i s e r u m p r e p a r e d against HSV-infected c e l l e x t r a c t s .  S e v e r a l o t h e r i n v e s t i g a t o r s have  demonstrated a t l e a s t f i v e s e p a r a t e immunofluorescent elements i n HSVinfected cells  (45,78,91,94).  I n t h e p r e s e n t study, f o u r d i f f e r e n t  f l u o r e s c e n t a n t i g e n s were d e t e c t e d 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 w i t h a commercial HSV a n t i s e r u m p r e p a r e d  i n guinea p i g s .  W i t h i n 4 hours o f i n f e c t i o n , f l u o r e s c e n c e was observed  i n the nucleus,  p e r i n u c l e a r r e g i o n , and cytoplasm  The e a r l y  o f both c e l l types.  n u c l e a r a n t i g e n s appeared as s m a l l , i r r e g u l a r l y shaped g r a n u l e s and the c y t o p l a s m i c a n t i g e n s as a d i f f u s e f l u o r e s c e n c e .  Geder and V a c z i  (22) d e s c r i b e d s i m i l a r immunofluorescent elements i n H S V - i n f e c t e d BSC-1  c e l l s with antiserum prepared a g a i n s t v i r u s - i n f e c t e d  culture extracts.  A f t e r 4 hours,  tissue  l a r g e f l u o r e s c e n t masses appeared  i n t h e n u c l e u s o f 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 , i n t e n s e s u r f a c e f l u o r e s c e n c e was observed  i n over 90% o f t h e c e l l p o p u l a t i o n .  These  l a t e r a n t i g e n s were p r e v i o u s l y d e s c r i b e d by Ross e t al^. (94) and Roane and Roizman (78), who a l s o d e t e c t e d a second g r a n u l a r a n t i g e n i n t h e cytoplasm  a f t e r 5-6 hours o f i n f e c t i o n .  The absence o f c y t o p l a s m i c  g r a n u l e s i n t h e p r e s e n t study may be r e l a t e d t o the use o f d i f f e r e n t v i r a l s t r a i n s o r d i f f e r e n t HSV  antiserums.  Ara-C and IDU f a i l e d t o p r e v e n t t h e f o r m a t i o n o f t h e n u c l e a r o r p e r i n u c l e a r a n t i g e n s i n i n f e c t e d c e l l s b u t d i d p r e v e n t t h e appearance of  surface fluorescence.  Geder and V a c z i  (22) r e p o r t e d s i m i l a r  i n BSC-1 c e l l s a f t e r treatment w i t h 10 U.g/ml ara-C.  This  results  evidence  90  i n d i c a t e s t h a t the n u c l e a r g r a n u l e s and p e r i n u c l e a r elements due  t o e a r l y a n t i g e n i c components o r p r o d u c t s o f HSV  the s u r f a c e f l u o r e s c e n c e  i s dependent on v i r a l DNA  are  i n f e c t i o n while  synthesis  and  assembly. I t i s apparent from t h i s and type o f v i r u s - s p e c i f i c a n t i g e n s  other  s t u d i e s t h a t the number  found i n H S V - i n f e c t e d c e l l s i s depen-  dent on the immunofluorescent t e c h n i q u e , the v i r u s s t r a i n , and preparation  o f the a n t i s e r u m .  Moreover, most o f the  a n t i g e n i c components a r e not s y n t h e s i z e d  information  cultured c e l l s .  the morphology and  However, v a r i o u s  does not p e r m i t the m i c r o s c o p i c noninfectious  particles.  much u s e f u l  development o f HSV  in  t e c h n i c a l problems have a l s o  posed c e r t a i n r e s t r i c t i o n s on the t e c h n i q u e . o f h i g h t i t r e samples c o n t a i n i n g  virus-induced  IDU.  e l e c t r o n m i c r o s c o p y has p r o v i d e d  concerning  the  a f t e r in_ v i t r o exposure of  the c e l l s t o the a n t i v i r a l agents ara-C and In the p a s t /  and  For  example, the  imuse  l a r g e numbers of d e f e c t i v e v i r u s e s d i f f e r e n t i a t i o n o f i n f e c t i o u s and  Moreover, asynchrony o f v i r u s development  makes i t d i f f i c u l t t o deduce the k i n e t i c sequence o f 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 o f t h e s e d i s a d v a n t a g e s ,  e v a l u a t i o n of e l e c t r o n m i c r o s c o p e s t u d i e s must be tempered w i t h  any  the  knowledge t h a t the r e s u l t s c o n s t i t u t e a b i a s e d view o f v i r u s s t r u c t u r e and  replication.  The  HSV  p a r t i c l e s observed i n t h i s study a f t e r n e g a t i v e  stain  p r e p a r a t i o n were s i m i l a r i n s i z e and morphology t o the v i r i o n s described  by Watson e t a l . (125) , S p r i n g  and Moss (13).  the dense c o r e .  second i n n e r envelope and HSV  (107)  Present e l e c t r o n microscopy revealed  elements o f the v i r u s - the o u t e r c a p s i d and  e t al_.  envelope, the o u t e r  and  Darlington  four s t r u c t u r a l c a p s i d , the  Other s t u d i e s have a l s o d e s c r i b e d a middle capsid  (85).  The  majority  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 t h a t  preparation  used i n t h i s r e s e a r c h  l o g i c a l l y mature v i r i o n s .  contained  a of  the  the  a l a r g e number o f morpho-  I n 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 o f  p a r t i c l e s t r u c t u r e observed a t room temperature c o n f i r m e d r e p o r t s o f HSV  inner  thermosensitivity  earlier  (122).  Under normal growth c o n d i t i o n s , t h i n - s e c t i o n e d H.Ep.2 and 21 c e l l s appeared q u i t e h e a l t h y  and  intact.  BHK-  A t the same time, s e v e r e  m o r p h o l o g i c a l changes were commonly o b s e r v e d i n BHK-21 c e l l s m a i n t a i n e d i n serumless growth medium.  These changes i n c l u d e d the development  o f abnormal p a r t i c l e s and marked c y t o p l a s m i c cells.  disruption i n affected  Such a l t e r a t i o n s have been p r e v i o u s l y d e s c r i b e d  c o n t i n u o u s and  oncogenic c e l l c u l t u r e s .  Bernhard and  i n many  Tournier  were the f i r s t t o d e t e c t v i r u s - l i k e p a r t i c l e s i n an a p p a r e n t l y BHK-21 (clone 13)  line.  The  s p h e r i c a l 85 nm  s i n g l y o r i n groups w i t h i n c y t o p l a s m i c t e r n a e o f the endoplasmic r e t i c u l u m .  (5) normal  s t r u c t u r e s were o b s e r v e d  v a c u o l e s or the s w o l l e n c i s The  p a r t i c l e s were f u r t h e r  d i s t i n g u i s h e d by t h e presence o f 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 s t r u c t u r e s t h a t appeared sequent  t o emanate from the n u c l e o i d .  s t u d i e s have s i n c e c o n f i r m e d t h e p r e s e n c e o f s i m i l a r  Sub"R"  ( r a d i a l ) 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 t r a n s f o r m e d by  (71). S V  ^Q  P a r t i c l e s have a l s o been found i n BHK-21 c e l l s  and polyoma v i r u s e s , i n hamster tumors induced  by polyoma-transformed cell  line  widespread  (6).  BHK-21 c e l l s , and i n a c o n t i n u o u s c a l f  Thus, abnormal p a r t i c l e development appears  phenomenon.  P r e v i o u s l y , however, the "R"  n o r m a l l y d e t e c t e d i n a c t i v e l y growing  kidney  t o be a  p a r t i c l e s were  c e l l s w h i l e i n t h e p r e s e n t study,  they were observed o n l y a f t e r serum s t a r v a t i o n .  Thus, i t can  be  concluded t h a t t h e c o n d i t i o n s o f serum d e f i c i e n c y induced o r a t l e a s t s t i m u l a t e d the f o r m a t i o n o f the abnormal v i r u s - l i k e s t r u c t u r e s i n the BHK-21 system.  A s i m i l a r s t i m u l a t o r y e f f e c t has been d e s c r i b e d i n  BHK-21 c e l l s i n f e c t e d w i t h r u b e l l a v i r u s  (71).  C u r r e n t t h e o r i e s t e n d t o r e g a r d the "R"  p a r t i c l e s and o t h e r  abnormal s t r u c t u r e s as m a n i f e s t a t i o n s o f l a t e n t v i r u s  infections.  However, t o d a t e , t h e r e i s l i t t l e e v i d e n c e t o support t h i s h y p o t h e s i s o t h e r than the obvious m o r p h o l o g i c a l s i m i l a r i t y o f the p a r t i c l e s t o known v i r u s e s .  The  s t r u c t u r e s may  t o some medium d e f i c i e n c y o r may agent.  e q u a l l y w e l l a r i s e i n response  r e p r e s e n t another unknown i n f e c t i o u s  Thin-section  e l e c t r o n microscopy of p r o d u c t i v e l y  infected  H.Ep.2 and B H K - 2 1 c e l l s r e v e a l e d t h a t HSV was assembled i n t h e n u c l e u s o f i n f e c t e d c e l l s and enveloped a t the n u c l e a r membrane. Previous investigations  have demonstrated a s i m i l a r c o u r s e o f  development i n a number o f mammalian 125).  cell lines  (13,53,63,99,102,  P r e s e n t o b s e r v a t i o n s showed t h a t a c t u a l v i r u s assembly was  p r e c e d e d by a l t e r a t i o n s i n t h e c e l l c h r o m a t i n and n u c l e o l i were p r o b a b l y r e l a t e d t o t h e e a r l y v i r u s - i n d u c e d DNA and RNA  synthesis  that  i n h i b i t i o n of host  ( 9 9 ) . The a l t e r e d n u c l e i a l s o e x h i b i t e d  number o f dense, g r a n u l a r aggregates p e c u l i a r  t o HSV-infected  S i m i l a r a g g r e g a t e s i n KB c e l l s tagged s p e c i f i c a l l y w i t h c o n j u g a t e d HSV a n t i b o d i e s  a cells.  ferritin-  and were thought t o be some form o f e a r l y  v i r a l antigen or precursor p a r t i c l e ( 6 4 ) . In t h i s s t u d y , naked v i r a l p a r t i c l e s were f i r s t observed i n t h e n u c l e u s o f i n f e c t e d c e l l s a f t e r 7 hours o f i n f e c t i o n .  These  scattered  p a r t i c l e s were soon s u p p l a n t e d by v i r i o n s w i t h a s i n g l e envelope and large aggregations of v i r a l capsids. p r o c e s s appeared t o be h i g h l y  In general, the v i r a l  asynchronous and i n e f f i c i e n t ,  assembly especially  during the l a t e r stages o f i n f e c t i o n ; The the  s i t e o f HSV envelopment has been p r e v i o u s l y  n u c l e a r membrane o f i n f e c t e d c e l l s  v i r u s p a r t i c l e s were o c c a s i o n a l l y lamella  (13,53).  r e p o r t e d t o be  In the present study,  seen budding t h r o u g h the i n n e r  o f t h e n u c l e a r membrane o f i n f e c t e d H.Ep.2 and B H K - 2 1  cells.  However, t h i s event was  relatively rare.  More common was  the  f i n d i n g o f e x t e n s i v e l y p r o l i f e r a t e d membranes e x t e n d i n g i n t o the cytoplasm or back i n t o the n u c l e a r m a t r i x . l i c a t i o n was  n e c e s s a r y f o r HSV  Whether t h i s  envelopment o r was  merely  redupthe p r o d u c t  of v i r u s - i n d u c e d a l t e r a t i o n s i n c e l l membranes i s n o t known.  Further-  more, the p o s s i b i l i t y o f o t h e r s i t e s o f v i r u s envelopment i n the cytoplasm c o u l d n o t be e n t i r e l y r u l e d o u t . HSV  budding  E p s t e i n (19)  demonstrated  i n t o c y t o p l a s m i c v a c u o l e s i n i n f e c t e d HeLa c e l l s w h i l e  S i m i n o f f and Menefee (102) o f the G o l g i a p p a r a t u s .  observed HSV  envelopment i n the v i c i n i t y  V i r u s envelopment may  thus o c c u r randomly  a t a number o f d i f f e r e n t c e l l membranes, i n c l u d i n g those o f t h e smooth endoplasmic  r e t i c u l u m and the G o l g i system,  although recent  e v i d e n c e s t r o n g l y i m p l i c a t e s the n u c l e a r membrane as the p r i m a r y Mature v i r u s p a r t i c l e s 170 nm i n membrane-bound v a c u o l e s and cells.  i n diameter were commonly observed  t u b u l e s i n t h e cytoplasm o f i n f e c t e d  These t u b u l e s resembled  s t r u c t u r e s d e s c r i b e d by Schwartz and  Roizman (98) i n H S V - i n f e c t e d H.Ep.2 c e l l s . mature HSV  p a r t i c l e s appeared  site.  I n the p r e v i o u s study,  t o move o u t o f the c e l l v i a a network  of f i n e , b r a n c h i n g t u b u l e s e x t e n d i n g from the n u c l e a r membrane t o the c e l l surface.  However, no e v i d e n c e o f a t r a n s p o r t system  seen i n the p r e s e n t s t u d y .  c o u l d be  V i r i o n s were r e l e a s e d from the c e l l s by a  form o f r e v e r s e p h a g o c y t o s i s s i m i l a r t o t h e method o f e g r e s s d e s c r i b e d by D a r l i n g t o n and Moss (13).  Budding v i r u s was  not observed a t the  cell  s u r  of  f a c e d e s p i t e r e p e a t e d attempts  t o confirm Epstein's early  report  HSV mode o f r e l e a s e from HeLa c e l l s ( 1 9 ) . The v a s t a r r a y s o f c r y s t a l s and membranous aggregates  i n BHK-21 c e l l s  found  a f t e r 20 hours o f i n f e c t i o n were v e r y s i m i l a r t o the  s t r u c t u r e s observed by N i i e t a l . (63) i n i n f e c t e d F L c e l l s . c a s e s , b i z a r r e v i r a l forms w e r e - r e s t r i c t e d t o c e l l s e x t e n s i v e v i r a l r e p l i c a t i o n and d e g e n e r a t i o n .  I n both  demonstrating  Moreover, t h e y  appeared  t o be composed t o numerous v i r a l components n o t i n c o r p o r a t e d 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  t o a l a r g e r number o f  m o r p h o l o g i c a l l y d e f e c t i v e v i r u s e s and a b e r r a n t v i r a l forms than d i d H.Ep.2 c e l l s .  P r e v i o u s growth s t u d i e s a l s o showed t h a t t h e hamster  c e l l s produced  lower t i t r e s  of infectious virus.  appear t h a t i n e f f i c i e n c i e s i n assembly ponents g i v e r i s e  Thus, i t would  and p r o d u c t i o n o f v i r a l com-  t o t h e reduced y i e l d s o f HSV found i n BHK-21 c e l l s .  The p r e s e n t study a l s o r e v e a l e d t h a t t h e a d d i t i o n o f 10 yg/ml ara-C a t t h e time o f i n f e c t i o n p r e v e n t e d t h e f o r m a t i o n o f 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, t h e drug d i d n o t  prevent the e a r l y chromatin displacement or the p r o d u c t i o n o f g r a n u l a r aggregates i n t h e n u c l e u s o f c e l l s  i n f e c t e d w i t h HSV.  These r e s u l t s  c o n f i r m p r e v i o u s b i o c h e m i c a l s t u d i e s c o n c e r n i n g t h e e f f e c t o f 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 t o  p e r m i t f o r m a t i o n o f HSV s t r u c t u r a l u n i t s and a n t i g e n s b u t c o m p l e t e l y 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 t o p r e v e n t n u c l e a r d i s o r g a n i z a t i o n and  p r e c u r s o r s y n t h e s i s i n H S V - i n f e c t e d H.Ep.2 and BHK-21 c e l l s .  How-  ever, ragged, m o r p h o l o g i c a l l y abnormal p a r t i c l e s were o c c a s i o n a l l y d e t e c t e d i n the cytoplasm o f t r e a t e d c e l l s .  Smith  (104) r e p o r t e d  t h e presence o f s i m i l a r d e f e c t i v e p a r t i c l e s i n H S V - i n f e c t e d t r e a t e d w i t h IDU.  cells  B i o c h e m i c a l s t u d i e s have a l s o shown t h a t IDU i s  d i r e c t l y i n c o r p o r a t e d i n t o HSV DNA and t h a t d e f e c t i v e l a t e p r o t e i n s a r e subsequently produced  (37).  As a r e s u l t , v i r u s assembly i s  s e v e r e l y i m p a i r e d , and a l t h o u g h a few abnormal p a r t i c l e s a r e produced, no i n f e c t i o u s v i r u s c a n be d e t e c t e d .  In c o n t r a s t , HSV-infected  c e l l s t r e a t e d w i t h another halogenated p y r i m i d i n e , BudR,  produced  v i r u s - s p e c i f i c a n t i g e n s b u t no p a r t i c l e s o f 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 o f ara-C and IDU observed p r e v i o u s l y i n t h i s study under t h e 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 t h e t h i n  s e c t i o n s o f H.Ep.2 and BHK-21 c e l l s .  A f t e r 48 hours o f drug  exposure,  c e l l s e x h i b i t e d marked a l t e r a t i o n s i n c l u d i n g d i s t o r t i o n o f t h e m i t o c h o n d r i a , s w e l l i n g o f t h e endoplasmic t h e cytoplasm. and extended  r e t i c u l u m and v a c u o l i z a t i o n o f  Thus, t h e p r e s e n t e l e c t r o n microscope  work c o n f i r m e d  p r e v i o u s o b s e r v a t i o n s o f ara-C and IDU-induced c y t o -  pathology. HSV i n f e c t i o n r e s u l t e d i n an immediate r e d u c t i o n o f thymidine  uptake i n t o t h e DNA o f H.Ep.2 and BHK-21 c e l l s .  This early  inhibition  was f o l l o w e d by a sharp i n c r e a s e i n DNA s y n t h e s i s a t 5 hours o f i n f e c t ion.  S i m i l a r r e s u l t s have been o b t a i n e d by Roizman e t a l .  i n H.Ep.2 c e l l s and R u s s e l l e t a l .  (96) i n BHK-21 c e l l s .  (86,89) Previous  work has a l s o shown t h a t HSV i n h i b i t s h o s t c e l l DNA s y n t h e s i s w i t h i n 2-3 hours and t h a t v i r a l DNA i s f i r s t d e t e c t e d a f t e r 4-5 hours o f infection. i n most c e l l  V i r a l DNA s y n t h e s i s i s v i r t u a l l y completed by 14 hours systems.  I n t h e p r e s e n t s t u d y , t h e e a r l y i n h i b i t i o n o f h o s t DNA appeared c h r o n o l o g i c a l l y r e l a t e d t o t h e n u c l e a r d i s r u p t i o n and c h r o m a t i n d i s placement observed i n i n f e c t e d c e l l s w i t h t h e l i g h t and e l e c t r o n microscope.  Furthermore, the o n s e t o f v i r a l DNA s y n t h e s i s  closely  preceded t h e appearance o f immunofluorescent a n t i g e n s and naked v i r a l capsids i n the nucleus.  Maximum c y t o p a t h i c e f f e c t s were u s u a l l y  seen  a f t e r t h e b u l k o f v i r a l DNA had been s y n t h e s i z e d i n H.Ep.2 and BHK-21 cells. Ara-C and IDU c o m p l e t e l y i n h i b i t e d DNA s y n t h e s i s as measured by 3 H-thymidine uptake i n u n i n f e c t e d and H S V - i n f e c t e d c e l l s  ( F i g . 30).  These r e s u l t s c o r r e s p o n d w i t h e a r l i e r i n v i t r o s t u d i e s by Smith Smith and Dukes (105), L e v i t t and Becker  (104) ,  (46), and Cohen ( 1 1 ) .  The m i t o t i c index o f H.Ep.2 and BHK-21 c e l l s i n c r e a s e d  sharply  a f t e r i n f e c t i o n w i t h HSV, r e a c h i n g i t s peak a t 6 hours and t h e n dec l i n i n g t o a l e v e l o f complete i n h i b i t i o n a t 24 hours o f i n f e c t i o n .  98  In t h e f i r s t hours o f i n f e c t i o n f o l l o w i n g i n h i b i t i o n o f h o s t synthesis, c e l l s already  i n m i t o s i s appeared t o be p r e v e n t e d from  c o m p l e t i n g t h e d i v i s i o n c y c l e and were thus d e t e c t e d numbers a t metaphase. and  DNA  i n increasing  However, a f t e r i n i t i a t i o n o f v i r u s r e p l i c a t i o n  development o f c y t o p a t h i c  e f f e c t s , t h e number o f c e l l s  d e c r e a s e d u n t i l c e l l d i v i s i o n ceased a l t o g e t h e r In c o n t r a s t , S t o k e r and Newton (112) r e p o r t e d  i n mitosis  i n infected cultures.  t h a t m i t o s i s was r a p i d l y  i n h i b i t e d i n parasynchronous HeLa c u l t u r e s i n f e c t e d w i t h HSV 2 hours p r i o r t o t h e c a l c u l a t e d time o f c e l l d i v i s i o n . Ara-C and IDU d i d n o t appear t o a l t e r t h e number o f H S V - i n f e c t e d cells  i n mitosis.  M i t o t i c e f f e c t s were t h e r e f o r e an e a r l y v i r a l  f u n c t i o n independent o f ensuing v i r a l DNA s y n t h e s i s .  On t h e o t h e r  hand, ara-C and IDU c o m p l e t e l y i n h i b i t e d m i t o s i s i n u n i n f e c t e d and  BHK-21 c e l l s w i t h i n 6-8 hours  ( F i g . 3 1 ) . S i m i l a r drug-induced  e f f e c t s have been o b s e r v e d i n r a b b i t k i d n e y  (9) and HeLa c e l l s ( 4 3 ) .  A number o f mammalian v i r u s e s a r e c a p a b l e o f i n d u c i n g irregularities  i n cultured c e l l s .  H.Ep.2  chromosome  The b i o l o g i c a l s i g n i f i c a n c e o f  t h e s e changes i s n o t y e t c l e a r b u t i s o f p o t e n t i a l importance i n c e l l death, carcinogenesis, c e l l mutation  (60).  some a b n o r m a l i t i e s 97,110).  aging,  teratogenesis,  and somatic and germ  HSV has been p r e v i o u s l y r e p o r t e d i n a v a r i e t y o f mammalian c e l l s  In the present  c e l l s resulted i n v i s i b l e  t o cause chromo-  (7,27,28,34,68,  study, HSV i n f e c t i o n o f H.Ep.2 and BHK-21 damage t o c e l l chromosomes.  The v i r u s -  induced aberrations  included  chromatid gaps, b r e a k s , secondary con-  s t r i c t i o n s , f r a g m e n t a t i o n , e r o s i o n and e n d o r e d u p l i c a t i o n . cytogenetic  terms commonly r e f e r t o v a r i o u s  s t a i n e d metaphase p r e p a r a t i o n s  These  l e s i o n s observed i n  o f c e l l chromosomes.  Thus, chromatid  gaps appear as u n s t a i n e d , l o o s e l y c o i l e d chromosome segments w h i l e breaks c o n s t i t u t e a c t u a l i n t e r r u p t i o n s o f t h e chromosome t h a t t o 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  lead  represent  p a r t i a l chromosome l e s i o n s and f r a g m e n t a t i o n , a s e r i e s o f m u l t i p l e breakages.  The b l u r r e d o u t l i n e s o f eroded chromosomes a r e a p p a r e n t l y  caused by t r a n s i e n t c o i l i n g anomalies and e n d o r e d u p l i c a t i o n r e p l i c a t i o n i n t h e absence o f a s p i n d l e . virus-induced  interference with c e l l protein synthesis,  e f f e c t s mediated by d i s r u p t e d  enzyme  lysozomes, and a d i r e c t combination o f  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 y e t , however, no one mechanism  been e s t a b l i s h e d on t h e s i n g l e cause o f chromosome Any  o f these  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 t o i n h i b i t i o n o f  c e l l DNA s y n t h e s i s ,  has  The p r o d u c t i o n  by chromosome  abnormalities.  o r a l l o f t h e aforementioned l e s i o n s may o c c u r i n one o r both  chromatids o f a g i v e n structures  c e l l chromosome.  Chromosomes behave as s i n g l e  i n t h e G l phase o f t h e c e l l c y c l e b e f o r e DNA s y n t h e s i s has  taken p l a c e .  Therefore,  i f a defect  i s produced a t t h i s time, t h e  l e s i o n i s r e p l i c a t e d a l o n g w i t h t h e second c h r o m a t i d d u r i n g  the S  or DNA s y n t h e s i s p e r i o d and t h e r e s u l t i s a f u l l chromosome abnormality.  However, i f t h e i n j u r y  o c c u r s a f t e r t h e chromosome has  s y n t h e s i z e d i t s DNA  i n the  l a t e S or G2  i s already a dual structure, result.  l a t e S or G2  phase o f the c e l l c y c l e  addition, virus-infected cultures o f exchanges or t r a n s l o c a t i o n s . suming t h a t h o s t DNA  synthesis  synthesis  involved  chromosomes (60).  r a r e l y g i v e r i s e to large If Taylor  (115)  i s correct  In  numbers in  as-  i s n e c e s s a r y t o a r e u n i o n of broken  chromosomes, many o f the v i r u s e s p r e v e n t exchanges by DNA  usual  observed t o date i n v i r u s - i n f e c t e d  chromatid v a r i e t y , i n d i c a t i n g t h a t the  are a f f e c t e d i n the  chromosome  a s i n g l e chromatid l e s i o n i s the  Most o f the a b e r r a t i o n s  c e l l s a r e o f the  p e r i o d when the  inhibiting cell  i n repair.  In the p r e s e n t study, p r e v i o u s o b s e r v a t i o n s c o n c e r n i n g e f f e c t o f v i r u s dose, t y p e of h o s t c e l l and  the  time of i n f e c t i o n  on  the amount o f 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. and  number o f chromosome a b e r r a t i o n s  BHK-21 c e l l s i n c r e a s e d  ion. by  The  w i t h time and  D e f e c t s were d e t e c t e d as e a r l y as  20 hours, 100%  hamster c u l t u r e s  errations  v i r u s m u l t i p l i c i t y of i n f e c t 2 hours a f t e r i n f e c t i o n  o f the c e l l s observed a t metaphase e x h i b i t e d  type o f chromosome a b n o r m a l i t y . and  i n i n f e c t e d H.Ep.2  S i m i l a r damage was  and some  observed i n human  a l t h o u g h the v i r u s a p p a r e n t l y produced more  a t an e a r l i e r time i n H.Ep.2 c e l l s .  chromosome a l t e r a t i o n s was  No  evidence of s p e c i f i c  seen i n e i t h e r c e l l l i n e d e s p i t e  the  t h a t a number o f i n v e s t i g a t o r s have r e p o r t e d non-random l e s i o n s H S V - i n f e c t e d Chinese hamster c e l l s  (110)  and  ab-  mastomys (34)  fact in  cultures.  101  P r o l o n g e d UV i r r a d i a t i o n o f HSV p r e v e n t e d t h e i n d u c t i o n o f a l l aberrations  i n infected c e l l s .  Moreover, t h e c a p a c i t y o f t h e v i r u s  t o damage h o s t 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 t o UV i n a c t i v a t i o n than was t h e i n f e c t i o u s p r o p e r t y . reported  Waubke e t al_. (126)  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 o f l a b e l e d HSV.  Upon  f u r t h e r i n v e s t i g a t i o n , they found e v i d e n c e o f i m p a i r e d v i r u s ads o r p t i o n a f t e r i r r a d i a t i o n a l t h o u g h they were n o t a b l e t o conclude t h a t t h e absence Waubke's group  o f chromosome damage was e n t i r e l y due t o t h i s  effect.  a l s o showed t h a t t h e i n d u c t i o n o f chromosome l e s i o n s  preceded and was thus independent o f v i r a l DNA  replication.  E v i d e n c e f o r t h e i n t e r f e r e n c e o f normal p r o t e i n s y n t h e s i s as a mechanism f o r i n d u c t i o n o f chromosome breaks has been o b t a i n e d from various  systems employing  d e f i c i e n t media and mycoplasma ( 6 0 ) .  Mycoplasma-induced breaks i n c u l t u r e d c e l l s were e f f e c t i v e l y p r e v e n t e d by the a d d i t i o n o f excess a r g i n i n e which i s known t o be d e p l e t e d from i n 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  itself  caused chromosome damage i n a number o f a c t i v e l y growing  cells.  Thus,  a l t h o u g h no e v i d e n c e o f mycoplasma i n f e c t i o n was found i n t h e H.Ep.2 or BHK-21 c e l l s used i n t h i s r e s e a r c h , if  i t was o f i n t e r e s t t o determine  t h e HSV-induced chromosome a b n o r m a l i t i e s  r e s u l t e d from  arginine  d e p l e t i o n , s i n c e i t i s known t h a t t h e amino a c i d i s r e q u i r e d f o r i n v i t r o v i r u s maturation increases  (3).  However, i n t h e p r e s e n t study, l a r g e  i n arginine f a i l e d t o prevent aberrations  i n HSV-infected  cells.  Thus, i t must be concluded that the v i r u s does not injure c e l l  chromosomes by i n t e r f e r i n g with the metabolism of arginine and the protein synthesis necessary f o r r e p a i r . Ara-C has been previously reported to induce chromosome breakage i n human leukocytes (8) and WI-38 c e l l s (61) as early as 1-3 hours a f t e r inoculation.  The present study confirms the production of  chromatid breaks and secondary constrictions a f t e r 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 s i g n i f i c a n t l y 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 i n h i b i t o r of DNA synthesis, the aberrations may have been related to the drug-induced i n h i b i t i o n of repair DNA synthesis i n mammalian c e l l s (60). In HSV-infected c e l l s treated with ara-C, the number of chromosome abnormalities was f a r greater than the combined effects of the two p o t e n t i a l mutagens.  O'Neill and Rapp (69) reported a similar  synergism i n HSV-infected human embryonic lung cultures.  The drug  and v i r u s apparently a c t together to produce c e l l s containing many multiple breaks.  Simultaneous autoradiography performed i n this  study on uninfected and HSV-infected c e l l s showed that ara-C  complete-  l y blocked DNA synthesis. Therefore, since ara-C d i d not prevent the chromosome abnormalities induced by HSV, i t can be concluded that  the l e s i o n s o c c u r r e d  i n the absence o f v i r a l DNA s y n t h e s i s .  Previous  s t u d i e s have suggested t h a t t h e a n t i m e t a b o l i t e and v i r u s a c t t o prevent  t h e r e p a i r o f c e l l DNA and thus induce v i s i b l e  chromosome  damage. In c o n t r a s t t o ara-C t r e a t e d c u l t u r e s , c e l l s exposed t o v a r i o u s concentrations  o f IDU d i d n o t g e n e r a l l y e x h i b i t a s i g n i f i c a n t  increase  i n t h e number o f chromosome a b e r r a t i o n s e x c e p t a t v e r y h i g h drug levels.  Thus, d e s p i t e i t s c a p a c i t y t o i n h i b i t c e l l DNA s y n t h e s i s as 3  measured by  H-thymidine uptake, t h e a n t i - v i r a l drug i s n o t an a c t i v e  agent o f chromosome damage. S i m i l a r l y , i n the present IDU  showed no evidence  study, H S V - i n f e c t e d  o f synergism.  c e l l s exposed t o  The number o f chromosome ab-  n o r m a l i t i e s i n such c e l l s e q u a l l e d t h e sum o f t h e l e s i o n s produced by HSV and IDU a l o n e . occurred  As w i t h ara-C, t h e v i r u s - i n d u c e d  defects  i n the absence o f c e l l and v i r u s DNA s y n t h e s i s .  Similar r e -  s u l t s were o b t a i n e d by O ' N e i l l and Rapp (70) i n human embryonic lung  cells. The  p r e s e n t work has r e v e a l e d t h a t u n i n f e c t e d and  HSV-infected  c e l l s t r e a t e d w i t h a combination o f ara-C and IDU showed a a d d i t i v e type o f chromosome damage.  simple  I t thus appeared t h a t t h e mole-  c u l a r i n t e r a c t i o n s r e s p o n s i b l e f o r t h e synergism o f ara-C and HSV were n o t p r e s e n t  i n t h e ara-C/IDU system.  A l t h o u g h t h e s i g n i f i c a n c e o f a b e r r a t i o n s induced  by v i r u s e s and  c h e m i c a l s i s not known w i t h c e r t a i n t y , t h e r e a r e a t l e a s t f o u r a r e a s o f a c t u a l o r p o t e n t i a l importance i n mammalian c e l l s .  These a r e c e l l  death, g e n e t i c damage t h a t p r e v e n t s f u r t h e r c e l l d i v i s i o n , changes i n chromosome number, and somatic and g e r m l i n e m u t a t i o n s .  Virus-induced  c e l l d e a t h and m i t o t i c i n h i b i t i o n may be o f g r e a t e s t s i g n i f i c a n c e i n the f i e l d o f t e r a t o g e n e s i s where i t c o u l d l e a d t o f e t a l t i e s and spontaneous a b o r t i o n . are n o r m a l l y  abnormali-  Most o f the c e l l s i n f e c t e d w i t h  expected t o cease d i v i d i n g and d i e .  small percentage o f c e l l s t h a t e x h i b i t v i s i b l e  HSV  These i n c l u d e t h e  chromosome d e f e c t s  a t a metaphase and t h e l a r g e m a j o r i t y o f c e l l s t h a t do n o t undergo mitosis.  Thus, i n l i g h t o f t h e c a p a c i t y o f t h e v i r u s t o cause human  g e n i t a l i n f e c t i o n s (57), t h e mutagenic a c t i o n o f HSV c o u l d a b l y be i n v o l v e d i n c e l l damage d u r i n g Virus-induced  conceiv-  embryogenesis.  aberrations t h e o r e t i c a l l y could a l s o r e s u l t i n  changes i n chromosome number o r somatic m u t a t i o n s t h a t s u b s e q u e n t l y might l e a d t o malignancy. transformation carcinoma  HSV has a l r e a d y been i m p l i c a t e d i n c e l l  (14) and p o s s i b l y i n the p r o d u c t i o n  (56,76).  n o r m a l i t i e s induced  o f human c e r v i c a l  Thus, t h e v i s i b l e and s u b v i s i b l e metaphase abby t h e v i r u s may indeed  be r e l a t e d t o t h e p r o c e s s  o f oncogenesis and p o s s i b l y i n t e g r a t i o n and l a t e n c y . i n t e r e s t a t present,  Of even more  i s t h e secondary f i n d i n g t h a t t h e chemotherapeutic  agents used t o combat HSV i n f e c t i o n i n d u c e s i m i l a r chromosome damage and  thus a l s o 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  o f normal  c e l l growth and d i v i s i o n . In summary, HSV was found t o cause p r o f o u n d m e t a b o l i c and morphological alterations  alterations included  i n i n f e c t e d H.Ep.2 and BHK-21 c e l l s . These  an i n h i b i t i o n o f h o s t DNA s y n t h e s i s and m i t o s i s  f o l l o w e d by c h a r a c t e r i s t i c n u c l e a r d i s r u p t i o n ,  formation of i n t r a -  n u c l e a r i n c l u s i o n b o d i e s , and p r o d u c t i o n o f v a r i o u s a n t i g e n s and chromosome a b n o r m a l i t i e s .  Ara-C and IDU f a i l e d t o p r e v e n t t h e v i r u s -  induced c y t o p a t h i c e f f e c t s i n v i t r o b u t d i d i n h i b i t v i r a l DNA t h e s i s and assembly o f i n f e c t i o u s p a r t i c l e s . the  Moreover, exposure o f  c e l l s t o t h e a n t i - v i r a l agents themselves r e s u l t e d  pathic  alterations involving  o f DNA s y n t h e s i s and m i t o s i s , chromosome damage.  i n severe c y t o -  cytoplasmic disorganization, and i n d u c t i o n  syn-  inhibition  of various levels of  BIBLIOGRAPHY  Baserga, R. 1965. The r e l a t i o n s h i p o f t h e c e l l c y c l e t o tumor growth and c o n t r o l o f c e l l d i v i s i o n . Cancer Res. 25: 581-595. Becker, Y., Dym, H., and Sarov, DNA. V i r o l o g y 36: 184-192.  I.  1968.  Herpes simplex  virus  Becker, Y., Olshevsky, U., and L e v i t t , J . 1967. The r o l e o f a r g i n i n e i n the r e p l i c a t i o n o f herpes simplex v i r u s . J . gen. V i r o l . 1: 471-478. Ben-Porat, T. and Kaplan, A. 1962. The c h e m i c a l c o m p o s i t i o n o f herpes simplex and p s e u d o r a b i e s v i r u s e s . V i r o l o g y 16: 261-266. Bernhard, W. and T o u r n i e r , P. 1964. I n f e c t i o n v i r a l e i n a p p a r e n t e de c e l l u l e s de hamster d e c e l e e p a r l a m i c r o s c o p i e e l e c t r o n i g u e . Ann. I n s t . P a s t . 107: 447-455. Beswick, T.S.L. 1962. Med. H i s t . 6_: 214-232.  The o r i g i n and use o f t h e word h e r p e s .  B o i r o n , M., Tanzer, J . , Thomas M. and Hampe, A. 1966. Early d i f f u s e chromosome a l t e r a t i o n s i n monkey k i d n e y c e l l s i n f e c t e d w i t h herpes simplex v i r u s . Nature 209: 737-738. Brewen, J.G. and C h r i s t i e , N.T. 1967. S t u d i e s on t h e i n d u c t i o n o f chromosomal a b e r r a t i o n s i n human l e u k o c y t e s by c y t o s i n e a r a b i n o s i d e . E x p t . C e l l . Res. 46_: 276-291. B u t h a l a , D.A. 1964. C e l l c u l t u r e s t u d i e s o f a n t i v i r a l a g e n t s . I . A c t i o n o f c y t o s i n e a r a b i n o s i d e and some comparisons w i t h 5iodo-2-deoxyuridine. P r o c . Soc. Expt. B i o l . Med. 115: 69-77. Chang, T. 1971. 284: 765-773.  Recurrent v i r a l i n f e c t i o n s .  New. Eng. J . Med.  Cohen, S. 1966. I n t r o d u c t i o n t o t h e b i o c h e m i s t r y o f the D-arabi n o s y l n u c l e o s i d e s . P r o g r . N u c l . A c i d Res. 5_: 1-88.  107  12.  Compans, R.W., Holmes, K.V., Dales, S., and Choppin, P.W. 1964. An electron microscope study of moderate and v i r u l e n t v i r u s - c e l l interactions of the parainfluenza virus SV5. Virology 30: 411-426.  13.  Darlington, R.W. and Moss, L.H. J . V i r o l . _2: 48-55.  14.  Duff, R. and Rapp, F. 1971. Properties of hamster embryo f i b r o blasts transformed i n v i t r o 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. J . V i r o l . 8j 469-477.  15.  E j e r c i t o , P.M., K i e f f , E.D. and Roizman, B. 1968. Characterizat i o n of herpes simplex virus strains d i f f e r i n g i n t h e i r e f f e c t on the s o c i a l behavior of infected c e l l s . J . gen. V i r o l . 3^: 357-364.  16.  E l f o r d , W.J., Perdrau, J.R., and Smith, W. 1933. The f i l t r a t i o n of herpes virus through graded collodion membranes. J . Path. Bact. 36: 49-54.  17.  E l l i s o n , S.A., Carton, C.A., and Rose, H.M. 1959. Studies of recurrent herpes simplex infections following section of the trigeminal nerve. J . Infect. Dis. 105: 161-173.  18.  Epstein, M.A. 1961. Observations on the f i n e structure of mature herpes simplex virus and on the composition of i t s nucleoid. J . Expt. Med. 115: 1-12.  19.  Epstein, M.A. 1962. Observations on the mode of release of herpes virus from infected HeLa c e l l s . J . C e l l B i o l . 12: 589-597.  20.  Figueroa, M.E. and Rawls, W.E. 1969. B i o l o g i c a l markers f o r d i f f e r e n t i a t i o n of herpesvirus strains of o r a l and g e n i t a l o r i g i n . J . gen. V i r o l . 4: 259-267.  21.  Flanagan, J . 1967. V i r u s - s p e c i f i c ribonucleic acid synthesis i n c e l l s infected with herpes ,simplex v i r u s . J . V i r o l . 1_: 583-590.  22.  Geder, L. and Vaczi, L. 1968. L o c a l i z a t i o n of nuclear and cytoplasmic herpes simplex antigens i n infected c e l l s by immunofluorescence. Acta V i r o l . 12^ 97-105.  23.  Goodheart, G., Plummer, G. and Waner, J.L. 1968. Density d i f ference of DNA of human herpes simplex viruses types I and I I . Virology 35: 473-475.  24.  G r a v e l l , M., Granoff, A. and Darlington, R.W. 1968. Viruses of renal carcinoma of Rana pipiens. VII. Propagation of a herpestype frog v i r u s . Virology 36: 467-475.  1968.  Herpesvirus envelopment.  108  25.  Green, M. 1966. B i p s y n t h e t i c m o d i f i c a t i o n s induced by animal v i r u s e s . Ann. Rev. M i c r o . 20_: 209-211.  26.  Greenberg, M. and Brightman, V. 1972. Serum immunoglobulins p a t i e n t s w i t h r e c u r r e n t i n t r a o r a l herpes s i m p l e x i n f e c t i o n s . J . Dent. Res. 50_: 781.  27.  Hampar, B. and E l l i s o n , S. 1961. Chromosomal a b e r r a t i o n s i n d u c e d by an animal v i r u s . Nature 192: 145-147.  28.  Hampar, B. and E l l i s o n , S. 1963. C e l l u l a r a l t e r a t i o n s i n t h e MCH l i n e o f Chinese hamster c e l l s f o l l o w i n g i n f e c t i o n w i t h herpes simplex v i r u s . P r o c . Nat. Acad. S c i . 49_: 474-480.  29.  H a r l e y , E.H., Rees, K.R., and Cohen, A. 1970. HeLa c e l l n u c l e i c a c i d s y n t h e s i s : The e f f e c t o f mycoplasma c o n t a m i n a t i o n . Biochim. B i o p h y s . A c t a 213: 171-182.  30.  Hochberg, E. and Becker, Y. 1968. u n c o a t i n g o f herpes simplex v i r u s .  31.  Holden, M. 1932. The n a t u r e and p r o p e r t i e s o f the v i r u s o f h e r p e s . J . I n f e c t . D i s . 50: 218-236.  32.  Holmes, I . and Watson, D.H. 1963. An e l e c t r o n microscope study o f the attachment and p e n e t r a t i o n o f herpes v i r u s i n BHK-21 c e l l s . V i r o l o g y 21: 112-123.  33.  Huang, A.S. and Wagner, R.R. 1964. P e n e t r a t i o n o f herpes v i r u s i n t o human epidermoid c e l l s . P r o c . Soc. E x p t . B i o l . 116: 863-869.  34.  Huang, C C . 1967. I n d u c t i o n o f a h i g h i n c i d e n c e o f damage t o the X-chromosome o f R a t t u s (mastomys) n a t a l e n s i s by base analogues, v i r u s e s and chromosomes. Chromosoma 23_: 162-179.  35.  Johnson, R. 1964. The p a t h o g e n e s i s o f herpes v i r u s J . E x p t . Med. 119: 343-355.  36.  Kaplan, A. 1969. Herpes simplex and p s e u d o r a b i e s V i r o l . Monogr. _5: 1-115.  37.  Kaplan, A. and Ben-Porat, T. 1966. Mode o f a c t i o n o f 5 - i o d o u r a c i l d e o x y r i b o s i d e . J . M o l . B i o l . 19_: 320-332.  in  A d s o r p t i o n , p e n e t r a t i o n and J . gen. V i r o l . 2i 231-240.  i  simplex Med.  encephalitis.  viruses.  109  38.  Kaplan, A., Brown, M., and Ben-Porat, T. 1968. E f f e c t o f l - g D a r a b i n o f u r a n o s y l c y t o s i n e on DNA s y n t h e s i s . M o l . Pharm. 4_: 131-138.  39.  Kaufman, H. 1962. C l i n i c a l c u r e o f herpes s i m p l e x k e r a t i t i s by 5-iodo-2-deoxyuridine. P r o c . Soc. Expt. B i o l . Med. 109: 251-252.  40.  Kaufman, H., Brown, D., and E l l i s o n , E . 1967. R e c u r r e n t herpes i n the r a b b i t and man. S c i e n c e 156: 1628-1629.  41.  K i e f f , E . , Bachenheimer, S., and Roizman, B. 1971. S i z e , compos i t i o n and s t r u c t u r e o f t h e DNA o f HSV Subtypes 1 and 2. J . V i r o l . 8: 125-132.  42.  K i e r , H.M., Subak-Sharpe, H., Shedden, W.I.H., Watson, D.H., and Wildy, P. 1966. Immunological evidence f o r a s p e c i f i c DNA polymerase produced a f t e r i n f e c t i o n by herpes simplex v i r u s . V i r o l o g y 30_: 154-157.  43.  Kim, J.H. and E i d i n o f f , M. 1965. A c t i o n o f 1-3-D a r a b i n o f u r a n o s y l c y t o s i n e on t h e n u c l e i c a c i d metabolism and v i a b i l i t y o f HeLa cells. Cancer Res. 23: 698-702.  44.  Klemperer, H.G., Haynes, G.R., Shedden, W.I.H., and Watson D.H. 1967. A v i r u s - s p e c i f i c thymidine k i n a s e i n BHK-21 c e l l s i n f e c t e d w i t h herpes simplex v i r u s . V i r o l o g y 31: 120-128.  45.  L e s s o , J . and Szanto, J . 1969. R e p r o d u c t i o n o f HSV i n HeLa c e l l s s t u d i e d by immunofluorescence and c y t o c h e m i c a l methods. A c t a V i r o l . 13: 278-284.  46.  L e v i t t , J . and Becker, Y. 1967. The e f f e c t o f c y t o s i n e a r a b i n o s i d e on t h e r e p l i c a t i o n o f herpes simplex v i r u s . V i r o l o g y 31_: 129-134.  47.  M a r s h a l l , W.J.S. 1967. Herpes simplex e n c e p h a l i t i s t r e a t e d w i t h i d o x u r i d i n e and e x t e r n a l decompression. The L a n c e t , Sept. 16: 579-580.  48.  Melendez, L., Hunt, R., D a n i e l , M., G a r c i a , F. and F r a s e r , C. 1969. Herpesvirus s a i m i r i . I I . E x p e r i m e n t a l l y induced m a l i g n a n t lymphoma i n p r i m a t e s . Lab. Animal Care 19: 378-386.  49.  M e l n i c k , J . , M i d u l l a , M., Wimberly, I . , B a r r e r a - O r o , J . , and Levy, B. 1964. A new member o f the h e r p e s v i r u s group i s o l a t e d from South American marmosets. J . Immunol. 92: 595-601.  110  50.  Miyamoto, K. and Morgan, C. 1971. S t r u c t u r e and development o f v i r u s e s as observed i n the e l e c t r o n m i c r o s c o p e . X I . E n t r y and u n c o a t i n g o f herpes simplex v i r u s . J . V i r o l . 8j 910-918.  51.  Moore, A.E., Sabachewsky, L., and T o o l a n , H. 1955. C u l t u r e c h a r a c t e r i s t i c s o f f o u r permanent l i n e s o f human cancer c e l l s . Cancer Res. 15: 598-604.  52.  Morgan, C , E l l i s o n , S.A., Rose, H.M., and Moore, D.H. 1954. S t r u c t u r e and development o f v i r u s e s as observed i n t h e e l e c t r o n microscope. I . Herpes simplex v i r u s . J . E x p t . Med. 100: 195-202.  53.  Morgan, C , Rose, H.M., Holden, M., and Jones, E . 1959. E l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s on the development o f herpes simplex virus. J . E x p t . Med. 110: 643-656.  54.  M o r r i s o n , J.M. and K e i r , H.M. 1968. A new DNA-exonuclease i n c e l l s i n f e c t e d w i t h herpes v i r u s : P a r t i a l p u r i f i c a t i o n and p r o p e r t i e s o f the enzyme. J . gen. V i r o l . 3_: 337-347.  55.  Munk, K. and Sauer, G. 1964. R e l a t i o n s h i p between c e l l DNA metabolism and n u c l e o c y t o p l a s m i c a l t e r a t i o n s i n herpes v i r u s infected c e l l s . V i r o l o g y 22} 153-154.  56.  Nahmias, A . J . , Naib, Z., and J o s e y , W. 1971. H e r p e s v i r u s hominis type 2 i n f e c t i o n - A s s o c i a t i o n w i t h c e r v i c a l c a n c e r and p e r i n a t a l d i s e a s e . P e r s p e c t i v e s i n V i r o l o g y V I I : 73-89.  57.  Naib, Z., Nahmias, A . J . , J o s e y , W., and Kramer, J . herpetic infection. Cancer 23: 940-945.  58.  Nardone, B., Todd, J . , Gonzalez, P., and G a f f n e y , E.V. 1965. Nucleoside incorporation i n t o s t r a i n L c e l l s . I n h i b i t i o n by p l e u r o p n e u m o n i a - l i k e organisms. S c i e n c e 149: 1100-1101.  59.  N a z e r i a n , K., Solomon, J . , W i t t e r , R., and Burmester, B. 1967. S t u d i e s on t h e e t i o l o g y o f Marek's d i s e a s e . I I . F i n d i n g o f a herpes v i r u s i n c e l l c u l t u r e . P r o c . Soc. E x p t . B i o l . Med. 127:  1969.  Genital  177-182. 60.  N i c h o l s , W. 1970. V i r u s - i n d u c e d chromosomal a b n o r m a l i t i e s . Ann. Rev. M i c r o b i o l . 24: 479-499.  61.  N i c h o l s , W. and Hengen, W. 1964. Chromosomal e f f e c t s o f a r a b i n o s y l c y t o s i n e i n a human d i p l o i d c e l l s t r a i n . H e r e d i t a s 52: 402-410.  Ill  62.  N i i , S. and Kamahora, J . 1961. C y t o p a t h i c changes i n d u c e d by herpes simplex v i r u s . Biken's J . £ : 255-270.  63.  N i i , S., Morgan, C , and Rose, H.M. 1968. E l e c t r o n microscopy of herpes simplex v i r u s . I I . Sequence o f development. J . V i r o l . 2: 517-536.  64.  N i i , S., Morgan, C , Rose, H.M., and Hsu, K.C. 1968. microscopy o f herpes simplex v i r u s . IV. S t u d i e s w i t h c o n j u g a t e d a n t i b o d i e s . J . V i r o l . 2_: 1172-1183.  65.  N i i , S., Rosenkranz, S., Morgan, C , and Rose, H.M. 1968. E l e c t r o n microscopy o f herpes simplex v i r u s . I I I . E f f e c t o f hydroxyurea. J . V i r o l . 2i 1163-1171.  66.  Olshevsky, U. and Becker, Y. 1970. Herpes simplex v i r u s s t r u c t u r a l p r o t e i n s . V i r o l o g y 40_: 948-960.  67.  O l s h e v s k y , U., L e v i t t , J . , and Becker, Y. 1967. S t u d i e s on the s y n t h e s i s o f herpes simplex v i r i o n s . V i r o l o g y 33: 323-334.  68.  O ' N e i l l , F . J . and M i l e s , C. 1969. Chromosome changes i n human c e l l s i n d u c e d by herpes simplex v i r u s , types 1 and 2. Nature 223: 851-852.  69.  O ' N e i l l , F . J . and Rapp, F. 1971. S y n e r g i s t i c e f f e c t o f herpes simplex and c y t o s i n e a r a b i n o s i d e on human chromosomes. J . V i r o l . 7_: 692-695.  70.  O ' N e i l l , F . J . and Rapp, F. 1971. E a r l y events f o r i n d u c t i o n o f chromosome a b n o r m a l i t i e s i n human c e l l s by herpes simplex v i r u s . V i r o l o g y 4£: 544-553.  71.  Payment, P., Chagnon, A., C o t e , J.R., A j d u k o v i c , D. and P a v i l a n i s , V. 1972. Morphology o f a l a t e n t v i r u s a s s o c i a t e d w i t h hamster c e l l s BHK-21. Can. J . M i c r o b i o l . 113: 369-371.  72.  Plummer, G., Waner, J . L . , Phuangsab, A., and Goodheart, C.R. 1970. Type 1 and type 2 herpes simplex v i r u s e s : S e r o l o g i c a l and b i o l o g i c a l d i f f e r e n c e s . J . V i r o l . 5_: 51-59.  73.  P r u s o f f , W.H. 1963. A r e v i e w o f some a s p e c t s o f 5-iododeoxyu r i d i n e and a z a u f i d i n e . Cancer Res. 23: 1246-1259.  Electron ferritin-  112  74.  P r u s o f f , W.H. 1967. Recent advances i n chemotherapy o f v i r a l d i s e a s e s . Pharmacol. Rev. 19: 209-250.  75.  Rapp, F. and Hsu, T.C. 1965. V i r u s e s and mammalian chromosomes. IV. R e p l i c a t i o n o f herpes simplex v i r u s i n d i p l o i d Chinese hamster c e l l s . V i r o l o g y 215: 401-411.  76.  Rawls, W., Tompkins, W., F i g u e r o a , M. and M e l n i c k , J . 1968. H e r p e s v i r u s type 2: A s s o c i a t i o n w i t h carcinoma o f t h e c e r v i x . S c i e n c e 161: 1255-1256.  77.  Reed, L . J . and Muench, H. f i f t y percent end-points.  78.  Roane, J r . , P. and Roizman, B. 1966. D i f f e r e n t i a t i o n o f n u c l e a r and c y t o p l a s m i c h e r p e s v i r u s a n t i g e n s i n i n f e c t e d c e l l s . Virology 29: 668-670.  79.  Roizman, B. 1961. V i r u s i n f e c t i o n o f c e l l s i n m i t o s i s . I . O b s e r v a t i o n s on the r e c r u i t m e n t o f c e l l s i n k a r y o k i n e s i s i n t o g i a n t c e l l s induced by herpes simplex v i r u s and b e a r i n g on the s i t e o f v i r u s a n t i g e n f o r m a t i o n . V i r o l o g y 13: 387-401.  80.  Roizman, B. 1962. The r o l e o f hormones i n v i r a l i n f e c t i o n s . I . S u p p r e s s i o n o f v i r a l a d s o r p t i o n and p e n e t r a t i o n i n t o c e l l s t r e a t e d w i t h p a r a t h y r o i d hormone i n v i t r o . P r o c . Nat. Acad. S c i . 48: 795-803.  81.  Roizman, B. 1962. The r o l e o f hormones i n v i r a l i n f e c t i o n s . I I . A c c e l e r a t i o n o f v i r a l a d s o r p t i o n and p e n e t r a t i o n i n t o c e l l s t r e a t e d w i t h t h y r o i d hormone i n v i t r o . P r o c . Nat. Acad. S c i . 48_: 973-977.  82.  Roizman, B. 1963. The programming o f herpes v i r u s m u l t i p l i c a t i o n i n d o u b l y - i n f e c t e d and i n p u r o m y c i n - t r e a t e d c e l l s . P r o c . Nat. Acad. S c i . 49: 165-171.  83.  Roizman, B. 1965. An i n q u i r y i n t o the mechanisms o f r e c u r r e n t herpes i n f e c t i o n s o f man. P e r s p e c t i v e s i n V i r o l o g y IV: 283-304.  84.  Roizman, B. 1969. H e r p e s v i r u s e s , membranes and t h e s o c i a l behavior of infected c e l l s . I n " V i r u s e s A f f e c t i n g Man and A n i m a l s " (M.Saunders and M . S c h a e f f e r , e d s . ) , pp. 37-72. Warren H. Green, Inc., S t . L o u i s .  1938. A simple method f o r e s t i m a t i n g Am. J . Hygiene 27_: 493-497.  113  85.  Roizman, B. 1969. The h e r p e s v i r u s e s : A b i o c h e m i c a l d e f i n i t i o n of the group. C u r r . T o p i c s M i c r o b i o l . Immunol. 49: 1-79.  86.  Roizman, B. and Roane, J r . , P. 1964. The m u l t i p l i c a t i o n o f herpes simplex v i r u s . I I . The r e l a t i o n between p r o t e i n s y n t h e s i s and t h e d u p l i c a t i o n o f v i r a l DNA i n i n f e c t e d H.Ep.2 cells. V i r o l o g y 22: 262-269.  87.  Roizman, B. and Spear, P. 1971. H e r p e s v i r u s a n t i g e n s on c e l l membranes d e t e c t e d by c e n t r i f u g a t i o n o f membrane-antibody complexes. S c i e n c e 171: 298-300.  88.  Roizman, B. and Spear, P. 1971. H e r p e s v i r u s e s : C u r r e n t i n f o r m a t i o n on t h e c o m p o s i t i o n and s t r u c t u r e . I n "Comparative V i r o l o g y " (K. Maramorosch and E . K u r s t a k , e d s . ) , pp. 135-168. Academic P r e s s , New York.  89.  Roizman, B., A u r e l i a n , L., and Roane, J r . , P. 1963. The m u l t i p l i c a t i o n o f herpes simplex v i r u s . I . The programming o f v i r a l DNA d u p l i c a t i o n i n H.Ep.2 c e l l s . V i r o l o g y 21: 482-498.  90.  Roizman, B., Borman, G. and Rousta, M. 1965. Macromolecular s y n t h e s i s i n c e l l s i n f e c t e d w i t h herpes simplex v i r u s . Nature 206: 1374-1375.  91.  Roizman, B., S p r i n g , S., and Roane, J r . , P. 1967. C e l l u l a r compartmentalization of herpesvirus antigens during v i r a l r e p l i c a tion. J . V i r o l . 1: 181-192.  92.  Roizman, B., S p r i n g , S., and Schwartz, J . 1969. The h e r p e s v i r i o n and i t s p r e c u r s o r s made i n p r o d u c t i v e l y and i n a b o r t i v e l y i n f e c t e d cells. F e d . P r o c . 28: 1890-1898.  93.  Roizman, B., K e l l e r , J . , Spear, P., T e r n i , M., Nahmias, A., and Dowdle, W. 1970. V a r i a b i l i t y , s t r u c t u r a l g l y c o p r o t e i n s and c l a s s i f i c a t i o n o f herpes simplex v i r u s e s . Nature 227: 1253-1254.  94.  Ross, L., Watson, D.H., and W i l d y , P. 1968. Development and l o c a l i z a t i o n of v i r u s - s p e c i f i c antigens during the m u l t i p l i c a t i o n o f herpes simplex v i r u s i n BHK-21 c e l l s . J . g e n . V i r o l . 2i 115-122.  95.  R u s s e l l , W.C.  96.  R u s s e l l , W.C, G o l d , E., K e i r , H., Omura, H., Watson, D.H., and W i l d y , P. 1964. The growth o f herpes simplex v i r u s and i t s n u c l e i c a c i d . V i r o l o g y 22: 103-110.  1962.  Herpes v i r u s n u c l e i c a c i d .  Virology  16: 355-357.  114  97.  Samso, A. and Karpas, A. 1971. Comparative study o f t h e e f f e c t of equine herpes 3 v i r u s and herpes simplex v i r u s on t h e chromosomes o f r a b b i t k i d n e y c e l l s . Z b l . Bakt. Hyg. 218: 417-433.  98.  Schwartz, J . and Roizman, B. 1969. Concerning t h e e g r e s s o f herpes simplex v i r u s from i n f e c t e d c e l l s : E l e c t r o n and l i g h t m i c r o s c o p e o b s e r v a t i o n s . V i r o l o g y 38: 42-49.  99.  Schwartz, J . and Roizman, B. 1969. S i m i l a r i t i e s and d i f f e r e n c e s i n the development o f l a b o r a t o r y s t r a i n s and f r e s h l y i s o l a t e d s t r a i n s o f herpes simplex v i r u s i n H.Ep.2 c e l l s : E l e c t r o n m i c r o scopy. J . V i r o l . 4_: 879-889.  100.  Shipman, J r , C , Vander Weide, G.C., and l i m a , B. 1969. P r e v a l e n c e o f type R v i r u s - l i k e p a r t i c l e s i n c l o n e s o f BHK-21 c e l l s . V i r o l o g y 38_: 707-710.  101.  S i l a g i , S. 1965. Metabolism o f l-$-D a r a b i n o f u r a n o s y l c y t o s i n e in L cells. Cancer Res. 25_: 1446-1453.  102.  S i m i n o f f , P. and Menefee, M. u r i d i n e i n h i b i t e d development C e l l Res. 44: 241-255.  103.  S i r t o r i , C. and B o s i s i o - B e s t e t t i , M. 1967. N u c l e a r changes i n KB tumor c e l l s i n f e c t e d w i t h herpes simplex v i r u s . Cancer Res. 27: 367-376.  104.  Smith, K. 1963. Some b i o l o g i c a s p e c t s o f h e r p e s v i r u s - c e l l i n t e r a c t i o n s i n t h e p r e s e n c e o f 5 - i o d o - 2 - d e o x y u r i d i n e (IDU). J . Immunol. 91: 582-590.  105.  Smith, K. and Dukes, C. 1964. E f f e c t o f 5 - i o d o - 2 - d e o x y u r i d i n e (IDU) on h e r p e s v i r u s s y n t h e s i s and s u r v i v a l i n i n f e c t e d c e l l s . J . Immunol. £ 2 : 550-554.  106.  Spear, P. and Roizman, B. 1972. P r o t e i n s s p e c i f i e d by herpes simplex v i r u s . V. P u r i f i c a t i o n and s t r u c t u r a l p r o t e i n s o f the herpesvirion. J . V i r o l . 9_: 143-159.  107.  S p r i n g , S., Roizman, B., and Schwartz, J . 1968. Herpes simplex v i r u s p r o d u c t s i n p r o d u c t i v e and a b o r t i v e i n f e c t i o n . I I . Electron m i c r o s c o p i c and immunological e v i d e n c e f o r f a i l u r e o f v i r u s envelopment as a cause o f a b o r t i v e i n f e c t i o n . J . V i r o l . 2_: 384-392.  1966. Normal and 5-bromodeoxyo f herpes simplex v i r u s . Expt.  115  108.  Stevens, J.G., Nesburn, A.B., and Cook, M.L. 1972. Latent herpes simplex v i r u s from trigeminal ganglia of rabbits with recurrent eye i n f e c t i o n . Nature 235; 216-217.  109.  Stich, H.F. and Yohn, D.S. 1970. Progr. Med. V i r o l . 12: 78-127.  110.  S t i c h , H.F., Hsu, T.C., and Rapp, F. 1964. Viruses and mammalian chromosomes. I. L o c a l i z a t i o n of chromosome aberrations a f t e r i n f e c t i o n with herpes simplex v i r u s . Virology 22: 439-445.  111.  Stoker, M. and Macpherson, I. 1964. Syrian hamster f i b r o b l a s t c e l l l i n e BHK-21 and i t s derivatives. Nature 203: 1355-1357.  112.  Stoker, M. and Newton, A. 1959. The e f f e c t of herpes virus on HeLa c e l l s d i v i d i n g parasynchronously. J . V i r o l . 7_: 438-448.  113.  Sydiskis, R. and Roizman, B. 1966. i n c e l l s infected with a DNA v i r u s .  114.  Tanaka, S. and Southam, C. 1965. J o i n t action of herpes simplex virus and 3-methylcholanthrene i n production of papillomas i n mice. J . Nat. Cancer Inst. 34_: 441-458.  115.  Taylor, J.H. 1963. DNA synthesis i n r e l a t i o n to chromosome r e production and the reunion of breaks. J . C e l l Comp. Phys. 62: 73-86.  116.  Tweedell, K. 1967. Induced oncogenesis i n developing frog kidney c e l l s . Cancer Res. 27^ 2042-2050.  117.  Underwood, G. 1962. A c t i v i t y of ara-C against herpes simplex k e r a t i t i s . Proc. Soc. Expt. B i o l . Med. I l l : 660-664.  118.  Waddell, G. and S i g e l , M. 1966. Factors associated with the response of the c e l l to herpes simplex virus i n f e c t i o n . Arch. Virusforsch. 19_: 130-142.  119.  Wagner, E. 1972. Evidence f o r t r a n s c r i p t i o n a l control of the herpes simplex v i r u s genome i n infected human c e l l s . Virology 47: 502-506.  120.  Wagner, E. and Roizman, B. 1969. RNA synthesis i n c e l l s infected with herpes simplex v i r u s . I. Patterns of RNA synthesis i n productively infected c e l l s . J . V i r o l . 4_: 36-46.  Viruses and chromosomes.  Polysomes and protein synthesis Science 153: 76-78.  116  121.  Wagner, E . and Roizman, B. 1969. RNA s y n t h e s i s i n c e l l s i n f e c t e d w i t h herpes simplex v i r u s . 2. E v i d e n c e t h a t a c l a s s o f v i r a l mRNA i s d e r i v e d from a h i g h m o l e c u l a r weight p r e c u r s o r s y n t h e s i z e d i n t h e n u c l e u s . P r o c . N a t . Acad. S c i . (Wash) 6_4: 626-633.  122.  W a l l i s , C. and M e l n i c k , J . L . 1965. T h e r m o s t a b i l i z a t i o n and s e n s i t i z a t i o n o f h e r p e s v i r u s . J . B a c t . 90: 1632-1637.  123.  W a l l i s , C , T r u l o c k , S., and M e l n i c k , J . L . 1969. I n h e r e n t p h o t o s e n s i t i v i t y o f herpes v i r u s and o t h e r enveloped v i r u s e s . J . gen. V i r o l . _5: 53-61.  124.  Watson, D.H., Shedden, W.I.H., E l l i o t , A., T e t s u k a , T., Wildy, P., Bourgaux-Ramoisy, P., and G o l d , E . 1966. V i r u s s p e c i f i c a n t i g e n s i n mammalian c e l l s i n f e c t e d w i t h herpes simplex v i r u s . Immunology 111 399-408.  125.  Watson, D.M., Wildy, P., and R u s s e l l , W.C. 1964. Q u a n t i t a t i v e e l e c t r o n m i c r o s c o p e s t u d i e s on t h e growth o f herpes v i r u s u s i n g the t e c h n i q u e s o f n e g a t i v e s t a i n i n g and u l t r a m i c r o t o m y . V i r o l o g y 24: 523-538.  126.  Waubke, R., z u r Hausen, H., and Henle, W. 1968. Chromosomal and a u t o r a d i o g r a p h i c s t u d i e s o f c e l l s i n f e c t e d w i t h herpes simplex v i r u s . J . V i r o l . 2j 1047-1054.  127.  Wildy, P. 1967. The p r o g r e s s i o n o f herpes simplex v i r u s t o t h e c e n t r a l nervous system o f t h e mouse. J . Hygiene 65: 173-192.  128.  W i l t o n , J.M.A., I v a n y i , L. and Lehner, T. 1972. C e l l - m e d i a t e d immunity i n H e r p e s v i r u s hominis i n f e c t i o n s . B r i t . Med. J . 1_: 723-726.  

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