The surgical significance of viruses

THE SURGICAL SIGNIFICANCE OF VIRUSES RICHARD J. HOWARD HENRY H. BALFOUR, JR. RICHARD L. SIMMONS

TABLE OF CONTENTS

G e n e r a l Properties of Viruses . . . . . . . . . . . . . .

5

Classification of Viruses . . . . . . . . . . . . . . . .

8

. . . . . . . . .

8

How Viruses Cause Disease

Principles of Diagnosis of Viral Infections

. . . . . . . . . . . . . .

14

P a t t e r n s of Viral Infections

. . . . . . . . . . . . . .

18

. . . . . . . . . . . . . . . . . . .

20

Viral Infections as the Cause of Surgical Illness . . . . . . .

26

Viral Infections C a u s i n g Congenital Malformations T h a t Require Surgical Correction . . . . . . . . . . . . .

36

Herpesvirus Infections Following Open H e a r t Operation a n d Blood T r a n s f u s i o n . . . . . . . . . . . . . . . . .

40

Viral Infections in Compromised Hosts after Operation

42

Viral Hepatitis

. . . .

P r e v e n t i o n a n d T r e a t m e n t of Viral Infections . . . . . . . .

51

Viruses and Cancer

57

. . . . . . . . . . . . . . . . .

QUESTIONS OF CLINICAL USEFULNESS ANSWERED IN THIS ISSUE 1. How do viruses differ from bacteria, fungi and other organisms? 2. Describe the essential steps in virus replication. 3. What are the methods of diagnosing viral infections? 4. What are the various methods by which viruses cause disease? 5. What are inapparent virus infections? 6. What is the Dane particle? 7. W h a t are the relationships of hepatitis B surface antigen (HBsAg), the core antigen (HBeAg) and HBeAg (DNA polymerase) to the structure of hepatitis B virus? 8. What is the relationship to HBsAg and HBeAg to infectivity? 9. What is the risk to patients from surgeons who are chronically positive for HB~Ag? 10. What effective treatment is available for hepatitis B infection? 11. What prophylactic measures can be used to prevent or a t t e n u a t e hepatitis B? 12. The etiologies of what surgical illnesses have been related to viral infections? 13. Why are viruses so difficult to prove as the etiologic agents of surgical illness? 3

14. W h a t viruses have been linked to congenital malformations? 15. W h a t factors determine whether a virus m a y be a teratogen? 16. W h a t group of viral infections occurs most commonly in compromised hosts with cancer and after transplantation? 17. W h a t are the various means of preventing viral infections? 18. W h a t agents are available for t r e a t m e n t of viral infections? 19. Viruses have been implicated in the etiology of which h u m a n cancers? 20. The greatest a m o u n t of evidence links which virus as the cause of which tumor? 21. Describe the methods used to link viruses and h u m a n neoplasms.

4

is Assistant Professor of Surgery at the University of Minnesota

Health Sciences Center. After graduation from the Yale University School of Medicine, he received his education in surgery at the University of Minnesota, where he also received a Ph.D. He is the recipient of a Research Career Development Award from the National Institutes of Health. His main research interests include transplantation immunobiology and surgical infections.

is Associate Professor of Pediatrics and Laboratory Medicine and Pathology, founder and Chief of the Section of Clinical Virology and Director of the Division of Clinical Microbiology, University of Minnesota Health Sciences Center. After graduation from Columbia College of Physicians and Surgeons, he received postgraduate training in pediatrics and infectious diseases at the University of Minnesota, the Minnesota Department of Health, and Columbia-Presbyterian Medical Center. He is certified by the American Board of Pediatrics. Doctor Balfour's interests are in clinical virology and include California (La Crosse virus) encephalitis and cytomegalovirus infections in renal transplant recipi.ents.

is Professor of Surgery and Microbiology at the University of Minnesota and President of the Society of University Surgeons. Previously, he served on the surgical faculty at Columbia University and on the U.S. Army Surgical Research Team in Vietnam. Doctor Simmons graduated from Boston University Medical School and received his postgraduate training at ColumbiaPresbyterian Medical Center in New York. His clinical and investigational interests for the past 15 years have centered around surgical aspects of immunology and host defense.

GENERAL PROPERTIES OF VIRUSES ANATOMY OF VIRUSES VIRUSES are a unique class of infectious agents, ubiquitous in nature. Viruses infect all types of plants and animals. Originally they were distinguished by their extremely small size and because they are obligate intracellular parasites. However, some NOTE: This work was supported in part by USPItb grants HL 06314 and AM 18883. 5

small bacteria share these features. Animal viruses range in diameter from 18 nm to 230 • 300 nm. By contrast, a n E . coli cell is 700 • 1500 nm and an animal cell is on the order of 10,000 • 10,000 nm. The distinctive nature of viruses lies in their rather simple composition and organization and their mode of replication. A complete virus particle or virion m a y be simply regarded as genetic material surrounded by a coat that protects the internal genetic information from the environment and functions as a vehicle for transmission from one host to another. The genetic material of viruses is either D N A or RNA. Unlike bacteria and higher organisms that contain both RNA and DNA, viruses have either DNA or R N A - never both. Viral nucleic acid codes for coat proteins and enzymes essential for synthesis of viral nucleic acid and viral proteins. One or more viral proteins form a unit (capsomere) of the protein coat (capsid) of the virus. The protein coat may give the virion a variety of shapes, depending on the arrangement of the protein subunits. The capsid and internal nucleic acid are termed the nucleocapsid. Viruses that exit from cells by budding rather than by cell lysis m a y attain an additional coat of nuclear or cytoplasmic membrane. Viruses with a membrane surrounding the nucleocapsid are said to be enveloped. ~ i r i o n s m a y also contain enzymes not provided by host cells b u t required for viral replication. Viruses differ from bacteria, plants and animals in that they lack the machinery for carrying out the basic processes of organisms. They have no ribosomes and hence cannot synthesize proteins. They cannot respire, because they lack mitochondria. Their Fig 1 . - A diagram of a typical virus particle. The central core contains either DNA or RNA. The core is surrounded by a layer of protein termed the capsid. The nucleic acid and the capsid make up the nucleocapsid. The capsid is composed of individual units called capsomeres. Enveloped viruses are surrounded by a membrane layer. DNA viruses usually derive this membrane layer, or envelope, from the nuclear membrane whereas RNA viruses usually get their envelope from the outer cell membrane.

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almost complete lack of enzymes means that they cannot metabolize compounds to generate energy or build complex molecules from simple ones. For these reasons, they must use the metabolic machinery of the host cells and hence are obligate intracellular parasites. REPLICATION OF VIRUSES Replication of viruses involves a series of ordered sequential steps: (1) The first step is adsorption of the virus onto the cell membrane. For enveloped viruses, such as the herpesviruses, the

Fig 2.-Envelopment and release of herpesvirus. Replication of viral DNA occurs in the nucleus. Viral proteins are synthesized in the cytoplasm and transported to the nucleus, where assembly takes place. Virus approaches the nuclear membrane (A), which thickens and progressively envelops the nucleocapsid (B). The viral envelope pinches off, leaving the nuclear membrane intact and the enveloped virus free in the perinuclear cisterna. The nucleocapsid can also acquire an envelope by budding into nuclear vacuoles (B). These vacuoles appear to be indentations of the nuclear membrane and are continuous with the perinuclear cisterna (C). The enveloped virus then is transported through the cytoplasm in vacuoles (D), which fuse with the surface membrane to release the virus to the extracellular space (G). Alternatively, the virus reaches the cell surface by traversing the cisternae of the endoplasmic reticuTum (E, F) to the cell surface (H). (Reproduced, with permission, from Jawetz, E., Melnick, J. L., and Adelberg, E. A., Review of Medical Microbiology [12th ed.; Los Altos, Califo: Lange Medical Publications, 1976].) 7

outer membrane of the virus appears to fuse with the cell membrane. (2) The next step is penetration of the virus into the cell. Penetration can occur by pinocytosis or by fusion of the viral and cell membranes, leaving the nucleocapsid inside the cell. (3) The third step is u n c o a t i n g - r e m o v a l of the viral protein coat by host enzymes, leaving the nucleic acid. (4) Reproduction of viral RNA usually occurs in the cytoplasm whereas reproduction of viral DNA occurs in the nucleus. (5) Synthesis of the proteins of the viral coat or capsid occurs in the cytoplasm. The viral DNA or RNA has the necessary information for the protein coat, and messenger RNA (mRNA) is synthesized (translated) from the viral DNA or RNA. Alternatively, viral RNA actually m a y be mRNA. (6) The proteins of the viral coat (capsid) are assembled in the nucleus for DNA viruses and in the cytoplasm for RNA viruses, and the nucleic acid is inserted into the empty capsid, forming the m a t u r e nucleocapsid. The m a t u r e virus then leaves the cell when the cell lyses or it m a y acquire a host membrane coat as it buds from the surface of the cell. When a cell lyses, it literally bursts apart, releasing thousands of virus particles at once. Lysis always results in death of the cell; budding viruses m a y be released over a prolonged period and the cell does not necessarily die. Replication of DNA viruses is similar to that of RNA viruses with the exception that virus reproduction generally takes place in the nucleus. The viral DNA must be transcribed into mRNA, which then is translated on host ribosomes in the cytoplasm.

CLASSIFICATION OF VIRUSES Animal viruses are classified according to their chemical and physical properties. Viruses are divided into two groups according to the type of nucleic a c i d - R N A or DNA. They are further divided according to the s y m m e t r y of their capsid and subdivided by whether the nucleocapsid is naked or is surrounded by a membrane envelope. Enveloped viruses are sensitive to ether and other organic solvents, because lipids in the membrane are dissolved by the solvent and the virus no longer can adsorb onto the host cell. Viruses are further divided according to size. Viruses grouped according to these criteria generally have similar antigenic determinants, growth characteristics and host range.

PRINCIPLES OF DIAGNOSIS OF VIRAL INFECTIONS Several techniques are available for the direct and indirect identification of viruses. These include: (1) viral isolation; (2) m e a s u r e m e n t of antibodies during the course of an infection; (3) histologic examination of infected tissues; (4) detection of viral antigens in lesions; (5) electron microscopic examination of fluids 8

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and tissue extracts for virus. Finding evidence of a virus by any of these techniques does not prove that it is the etiologic agent for a given disease. Some viruses, such as the herpesviruses, may persist in human hosts for long periods and some, such as the enteroviruses, may be isolated from asymptomatic people. In patients which clinical illness, these viruses may be innocent bystanders. Specimens must be handled properly to ensure the greatest likelihood of viral infection. The normal procedures of most operating rooms are geared for bacterial isolation only, and special arrangements must be made by the surgeon. Specimens for isolation should be promptly inoculated onto the appropriate cell line. Alternatively, they may be stored at refrigerator temperature (4~ C). The virologist should be told what viruses are suspected. For detection of viral antigens in tissues by immunofluorescence, small pieces of tissue must be immediately "snap" frozen in liquid nitrogen. This technique requires having liquid nitrogen in the operating room or very close by so that no time is lost in freezing the tissue. For electron microscopy, the tissue specimen must be placed into a proper fixative such as glutaraldehyde, which is not commonly available in the operating room unless special arrangements have been made. VIRUS ISOLATION AND IDENTIFICATION.--The isolation of virus requires the proper collection of specimens, transportation to the laboratory and inoculation into animal or cultured cell capable of being infected by the virus in question. Blood, sputum, throat and nasal washings, stool, urine, unfixed biopsy material and autopsy specimens, etc. all serve as sources of virus from clinical cases. The sample should be promptly inoculated into animals or cell cultures. Alternatively, the virus may be preserved by freezing, lyophilization or suspension in glycerol until inoculation. Bacteria must be removed from potentially contaminated material by addition of antibiotics, filtration or centrifugation. Tissue culture methods are most widely used for virus identification. Many cell lines (human, monkey, hamster, etc.) are used, since some viruses replicate on some cell lines and may not grow at all on others. Virus identification depends on the effects produced in tissue culture. Viruses may cause histologic alterations of the host cells called cytopathic effects (CPE), i.e., lysis of cells, rounding of cells, multinucleated cells, etc. Some viruses (e.g., rubella) produce no direct CPE but can be detected by their interference with the CPE of a second challenge virus (viral interference). Other viruses (myxoviruses) cause changes in the cell membrane so that certain species of red cells will stick to the cell surface (hemadsorption). The identity of a virus isolated is conlo

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Fig 4 . - C y t o p a t h i c effects p r o d u c e d by herpesviruses. A, cytomegalovirusinfected fibroblasts. Infected ceils are enlarged and rounded ( a r r o w s ) (X80). B, rhesus monkey kidney cells infected with H e r p e s v i r u s h o m i n i s . The infected cells are rounded. Several cells have died and have lifted off the flask, leaving clear spaces where a confluent monolayer had been (X80). C, plaques produced by poliovirus ( l e f t ) and by an echovirus ( r i g h t ) . (After Hsuing and Melnick.) (Reproduced, with permission, from Jawetz, E., Melnick, J. L., and Adelberg, E. A., R e v i e w o f M e d i c a l M i c r o b i o l o g y [12th ed.; Los Altos, Calif.: Lange Medical Publications, 1976].)

firmed by the use of specific antiserum, which inhibits viral growth and biologic effects. Animal inoculation frequently is used to isolate neurotropic viruses causing encephalitis. Infant mice are inoculated intracerebrally. If the mouse dies and if bacteria are not cultured from the brain, the virus can be identified by specific antiserum, which neutralizes the virus and prevents death of the infant mouse. 11

HISTOLOGIC EXAMINATION OF TISSUE. -- H i s t o I o g i c examination of biopsy and autopsy tissues m a y reveal cellular inclusion bodies typical of viral infection. DNA viruses usually produce intranuclear inclusions surrounded b y a clear halo from the nuclear membrane. With RNA viruses, inclusion bodies are seen in the cytoplasm where RNA viruses replicate. Some viruses (e.g., measles) produce both intranuclear and intracytoplasmic inclusions. Inclusion bodies are believed to be either masses of closely packed virus particles that can be seen by electron microscopy to mature within the inclusion body or a r e m n a n t of earlier virus replication. Inclusion bodies m a y be of considerable diagnostic aid. For instance, the Negri body is an intracytoplasmic inclusion in nerve cells that is pathognomonic for rabies.

ELECTRON MICROSCOPIC EXAMINATION OF BODY FLUIDS, TISSUES AND TISSUE E X T R A C T S . - - T y p i c a l virus particles can be seen by electron microscopy in body fluids, tissues and tissue extracts. Fig 5 . - A , Jntranuclear inclusions (arrows) are found in cells infected with cytomegalovirus. The eosinophilic intranuclear inclusions are surrounded by a clear halo (X400). B, cytoplasmic inclusions found in ganglion cells infected with rabies virus. These inclusions commonly are called Negri bodies (X600). (Courtesy of Dr. J. H. Sung.)

12

They are observed more easily in body fluids and tissue extracts after concentration of virus by ultracentrifugation, evaporation or ultrafiltration. Only after virus concentration was hepatitis B virus observed in urine specimens of patients with serum hepatitis. Electron microscopic examination permits diagnosis within a m a t t e r of hours in patients with vesicular lesions such as those produced by the poxviruses or herpesviruses. Myxoviruses (influenza) m a y be identified j u s t as rapidly in respiratory secretions. DETECTION OF VIRAL ANTIGENS IN BIOPSY LESIONS.-- Viral antigens can be detected in biopsy and autopsy specimens by Fig 6.--Electron micrographs of virus particles. A, top, herpesvirus particles from human vesicle fluid, stained with uranyl acetate to show DNA core (X140, 000). Bottom, virions stained to show protein capsomeres of the virus coat (X140,000). (After Smith and Melnick.) B, vaccinia virus particles within the cytoplasm of an infected cell (X74,000). (After Morgan, Rose and Moore.) O, influenza virus along the cell membrane, which passes diagonally across the field with the cytoplasm to the right. Several particles just beneath the cell membrane seem to be undergoing differentiation toward the mature extracellular form (Xl16,000). (After Morgan, Rose and Moore.) (Reproduced, with permission, from Jawetz, E., Melnick, J. L., and Adelberg, E. A., Review of Medical Microbiology [12th ed.; Los Altos, Calif.: Lange Medical Publications, 1976].)

13

reacting frozen sections with fluorescein-labeled antiviral antibody (direct immunofluorescence) and examining the specimen for fluorescence under ultraviolet light. Since formalin fixation m a y destroy viral antigens, fresh frozen (preferably in liquid nitrogen) specimens must be used. Alternatively, antiviral antibody can be applied to the frozen tissue section followed by a fluorescein-labeled a n t i g a m m a globulin (indirect immunofluorescence). The specimen then is examined for fluorescence under ultraviolet light. This technique is used today for the detection of variola antigen in skin lesions and for m a n y other viruses in' a variety of specimens. It also has great potential for the detection of many other viral antigens and for identification of the cellular sites of production of various viral components.

SEROLOGIC DIAGNOSIS.--Several procedures are available for detection of serum antibody against virus. The neutralization test generally is not used for routine laboratory testing, because other tests (complement fixing, hemagglutination inhibition) are much easier to perform and require less time (see the accompanying table). Two or more serum samples taken several days or weeks apart are required to establish proof of an active viral infection, since a rising titer to the virus must be demonstrated. Antibody to a virus detected in a single serum sample only means that the individual has been infected with that virus at some time, past or present. HOW VIRUSES CAUSE DISEASE Viruses cause disease through a variety of mechanisms. Moreover, a single virus m a y cause one or several illnesses by different mechanisms. The status of the host as to age, immune competence, etc., as well as the virus itself, including virulence, size of inoculum, route of infection, etc., are important in determining the mechanism by which the virus causes disease and the signs and symptoms and severity of the illness. CELL LYSIS.--When the replication of nonbudding viruses is completed, cells m a y lyse to release new virions. This lysis results in cell death. Death of sufficient cells in an organ can cause such severe organ dysfunction that the host dies (e.g., viral hepatitis). Cell lysis m a y be confined to one cell type or to numerous cell types in many different organs, depending on the virus. ALTERED CELL FUNCTION.--During cellular infection, various metabolic processes may be altered in order to replicate virus. Thus, cell DNA and RNA synthesis m a y cease or be altered, with cessation of production of cell proteins. This interference by virus 14

SEROLOGIC TESTS FOR DIAGNOSIS OF VIRAL INFECTIONS TEST

1. Complement fixation

2. Neutralization 3. Hemagglutination inhibition

4. Precipitation

5. Immunofluorescence

6. Radioimmunoassay

BASIS OF TEST

Virus-antibody complexes bind complement, making it unavailable for lysis of sheep red blood cells, which have been sensitized by addition of anti-sheep red blood cell antibody Addition of specific antibody to virus neutralizes its infectivity for animals or for cells in tissue culture Some viruses can agglutinate red blood cells. Addition of antiviral antibody to the virus prevents them from agglutinating erythrocytes Antiviral antibody and virus results in a solid precipitate. In the immunodiffusion or counter electrophoresis system, the precipitation reaction takes place in agar, forming a visible line Antiviral antibody will react with viral antigen in tissue culture cells in which the virus is growing. If rabbit anti-human gamma globulin labeled with fluorescein isothiocyanate is added to the cells, they will fluoresce under ultraviolet light. They will not fluoresce if antiviral antibody was not present in the test serum A radioactive antigen forms antigenantibody complexes, which can be separated from the uncomplexed antigen. The quantity of uncomplexed antigen then is detected by the amount of radioactivity it emits. The presence of antibody reduces the amount of radioactivity in the unbound fraction

w i t h n o r m a l cell m a c h i n e r y c a n c a u s e altered cell function a n d e v e n death. F o r example, influenza a n d c y t o m e g a l o v i r u s infect polymorphonuclear leukocytes and macrophages and inhibit t h e i r p h a g o c y t i c ability2 T h i s decreased p h a g o c y t i c ability renders t h e infected host m o r e susceptible to b a c t e r i a l invasion, w i t h t h e r e s u l t t h a t d e a t h of t h e host a c t u a l l y m a y be due to b a c t e r i a l superinfection. CONGENITAL MALFORMATIONs.-- S e v e r a l v i r u s e s are k n o w n or are s u s p e c t e d to c a u s e c o n g e n i t a l m a l f o r m a t i o n s in h u m a n s a n d e x p e r i m e n t a l animals. T h e e m b r y o or fetus m a y be infected by t r a n s p l a c e n t a l t r a n s f e r from t h e m o t h e r . Since c o n g e n i t a l defects are f o r m e d d u r i n g the a n a t o m i c d e v e l o p m e n t of o r g a n s y s t e m s a n d this d e v e l o p m e n t is l a r g e l y completed d u r i n g t h e first t r i m e s ter of p r e g n a n c y , c o n g e n i t a l defects u s u a l l y o c c u r with e x p o s u r e 15

to the teratogen during the early period of fetal development. Most viruses causing congenital malformations affect the nervous system, but some (e.g., rubella) cause defects requiring surgical correction (e.g., cardiac defects). Obviously, not all congenital abnormalities are due to viral infection, since there is a variety of teratogens; others include radiation, chemicals and drugs. Genetic factors as well as maternal age and disease states also affect the development of congenital malformations. CHROMOSOME DAMAGE. - - C h r o m o s o m e damage such as bre~kage, fragmentation and rearrangement can be caused by viral infection. Abnormal chromosomes and changes in chromosome number have been reported also. Several viruses of various classifications (i.e., measles, rubella, herpesviruses, influenza, mumps, adenoviruses) have been demonstrated to cause chromosomal injury. For most viruses, this chromosomal injury is observed only in infection of primary cell cultures. Chromosome breaks during natural infection have been observed in peripheral blood leukocytes of patients with measles and chickenpox. Chromosome damage may be important in the cause of congenital malformations by viruses. ALTERED IMMUNE FUNCTION.--Some viruses cause an alteration of immune function of the host, including changes in serum gamma globulin concentrations, increased and decreased humoral and cell-mediated immunity and increased and decreased phagocytic capacity of polymorphonuclear leukocytes and macrophages. 2 Many human viruses such as cytomegalovirus, rubella and measles produce these variations in immune capacity. On the one hand, decreased immune function can lead to increased susceptibility to bacterial and fungal superinfection; on the other hand, it has been hypothesized that the increased humoral and cell-mediated immunity caused by recurrent cytomegalovirus infection may cause increased susceptibility to renal homograft rejectionY"5 IMMUNE RESPONSE TO THE VIRUS AS A CAUSE OF D I S E A S E . A l t h o u g h the immune response to infectious agents such as vi-

ruses usually serves to rid the host of the infection, such immune responses paradoxically can lead to disease. Circulating virus can persist until the host mounts an immune response against it. The resultant antibody binds the virus and these virus-antibody complexes are filtered out by the reticuloendothelial system. Some circulating virus-antibody complexes are not removed from the blood but remain in the circulation for the life of the host. These complexes can attach to basement membranes, notably in the renal glomeruli and small arterioles. Here, they activate the 16

complement system, leading to an inflammatory response. By this complement activation, circulating virus-antibody complexes can cause glomerulonephritis (viz., lymphocytic choriomeningitis virus and lactic dehydrogenase virus in mice) and arteritis (viz., Aleutian mink disease). Proof of the participation of virusantibody complex in the pathogenesis of disease exists most convincingly in experimental animals, b u t hepatitis has been reported to cause immune complex glomerulonephritis in man.6, 7 INCREASED CELL PROLIFERATION AND O N C O O E N E S I S . - - A n o t h e r

mechanism whereby viruses can cause disease is through cellular proliferation-controlled or uncontrolled. Controlled cellular proliferation occurs in lymphatic tissue during infection with Epstein-Barr virus (the etiologic agent of infectious mononucleosis) and other viruses. This enlarged lymphoid tissue may cause surgical illness by blocking the lumen of hollow viscera (causing appendicitis) or m a y serve as the lead point in intussusception. Benign proliferation of virus-infected duct epithelial cells m a y be a cause of congenital biliary atresia. Viruses m a y also cause tumors through the induction of uncontrolled cellular proliferation. Viruses have been proved to cause a variety of tumors in animals and have been strongly implicated in a n u m b e r of h u m a n tumors as well. The best evidence for the viral etiology of h u m a n cancer exists in the cases of nasopharyngeal carcinoma and Burkitt's lymphoma, both of which are believed to be due to Epstein-Barr virus. Virally produced tumors m a y not all be m a l i g n a n t - for example, it is well established that h u m a n warts are caused by a virus. Difficulties arise in studying oncogenic viruses, since some animal and h u m a n viruses m a y cause in vitro malignant cell transformation only in cells from species other than the one naturally infected by the virus. Cell transformation is a term referring to in vitro cell cultures in which the cells take on properties akin to t u m o r s - i . e . , uncontrolled growth, lack of contact inhibition, increased rate of multiplication, chromosomal abnormalities, lack of uniform cell size and shape, etc. Yet, viruses that cause in vitro cell transformation m a y not cause any tumors in intact animals. Conversely, some viruses that cause tumors in vivo do not cause in vitro celt transformation. The process whereby viruses cause cell transformation and tumors involves changes found in the early stages of other viral infections, such as adsorption, penetration and uncoating. However, the DNA or RNA is not replicated. Instead, viral DNA is physically integrated in intact form into cellular DNA. The integrated viral DNA (provirus) is replicated along with cellular DNA during cell division. The viral nucleic acid of oncogenic 17

R N A viruses cannot be incorporated into host DNA. Instead, an RNA-directed D N A polymerase (reverse transcriptase) intrinsic to the virion allows D N A to be synthesized using the viral RNA as a template. This D N A then is integrated into the cellular D N A and it is replicated during cell division. P A T T E R N S OF V I R A L INFECTIONS Viruses can cause at least three different patterns of infection: acute, persistent and slow. Some viruses can cause more than on~

type of infection under different conditions. ACUTE VIRAL INFECTIONS.-Viruses can cause three basic patterns of acute infection: localized, disseminated and inapparent.

Localized infection.- In a localized infectionl virus remains localized near the site of e n t r y - s k i n , respiratory tract or gastroinFig 7 . - T h e virus enters the cell and the protein coat is removed, leaving the viral nucleic acid. The nucleic acid can become integrated into the host DNA, which can lead to cell transformation and tumor production. For RNA viruses, a DNA is synthesized using the viral RNA as a template by means of an RNAdependent DNA polymerase. The oncogenic viruses follow this path. Alternatively, the viral nucleic acid can be replicated. If the protein coat is also synthesized, mature virus is produced. This mature virus is released suddenly by cell lysis with death of the cell or continuously by cell budding. Release by budding usually does not cause cell death. The viral nucleic acid can also be replicated in phase with cell division without production of mature virus, leading to a latent state. Certain stimuli (e.g., radiation, heat) may reactivate this latent virus, causing mature virus to be produced.

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Fig 8.-Patterns of viral infections. Dashed line-threshold of apparent infection (clinical disease). Solid line-course of disease. Stippled area-presence of occult virus. Arrows-activation of virus in original host. A, acute viral infection with acute clinical course followed by long-lasting immunity (measles, poliovirus). Some acute viral infections may not become clinically evident but still may lead to long-lasting immunity (polio). B, viral infections can also be clinical or subclinical. Virus may persist after the illness or there may be a long latent infection during which virus is present in small quantity (adenoviruses. influenza). C, viral infection can be latent for long periods before it is activated. These "slow" viral infections are characterized by kuru and cancer viruses. D, viruses can be reactivated periodically, giving rise to clinical disease. In between clinical manifestations, the virus is latent (fever blisters [Herpesvirus hominis] and zoster or "shingles" [varicella-zoster]. (Drawn, with permission, from Jawetz, E., Melnick, J. L., and Adelberg, E. A., Review of Medical Microbiology[12th ed.; Los Altos, Calif.: Lange Medical Publications, 1976].)

testinal tract. Virus may spread to neighboring cells by diffusion and cell-to-cell contact, forming a single lesion or group of lesions, such as warts. At no time does it enter the bloodstream. The common cold and viral gastroenteritis can be regarded as localized infections.

Disseminated Infection.-Many viruses cause clinical illness only after a sequential series of steps. The virus usually undergoes multiplication at its site of entry. Progeny virus then enter the blood and lymphatics and are disseminated throughout the body. The virus then may reach the target organ (e.g., the skin in exanthematous diseases) or it may undergo a further multiplication throughout the body. A secondary viremia then takes place and the virus then goes to the target organ and produces clinical disease. Usually clinical signs and symptoms of illness begin only after the virus is disseminated and has attained maximal titers in the blood. Measles and rubella are examples of this type of acute infection. Inapparent Infection.-Transient viral infection can occur without causing clinicalillness. People with inapparent infections represent an unrecognized source of viral spread throughout a population. Inapparent infections also confer immunity on the host 19

and represent an important source of "natural" immunity. Examples of inapparent infection include anicteric viral hepatitis and poliomyelitis. Prior to the use of the polio vaccine there were an estimated 100 inapparent infections for every paralytic case of poliomyelitis. PERSISTENT VIRAL INFECTIONS (LATENT INFECTIONS).-- In persistent viral infections, clinical disease does not occur, or occurs infrequently. An equilibrium is achieved between virus and host in different ways, depending on the virus and host. Since virus replication occurs within cells, it is unavailable to the host's protective immune response until it leaves the cell. Thus, persistence can continue as long as the virus does not destroy the cell or interfere with its essential cellular functions. Under these circumstances, cells can continue to replicate virus. Persistence m a y also occur if the viral genome is incorporated into and replicates with the cellular genome, or if replication of separate viral genomes occurs without viral maturation. The viral genome persists in the host cell but it is not entirely transcribed and no virus is produced. Oncogenic (tumor-producing) viruses exist in the cell in this manner. The herpesviruses are the best known persistent h u m a n viruses that can be reactivated by different stimuli. Herpesvirus hominis (herpes simplex virus) is reactivated by stimuli such as heat (fever blisters), actinic radiation and stress. Cytomegalovirus, a member of the herpesvirus family, is reactivated by immune reactions, immunosuppressive drugs such as those used to prevent transplant rejection and cancer chemotherapeutic agents. Varicella-zoster, which causes shingles, is latent in the dorsal root ganglia. When appropriately stimulated, it migrates along the dorsal nerve to the skin epithelial cells, where it proliferates and causes the vesicular lesions typical of shingles. SLOW VIRAL INFECTIONS.--Slow viral infections refer to those viruses that require prolonged p e r i o d s - u p to several y e a r s - b e fore disease becomes clinically apparent. Virus multiplication is not necessarily slow, but the disease develops over a prolonged period. In man, these are all neurologic illness. One of them, subacute sclerosing panencephalitis, is due to measles virus.

VIRAL HEPATITIS A brief discussion of viral hepatitis is in o r d e r - not because of its significance as a disease amenable to surgical therapy b u t because of its importance as a disease of surgeons. Two major forms of viral hepatitis have been recognized and currently are designated hepatitis A and B. These two illnesses can be specifically identified in the clinical laboratory, b u t be20

cause some cases of hepatitis are found to be neither A nor B, a third type, "hepatitis C," has been proposed. It is likely that hepatitis C subsequently will be found to have multiple etiologies. Hepatitis A, which formerly was known as "infectious hepatitis," is not of special importance to surgeons except in the differential diagnosis of the jaundiced patient. The agent is a small RNA virus (average diameter 27 nm) that can be propagated in primates, particularly marmosets, s Serologic methods, both complement fixation and radioimmunoassay, have been developed for the laboratory diagnosis. The disease is spread by the fecal-oral route, and foodborne or waterborne epidemics occur frequently. The disease rarely is transmitted by blood products, and surgeons do not appear to have a greater frequency of hepatitis A infections than the general population. In contrast, hepatitis B is a grave threat to s u r g e o n s - i n d e e d , 12.2% of surgeons have a history of hepatitis. Previously called serum or transfusion hepatitis, this infection is caused by hepatitis B virus, a DNA virus (average diameter 42 nm) demonstrable by electron microscopy in the serum of infected individuals. 9 Electron microscopy has revealed three major morphologic forms of hepatitis B virus in infected bloody -1~ These forms include spherical particles with a diameter of 22 nm, filaments with a diameter of 22 nm and length of 2 0 0 - 4 0 0 nm and the so-called Dane particle with a diameter of 42 nm, which is a doughnut-like structure with an inner core. The Dane particles represent the complete virus and the smaller spheres and filaments represent excess coat or surface protein. The surface protein contains HBsAg (hepatitis B surface antigen) whereas the core material has HBcAg (hepatitis B core antigen) on its surface. The core of the virus contains the DNA and DNA pelymerase. The "e" antiFi9 9 . - A schematic drawing of hepatitis B virus (diameter 42 nm). Outer rings constitute the surface antigen (HB,Ag). The inner 28-nm hexagonal core (HBcAg) contains the DNA polymerase, which is closely associated with the "e" antigen (HBoAg) and the circular DNA. (Drawn after Krugman, S., Hepatitis: Current status of etiology and prevention, Hosp. Prac., November, 1975.)

HBsAg\ I ~

( HBcAg

' Polymerose (HBe Ag )

21

Fig 10.-Electron micrograph of hepatitis B virus. The large 42-nm particle (the Dane particle) is believed to represent the complete virion, with the outer circular layer containing the surface antigen and the inner 28-nm hexagonal core containing the DNA, DNA polymerase or "e" antigen (HB,Ag) and the core antigen (HBcAg). The smaller (22 nm) circular and filamentous forms (200400 nm long) contain HBAg and may represent defective (incomplete) virus (X140,000). (Courtesy of Dr. D. S. Dane.)

gen is closely related to the polymerase or is the polymerase itself. Most recently, the infectivity of blood and blood products has been shown to be directly related to the presence of DNA polymerase or "e" antigen (HBeAg). 12, 13 In the future, it will be essential to assay for "e" antigen because patients who have this are clearly infectious and a definite risk for other patients and hospital personnel. Individuals who have HB~Ag b u t do not have "e" antigen represent little or no contagious risk, and isolation procedures accordingly can be modified. EPIDEMIOLOGY.-Hepatitis B virus is present in highest concentrations in h u m a n serum and is most readily transmitted by blood or blood p r o d u c t s - w h o l e blood, h u m a n thrombin and fibrinogen preparations. It was estimated ~4that 6.5 million units of blood were transfused in the United States in 1971. From these 22

transfusions there were an estimated 120,000 instances of jaundice and 14,000 deaths. The number of cases of transfusion hepatitis m a y be substantially greater, since it has been estimated that 9 0 - 99% of the instances are anicteric. TM ~6 Most cases of transfusion hepatitis can be prevented by routine screening of blood donors for HB~Ag. ~4, 17The incidence of HBsAg among blood donors varies with geographic location and with whether the donors are paid. 18, 19 In a study from five medical centers, the incidence of HB~Ag-positive samples varied from 0.06% to 1.47%. The incidence of post-transfusion hepatitis varied directly with the incidence of HBsAg-positive blood units transfused. Since m a n y and perhpas m o s t - o f the P0st-transfusion instances of hepatitis B are anicteric, the incidence of Post-transfusion hepatitis also varied with the vigor with which it was sought through routine screening. 2o-22 Hepatitis B is transmitted most readily by the parenteral route because serum contains the greatest concentration of virus particles. Patients on chronic hemodialysis and immunosuppressed patients have a higher incidence of hepatitis B than does the general population and constitute a significant risk to the staffs of renal transplant servicesY 3"27 Uremic patients may have persistent HB~Ag in their blood even though the clinical manifestations of hepatitis are minimal. 23, 2s, 29 HB~Ag can also be found in stool, urine, semen and saliva. 3~ h u m a n bite and sexual contact have been implicated in the transmission of hepatitis B22, 33 Other viruses, such as cytomegalovirus or Epstein-Barr virus, occasionally are responsible for hepatitis, especially in immunosuppressed patients. However, the vast majority of cases of hepatitis among surgical patients and surgeons are due to hepatitis B virus. This agent poses, in our opinion, the single most dangerous infectious risk to surgeons. Once thought to be relatively uncommon, hepatitis B now is believed to cause between 18% and 46% of all reported hepatitis. 34 The over-all case fatality rate is approximately 1%, the disease being more severe with each increasing decade of life. The incubation period is approximately 2 - 3 months, with a range of 6 weeks to 6 months. The prodromal phase begins with malaise, anorexia, nausea, vomiting, fatigue and sometimes mental depression. Urticarial rash and arthralgia, which m a y be manifestations of circulating antigen-antibody complexes, m a y occur. 35 Symptoms of hepatitis B, in contrast to hepatitis A, often begin very subtly. Clinical jaundice usually lags 1 - 2 weeks behind the other symptoms and persists for several weeks after the patient appears to improve. At the time the patient becomes jaundiced, the liver frequently is tender and enlarged. Complete recovery m a y take 2 - 3 months or longer. During " e period of recovery, the patient often has easy fatigability b u t otherwise is well. It is estimated that up to two-thirds of patients with hepatitis B infec23

tions are anicteric and would go undiagnosed if serum testing were not done. Eighty per cent to 85% of patients recover completely. However, some have a protracted icteric clinical illness that m a y last for 4 - 6 months. In addition, as m a n y as 21/2% of patients may have fulminant hepatitis. The case fatality rate in this group is related to age; the older the patient the higher the fatality rate. 36 Two types of disease are encompassed in the term chronic hepatitis. The more benign type, which has been referred to as chronic persistent hepatitis (or unresolved hepatitis), is characterized b y the absence of clinical disease, recurrent or persistent elevation of the serum aminotransferases (SGOT and SGPT) and histologic evidence of persistent inflammation of the liver. These patients are not jaundiced, although they m a y have mild hepatomegaly. Patients with the more severe disease termed chronic active hepatitis m a y have weakness and malaise with chronic or intermittent jaundice, elevation of liver enzymes and ultimate progression to cirrhosis. 3G Approximately a month after exposure, which is 1 - 2 months before the clinical manifestations described above, the presence of hepatitis B surface antigen (HBsAg) and DNA polymerase of the hepatitis B virus can be measured by various techniques. The most sensitive test for HBsAg appears to be radioimmunoassayW DNA polymerase activity has been correlated with the presence of "e" antigen, a soluble protein detected by precipitin techniques. 3s Approximately 1 - 3 weeks before the clinical illness, serum aminotransferases (SGOT and SGPT) become elevated. These enzymes remain elevated for the duration of clinical illness. HBsAg remains positive for about 3 months after the onset of clinical hepatitis. When the HBsAg disappears, antihepatitis B antibody develops. This antibody can be measured either by complement-fixation or radioimmunoassay methods, and it persists for a relatively short time after the acute illness. PREVENTION AND T R E A T M E N T . - Prevention of hepatitis B depends on early and rapid detection of individuals who have hepatitis B infections. All potential blood donors must be screened for the presence of HB~Ag and most likely in the future for "e" antigen as well. Those who are positive must be excluded from donation of blood or plasma products. Patients who are in high-risk groups, such as those on hemodialysis or those who have received frequent transfusions, as well as all who have clinical manifestations of hepatitis, should be screened for HB~Ag. Positive patients should be isolated for the duration of their hospitalization or until the acute illness subsides. In our hospital, hepatitis isolation consists of placing the patient in a room that is private or occupied by other patients whose sera contain HB~Ag. The patient's personal articles are used only by him, and all linen and other materials 24

that come into contact with secretions from the patient are double-bagged before autoclaving. Personnel wear gowns and gloves for direct care or when drawing blood or handling other body fluids. In addition to gowns and gloves, masks are worn when manipulating arteriovenous shunts or during other procedures that m a y result in aerosolization of blood. In the operating room, double gowning is recommended; in addition, the use of two pairs of gloves is recommended, whenever possible, to minimize parenteral exposure. Tissue penetration by contaminated surgical instruments is the route of acquisition of hepatitis B for most surgeonsY 9 Any means of preventing inadvertent needle or scalpel cuts (such as the use of retractors instead of hands) is of value. The staff members are not permitted to eat, drink or smoke in any areas of patient care or in the clinical laboratory, where ser u m specimens from hepatitis patients are received. Specimens sent to the clinical laboratory are identified as coming from a high-risk source and are sent in ziplock bags. If spills of blood or body fluids from HBsAg-positive patients occur, these are cleaned up with 1% sodium hypochlorite solution. One question that arises repeatedly is what is the risk to patients from antigen-positive medical personnel? In a recent commentary, Redeker 4~ pointed out that with the single exception of dentists, "there has been no recorded instance wherein antigenpositive medical personnel have been clearly implicated in the transmission of infection to patients." However, outbreaks of hepatitis B have been linked to medical personnel. 41, 42 Although few studies have been carried out, one study cited by Redeker investigated 209 patient contacts of 4 antigen-positive personnel at the Clinical Center, NIH, Bethesda, Maryland. These were 2 physicians with chronic hepatitis, a nurse who was a chronic HBsAg carrier and a food handler with acute hepatitis B. The patients were followed for 9 months and no evidence of transmission of hepatitis B could be demonstrated. Despite such reassurance, it is likely that without certain precautions transmission from an infected surgeon to a patient will occur. The discovery that the "e" antigen in the blood is indicative of the contagious state is important, since surgeons with "e" antigen in their blood probably should not operate until it no longer is detectable. HBsAg probably is not important in this regard. At present, no effective t r e a t m e n t is available for hepatitis B infections. Recent studies have described the use of interferon in eliminating antigenic components in patients with chronic active hepatitis. 43 The disappearance of the antigens will have to be related to clinical improvement before we can truly become enthusiastic over the use of interferon or interferon inducers in the treatment of hepatitis B virus infections. We should not be too pessimistic, however, about the future, 25

because other DNA viruses such as the herpes group have been shown to be susceptib]e to antiviral chemotherapy, and it certainly is a reasonable expectation that an antiviral agent specifically directed against hepatitis B virus can be found. In the meantime, however, the most logical approach toward control of hepatitis B lies in careful preventive measures that include rapid recognition of the patients and precise isolation techniques in areas such as hemodialysis units, where the prevalence of hepatitis B is greatest. (For a discussion of vaccines for hepatitis B, see the section on Prevention and T r e a t m e n t of Viral Infections.)

VIRAL INFECTIONS AS THE CAUSE OF SURGICAL ILLNESS Viruses are alleged to cause several illnesses that can require operative treatment. For m a n y of these diseases-appendicitis, Crohn's disease, ulcerative colitis, pancreatitis, e t c . - v i r u s e s are b u t one of the etiologic agents that can cause the disease. (Viruses not only m a y be etiologic but may be involved in complex p a t h o g e n e s i s - e x a c e r b a t i o n s or slow progression.) Proving that a surgical illness is caused by a virus can be difficult. The most obvious method is to culture the virus from the p a t i e n t - especially from the affected tissues. However, live virus no longer may be available for culture by the time operation or autopsy is performed. It already m a y have been eliminated from the tissues or subsequent overgrowth by bacteria m a y make detection of viable virus impossible. Neither culture nor histologic evidence of tissue infection is proof that the virus caused the actual illness, since it m a y have found the diseased tissue a fertile soil in which to replicate. Similarly, viruses cultured from other tissues, secretions, urine or feces, etc. m a y not be related to the disease being studied but m a y be innocent passengers. Some viruses m a y even be present in the individual in a latent form and be reactivated by the surgical illness rather than cause it. Thus, fever blisters, due to Herpesvirus hominis, commonly can be found in patients with a variety of illnesses. Epidemiologic studies can provide evidence of a correlation between virus infection and a disease process b u t they do not prove causation. Nevertheless, carefully performed epidemiologic studies undoubtedly are one of the best methods of establishing viruses as the cause of surgical illness. Properly performed studies require extensive viral isolation and serologic and pathologic studies. These require careful collection and handling of specimens with inoculation onto a variety of cell lines, since some viruses grow on one cell line b u t not on another. These kinds of epidemiologic studies can be very expensive and extremely timeconsuming. It is not surprising, therefore, that few carefully performed epidemiologic studies of viral disease have been carried 26

out. In fact, many of the reports to be cited present only one-or a few cases showing some evidence of viral infection. These isolated reports, however, can indicate where epidemiologic studies should focus. One reason that epidemiologic investigations must be relied on is that Koch's postulates are not easy to fulfill for viral infections. Some viruses (hepatitis virus A and B) have not been successfully grown in tissue culture. Many viruses causing disease in humans do not produce illness in common laboratory animals. Finally, attempting to produce the original illness in humans by inoculating the original virus is unacceptable, since it may produce a chronic illness, cancer, etc. APPENDICITIS. -- Viruses can cause appendicitis in different ways. Virus m a y infect the appendiceal mucosa or lymphoid tissue, producing the full-fledged disease. Virus may also cause hyperplasia of appendiceal lymphoid tissue to such a degree as to occlude the lumen. Bacteria then proliferate in the occluded appendiceal lumen, resulting in appendicitis. Alternatively, viruses may infect cells in the mucosa, resulting in their death. The denuded appendix then is a ready portal of entry for bacteria that cause appendicitis. Bonard e t al. 44 attempted to isolate virus from appendixes and mesenteric lymph nodes of 58 patients undergoing laparotomy to treat presumed appendicitis. An enterovirus was cultured from the appendix and lymph node of 1 patient and an adenovirus was recovered from the appendix, lymph node, or both, of 4 others. All 5 appendixes were grossly hyperemic. B u t histologically 3 were normal, 1 had mild inflammation and only 1 had mucosal necrosis. All 4 lymph nodes were histologically normal. Three of 5 patients had pharyngitis when abdominal symptoms began or shortly before. Something less than full-blown acute appendicitis frequently is found on microscopic examination of appendixes from which viruses have been cultured and some, as in this report, are normal. Viruses m a y cause a milder form of appendicitis than that due to other causes. Virus isolated from lymph nodes may indicate that the patient's symptoms were due not to acute appendicitis b u t to mesenteric adenitis, which can be caused by a viral infection254s ' I n another report, Bonard and Paccaud a6 tried to isolate virus from 99 appendixes and 132 mesenteric lymph nodes, or both, from 158 patients who underwent laparotomy for the clinical diagnosis of acute appendicitis. Adenovirus was isolated from appendixes and/or lymph nodes of 15 patients. More than one adenovirus type was found in some instances. At operation, 7 patients had mesenteric adenitis, 1 had acute appendicitis, 1 had both acute appendicitis and adenitis, 3 had mild appendicitis and adenitis, 1 had ileitis and adenitis and 2 had no discernible disease. 27

Seven of these 15 patients who had virus cultured had complained of abdominal pain for several weeks before laparotomy. Eleven had pharyngitis preceding the onset of abdominal pain. In another study, Bonard ~9 isolated an adenovirus from the appendix or adjacent lymph node from three instances of acute appendicitis. Appendicitis occurs in children more frequently in the months when upper respiratory infections are most prevalent. 5~Jackson 5~ found that 20% of 313 children with acute appendicitis had an upper respiratory infection in the 2 weeks before admission to the hospital, and 24% of the children had an upper respiratory infection at the time of admission. As a result of this observation, Jackson et al. 52 attempted but failed to isolate virus from 51 appendixes removed for presumed appendicitis. The fact that antibody titers to mumps virus were significantly higher in patients with appendicitis than in a control group proves nothing, since the authors did not demonstrate a rising mumps antibody titer in paired sera from any of the patients with appendicitis. Furthermore, the culture methods used were not appropriate for mumps virus isolation. Again, this report points out the need for careful, thorough methods when screening for viruses. Tobe'~3 noted an association between "mild" acute appendicitis and lymphoid hyperplasia due to enterovirus infection. The patients had right lower quadrant pain, which was relieved by appendectomy, but the histologic finding was lymphoid hyperplasia rather than acute appendicitis. Serum drawn before appendectomy had high complement-fixing antibody titers against coxsackievirus B5 and adenovirus. Tobe'~3 also used fluorescent antibody against coxsackievirus B5 and adenovirus to stain appendiceal specimens from 30 patients with acute focal appendicitis or acute diffuse appendicitis. He found evidence of coxsackievirus B in 20 mucous membranes and 5 lymphoid follicles of the 30 specimens compared to 5 of 38 mucous membranes from control specimens. Adenovirus type 7 was found in 7 mucous membranes and 2 lymph follicles of 14 appendiceal specimens. None of 20 normal appendixes had adenovirus by the fluorescent antibody technique. When Tobe e t al. ~4 instilled coxsackievirus B into the intestine of crab-eating monkeys, hyperplasia developed in the appendiceal lymphatic tissue. None of the monkeys developed acute appendicitis, howhver. Two children who had acute appendicitis shortly after the onset of infectious hepatitis were described by Geschke. 55 They both were jaundiced when appendicitis occurred. Viral studies were not performed. Evidence that viruses can cause lymphoid hyperplasia in the appendix was found by Bricout e t al., 56 who isolated enterovirus, adenovirus, coxsackievirus A2 and coxsackievirus B5 from the appendix and/or feces of 6 of 74 patients operated on with a clinical diagnosis of acute appendicitis. The complement28

fixing antibody titer increased fourfold to the isolated virus in 5 of 6 instances. On histologic examination, however, the appendixes showed marked lymphoid hyperplasia b u t no inflammation. No control patients were included. Measles can also cause mesenteric adenitis, which can be confused clinically with appendicitis. The bulk of the evidence, therefore, suggests that acute viral adenitis m a y simulate and in some cases contribute to the pathogenesis of acute appendicitis by inducing lymphoid hypertrophy. Whether viral infection of appendiceal mucosa paves the w a y for bacteria is less convincing, however. INTUSSUSCEPTION.- Ileocecal intussusception usually occurs in children less than 2 years of age. Enlargement of the mesenteric lymph nodes in the region of the terminal ileum frequently can be seen if operation is required. This enlargement has suggested that virus-induced lymphoid hyperplasia may be a significant factor in the cause of intussusception in that the ileocecal valve is ringed by lymphoid tissue, which often serves as the lead point of the intussusceptum. These lymphatic tissues are relatively larger in children less than 2 years of age than in older children. Intussusception frequently is preceded by an upper respiratory infection. Ross and Potter ~r found a 31.5% incidence of upper respiratory infection among 285 cases of intussusception. In careful study of 16 of these patients with intussusceptiofi, these authors found that 9 (56.2%) had a recent or concurrent respiratory infection, with serologic evidence of adenovirus infection in 11 of the 16 children. In addition, adenovirus was isolated from the pharynx, feces and/or mesenteric lymph node of every patient who seroconverted to adenovirus. Only 1 of 21 age-matched control subjects had adenovirus present in the feces. One-half of 24 children with intussusception had recent upper respiratory infections in a later study by Ross e t al. 5s Adenovirus w a s cultured from the feces of 15 patients, from the throat of 11 and from a mesenteric lymph node of 9. Virus was cultured from all 3 specimens of 7 children. All 15 patients seroconverted to adenovirus. Adenovirus was cultured from only 1 fecal specimen of 41 control patients (those having laparotomy for acute appendicitis) and none of 15 mesenteric lymph nodes cultured adenovirus. Adenovirus was grown from 1 of 20 throat swabs taken from Children with upper respiratory tract infection. Potter e t al. 59 also compared 48 children with intussusception with 171 age-matched controls for neutralizing antibody to adenovirus types 1, 2, 3, 5, 6 and 7. Significantly fewer children with intussusception had neutralizing antibody to types 1, 2, 5 and 6 than control children. Other investigators have also isolated adenovirus from children with intussusception more frequently than from control patients.6 o~ Bell and Steyn 45 used more thorough culture techniques and 29

were able to culture more types of virus from children with intussusception. Virus was cultured from 11 of 17 mesenteric lymph nodes removed at operation for intussusception. These investigators cultured adenovirus types 1, 2, 3, 5 and 6, echovirus types 9 and 11 and coxsackievirus B1 and B3. Eleven of 31 mesenteric nodes from children with mesenteric adenitis contained the same three viruses. These children were much older than those with intussusception, however. Only 5 of 50 mesenteric nodes from children undergoing laparotomy for other causes, including 32 instances of appendicitis, contained virus. These children were also older than the patients with intussusception. Seroconversion occurred in 10 of 17 instances of intussusception. Thus, evidence of viral infection by culture or serologic techniques was found in 13 of 17 patients with intussusception. The reports cited above have implicated adenovirus in the etiology of intussusception. Others G*have pointed out that several other factors are correlated with intussusception. Adenovirus is found frequently in children, so that some investigators have questioned whether there is an etiologic association between viral infection and intussusception. PANCREATITIS.- Viral infections are but one of many causes of pancreatitis. Two viruses implicated in the etiology of pancreatit i s - m u m p s and cytomegalovirus- grow mainly in acinar cells of the salivary gland during natural infection. That they also grow in the pancreas is not surprising, because of the similarity in structure of the salivary gland and the pancreas. The diagnosis of mumps pancreatitis is based on the clinical findings of nausea, vomiting, abdominal pain and abdominal signs during the course of mumps parotitis. Since parotitis can cause hyperamylasemia and because operation seldom is necessary in most cases of pancreatitis, diagnosis frequently is based on clinical criteria without substantiation by virus cultures or serologic studies. The incidence of clinical pancreatitis complicating mumps parotitis is low. MacDonald6~ found that symptoms of pancreatitis developed in only 3 of 500 patients with mumps parotitis. McGuiness and Gall 66 found only two instances ofpancreatitis among 1378 patients with mumps parotitis. Joske, 67 in a review of 90 patients with pancreatitis, found 2 who also had parotitis, presumably due to mumps virus infection. Renal transplant recipients are prone to both viral infections and pancreatitis. Acute pancreatitiS in 17 of 301 renal transplant recipients was reported by Penn et alY s Three patients developed pancreatitis during the course of acute hepatitis. Joske 6~ also found infectious hepatitis associated with 8 of 90 instances of acute pancreatitis. Tilney et al29 described two instances of fatal hemorrhagic pancreatitis in transplant recipients. They found 100,000 virus particles per gram of tissue in 1 patient, but the 3O

virus was not identified. No virus was cultured from the other patient, b u t cytomegalovirus was present in the lungs at postmortem examination. Van Geertruyden and Toussaint 7~ found intranuclear inclusions typical ofcytomegalovirus in cells of pancreatic acinar tissue of 1 patient dying with pancreatitis. Cultures and serologic studies were not done, however. We also have found fatal pancreatitis in a renal transplant recipient dying with disseminated cytomegalovirus. However, cytomegalovirus frequently can be found in renal transplant recipients. There are so m a n y causes of pancreatitis that the actual relationship ofcytomegalovirus to pancreatitis in these patients is not known. ULCERATIVE

LESIONS

OF

THE

GASTROINTESTINAL

TRACT. -- Vi-

ruses occasionally have been found in the base of ulcerative lesions of the esophagus, stomach, small intestine, colon and rectum. These studies, largely a compilation of case reports, superficially suggest that viruses m a y play some role in the etiology of ulcerative and inflammatory lesions of the gastrointestinal tract. Levine e t al. 7' described 2 otherwise healthy patients who had ulcers with cytomegalovirus inclusion bodies in the ulcer base. One patient developed a stomal ulcer following hemigastrectomy and gastrojejunostomy for duodenal ulcer disease. No free acid could be detected within the gastric r e m n a n t and inclusion cells were observed in the base of the ulcer. An ileal ulcer in a 54-yearold female also demonstrated intranuclear inclusions typical of cytomegalovirus in the ulcer base. The presence of a stomal ulcer in the absence of free acid and the rarity of ileal ulcers in an otherwise healthy female suggest that the virus m a y be of etiologic importance. Nakoneczna and K a y 72 reported superficial ulcerations of the rectum and anus with cytomegalic inclusion cells at their base in a 71-year-old man with disseminated cytomegalovirus infection. Evans and Williams TM identified cytomegalovirus inclusions in the base of mucosal ulcers of the cecum removed at postmortem examination from a 30-year-old female with discoid lupus erythematosus. The cells were also seen in vascular endothelium and cecal epithelium. A few inclusions were also observed in the liver and spleen. The colon pathology was not similar to ulcerative colitis macroscopically or microscopically. The rarity of cecal ulcers in all but immunosuppressed patients (who frequently have concomitant viral, bacterial and fungal infections) suggests that the virus played some part in their etiology. Toghill and McGaughey TM described a 35-year-old male with Hodgkin's disease. At postmortem examination he had disseminated cytomegalovirus infection and esophagitis and gastritis. Cytomegalovirus inclusion cells were seen in the areas of the esophagus affected by esophagitis. Two renal transplant recipients who died with ulcers of the esophagus, one of whom also had 31

It/ k

a

Fig 11.-A, gross pathology specimen of the colon from a 48-year-old female who had colectomy performed for acute ulcerative colitis. Numerous mucosal ulcerations and pseudopolyps can be seen. B, many cytomegalic inclusions (arrows) can be seen in capillary endothelium in the base of the colonic ulcers (X280). C, cytomegalic inclusions in vascular endothelium macrophages and smooth muscle cells in muscularis of colon (X380). (Courtesy of Dr. N. I= Warner.)

ulcers in the mouth, ileum and anogenital region, were reported by Montgomerie et al. 75 They cultured H e r p e s v i r u s h o m i n i s from the esophageal ulcer of 1 patient and from the ileal and anogenital ulcers of the other patient. Intranuclear inclusions typical of cytomegalovirus were seen in the base of the esophageal ulcer of the latter patient. Wong and Warner TM described 7 patients who died of leukemia or Hodgkin's disease and had gastric ulcers on postmortem examination. Intranuclear inclusions indicative of cytomegalovirus were found in the ulcer b a s e - o f t e n in the wall of blood vessels. Another patient had gastric and duodenal ulcers that were resistant to medical therapy. Gastrectomy was per32

formed but a stomal ulcer developed. She died following resection of the stomal ulcer. Autopsy revealed cytomegalic cells in the base of the jejunal ulcer. Cytomegalic inclusions in the base of esophageal, gastric, duodenal, small intestinal and colonic ulcers in 7 patients were described by Henson. 77 He also identified herpesvirus inclusions in esophageal ulcers of 2 other patients. These patients were debilitated, which most likely compromised their host defenses and led to invasion by cytomegalovirus. The virus merely may have found the base of these ulcers to be another fertile place to proliferate. Frequently we also have found cytomegalic inclusion cells in renal transplant recipients with duodenal and cecal ulcers. These patients usually had cytomegalovirus identified in other sites as well. One of the questions that is difficult to resolve is whether cytom e g a l o v i r u s - t h e virus observed so frequently in ulcerative lesions of the gastrointestinal t r a c t - i s related to the etiology of the ulcerative lesions or whether it is merely replicating in the ulcer base and/or adjacent mucosal cells. Cytomegalovirus is uncommon in healthy adults but it is found frequently in patients with compromised host defenses, such as transplant recipients, patients with lymphomas and leukemias, patients receiving chemotherapeutic agents for treatment of cancer and patients with chronic debilitating diseases. Such patients may shed virus for months or years without clinical evidence of infection and in the face of circulating antibodies directed against the virus. These patients may also have infections with a number of microbial agents and are receiving therapy with drugs (e.g., steroids) long associated with ulceration of the gastrointestinal tract. : Epidemiologic studies culturing diseased tissue specimens, body fluids and excretions for a variety of viruses and serologic studies are needed to better establish the role of viral infections in these surgical illnesses. Normal specimens from properly matched control populations always must be included. INFLAMMATORY LESIONS OF THE GASTROINTESTINAL TRACT-REGIONAL ENTERITIS AND ULCERATIVE COLITIS.-Regional enteri-

tis and ulcerative colitis have been attributed to a wide variety of causes, viral infectionsamong them. In fact, more than one etiologic agent may cause these diseases. Granulomas have been produced in rabbits and in mousefoot-pads by filtrates of ileum of patients with regional enteritis but not by filtrates of normal ileum. 7s-s~ These reports suggest that a virus may be involved. Virus has been recovered from the affected tissue of patients with regional enteritis by Gitnick et al., s' Gitnick and Rosens2 and Aronson et al. ~ These studies used new cell lines, human diploid lung cells and continuous cultures of rabbit ileal mucosa to cul33

ture viruses from diseased ileum removed at operation. Aronson e t al. ~ were able to elicit cytopathic effects (CPE) in cell culture from such tissue specimens. CPE were produced from 10 of 18 specimens from patients with regional enteritis compared to 4 of 30 control patients, 1 of whom had ulcerative colitis. The agent passed through a 50-nm filter and apparently was an RNA virus, since its growth was prevented by RNA inhibitors but not by DNA inhibitors. Serum from 3 regional enteritis patients neutralized their own viral isolates. Gitnick e t al. 8' also cultured diseased ileum from 4 patients with regional enteritis on continuous rabbit ileum and h u m a n diploid lung cells. CPE were produced by diseased ileum b u t not by ileum from 4 control patients with normal ileum. The CPE produced were similar to those described by Aronson e t a l . sa In a follow-up study, Gitnick and Rosen, s2 by electron microscopy, identified the virus as having a particle diameter of 30 nm, b u t it has not otherwise been classified. Cytomegalovirus has been implicated in the etiology of acute ulcerative colitis. Wong and Warner TM described 3 patients with ulcerative colitis who had cytomegalic inclusion cells in the colonic mucosa and base of the colonic ulcers. Two of the patients were young adults with chronic ulcerative colitis. One patient, however, was an otherwise healthy 60-year-old man who developed acute ulcerative colitis for the first time and required colectomy. T a m u r a s4 reported acute ulcerative colitis in a 65-year-old man with arteriosclerotic heart disease. He underwent colectomy for diarrhea and bleeding b u t died 7 days later of sepsis. Cytomegalic cells were seen in the base of the colonic ulcers. They were also observed in erosions of the small intestine removed at postmortem examination. Since it is unusual for ulcerative colitis to ap9 pear for the first time in the elderly, cytomegalovirus was believed to be important in the pathogenesis of these last two illnesses. Levine e t a l J ' provided additional examples of cytomegalic cells in the base of colonic ulcers of patients with acute ulcerative colitis. Two patients, ages 18 and 60, underwent operative resection when they did not respond to medical management. In addition, they both seroconverted to cytomegalovirus. Powell e t a l . ~ reported an 18-year-old male who had colon resection for ulcerative colitis that was unr.esponsive to adrenocorticotropic hormone therapy. Pathologic examination revealed changes typical of ulcerative colitis. Microscopically, cytomegalic cells were observed in the base of the colonic ulcers and in the mesenteric vascular endothelium. Virus could not be cultured from a specimen of frozen colon, and serologic studies were not done. Since cytomegalovirus is described only rarely in association with ulcerative colitis, it probably is at best an uncommon cause. The ulcerative disease of the colon in these rare patients likely is due to cytomegalovirus 34

infection, b u t the disease process is distinct from the commonly encountered ulcerative colitis. PORTAL HYPERTENSION.- Since hepatitis is the result of a viral infection, the surgical complications of viral hepatitis should be mentioned. Posthepatitic cirrhosis m a y require portasystemic shunting to correct portal hypertension. Posthepatitic cirrhosis accounts for up to 31% of all instances of cirrhosis s6, 87 and for up to 20-42% of patients requiring portasystemic shunting for portal hypertension. 88-9~ Baggenstoss and Stauffer 9' stated that portal hypertension with esophageal varices and ascites was more common in Laennec's cirrhosis whereas hepatic insufficiency was a more common sequel of posthepatitic cirrhosis. However, 15 of 45 patients with posthepatitic cirrhosis who had hematemesis during their hospital course were reported by Ratnoff and Patek22 Varices were found in 22 of 39 autopsies on these patients. Posthepatitic cirrhosis m a y develop only in patients who had a fulminant or chronic form of the disease. Boyer and Klatskin ~ believe that posthepatitic cirrhosis develops only in patients who had subacute hepatic necrosis. Sherlock et al24 likewise found cirrhosis in 8 of 17 patients with HB~Ag-positive serum and chronic liver disease. CONSTRICTIVE PERICARDITIS.-- Constrictive pericarditis is an inflammatory disease associated with pericardial thickening and calcification that restricts heart pulsations and can result in cardiac failure. In epidemics, acute pericarditis can be caused by coxsackie virus A and B, influenza virus A and B and varicellazoster virus b u t only coxsackievirus B has been implicated in the etiology of constrictive pericarditis. Gibbons et al. 95 presented a 4year-old girl whose pericardium was removed because of constrictive pericarditis. She had no history of acute pericarditis. No virus was cultured from the affected tissues, spinal fluid, pharynx or rectum. However, a fourfold increase in the neutralizing antibody titer to coxsackievirus B5 was found over a 3-month period. This seroconversion to coxsackievirus m a y have been incidental, since the etiologic agent causing constrictive pericarditis must have been present a long time before. The patient should have seroconverted to coxsackievirus previously if it had been the etiologic agent. Such is the case in a patient described by Howard and Maier. ~ They found serologic evidence for coxsackievirus B3 during an episode of acute pericarditis in a 51-year-old male. One year later, he required resection of the pericardium for constrictive pericarditis. Coxsackievirus could not be isolated from specimens of the pericardium. Robertson and Craig 9' reported a high incidence of constrictive pericarditis after an epidemic due to coxsackievirus B5. Twelve patients subsequently required pericardiectomy. 35

VIRAL INFECTIONS CAUSING CONGENITAL MALFORMATIONS THAT REQUIRE SURGICAL CORRECTION A variety of different agents m a y be important in the etiology of congenital malformations, including drugs, chemicals, infections, radiation and genetic factors. Maternal age and maternal disease are important also. From studies on viral teratogenesis in experimental animals it has been determined that: (1) Defects occur only in the presence of a fully infective virus. (2) Defects result from the death of cells in primordial tissues or from the inhibition of growth of specific tissues. For example, it is known that rubella and cytomegalovirus inhibit the division of cells in tissue culture and cause chromosome breaks. (3) If embryos are inoculated after a critical stage of development, the defect no longer occurs. For a defect to arise in a certain organ system, the viral infection must be present at the time that the organ system is actively undergoing differentiation and development. (4) Each virus produces a different range of host defects. This may be due to different viral receptor sites on developing cells. (5) The likelihood of a defect developing during embryonic viral infections is proportional to the size of the virus inoculum. 9s Whether a viral infection of a pregnant female and/or the developing fetus results in a congenital defect depends on a number of factors: (1) the maternal immune status; (2) the particular virus in question; (3) the strain of the virus; (4) the maternal-fetal host susceptibility; (5) the developmental stage of the fetus. Virus m a y gain access to the developing embryo transplacentally following maternal infection or via an ascending infection through the vagina. The lack of fetal immune response may allow proliferation of the virus in the developing fetus for a prolonged period without challenge from the host. Proving that a virus is a teratogen in humans can be difficult. Many studies have been carried out retrospectively comparing the incidence of illness during pregnancy in mothers of children with congenital defects with that of mothers of normal children. However, these are unreliable, since mothers of normal infants are more likely to minimize any illness that they m a y have had during pregnancy and it is difficult to remember months or years later what may have been a minor illness. Some viruses that have been demonstrated to cause congenital defects may produce completely asymptomatic infections in the mother, or infection of the fetus can occur without maternal infection2 ~ Serologic studies have also been used to provide retrospective evidence of viral infection in children with congenital defects. Although the presence of antibodies to viruses is proof of previous exposure to the virus, single serum specimens do not provide evidence of recent infection. Only a recent increase in antibody titer during pregnancy gives evidence of active infection. 36

Prospective studies are more reliable but more difficult. For valid studies, accurate recordings of illness, culture data from the mother and determination of serial antibody levels to detect subclinical infections during the early months of pregnancy must be obtained. Since major embryonic development is essentially complete during the first trimester, these prospective studies must focus their primary attention on this early period. By the time of the first visit to a physician, the viral infection already may have occurred. These studies require the inoculation of specimens into a wide variety of cell lines and a search for antibodies against a battery of viruses. Otherwise, the viruses causing anomalies may not be found. Prolonged follow-up examination of the infant is required, since anomalies may not be recognized at birth. Many viral infections that could possibly produce congenital defects fail to give rise to easily identifiable syndromes or a complex of malformations. Virus-caused congenital malformations that may require surgical correction later probably represent a minority of anomalies due to viral infections. Most congenital defects due to viral infections affect the central nervous system, producing microcephaly, mental retardation, deafness and other uncorrectable lesions. Congenital viral infection can also result in infection of the fetus without producing congenital anomalies. RUBELLA. Gregg '~176 was the first to call attention to the teratogenicity of rubella virus infection. Cataracts, deafness, mental retardation and congenital heart disease are the hallmarks of the rubella syndrome. The congenital rubella syndrome is characterized by one or more of the following problems in the neonate: hemolytic anemia, thrombocytopenia, hepatosplenomegaly, necrotizing myocarditis, pneumonitis, long bone lesions, central nervous system abnormalities and deafness. Potentially correctable heart defects include patent ductus arteriosus, pulmonary valve stenosis, supravalvar pulmonic stenosis, hypoplasia of the peripheral pulmonary artery branches, coarctation of the aorta, atrial septal defect, ventricular septal defect, tetralogy of Fallot, atrioventricular canal, transposition of the great vessels, Eisenmenger syndrome and aortic stenosis. '~176 Lesions that occur less frequently have been reported also. Singer e t a l . ' ' ~ described the right subclavian artery arising from the descending aorta in an infant with congenital rubella. They found bilateral inguinal hernias and undescended testicles in other infants from whom rubella virus was cultured. Hardy and Sever'" also described indirect inguinal hernias in 2 infants with congenital rubella. Unilateral stenosis of the renal artery ostium found at postmortem examination of an infant with congenital rubella was reported by Esterly and Openheimer. '~ Campbell '~ found the incidence of esophageal atresia in children with congenital rubella to be 6 times greater than that seen in 37 -

-

control children. The incidence of pyloric stenosis was 3 times greater than in controls. Esterly and Talbert 1~ reported 1-month-old twin females who required operation for jejunal atresia. Virus was not isolated from the infants and there was no clear seroconversion to the virus in the mother. Diagnosis of congenital rubella was presumed on the basis of an elevated hemagglutination inhibition antibody titer to rubella virus in the mother's serum. The infants did not have other stigmata of the rubella syndrome. An instance of a cleft palate in an infant whose mother had a clinical diagnosis of rubella in the fourth week of pregnancy was described by Beswick et al. ~12 Swan et al. u3 described an infant with talipes equinovarus whose mother had clinical rubella in the second month of pregnancy. However, these instances of isolated congenital defects occurring in children whose mothers had rubella may be independent events. CYTOMEGALOVIRUS.--Cytomegalovirus is the most common congenital infection of infants; 0.5-1.0% of all newborn infants excrete virus in the urine, u4 Most of these infants are and remain completely asymptomatic. The widespread use of a rubella vaccine has decreased the incidence of congenital defects due to the rubella virus so that cytomegalovirus now accounts for the largest number of virus-induced congenital defects. The full-blown clinical picture of congenital cytomegalovirus infection is characterized by small infant size, hepatosplenomegaly, purpura, ecchymoses, jaundice, respiratory illness, microcephaly, cerebral calcifications, thrombocytopenia, feeding difficulties and failure to thrive. Numerous intranuclear inclusion bodies can be seen in many organs, such as liver, lung, kidney and intestine. H4 This virus generally is clinically inapparent in the mother but pregnancy frequently is associated with serologic evidence of infection and the virus can be isolated from the urine and cervix. The diagnosis depends on the isolation of virus or the finding of typical intranuclear inclusions in the tissues of infected infants. Congenital defects of surgical importance due to cytomegalovirus have not been demonstrated frequently. Okamoto et al. n5 described 4 Japanese infants who had typical intranuclear inclusion bodies suggestive of cytomegalovirus infection in autopsy specimens of liver, kidney and stomach. The first infant had a Taussig-Bing deformity, coarctation of the aorta, hypoplasia of the ascending aorta and patent ductus arteriosus. The second infant had transposition of the great vessels and a patent ductus arteriosus. The third child had transposition of the great vessels, common ventricle, a left superior vena cava, total anomalous drainage of the pulmonary veins and absence of the spleen. The last infant had corrected transposition of the great vessels and a ventricular septal defect. 38

Twin infants were studied by McAllister e t al. ~6 Cytomegalovirus was cultured from one twin with pulmonary valve stenosis and mitral stenosis. The other twin died with clinical and histologic evidence of cytomegalovirus, but this Child did not have congenital cardiac defects. Ebert HT reported autopsy findings of several infants with typical inclusions in the parotid gland, a common site of infection of cytomegalovirus. Among the defects noticed were coarctation of the aortic isthmus, ventricular septal defect, patent ductus arteriosus and subvalvar aortic stenosis. Finally, Quan and Strauss Hs observed a ventricular septal defect in the postmortem examination of a stillborn infant. Intranuclear inclusions typical of cytomegalovirus infection were seen in many organs and in the placenta of the mother. Lang u9 examined 26 infants with cytomegalic inclusion disease. Eleven of 14 males had inguinal hernias. No congenital heart disease was reported. INFLUENZA.- Several reports document an increased incidence of congenital anomalies in children whose mothers had influenza early in pregnancy. Hamburger and Habel ~~ and Heath e t a l . ~21 showed that influenza virus is teratogenic for chick embryos. Pachaly and Schiiermann ~22 described congenital anomalies in infants following the influenza epidemic in 1957 in Chile. They described 4 infants with surgically important lesions: truncus arteriosus; pseudotruncus, pulmonary artery atresia and a hypoplastic right ventricle; a ventricular septal defect and Meckers diverticulum; and esophageal atresia. In a study covering three influenza outbreaks in Birmingham, England from 1959 to 1961, Leck ~3 found the incidence of esophageal atresia, and atresia and cleft lip more than doubled compared with preceding years. Esophageal atresia was especially common after each epidemic. COXSACKIEVIRUS B.--In retrospective and prospective studies, Evans and Brown ~24'~25found serologic evidence of maternal coxsackievirus B infection in the etiology of congenital anomalies. Significantly more mothers of infants with congenital heart disease had increased antibodies to coxsackievirus B during pregnancy than did matched controls. Patent ductus arteriosus, ventricular septal defect, atrial septal defect, transposition of the great vessels and tricuspid atresia were found. MUMPS.-A 13% incidence of congenital anomalies among infants whose mothers contracted mumps parotitiS during the first trimester of pregnancy was reported by Dumont. ~2G He found unspecified congenital heart defects, 1 patient with intestinal atresia, and an anal rectal malformation. OTHER vmUSES.--There have been reports suggesting that other maternal viral infections can lead to congenital defects in 39

the offspring. These viruses include varicella-zoster, echovirus, hepatitis, peliovirus, measles, smallpox, encephalitis virus, adenovirus and H e r p e s v i r u s h o m i n i s . "8, 99, ,24, ,~7 Most of the defects were not of surgical importance and are isolated descriptions. There is no clear syndrome of defects associated with maternal infection with these viruses as there is with rubella virus.

HERPESVIRUS INFECTIONS FOLLOWING OPEN HEART OPERATION AND BLOOD TRANSFUSION Following open heart procedures with extracorporeal perfusion, a syndrome that resembles infectious mononucleosis can develop, variously called the peostperfusion syndrome, post-transfusion syndrome or the postpump syndrome. Most often it is characterized by fever, erythematous rash, hepatomegaly, splenomegaly, eosinophilia and atypical lymphocytes in the peripheral blood. However, the heterophil test is negative. '28-'3~Usually the postperfusion syndrome appears within 3 - 5 weeks of operation and almost always is self-limited and not fatal. However, it may lead to unnecessary hospitalization and to an expensive search for a source of fever. The syndrome is unusual in adults, b u t as m a n y as 10% of children and young adults can be affected. In some centers, this syndrome has been traced to the use of fresh blood for perfusion, and a similar syndrome can be observed in patients receiving transfusions of fresh whole blood even though they do not undergo cardiac operation. '3'. ,32 Both Epstein-Barr virus and cytomegalovirus have been implicated in the etiology of the pest-transfusion and pestperfusion syndromes. These syndromes have also been attributed to re-exacerbation of rheumatic fever, an autoimmune response to traumatized cardiac tissue, an inflammatory response to blood in the pericardial sac and a cellular immunologic response to the transfused white blood cells. The most likely cause of this syndrome is infection with cytomegalovirus. Significant increases in cytomegalovirus antibody titers occur in as m a n y as 3 - 6 0 % of patients undergoing open heart operations even though the incidence of clinical postperfusion syndrome is significantly lower. '~176176 It is estimated that only I in every 10 patients who have postoperative cytomegalovirus develops the pestperfusion syndrome. '33 Still unanswered is whether the transfused blood is the source of virus or whether the blood transfusion and t r a u m a of the operative procedures lead to reactivation of endogenous latent virus in the patient. Several studies have implicated the transfused blood as the source of viral infection. Armstrong et al. TM were able to isolate cytomegalovirus for 28 days from whole blood stored at 4~ and from fresh frozen plasma for up to 97 days. Thus, fresh whole blood does not necessarily have to be used in order for live virus to be transfused. Diosi et al., '35 however, have noted that 40

cytomegalovirus normally does not survive storage under blood bank conditions. The anticytomegalovirus antibody levels of 187 blood donors for 24 patients undergoing open heart surgery were studied prospectively by Klemola et al. 136 None of the 24 blood recipients had prior detectable antibody to cytomegalovirus but 14 developed antibody after the operation. There was no significant difference in anticytomegalovirus antibody titers between the donors who gave blood to the 14 patients who had anticytomegalovirus antibody titers after operation and those who donated blood to the 10 patients who did not have postoperative antibody titers. However, all 14 patients who developed evidence of cytomegalovirus infection had received fresh blood from at least 3 seropositive donors. Paloheimo et al. ~37 found that 30% of 88 donors of fresh blood did not have antibodies to cytomegalovirus. In a study of open heart patients, these investigators found that 60 patients who underwent open heart procedures without receiving fresh blood had no increase in cytomegalovirus antibody titer following the operative procedure. On the other hand, 63 patients who underwent such operations and were perfused with fresh blood had an incidence of postoperative cytomegalovirus infection that was inversely proportional to the preoperative antibody titer of the recipient. They concluded that cytomegalovirus is destroyed by storage of blood and that fresh blood must be the source of virus. A number of similar epidemiologic studies of the postperfusion syndrome have been carried out. 13~,~3s-14sMost are consistent with the idea that cytomegalovirus and to a lesser extent Epstein-Barr virus and H e r p e s v i r u s h o m i n i s cause disease within the first 2 - 3 postoperative months. The virus itself sometimes can be cultured from the blood or urine, but proof of the infecting agent is most consistently found in change in antibody titer. Seroconversion to one or more viruses most often occurs without clinical illness, but the clinical syndrome almost always is followed by seroconversion after recovery. In contrast, viral infections only rarely have been reported following other operative procedures. Stevens et al. ~49 reported that 13 of 41 tumor patients undergoing a variety of operative procedures seroconverted to cytomegalovirus 8 - 1 6 weeks following operation. Cytomegalovirus was isolated from 1. The incidence of antibody increase was correlated directly with the volume of the blood received and not with the time the blood had been stored. No clear-cut clinical illness was associated with the viral infections. Murphy et al. ~5~ described a female patient who received 2 fresh units of blood while undergoing a hysterectomy and bilateral salpingo-oophorectomy. For 2 weeks she had fever and night sweats and atypical lymphocytes appeared on the peripheral blood smear. The antibody titer to cytomegalovirus increased eightfold over the preoperative values. It was discovered that one 41

of the blood donors had a positive antibody titer to cytomegalovirus. '~' Henson '52 described a 44-year-old female who underwent two operat!ons for extensive t r a u m a to the head,.chest and abdomen. She received 79 units of whole blood and 75 units of plasma before she died 2 months later. On postmortem examination she was observed t o have generalized cytomegalic inclusion disease without evidence of other infection. The majority of postoperative viral infections appear to be asymptomatic, but the illnesses reported are so nonspecific that the symptoms and signs might well be attributed to a variety of insignificant problems. Most have been detected by seroconversion, and no extensive culture studies have been done. Furthermore, there has been no attempt at an extensive prospective epidemiologic study examining the incidence of viral infections in a variety of surgical procedures and no careful attempt to correlate minor clinical illness with viral screening has been undertaken. Such a study would give a truer picture of the incidence and significance of viral infections in the postoperative period. In addition, the relative role of exogenous sources of virus and its reactivation from latent endogenous sites might be obtained. One cannot assume that such infections are not of clinical significance, because it is known that certain viruses so impair host defenses that seriou's and even lethal bacterial infections can supervene. Viral cultures and serologic studies are obtained only infrequently in patients who develop febrile illness in the postoperative course, however, whereas extensive bacteriologic and fungal studies usually are obtained.

VIRAL INFECTIONS IN COMPROMISED HOSTS AFTER OPERATION VIRAL INFECTIONS IN PATIENTS WITH MALIGNANT TUMORS.Patients with cancer represent a population ofcompromised hosts who are susceptible to a variety of opportunistic infections. Members of the herpesvirus family most commonly have been isolated from patients with neoplastic disease. Patients with malignancies of hematologic origin are particularly susceptible to infections with the herpesvirusesY,'53-'6' Cytomegalovirus occurs more commonly in patients with leukemias whereas varicellazoster is found more frequently in patients with Hodgkin's disease and non-Hodgkin's ]ymphomas.'5~-'6' Wright and Winer'G' recorded a 0.22% incidence of zoster infection among 51,292 patients who did not have neoplastic disease compared with an incidence of 0.85% among 3984 patients with a Varietyof neoplasms, almost a fourfoldincrease. Of the 34 cancer patients who had zoster, 16 had leukemias or lymphomas and 10 had Hodgkin's disease. Other patients had neoplasms of the kidney, prostate, bladder, bronchus, stomach, skin and colon. 42

Zoster (manifested clinically as shingles) occurs in 8 - 2 5 % of patients with Hodgkin's disease and in up to 97% of patients with non-Hodgkin's lymphomas. '57, ,60 The incidence of zoster increases dramatically in patients who have been splenectomized. '59 The incidence of zoster was 22% in splenectomized patients with.Hodgkin's disease compared to a 13.1% incidence in nonsplenectomized patients in a study of Goffinet e t al. '59 Chemotherapy increases the incidence (up to 29%) whether or not splenectomy is done. 159 Radiotherapy also leads to a high incidence. 15s Among those patients who developed zoster more than 6 months following the primary t r e a t m e n t there was a higher incidence (10 of 17) in those patients who subsequently developed recurrence than in those who did not (3 of 14). '~8 Wilson e t a l . 15s thought that late (more than 6 months following primary treatment) zoster was an important prognostic sign, since it could be a harbinger of recurrent disease. Cutaneous zoster is an extremely uncommon manifestation of Varicella-zoster infection in children. However, it has been observed in children with leukemia, Hodgkin's disease, Wilms' tumors and Ewing's sarcoma. '54, 162-164 Viral infections also occur in patients with solid tumors. Duvall e t al. 165 recovered cytomegalovirus from the urine, sputum or both in 11 of 32 adults with solid malignant neoplasms of unspecified types. Only 2 patients had seroconversion to cytomegalovirus. Ten of the 11 patients were receiving steroids or chemotherapeutic agents. These authors did not recover cytomegalovirus from 7 control patients who did not have neoplasms. Wong and Warner TM reported 2 patients with malignant melanoma and carcinoma of the lung who had cytomegalic inclusion disease found at autopsy. , The occurrence of viral infections in cancer patients is not known to be associated wi~h a poor prognosis except for those patients who have disseminated zoster. It is interesting that viral infections are discovered to be important contributors to complex illnesses only when they are associated with characteristic cliniCal pictures such as rashes (e.g), zoster). Viruses that cause malaise, hepatic malfunction or fever will easily be missed because the illness will be attributed to the neoplasm, bacterial infection, chemotherapeutic drugs, etc. Thorough postmortem examinations may not be performed in patients dying of cancer, and little note m a y be taken of the nonspecific inflammatory changes induced by viral illnesses. Only when careful systematic epidemiologic studies are carried out will the true importance of viral infections be determined. We have found a high incidence of herpesvirus infections in cancer chemotherapy patients when viral cultures and serology are done routinely in these compromised patients. VIRAL INFECTIONS FOLLOWING RENAL TRANSPLANTATION.Because they are receiving large doses of immunosuppressive 43

agents, renal transplant recipients represent a population of compromised hosts who are more subject to opportunistic infections. Immunosuppressive agents are known to increase the susceptibility to viruses. ~GG''72 Corticosteroids inhibit the formation and activity of viral interference proteins, and they have been shown to reactivate latent cytomegalovirus in guinea pigs. '53 Immunologic reactions to which these patients are subject can also reactivate latent viral infections. 'Ta Wu e t al. ~4 showed enhancement of cytomegalovirus infection in skin-grafted mice compared to ungrafted mice. Members of the herpesvirus family have been found most frequently following renal transplantation. Of the herpesviruses, cytomegalovirus has been reported most frequently. Hill et al. ~7~ and Hedley-Whyte and Craighead ~76were the first to report cytomegalovirus infection in renal allograft recipients. These and subsequent reports indicated a high incidence of cytomegalic inclusion cells in lung, parotid glands, lymph nodes, liver, pancreas, parathyroid and brain at autopsy after renal transplantation. Rifkind et al. '77 found cytomegalovirus in the lungs and in other organs of 27 of 51 autopsied renal transplant recipients. They also found cytomegalovirus in the urine of 17 of 26 living transplant recipients. In another early paper, Craighead et al. ~Ts found that 30 of 41 patients had serologic evidence or isolation ofcytomegalovirus post-transplantation. They also found that 22 (91%) of 24 patients with serologic evidence of cytomegalovirus pretransplant had culture or serologic evidence of infection posttransplant. However, only 8 (47%) of 17 patients who had not been exposed to cytomegalovirus before transplantation had subsequent infection. Cytomegalovirus infection in renal allograft recipients has been confirmed by many others, and infection rates of 72-93% have been recorded following renal transplantation. ~79"~89We have also confirmed that the incidence of infection in patients following transplantation is higher in those who had antibodies to cytomegalovirus prior to receiving their kidney allograft than in kidney recipients who did not. ~84,~s6Thus, in many instances, the infection may have resulted from reactivation of latent virus in renal transplant recipients. The source of infection in transplant patients thus may be different from that of patients having cytomegalovirus after open heart operations. In the latter instance, transfusion of fresh blood is the most likely source of the viral infection whereas transplant recipients appear to reactivate latent viral infection. The distinction is not entirely clear, however, since transplant recipients also receive many blood transfusions that can harbor the virus. In addition, Ho et al. ~~176 and Betts et al. T M have indicated that the donor kidney may be the source of virus. They showed a higher incidence of infections in the recipients whose living related donors had antibody to cytomegalovirus prior to transplantation than in those whose donors did not have 44

antibody. In a similar study, we were unable to culture virus from any donor kidney, and the very high incidence ofcytomegalovirus infection was inconsistent with the idea that most infections were transmitted from the donor. The common finding of cytomegalovirus in asymptomatic patients after transplantation has suggested to many observers that cytomegalovirus may be an incidental finding. Many of the early investigators were unable to find any clinical syndrome caused by cytomegalovirus even in patients dying of a variety of causes. However, several other reports have implicated cytomegalovirus in the etiology of rejection episodes as the principal cause of mild febrile illnesses, hepatitis, a mononucleosis-like syndrome, hemolytic anemia, acute inclusion retinitis, pneumonitis, encephalitis and deathJ 84"18~ Simmons et al. T M described 43 pulmonary complications among 212 renal allograft recipients. Among these were 14 instances of nodular alveolar pneumonitis. Nine of the 14 patients had cytomegalovirus, 6 had cytomegalovirus cultured from the lung in the absence of other bacterial or fungal pathogens, I had cytomegalovirus isolated from the kidney biopsy and 2 seroconverted to cytomegalovirus. One patient had influenzal pneumonia and another had Herpesvirus hominis infection. Five of 6 patients with true viral pneumonia died of bacterial and fungal superinFig 12.-Composite clinical course of 46 renal transplant recipients with known time of cytomegalovirus infection. Fever, renal malfunction and leukopenia were followed by the development of antibodies to cytomegalovirus. Viral isolation is not always possible until seroconversion occurs. (Reproduced, with permission, from Simmons, R. L, Cytomegalovirus: Clinical virological correlations in renal transplant recipients, Ann. Surg. 180:623, 1974.)

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Days Post-Transplant

45

Fig 13.-Cytomegalovirus pulmonary infection. A and B, a 29-year-old female diabetic received a kidney transplant from her sister on December 21, 1976. The serum creatinine was normal within 5 days. She was discharged 18 days after transplantation but returned on January 19, 1977 because of a fever of 103~ F. A, January 20, 1977, the chest x-ray was consistent with pulmonary edema. Cytomegalovirus infection can be impossible to differentiate from pulmonary edema based on chest x-ray alone. Both can produce low-grade fever, but only CMV infection results in low white blood cell count. B, chest x-ray on February 9, 1977, demonstrating diffuse alveolar infiltrates. The white blood cell count was 1700 per mm ~. This x-ray is typical but not diagnostic of CMV infection. She continued to have fevers to 104.6 ~ F. Normal renal function was maintained until 5 days prior to death despite the cessation of azathioprine and the reduction of prednisone to 10 mg per day. She died February 11, 1977. CMV was cultured from the lungs. No other organisms were identified. The anti-CMV antibody titer remained < 4 until her death. C, a 37-year-old male diabetic received a transplant from his sister on June 17, 1971. Eleven days post-transplant he had a fever of 103.4 ~ F. Chest x-ray revealed bilateral interstitial infiltrates. The white blood cell count fell to 1400 per mm 3. Chest x-ray on July 5, 1971 demonstrated bilateral infiltrates in an alveolar pattern; lung biopsy revealed cytomegalic inclusions and CMV was cultured from the biopsy specimen. Reduction of immunosuppression to very low levels resulted in clearing of the lung infiltrates. The fever gradually disappeared and the white blood cell count increased to 14,000. The serum creatinine was 1.2 mg/dl at the time of discharge on August 10, 1971.

fection. Thus, it appears that cytomegalovirus infection may also precede and predispose patients to bacterial and fungal superinfections. Recently, several large systematic epidemiologic investigations of renal transplant recipients have been carried out. Lopez et al. I~" systematically screened 61 renal transplant recipients for viral infections. Only herpesviruses (cytomegalovirus, H e r p e s v i r u s h o m i n i s and varicella-zoster were implicated consistently. Fifty-three (87%) patients had at least one viral infection and 47 46

(77%) had cytomegalovirus. Fifteen (24.6%) had H e r p e s v i r u s h o m i n i s and 7 (11.4%) had varicella-zoster virus. These authors were able to make a clear correlation of laboratory evidence of viral infection with clinical findings. The clinical syndrome associated with viral infection consisted of fever, leukopenia and usually reversible renal allograft rejection. Many patients continued to excrete virus even after they had made an antibody response and had totally recovered from the clinical illness. Lopez et al. 192 frequently identified a clear-cut association between viral infection and rejection episodes. The two mechanisms that seemed to explain the relationship best were (1) the viral infection acts as an immunologic adjuvant and triggers the rejection of the allograft and (2) allograft rejection activates a latent viral infection. There are experimental models that support both explanations. Viral infections are known to have profound effects on immune responses, and experimental cytomegalovirus under appropriate conditions has been shown to act as an immunologic adjuvant causing decreased graft survival times in mice.5 On the other hand, graft rejection and other vigorous immunologic reactions have been shown to reactivate latent viruses, including cytomegalovirus. Simmons et al. Is2 have speculated on the role of mild viral and bacterial infections in triggering episodes of renal allograft rejections. They have suggested that these infections are capable of altering the delicate immunologic balance between the host and graft, thus leading to rejection episodes. It is important to note that most such rejection episodes respond to antirejection therapy and that the febrile illness most often is self-limited. A few such patients die, however, and the various syndromes have been studied further. Simmons et al. T M showed that 110 of 132 renal transplant recipients had evidence of infection with herpesviruses post-transplant. Eighty-nine of the 132 patients had serologic or culture evidence of cytomegalovirus. Of the 89 patients with cytomegalovirus, 6 died within a month of virus isolation. These 6 patients were distinguished from those who recovered by a decrease in antibody response to the virus, suppressed lymphocyte response to mitogens and absent histologic evidence of rejection of the renal allograft. The 83 patients who survived had a self-limited syndrome characterized by fever, increasing anticytomegalovirus antibody response and renal biopsies that demonstrated typical rejection that responded to antirejection treatment. Thus, in immunocompetent patients, infection was associated with allograft rejection, a prompt antibody response to the virus and recovery. However, the severely immunosuppressed patients who could not make an antibody response and did not exhibit allograft rejection were further immunosup-' pressed by the viral infection and were susceptible to opportunistic infections that could cause death. The clinical characteristics of the lethal form of this illness have been described in detail. 193 47

All the previous studies were retrospective or concurrent. Howard et al? 86 prospectively studied 112 recipients of first kidney allograft with serial culture and se~:ologic studies beginning prior to transplantation. They found that 104 (92.9%) had serologic evidence of infection with at least one of the herpesviruses before transplantation. Of the 104 patients, 79 (75.9%) became infected after transplantation. Five of 8 patients who were not infected prior to transplantation became infected after transplantation. Cytomegalovirus was the most common of viruses detected (56 of the 112 patients). Forty-nine (89%) of the 55 patients who had antibody to cytomegalovirus prior to transplantation had cytomegalovirus infection pest-transplant, confirmed by seroconversion or isolation or both. On the other hand, only 17 (29.8%) of the 57 patients who did not have antibodies to cytomegalovirus prior to transplantation became infected with cytomegalovirus post-transplant. Following transplantation, 45 patients became infected with H e r p e s v i r u s h o m i n i s and 18 were infected with varicella-zoster. Three patients had infections with influenza A, peliovirus and measles virus. Almost half of the viral infections occurred between 30 and 60 days following transplantation and 88% of the. infections occurred within 125 days of transplantation. Clinical illness was associated with cytomegalovirus in 72.4% of the cases. Common findings were fever, leukopenia and rejection. Eight patients had cytomegalovirus associated with severe sytemic illness characterized by clotting disorders, hepatic dysfunction, pneumonitis, weakness, malaise and muscle wasting. Seven of these 8 patients died. A similar epidemiologic investigation of cytomegalovirus infection after transplantation was carried out by Fiala et al. ls5 They found that active cytomegalovirus infection appeared in 96% of 89 patients after renal transplantation. Infection with Herpesvirus h o m i n i s occurred in 35% of the patients. Varicellazoster occurred in 24%. They found no evidence of Epstein-Barr virus infection in any of their patients. They found cytomegalovirus viremia in 42% of their patients an average of 2 months after transplantation, and it was followed by chronic viruria. On the other hand, we 1~, ls~. 186 were unable to detect viremia routinely in renal transplant recipients. Fiala et al. ~85 found cytomegalovirus infection most frequently 1 - 3 months after transplantation, characterized by fever, pneumonitis, leukopenia and arthralgia. Patients developing cytomegalovirus later than 4 months after transplantation developed hepatitis and retinitis. The cytomegalovirus did not seem to originate during hemodialysis, nor was the hospital staff a source of infection, since the virus could not be isolated from any staff member. These authors concluded that an endogenous reactivation in the recipients of allografts was the most likely mechanism of viral infection. They also found an increased incidence of rejection in patients who were infected with 48

cytomegalovirus. Immunofluorescence studies indicated that cytomegalovirus antigens could be present in the tubular cells and interstitium without the presence of inclusions detectable by light microscopy. Since some viruses share certain antigenic markers with histocompatibility antigens, the presence of viral antigens in the renal cells might provide a mechanism for initiating the rejection reaction. There is no evidence yet that herpesviruses have antigens that cross-react with human histocompatibility antigens. Although most emphasis has been placed on cytomegalovirus by transplant groups, it is obvious that the other herpesviruses are clinically important in transplant recipients as well. Cutaneous zoster is common following transplantation, but it usually is a self-limited illness with mild transient fever (or none at all) and without leukopenia or signs of rejection. However, disseminated chickenpox has occurred in several patients never previously exposed to the virus, with 1 death. A few children have developed mild chickenpox despite a previous episode of the disease. Previously uninfected children should receive zoster immune plasma if they are exposed to chickenpox. 191,~95 As noted above, H e r p e s v i r u s h o m i n i s is common in transplant recipients. It usually appears in the early post-transplant months and is characterized by "cold sores" or vesicular eruption of the buccal mucosa, pharynx or genitalia. Herpes esophagitis has been found associated with dysphagia, and Montgomerie et al. ~5 cultured the virus from ulcers of the esophagus, ileum and anogenital region. Even the "cold sores" can become necrotic and can be slow to heal, but recovery is the rule. There is some suggestion that episodes of rejection are associated with local labial herpes lesions, but the association is not as well founded as that observed with cytomegalovirus. Rare cases of disseminated herpes, herpes encephalitis and herpes hepatitis have occurred in transplant recipients. Like most viral infections in transplant recipients, the development of antibodies can occur in the absence of clinical illness. Strauch et al. ~96 recovered Epstein-Barr virus from 52% of renal allograft recipients. Virus was not associated with clinical illness. However, we recently encountered 2 patients who had a significant rise in antibody to Epstein-Barr virus at the same time lymphomas became clinically apparent (see below). Viruses have also been implicated in the pathogenesis of complications previously thought to be due to technical error. Coleman~97 observed a new papovavirus, designated as the BK virus, by electron microscopy from the urine of 5 renal transplant recipients and cultured the virus from 2 of these. Four patients remained healthy but 1 had ureteral obstruction. This last patient and 1 of 5 other patients had ureteral obstruction more than 100 days after transplantation and had viral inclusions in the 49

ureteral endothelium. The virus was not identified, however. Coleman '97 also described 3 recipients of liver homografts who required revision of the cholecystojejunostomy because of cystic duct obstruction. Cytomegalovirus was isolated from the cystic duct in all 3 instances. Whether it had any role in the etiology of the cystic duct obstruction is not known. In addition to causing morbidity during the period after transplantation, viral infection may be responsible for the development of renal failure, which ultimately leads to the need for transplantation developed hepatitis and retinitis. The cytomegalSgitis virus and lactic dehydrogenase virus have been shown to cause glomerulonephritis in mice. '98 In humans, varicella-zoster, echovirus type 9, hepatitis B virus, coxsackievirus B4, vaccinia and mumps viruses have been implicated in the cause of nephritis and glomerulonephritis.'99-2~ VIRAL INFECTIONS AND CANCER IN TRANSPLANT R E C I P I E N T S . A n increased incidence of malignancy has been reported in trans-

plant recipients. The pathogenesis of this increase originally was attributed to the loss of immunosurveillance due to administered immunosuppressive drugs. However, not all tumors are increased in proportion to their occurrence in the general population-for example, 75% of reported tumors in transplant recipients are lymphoproliferative tumors or carcinoma of the skin, lip or cervix. These findings cannot be totally explained by impaired immunosurveillance and an alternative must be considered. Matas e t a l . 2~ suggested that the pathogenesis of cancer in transplant recipients may be related to the chronic antigenic stimulation from the graft plus herpesvirus infection. They argued that since 90% of transplant recipients develop clinical or serologic evidence of herpesvirus infection, and since herpesviruses have been implicated in the pathogenesis of lymphoproliferative tumors and carcinoma of the skin or cervix, the herpesviruses may be important pathogenic factors in the development of cancer in transplant recipients. Furthermore, the herpesviruses can remain in latent form and be reactivated by allogeneic stimulation (i.e., from the graft) and/or immunosuppression. Since the herpesviruses produce lesions of the skin, cervix and neural tissue, i.e.,, exactly those sites where cancer develops in transplant recipients, herpesviruses may well be responsible for the increased incidence of both lymphoproliferative tumors and carcinoma of the skin, tip and cervix in transplant recipients. Recently we have encountered 2 transplant recipients with lymphoproliferative tumors. Such tumors commonly have been classified as reticulum cell sarcomas in the past but actually appear to be due to a proliferation of B lymphocytes, i.e., those lymphocytes that secrete immunoglobulins.2~ Both patients demonstrated cells that produced immunoglobulin, and 1 such patient 50

had hyperglobulinemia with IgG, IgM and IgA. If these were true neoplasms, one would expect that only one of the immunoglobulins (for example, IgG) would be produced by these tumors, but immunohistologic studies have demonstrated that several types of immunoglobulins are produced by these tumors, suggesting that this is a pseudotumor and represents not a monoclonal expansion of a single cell (i.e., cancer) but a polyclonal expansion of the B lymphocyte population (i.e., a hyperplastic disease). One patient recently had been exposed to infectious mononucleosis and demonstrated recent seroconversion to the Epstein-Barr virus antigens, suggesting that the Epstein-Barr virus in some way caused the proliferation of the B lymphocyte population, which led to death from the pseudomalignancy. The other patient had seroconversion to the Epstein-Barr virus as well, and has shown regression of the tumor after cessation of immunosuppression and removal of the graft. Thus, our original hypothesis, at least in the case of the post-transplant lymphoproliferative tumors, may be substantiated and these patients may serve as a "missing link" between the viral causation of cancer and viral infection.

PREVENTION AND TREATMENT OF VIRAL INFECTIONS VIRAL VACCINES.-Vaccines are preparations of live attenuated, or killed, virus that elicit a protective immune response. Generally, attenuation of virus is accomplished by multiple passages in tissue culture, which permits selection of mutant virus strains whose pathogenicity is lowered. Live viral vaccines have the advantage of eliciting a more vigorous immune response due to some limited multiplication of virus within the host. In addition, live vaccines may be administered orally or intranasally, thereby facilitating an immune response at the natural site of infection. Killed viral vaccines must be administered parenterally but have the advantage of eliminating the danger of transmitting active viral infection. The viral vaccines that have been developed thus far are against acute infections (i.e., rubella, smallpox, measles, poliovirus). Viral vaccines against members of the herpesvirus family and against hepatitis, the infections seen most commonly by the surgeon, are in the early stages of development. Krugman et al. 2~176 tested a vaccine against hepatitis B in children. They prepared a vaccine from serum containing hepatitis B antigen by simply diluting the serum 1:10 in distilled water and heating at 98~ C for 1 minute. They administered the vaccine to 29 children and after 4 - 8 months administered untreated MS-2 serum ( a serum known to transmit hepatitis B). Hepatitis B antigenemia and elevated serum glutamic oxaloacetic transaminase (SGOT) was detected in 12 of the 29 children. Only 1 child had evidence of jaundice. Persistent hepatitis B antigenemia developed in only 3..Thus, the vaccine prevented or modified the 51

hepatitis in 69% of the children. In contrast, 25 susceptible children were left unimmunized and were challenged with MS-2 serum. All 25 children developed hepatitis B antigenemia and 24 of the 25 had elevated SGOT values. Six of the 25 children had abnormal serum bilirubin values. In addition, a hepatitis B carrier rate of 37.5% subsequent.ly was found in the unimmunized children. B u y n a k e t al. 2~ also prepared a vaccine against hepatitis B virus. They prepared a highly purified HBsAg antigen b y ultracentrifugation. The antigen was concentrated and inactivated with 1:4000 formaldehyde solution for 72 hours at 36 ~ C. With this vaccine preparation, they were able to induce antibody in guinea pigs, monkeys and chimpanzees. The chimpanzees were protected against a subsequent challenge of live hepatitis B virus. B u y n a k and colleagues have not used this vaccine in humans yet. Maupas e t a l . 2~ have also prepared a hepatitis B vaccine from HBsAg. Thirty-five of 46 vaccinated subjects made anti-HBs antibody, but challenge with live virus was not carried out. Without doubt, one or more of these vaccines will be available in the not-too-distant future. Elek and Stern 2~~in England developed an attenuated live vaccine against cytomegalovirus by repeated passage in tissue culture, using several cell lines. After subcutaneous administration, neutralizing and complement-fixing antibody were elicited without side-effects. In the United States as well, 21~ a cytomegalovirus vaccine points out one of the problems of making a virus vaccine with a herpesvirus or other relatively slow-growing virus that can remain latent in vivo. There always is the concern that the virus given by vaccination m a y persist in host cells for a prolonged time and m a y revert to its wild type form and cause disease. In addition, the implications of having an attenuated virus persist for a prolonged time in the body are as yet uncertain. One of the reasons for the massive support given to studies of the viral etiology of h u m a n tumors by the National Cancer Institute was the possibility of producing a viral vaccine against these oncogenic viruses. Epstein 2~2 has proposed that a vaccine be prepared against Epstein-Barr virus, since evidence is so strong that Epstein-Barr virus is the etiologic agent of Burkitt's lymphoma and nasopharyngeal carcinoma. Since oncogenic viruses m a y be in 4heir host for a long time before a tumor becomes evident, it m a y be difficult to test such a viral vaccine because prolonged follow-up of patients would be required. It m a y be many years before the efficacy of such a viral vaccine is known. Also, it is difficult to know ira truly safe vaccine that lacks malignant potential can be developed. That such a vaccine can be developed is suggested by evidence that Marek's disease, a lymphoma of chickens, can be prevented by vaccination with the live attenuated herpesvirus that causes it. Killed herpesvirus vaccines are also being pre52

pared from viruses that cause lymphoma and leukemia in monkeys. Such vaccines elicit high titers of antibodies, and the recipients are protected from challenge with live tumor virus. Using only purified viral antigens required for antibody production, development of vaccines containing no viral nucleic acid, and thus unable to replicate in the host or vaccines, would eliminate the possibility of chronic infection. These vaccines would be safe; effectiveness, of course, still would have to be evaluated. PASSIVE IMMUNIZATION.-- Passive immunization utilizes infusions of plasma containing protective antibody or purified immunoglobulin prepared from this plasma. Although passive immunization is effective in the prevention of several viral infections, the protection is short-lived, because of the normal degradation of g a m m a globulin. Several studies 2~ 2~4,2~6 have shown that specific immunoglobulin can prevent hepatitis B in susceptible subjects and animals. K r u g m a n and Giles 2~ showed that hepatitis B immune serum globulin was able to protect children against challenge with a serum that transmitted hepatitis B; pooled normal immunoglobulin did not afford any protection. Szmuness et a l 3 ~3 found no significant difference in the incidence of hepatitis among two groups of children given either high-titer anti-HBs immunoglobulin or low-titer anti-HBs immunoglobulin at 4-month intervals. Persistent hepatitis B antigenemia developed in 7 of the 52 untreated children b u t in none of the 81 passively immunized children. Hepatitis B is a common problem in hemodialysis and transplant units because of the requirement for multiple blood transfusions in these patients. Desmyter et al315 performed a doubleblind study on hemodialysis patients. Patients given two doses of high-titer anti-HBs immunoglobulin at 6-month intervals had a significantly lower hepatitis B infection rate than did patients given serum globulin containing no anti-HBs immunoglobulin. Similarly, Prince et al3 ~6 showed that dialysis patients receiving high-titer "anti-HBs immunoglobulin had a significantly lower infection rate than did patients receiving intermediate or lowtiter material. The widespread use of hemodialysis, the requirement for surgeons to establish vascular access and the growing number of surgeons dealing with transplant recipients have made hepatitis an occupational disease among surgeons. In a recent study, 12.2% of the surgeons had a history of hepatitis infection. 4~ Periodic administration of high-titer immunoglobulin to antibody-negative surgeons no doubt would reduce the incidence of new cases of hepatitis. Two controlled trials in hospital staff accidentally exposed to HB~Ag have demonstrated a lower attack rate in those who received hepatitis B immunoglobulin within I week of exposure.217, 2Is 53

Zoster immune plasma and zoster immune globulin have been demonstrated to be highly effective in decreasing the incidence and severity ofchickenpox in children with renal transplants and neoplastic disorders, if given shortly after exposure to chickenpox.l~, 1,5 Since chickenpox can be fatal in these children, zoster immune plasma or globulin should be given soon after exposure to prevent or attenuate the infection. The antibody is ineffective once the disease has become established. CHEMOTHERAPY OF VIRAL INFECTIONS.--The chemotherapy of bacterial infection takes advantage of the differences in biochemical make-up and metabolic requirements of bacterial and mammalian c e l l s - o n e can be killed or inhibited and the other remains unharmed. Until recently, many scientists believed that a safe and effective antiviral agent could not be produced, because viruses use the host cells' own synthetic and metabolic machinery for replication. It has been difficult to find agents that are effective against viral infections and are not highly toxic to the m a m m a l i a n cells. Recently, however, investigators have identified many differences between the replicative cycle of viruses and of mammalian cells. Some viruses rely on unique enzymes not present in the uninfected cell; some have nucleotides in their nucleic acid that are not normal components of m a m m a l i a n nucleic acids. Still, there are few effective and safe antiviral agents availableY~9, 220 The first commercial antiviral agent was idoxuridine (Stoxil), which is effective in the topical treatment or herpes keratitis. Its side-effects, however, prevent its parenteral use. Another antiviral agent is methisazone, which is effective against smallpox (variola) and cowpox (vaccinia). However, with the elimination of smallpox from the world, methisazone has become a cure looking for a disease. Amantadine hydrochloride is licensed for prophylaxis of influenza A. 2zI Amantadine attaches to the surface membrane of the cells and interferes with the adsorption of influenza onto cells and decreases or prevents the engulfment of virus in phagocytic vacuoles. More than 200 analogues of purines or pyrimidines have been examined for antiviral activity. Adenine arabinoside is a more recently investigated agent, currently being used widely for the t r e a t m e n t of herpesvirus infections, including varicella-zoster, H e r p e s v i r u s h o m i n i s and cytomegalovirus. Most of the studies thus far have been uncontrolled, although Whitley et al. 222 have presented a controlled study of the t r e a t m e n t of herpes zoster with adenine arabinoside in immunosuppressed patients. They showed that patients had accelerated clearing of virus from the vesicles, cessation of new vesicle formation and a shorter time to pustulation. Adenine arabinoside has also been shown to be effec-

54

tive for herpes keratitis. Cytosine arabinoside originally was thought to be an effective agent against members of the herpesvirus family, but recent controlled trials have failed to reveal any therapeutic effect except for herpes keratoconjunctivitis. INTERFERON.- Interferons are cellular proteins produced in response to viral infections or chemical agents that confer resistance to viral infection. To be effective, cells must he exposed to interferon before infection. This exposure establishes an "antiviral state" that is mediated by a second protein induced by interferon. This second protein inhibits viral replication by interfering with transcription of the viral genome or the translation of messenger RNA into viral proteins. Although not virus specific, interferon is species specific, so that any attempt at using interferon in the prevention or treatment of viral infections required large-scale production from human cell lines, with a resultant great production cost. Also, the half-life of interferon after injection is short (10 minutes). Experimental studies, however, have shown interferons to be effective in prevention of viral infections in animals, and they may prove of use in the prevention of viral infections in persons at high risk. A second possible approach is the use of synthetic agents that can elicit endogenous interferon production. Few clinical trials with interferon have been attempted, and no convincing success of interferon therapy has been demonstrated. Jordan e t a l . =23 administered interferon from human leukocytes to cancer patients with disseminated zoster. They showed its safety, demonstrated increases in circulating interferon and host antiFig 14.-Production of interferon. Viral infection can lead to derepression of a repressor gene, R. This derepression leads to transcription of the I gene, which codes for interferon production. The I gene can be directly induced with synthetic nucleotides (i.e., a polymer of inosine and cytosine, poly I:C). Interferon induces the AV gene, which codes for an antiviral protein. This antiviral protein confers viral resistance on the producing cell and on other cells as well. Interferon also induces the R gene, which leads to repression of the I gene, shutting off production of interferon. Viruses that replicate slowly and do not markedly damage the cell are good stimulators of interferon production. Viruses that multiply rapidly are poor producers of interferon. Virus

An//viral

Protein Resistance

".~

Anh'vira/

Prote~__~ Resistance 55

body response to varicella-zoster in treated patients but failed to demonstrate any difference in clinical course. Similarly, chronic hepatitis B antigenemia was partially ameliorated in chimpanzees bY Purcell e t a l Y 24 and in humans by Greenberg e t al. 22~ Interferon inducers or exogenous interferon decreased hepatitis B antigenemia. The "e" antigen disappeared in 2 of 2 patients. The suppressive effect was transient when the interferon was given to humans for 10 days or fewer b u t appeared to be more sustained when administration was prolonged for a month or more. No comment was made about the clinical course of the patients. From what is understood of its mechanism of action, interferon should be effective only when given before viral infection. How can it be expected to be efficacious in chronic viral infections? Ho 2-~Ghas suggested that with some chronic viral infections, such as chronic hepatitis B, new cells must be constantly infected to maintain the production of hepatitis B virus. By protecting these new, previously uninfected, cells from viral replication, the amount of virus detected in the serum would diminish. TRANSFER FACTOR.--Transfer factor is one of the m a n y humoral agents (lymphokines) derived from lymphocytes of man. It can be extracted from lymphocytes in vitro and can transfer immunity from an immune cell to a nonimmune one. It is dialyzable, stable on prolonged storage and has a molecular weight of less than 10,000. It is inactivated by heat (56 ~ C for 30 minutes) b u t not by trypsin or DNAse. Its effects appear to be antigen specific, i.e., it will confer specific immunity to a given antigen to a population of nonimmune cells. Transfer factor transfers cell-mediated but not humoral immunity. The understanding of the chemical nature and mode of action of transfer factor has been hampered because there is no reliable animal model or in vitro assay, so that all testing of purified transfer factor preparations must be done in human volunteers. Transfer factor currently is being used clinically to treat patients with infections, malignancies and immunodeficiency diseases. It has been used to treat patients with chronic hepatitis B 22v231 and 1 patient with cytomegalovirus retinitis/3-~ Evaluating the efficacy of transfer factor therapy in these few case reports is difficult because the clinical course of the diseases can vary with no treatment at all. Trepo and Prince e2z were able to induce delayed hypersensitivity to HBsAg in chimpanzees and humans with chronic HBsAg antigenemia. Tong e t a l . 229 were unable to influence HB~Ag antigenemia in a patient with inactive chronic hepatitis. On the other hand, Shulman e t al. 22s did find chemical and histologic improvement in 4 patients with chronic aggressive hepatitis treated with transfer factor. However, 2 of 4 control patients treated with a placebo improved also. Rytel e t al. 232 treated with transfer factor a renal transplant 56

recipient who had cytomegalovirus retinitis. The amount of virus in the urine fell and the inflammation in the eye subsided. Although this and other case reports leave the effectiveness of transfer factor therapy far from proved, eventually it m a y be found to be effective in the t r e a t m e n t of viral infections t h a t do not respond to other modes of therapy.

VIRUSES AND CANCER It is obvious that the etiology of cancer is of great interest to surgeons. For this reason, a brief review of the possible viral etiology of cancer is included here. The discovery that many animal tumors are caused by viruses has led to the search for similar agents in h u m a n neoplasms. 233-239 Although to date there is not clear proof that a virus is the etiologic agent of any given h u m a n tumor, viruses have been indirectly implicated in the etiology of m a n y h u m a n neoplasms, including rhabdomyosarcoma, 24~ liposarcoma,245.246 breast cancer,234, 23s,24~-252 nasopharyngeal carcinoma, 253-256 Burkitt's lymphoma, 234,249,257-26~ carcinoma of the cervix, 261-267 leukemia,236,23s, 26~-269 lung carcinoma, 27~ osteogenic sarcoma, 245 fibrosarcoma, 245 Hodgkin's disease, 271 leiomyosarcoma, 245 neurofibrosarcoma, 245 lymphoma, 2~2,273 renal pelvis carcinoma 239,2T4 and melanoma.2~5. 276 The critical experiment, of course, cannot be performed in h u m a n subjects, i.e., administering a purified preparation of a putative oncogenic virus to a h u m a n subject and observing for the induction of a neoplasm. Koch's postulates, therefore, cannot be fulfilled. Even if one could perform such an experiment, one might have to wait years for the tumor, or one might not be able to provide the right circumstances that permit the virus to induce cell transformation. One could also administer the putative h u m a n oncogenic virus to a closely related primate, but failure to elicit a neoplasm might only mean that the virus did not produce tumors in the selected species. In an attempt to use this latter technique to implicate viruses in the etiology of h u m a n leukemias, bone marrow, blood and blood fractions have been administered to more than 1500 rhesus monkeys. No leukemias developed. No indirect method clearly proves that a given virus causes a given tumor. There is also the possibility, as has been shown for the murine sarcoma virus, that the oncogenic virus is defective and requires a "helper virus" for the tumor to be produced. Or the virus m a y exist in a latent form in various tissues and require activation by other a g e n t s - c h e m i c a l s , radiation, infectious agents, etc. These possibilities only compound the difficulty in searching for viruses as etiologic agents in h u m a n tumors. 57

TECHNIQUES FOR STUDYING VIRUS-TUMOR RELATIONSHIPS.S e v e r a l techniques have been used to study the relationship be-

tween viruses and h u m a n t u m o r s , including epidemiologic studies, tissue culture techniques, electron microscopy, immunologic studies and biochemical investigations. Epidemiologic methods correlate the presence of tumors with evidence of viral infection. The main tool is statistical analysis, and the findings are c o r r e l a t i v e - t h e y do not establish causality. Nevertheless, such studies m a y provide clues as to where to look for viruses by other techniques. A now-classic example is the observation by Denis B u r k i t t that a lymphoma, usually involving the mandibular and maxillary bones, generally was confined to a certain geographic distribution in central AfricaY 7 Other studies indicated that the disease was not found above an altitude of 5000 feet. Subsequent reports established that altitude was important only in that it selected a minimal temperature of 60~ in the coldest season of the y e a r Y s The fact that the tumor distribution appears dependent on climatic factors led Burkitt to hypothesize that a v e c t o r - a m o s q u i t o - m a y be responsible for the transmission of an infectious agent, and subsequent studies have implicated the Epstein-Barr virus. 27s Epidemiologic evidence has long implicated a venereal factor in the etiology of squamous cell carcinoma of the cervix, thus suggesting an infectious agent364, 26~ More recently, seroepidemiologic studies have revealed a strong correlation between the incidence of antibodies to Herpesvirus hominis type 2 and cervical carcinoma, carcinoma in situ and premalignant atypia3612~, 2~, 2~7 These studies have shown antibodies to Herpesvirus hominis type 2 in 8 3 - 98% of women with carcinoma of the cervix compared with 3 5 - 50% for control patients matched for age, religion and socioeconomic status. These kinds of studies implicate Herpesvirus hominis type 2 in the etiology of carcinoma of the cervix by showing the high correlation of prior infection with this virus (as demonstrated by antibodies in the patients' sera) and carcinoma of the cervix. Alternative arguments can be made. Viral infection and malignant change m a y be independent e v e n t s - t h e r e is also a high incidence of antibody in normal patients. The virus m a y grow better in neoplastic cells, so these patients with neoplasms may have a higher incidence of infection. And the virus may act as a cocarcinogen, not being entirely (or even primarily) responsible for the tumor. Excess frequency of acute leukemia among children was found in Niles, Illinois, from 1957 to 1960. 279 Clusters such as this suggest person-to-person transmission or common exposure to an etiologic agent. Most epidemiologic studies, however, do not suggest a role for an infectious agent in the etiology of acute leukemia and the rare occurrences of case clustering m a y occur by chance alone3 s~ 58

B

A I

C I

O. lp

Fi9 15.-Schematic drawing of virus particles associated with many murine and human tumors. The type B particle is associated with mouse and human breast cancers. The significance of the type A particle is not known for sure but it is believed to be an immature form of the type B particle. The type C particle is found in murine and human leukemias and in several other human cancers. (Drawn after Dr. W. Bernhard.)

Electron microscopy has been used extensively to search for virus-like particles in neoplastic tissues, cell cultures from neoplastic tissues and certain body fluids (e.g., milk). The finding on electron microscopy of neoplastic tissues of structures that resemble virus particles and the correlation of their structure with that of viruses known to cause tumors in animals has been used to implicate viruses in the etiology of neoplasms. Bernhard 2sl has characterized the types of R N A tumor viruses observed on electron microscopy. The m a m m a r y - t u m o r virus (MTV) is termed a "B particle," its immature form being called the "A particle." The "C particle" is characteristic of leukemias and sarcomas. In most laboratory-induced animal tumors, C-type virus particles are found in large numbers and without difficulty. 2s2' 2~ But only a few particles can be found in spontaneous animal and h u m a n neoplasms. 2s4 That even in the most extensive series usually less than 35% of patients with various neoplasms have B- or C-type particles points to the difficulty of the electron microscopic approach. The finding of B or C particles, or what are termed "small virus particles," in tumors, tissue cultures or h u m a n milk does not prove that these structures are viruses. They m a y be cell debris, lysosomes, mycoplasma, platelet fragments or other extraneous structures that resemble viruses. If indeed they are viruses, their presence still is merely an association with the tissue studied and does not prove causality. On the other hand, viruses that are present m a y b e overlooked because the appropriate section of tissue was not examined, the virus was present b u t in too low a concentration or virus was not present in the form of m a t u r e virions but in some morphologic form not recognizable by electron microscopy. Numerous attempts have been made to demonstrate virus-like 59

Fig 16.-Electron micrograph of a mouse mammary tumor showing type A and type B particles. The doughnut-like type A particles with an electrondense outer layer can be seen within the cell cytoplasm. Type B particles with their eccentric electron-dense core can be seen budding at the cell surface and in the extracellular space (X97,000). (Courtesy of Dr. W. Bernhard.)

particles in h u m a n milk and b r e a s t tissue, 247,24s, 250,251, ~75 especially those resembling the B-type particles identified as the murine m a m m a r y t u m o r agent. B i t t n e r =s5 showed t h a t mouse m a m m a r y cancer is caused by a virus t h a t the m o t h e r passes to the offspring through the milk (vertical transmission). It is a Btype virus. Moore e t a l . , 24s, 2ss Schlom et a l . , 2~1 Feller and Chopra 2~ 60

and Sarkar and Moore2~~found a high incidence (40- 60%) of Btype particles by electron microscopy in milk from patients with breast cancer compared to normal females (5-12% incidence). The P a r s i s - a relatively inbred subpopulation of Bombay with a high incidence of carcinoma of the breast but a relatively low incidence of other types of t u m o r s - a l s o had in their milk a high incidence of B-type particles (39%) in 46 specimensY4s, 2s6,2s7 Many milk samples also exhibited a high incidence of C-type pa rticles and small virus particles. They also studied 101 milks from high-risk American families with a high family history of breast cancer and found a 31% incidence of B-type particles. By comparison, they only found a 12% incidence in the normal controls. However, following a stringent re-evaluation of their electron microscopic data, Sarkar and Moore2ss determined that only 13 of the total 381 milk specimens were positive for B-type particles, with only one or two particles in each case. Calafat and Hageman 289 could not detect any intact B- or C-type particles in milk samples from 43 Dutch women. They challenged the previous data of Moore e t a l . 2~s' 2s6,2s~ and Sarkar and Moore~s on technical grounds, pointing out that cell debris can look like C-type particles by the negative staining method used. However, Feller and Chopra 2~7used the same thin technique as Calafat and Hageman and identified virus-like particles in 9 of 59 women. Seman e t al. 252 examined biopsy specimens of 44 patients with breast cancer. They found B-type particles in 8 specimens, C-type particles in 4 biopsies and small particles in 13 instances. The nature of these small particles has not been determined. Up to 1971, this group had performed electron microscopic studies on biopsies of 84 breast cancers, 13 metastatic lymph nodes and 3 fibroadenomas.29~They found B- and/or C-type particles in 34 of 100 breast cancers and in 2 of 33 pleural effusion specimens from patients with breast cancer. There are many studies showing electron microscopic evidence of virus-like particles in patients with leukemia. These particles have been demonstrated in cell material, plasma and in the urine from patients with leukemia? 9x'2~ Large series were undertaken to compare the incidence of virus-like particles in leukemic patients and normal controls.29~,292 Approximately 30% of the blood cells from 382 patients with leukemia or lymphoma, 34% of the biopsies and 12% of the plasma pellets were positive for virus-like particles. Particles rarely were observed in similar specimens from normal controls. Other largescale studies 2~, 295 could not corroborate significant differences between leukemic and normal blood cell and plasma samples. Seman e t a l . 252, 29o also found electron microscopic evidence for Band C-type particles and small virus particles in neoplastic and benign tumor tissue and metastatic lymph node and in tissue culture samples from breast tissue. 61

Numerous other studies have also been reported. They have provided electron microscopic evidence of herpes-like virus particles in tumor specimens or tissue culture specimens from Burkitt's lymphoma,25s, 260 chronic myelogenous leukemia, 26s carcinoma of the cervix 257 and Hodgkin's disease. 271 C-type particles have been identified in biopsy specimens or tumor-derived cell lines from h u m a n osteogenic sarcoma, 235,246,266 liposarcoma,246, 296 fibrosarcoma, 246 renal papillary tumor, 274 rhabdomyosarcoma,243, 244,297 lymphoma,2:3 melanoma275 and transitional cell cancer of the bladderY* However, C-type particles have been identi-j fled in normal h u m a n tissue and a normal amniotic cell line. 29s,299 Tissue culture techniques have been used to grow viruses in vitro from neoplastic tissues. Other techniques then must also be used to actually demonstrate the presence of virus. Electron microscopy has been used most often but immunologic and biochemical techniques have been utilized also. The danger is t h a t viruses may be introduced by contamination of the cell cultures. Even if the virus is endogenous to the tissue, there is not proof t h a t the virus identified caused the neoplasm. McAllister e t al. 241. 242 administered cultured cells from a hum a n rhabdomyosarcoma to fetuses of pregnant cats. The kittens developed disseminated rhabdomyosarcomas, and C-type virus particles are observed in the tumors. In this case, the virus may have been picked up from the cats, since the parent tumor contained no detectable C-type particles. There are conflicting reports as to whether the virus is of h u m a n or feline origin. 243,300 Virus-like particles have also been observed in cells from other cultured tumors. 246,248,253,274,297 Elliott e t al. 274 were able to prepare virus from 3 renal pelvic papillary carcinomas and showed t h a t they all were antigenically similar. Moreover, the virus produced a cytopathogenic effect in h u m a n fibroblasts, testicle and chick embryo fibroblasts. Transformation of normal tissue cultured cells to a neoplastic form did not occur, however. Transformation is the modification of cells in culture so that the cells do not exhibit contact inhibition, grow in heaped-up layers and can be subcultured i n d e f i n i t e l y - c h a r a c t e r i s t i c s not present in normal, untransformed cells but usually found in cultured tumor cells. For this reason, transformation commonly is viewed as an in vitro correlate of tumor induction and has been used to study the process of viral oncogenesis. 186.189 But it has not been used extensively to determine whether a putative h u m a n oncogenic virus can cause in vitro transformation of h u m a n cells. Where so tested, 243,274 transformation by some putatively oncogenic viruses has not occurred. As Sabin 236 has pointed out, however, cells of the right genetic constitution are required for transformation to occur, and m a y not have been provided in the above experiments. Thus, some h u m a n viruses (adenovirus) can induce transformation and tumors in species other t h a n man but not in 62

man. Similarly, SV40 virus can transform h u m a n cells but not cells of its natural host (the monkey). Change in cultured lymphoid cells suggestive of transformation has occurred with Epstein-Barr virus, the possible cause ofBurkitt's lymphoma and nasopharyngeal carcinoma. The cell morphology has become blastoid, continuous proliferation is induced, chromosome changes occur and surface-neoantigens are induced. Tissue culture techniques are important from another standpoint in that they allow preparation of quantities of virus that can be used to prepare antisera for immunologic studies so that viruses obtained from similar kinds of tumors can be compared for antigenic similarityY 59, 273,2~4 Serologic methods have been used to identify virus-related antigens in h u m a n cancer by three principal techniques: (1) Animal tumor viruses and virus-like particles are used to produce antibodies that then are tested for reactivity with the same patient's or another patient's tumor cells. (2) Antibodies present in the patient's serum and assumed to be virus specific are tested against tumor cells of the same patient and of other patients. If crossreactivity is detected, a viral agent is suggested. The antibodies can also be used to neutralize known animal tumor viruses. (3) Antibodies synthesized against interspecies-specific antigens of mammalian RNA tumor viruses are used to look for cross-reactive antigens on h u m a n tumor cells. Many tumors develop antigens not found on normal cells. Tumors induced by the same virus, whether in the same individual, in different individuals of the same species or induced in different species, carry the same tumor-specific transplantation antigens (TSTAs). TSTAs induced by chemicals or radiation generally are distinct on each tumor, although some cross-reactivity has been demonstrated. Thus, the finding of antibody to one h u m a n tumor reacting with the tumor of another individual would be supportive evidence for a common viral etiology of the tumor. A n example of the first technique is the production of antibody in rabbits against the murine Rauscher leukemia virus, which was shown to cross-react with bone marrow and blood cells from i patients with leukemia. 3~176 Similarly, antiserum prepared in rabbits against virus-like particles in plasma pellets of patients with leukemia and Hodgkin's disease reacted with peripheral blood and bone marrow cells of patients with leukemia and lymphoma b u t did not react with cells of normal control individuals.a01, 3o4-306 The second technique was used by Morton and Malmgren 3~ in a study of osteosarcomas. They tested sera from patients with osteosarcoma, from their relatives and close associates and from normal donors against sarcoma cells of the same and other individuals, Using patient's sera, they found positive reactions in 100% with the patient's own tumor cells and in 80% with other 63

patients' tumor cells. Sera from family members and close associates reacted positively in approximately 90% and sera from normal blood bank donors were positive with only 29% of tumors. This high degree of cross-reactivity suggests that there are similar or identical antigens on different sarcomas. This study, however, does not prove that the reactive antigens were viral. Osteosarcomas were induced in Syrian hamsters by inoculating them at birth with cell-free extracts from h u m a n osteosarcomas. 3~ A common sarcoma-specific antigen in h u m a n osteosarcomas and the hamster osteosarcomas was found. In similar studies, con]mon cytoplasmic antigens have been demonstrated in melanomas. 309 Antibody in a patient's serum can also be used to neutralize known animal tumor viruses. Thus, Moore et al34s used h u m a n sera from 5 breast cancer patients to neutralize highly purified mouse m a m m a r y tumor virus (MTV). Five normal sera were used as controls. The "neutralized" virus then was assayed by inoculation into virus-free, tumor-free mice. Moore et al. reported what they considered to be a significant difference in infectivity incidence (69% after neutralization by sera from breast cancer patients vs 87% for controls). The specificity of this reaction was demonstrated by the ability of virus-positive milk, but not of virus-negative milk, to reduce the MTV neutralizing ability of the h u m a n sera. Thus, serologic studies support electron microscopic findings. The mouse MTV is a B-type particle; B-type particles have been most consistently identified in milk and tissues of h u m a n breast cancer patients, and antiserum to one neutralized the other. The same types of findings have been demonstrated with Burkitt's lymphoma and the animal leukemia viruses. Both are C-type particles by electron microscopy, and antiserum to the virus isolated from Burkitt's lymphoma patients reacts with leukemia viruses of the mouse, rat, hamster and cat3 ~9, 2r3 Sera of patients with Burkitt's lymphoma contain an immunoglobulin that reacts with autochthonous or allogeneic Burkitt's lymphoma cells but not with cells of non-Burkitt's tumors nor with bone marrow cells from Burkitt's lymphoma patients. 3'~ Similarly, Elliott et al. 2r~ were able to neutralize three viruses obtained from three different renal papillary tumors with antiserum against one of the viruses. B i o c h e m i c a l techniques are useful for looking for "fingerprints" of viruses or viral products in cells that m a y not produce infectious virus. Studies of this kind have been directly borrowed from work with animal viruses. One of the more recent approaches has been the search in h u m a n tumor tissues for DNA-directed RNA polymerase. The permanence of transformation of cells by oncornaviruses (oncogenic R N A viruses) and the finding that DNA synthesis and transcription are required for viral production and infection suggested that DNA synthesis must be important in 64

infection by oncornaviruses and subsequent tumor formation. The demonstration of homology between Rous-sarcoma virus RNA and DNA from infected cells led Temin3" to propose that RNA-directed DNA synthesis occurred and that a DNA "provirus" existed in infected cells. The R N A of RNA tumor viruses is transcribed into DNA by a RNA-directed DNA polymerase. This viral-coded DNA (provirus) then becomes integrated into the cell DNA and is replicated during cell division along with cellular DNA. This integrated DNA (provirus) contains the information for tumor induction. The undifferentiated tumor cells that have integrated viral DNA may not be producing complete or infectious virus that can be detected by electron microscopy or culture. In 1970, Temin and Mizutani 3'2 and Baltimore 3x3 provided evidence of an RNA-directed DNA polymerase (reverse transcriptase) in oncornaviruses. Subsequent studies indicated that almost all oncornaviruses contain this enzyme whereas nononcogenic RNA viruses do not33~ It has also been found, however, that visna virus and primate syncytium-forming virus (viruses not known to cause tumors) also contain reverse transcriptase, so that this property may not be specific for tumor viruses347 Reports have even indicated that RNA-directed DNA polymerase can be present in normal cells (i.e., those not affected by any virus) b u t others have consistently failed to find such evio dence34~, 3~2,314 A source of confusion concerning the presence of reverse transcriptase in normal cells and cancer cells stem.med from methodologic conditions required for its detection, such as separation of reverse transcriptase from normal DNA polymer~ ases, type of template used and reaction conditions. 233 Although an RNA-directed DNA polymerase provides a mechanism for the incorporation of genetic information from oncogenic R N A viruses into the genome of the cell, it does not explain the mechanism of oncogenicity. Nevertheless, because reverse transcriptase has been found to be associated with oncornaviruses and only rarely with nononcogenic RNA viruses, a demonstration of this enzyme in viruses or tissues obtained from h u m a n neoplasms can be used as indirect evidence of the presence of an oncogenic RNA virus. Thus, Schlom et al. T M found B-type particles in 5 of 13 milk specimens from patients with carcinoma of the breast and all 5 had RNAdirected DNA polymerase activity. None of the 8 samples without enzyme activity contained B-type particles. Sarkar and Moore 2~~ also found a correlation between the presence of reverse transcriptase activity and virus particles in the milk of h u m a n breast cancer patients. Reverse transcriptase activity has also been detected in h u m a n leukemic cells. Gallo e t a / . 269 found RNA-depondent DNA polymerase activity in extracts of white blood cells of 3 patients with acute lymphoblastic leukemia. None of 48 control specimens con65

tained reverse transcriptase activity even after stimulation with phytohemagglutinin. Whether the enzyme is of viral or cellular origin was not established, however..RNA-dependent DNA polymerase activity has also been found in viruses obtained from tissue-cultured cells of h u m a n rhabdomyosarcoma,243renal papillary tumors 274 and Burkitt's lymphoma.3~5 The association of a high molecular weight RNA and a reverse transcriptase associated with it is another line ofevidence for the presence of RNA tumor viruses in h u m a n tumors. Baxt and Spiegelman 3'6 found reverse transcriptase associated with high molecular weight RNA in the cells of 22 of 23 patients with leukemia. Eighteen normal white blood cell specimens were negative. High molecular weight RNA and reverse transcriptase were also present in 79% of 38 breast adenocarcinomas, 87% of Burkitt's lymphomas, 59% of 51 brain tumors, 72% of 25 gastrointestinal cancers and 70% of 10 lung cancers. They have also been found in melanomas and Hodgkin's lymphomas. Another biochemical approach in the search for presence of viruses in h u m a n cancer utilizes the technique of molecular hybridization. This technique looks for sequences of viral RNA (or DNA) or DNA synthesized from viral RNA t h a t are complement a r y to cellular RNA or DNA. Complementary viral RNA and DNA can form electrostatic bonds (hybridize) with cellular RNA or DNA t h a t can be separated from nonhybridized RNA and DNA and identified. The more complementary the sequences in the nucleic acids the greater the degree of hybridization. This technique has been used extensively in the past few years. Kufe et a/. 245 have shown t h a t RNA from h u m a n sarcoma, leukemia and lymphoma cells can hybridize to DNA homologous to the RNA of Rauscher leukemia virus but not to the mouse MTV. Twenty-four of 27 leukemia patients and 18 of 25 sarcoma patients contained RNA t h a t hybridized to DNA complementary to the RNA of Rauscher leukemia virus. Although these studies do not provide proof of a viral etiology of h u m a n leukemia or sarcoma, they do show t h a t certain h u m a n tumors have RNA similar to t h a t known to cause cancer in animals. Others ~17~2~also showed RNA in h u m a n leukemias, sarcomas and lymphomas to be homologous to RNAs of known animal RNA tumor viruses and suspected h u m a n cancer viruses. In the case of h u m a n leukemias, more t h a n 99% showed homology3'7, 3,s; more t h a n 50 normal control tissues did not. In similar studies, 19 of 29 h u m a n m a l i g n a n t breast tumors were shown to contain RNA in the cytoplasm t h a t could hybridize with DNA t h a t was complementary to mouse MTV. T M Fourteen normal or n o n m a l i g n a n t breast tissue specimens did not show homology. Other studies 249,254.25~.322 have examined by hybridization techniques cell lines obtained from Burkitt's lymphoma and nasopharyngeal carcinoma. Using DNA-DNA hybridization of c0m66

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Separate on CsCI Gradient Fig 17.--Typical hybridization experiment. RNA is isolated from human tumor cells and from a known murine RNA virus. DNA is synthesized from the viral RNA using an RNA-dependent DNA polymerase and ~H-thymidine, guanidine, adenine and cytosine. This radioactive DNA is allowed to react with RNA isolated from the tumor cells. Pairing (annealing) occurs where bases of the viral-coded DNA are complementary to those of the tumor RNA. The unpaired singlestranded DNA and RNA then are degraded with a nuclease that attacks only single-stranded DNA and RNA, leaving the paired hybrids, which are approximately 50-60 base pairs long. These hybrids then are separated on a CsCI gradient. Alternatively, DNA-DNA hybridization can be performed also.

plementary RNA-DNA hybridization, evidence was found of the presence of Epstein-Barr virus DNA in these cell lines. Moreover, multiple viral genomes were found per cell. Zur H a u s e n et al. TM also found that cell lines from normal patients probably were infected with Epstein-Barr virus, indicating that it m a y have been responsible for transformation of normal cells. No hybridization was found with white blood cells from newborn infants. Kufe et al. 3~s found that R N A from Burkitt's lymphoma and nasopharyngeal carcinoma hybridized to RNA of a murine leukemia virus. Various reports indicate that 4 8 - 91% of h u m a n ]eukemias, 6 9 - 75% of h u m a n lymphomas and sarcomas and 3 0 - 67% of h u m a n breast cancers contain nucleic acid that is homologous to some RNA sequences of viruses causing analogous diseases in mice and primates. 233 Investigators have also found evidence that cellular DNA from patients with leukemias, Hodgkin's disease, 67

Burkitt's lymphoma and melanoma can be hybridized to RNA of animal tumor viruses. 233 These findings provide evidence t h a t the RNA of RNA tumor viruses has a sequence similar to that of some DNA sequences of h u m a n cancers. Despite the numerous studies that have been carried out, no h u m a n tumor has been definitely proved to be caused by a virus. Much circumstantial evidence has been accumulated to implicate viruses in the etiology of h u m a n neoplasms, however. Evidence from many kinds of techniques has been brought to bear on a few t u m o r s - e s p e c i a l l y Burkitt's lymphoma and renal papillary carcinoma. The studies that provide indirect evidence of viral etiology of h u m a n tumors using several kinds of evidence and demonstrating the putative oncogenic virus with a high frequency in specific kinds of tumors indicate that at least some h u m a n tumors m a y be caused by viruses. The most evidence of the viral etiology of a h u m a n neoplasm has been gathered for Epstein-Barr virus as the cause of Burkitt's lymphoma and nasopharyngeal carcinoma. The lines of evidence are: (1) high antibody titers to Epstein-Barr virus in patients with Burkitt's lymphoma; (2) ability of Epstein-Barr virus to induce DNA synthesis in resting cells; (3) Epstein-Barr virus transforms normal lymphocytes into lymphoblastoid cells that contain Epstein-Barr virus-specific markers; (4) presence of Epstein-Barr virus particles and antigens in cell lines from patients with Burkitt's lymphoma; (5) Epstein-Barr virus DNA in tumor biopsy material from patients with Burkitt's lymphoma and nasopharyngeal carcinoma; (6) tumor induction in cottontail marmosets with Epstein-Barr virus transformed cells and cellfree Epstein-Barr virus suspensions; (7) presence of Epstein-Barr virus-associated nuclear antigen and membrane antigen in tumor specimens. To implicate viruses in the etiology of h u m a n cancers, one will have to use indirect techniques. The more indirect evidence one can accumulate for a relationship between a virus and a given tumor the more that virus can be implicated in the etiology of that tumor. No amount of indirect evidence, however, clearly proves that a given virus causes a given tumor. Furthermore, finding that viruses do cause cancer tells us nothing about the regulatory mechanisms of gene expression that exist in cancer cells. We still have little understanding of gene r e g u l a t i o n whether the DNA is of cellular or viral o r i g i n - and the mechanism of carcinogenesis. One of the hopes in searching for a viral etiology of cancer is that a vaccine perhaps could be developed against the oncogenic virus. Such a potential vaccine would be hard to test, since the virus m a y not grow in nonhuman cells and testing then would have to take place in humans. B u t because there m a y be a long time before virus infection and the appearance of a cancer, it 68

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