The Enteroviruses HERBERT A. WENNER,

Transcription

1 The Enteroviruses HERBERT A. WENNER, M.D. Departtnent of Pediatrics, University of Missouri-Kansas City School of Medicine and Children's Mercy Hospital, Kansas City, Missouri 6418 ABSTRACT Wenner, Herbert A.: The enteroviruses. Am. J. Clin. Pathol. 57: , Ponderable changes have taken place in the field of infectious diseases during the 2th century. Powerful weapons have been forged in prevention and treatment of bacterial infection. During the first half of the century, when formidable barriers prevailed in the virus laboratory, our knowledge of virus diseases, such as influenza and poliomyelitis, accrued slowly but steadily towards fundamental appreciations of pathogenesis and immunogenesis; ultimately, this knowledge contributed to clinical modulation and prevention of disease. The new look in virology has been tailored by modern tissue culture methods developed primarily by Enders and his associates. 6 The introduction of new tools (e.g., electron microscope, isotopes, and the ultracentrifuge) helped in large measure to provide useful information on the nature of viruses and intracellular synthetic events. Of equal significance was the recognition by tissue culture methods of many viruses heretofore unknown. During the past 25 years the clinic has found renewed interest in benign meningitis, selective cardiopathies, and viral exanthems. In 198, Landsteiner and Popper 13 recovered poliovirus from monkeys inoculated with emulsified central nervous system (CNS) tissues from a 9-year-old boy dying of paralytic poliomyelitis. After almost 4 years of detailed studies in the clinic and the laboratory, amid conflicting data, evidence of the existence of three distinct types of polioviruses was obtained. In 1949, Dalldorf and associates 4 reported recovery of enteroviruses unlike poliovirus from patients with nonparalytic infections of the CNS. These viruses produced distinctive disease in newborn mice. They were named "coxsackieviruses," after the village in New York State where they were first isolated. Dalldorf segregated these viruses into two subgroups, A and B. In mice, Received January 6, Reprints of this entire Research Symposium are available from the ASCP Convention Department, 21 West Harrison Street, Chicago, Illinois 6612, for $3. per copy. Group A virsuses cause generalized myositis accompanied by flaccid paralysis. Group B viruses cause focal myositis and panniculitis, along with inflammatory lesions in the pancreas, liver, and heart. There are 23 Group A and 6 Group B coxsackieviruses, each distinguishable serologically. The advent of tissue cultures brought to the fore many viruses antigenically unrelated to polioviruses or coxsackieviruses. Initially called "orphan viruses," or viruses in search of disease, they were finally called "echoviruses" (ECHO enteric cytopathic human orphan). At least 32 serotypes are known. 2 Many have now been associated with clinical disease. A listing of the subclasses of enteroviruses appears in Table 1. Definition Enteroviruses are members of the family of picornaviruses. Rhinoviruses and small 751 on 14 March 218

2 754 WENNER A.J.CP. Vol. 57 F yrs. NECK PAIN SORE THROAT HEADACHE VOMITING INCUBATION PERIOD * 2tolOdoys >- DAYS I CLINICAL WBC 52 ^ LABORATORY PLEOCYTOSIS DATA 6, (85% lymphocytes) VIRUS PRESENT IN BLOOD * STOOL OROPHARYNX CSF ANTIBODIES < CLINICAL VARIAT IONS In the Kalian epidemic(1954) cutaneous lesions were not observed E9). An enanthema consisting of yellowish or grayish white I-3mm "spots" may appear on tonsillar fauces or buccal mucosa (23). 1 probably occurs prior to demonstrable illness! Fie. 2. The clinical course and laboratory findings during and following aseptic meningitis caused by echovirus type 9. symptoms: headache, sore throat, abdominal pain, nausea, vomiting, and occasionally myalgia. Nuchal and spinal rigidity of varying intensity intervenes. All three polio-, all Group B, and at least 27 of the 32 echoviruses have been implicated (Fig. 2; echovirus type 9). Muscular Weakness and Paralysis. Enteroviruses may attack cells within the CNS and simulate poliomyelitis. The kinds of illnesses include mild and transitory muscle weakness, spinal paralysis, and bulbospinal disease. Of all the enteroviruses, coxsackievirus A 7 rates next to poliomyelitis with respect to injury of anterior horn cells. Echovirus types 4, 9, and 14 may also produce flaccid limb paralysis. During an outbreak of infection with echovirus type 3, 5% of patients developed limb and trunk paralysis. Echovirus types 2, 6, 7, 11, 14, and 16 have been associated with transient episodes of muscle weakness. Other syndromes less frequently encountered are acute cerebellar ataxia and encephalomyelitis. Myalgia. Five of the six Group B coxsackieviruses have been associated with epidemic myalgia. The syndrome occasionally has been found in association with several Group A coxsackie- and echoviruses (Table 2). In addition to excruciating pain, involving largely the chest and abdomen, these patients may develop concomitantly aseptic meningitis, pericarditis, myocarditis, or orchitis. Myocarditis and Pericarditis. The heart may be involved, either during isolated infections or episodically during epidemics. The Group B coxsackieviruses have been more commonly encountered than others, although several Group A coxsackie- and echoviruses have been associated with cardiopathies also. Onset of disease in isolated cases is preceded by respiratory or "viral" illnesses. Patients may have features predominantly of myocarditis, pericarditis, or both. Experimentally, endocarditis and myocarditis may be induced by coxsackieviruses in mice and monkeys. Recently Burch and colleagues 2 examined the hearts on 14 March 218

3 June 1972 THE ENTEROVIRUSES 755 Table 2. Tissue Cultures Used for Detection of Enteroviruses* Enterovirus Serotypes Poliovirus Coxsackievirus Echovirus Group A Group B Muscular paralysis "Aseptic" meningitis {Herpangina Hand-foot-mouth disease Papulovesicular disease Maculopapular disease Pleurodynia Cardiopathies Respiratory Miscellaneous Neonatal CNS-ataxia Orchitis Diarrhea 1, 2,3 1,2,3 Rare 7,9 2, 4, 7, 9, 1 1 through 6 & 8, 1, 5, 1, 16 5, 9, 1, 16 4,9 4, 16 9, 16, 21, 24? 2,4,9 Rare 22 2, 3, 4, 5 All 6 1, 2, 3, 4, 5 3 1,3, S Ibid. 1,2,3,4,5 1, 2, 3, 4, 5 1, 3, 4, 5 1, 2, 3, 4, 5 2,3,4 2,4,6,9,11,3 All, except 24, 26, 29, 32, 34 9, 16, 17 2, 4, 6, 9, 11, 16, 18 1,6,9 1,6,8,9, 19 4, 9, 11, 2, 25 Rare , 14, 18 * = not reported at this time; = association not validated. For simplicity, only those serotypes firmly associated with specific disease syndromes are listed. Omitted are a number of echovirus serotypes associated rather loosely as yet with encephalitis, radiculitis, aseptic meningitis, hepatitis, etc. Enteroviruses are also encountered in mild febrile illnesses. from 5 infants and correlated histologic lesions with Group B coxsackieviruses by staining with fluorescein-labelled antibody. Twelve (24%) of the myocardial specimens from the infants were specifically labelled; all showed histologic evidence of interstitial myocarditis. Eruptions of the Skin and Mucous Membranes. These include a variety of skin lesions and a large number of enteroviruses. Coxsackieviruses. Herpangina. Herpangina is an acute febrile disease involving young children, particularly during the late spring and summer. The lesions appear as minute vesicles or small punchedout ulcers principally on the anterior pillar of the fauces, the tonsils, the pharynx, and the edge of the soft palate. At least 9 Group A viruses are regularly associated with the disease. Several other Group A viruses (types 7 and 9), four Group B viruses, and three echoviruses have been recovered in this disease. Hand-foot-and-mouth Disease. This unique syndrome is a combination of herpangina and vesiculo-ulcerative lesions involving the hands and feet, buttocks, and sometimes other cutaneous surfaces. The lesions differ from herpangina by the frequent appearance of lesions on the buccal mucosa, tongue, and gingiva; superficially, the syndrome may be confused clinically with gingivostomatitis and cutaneous lesions of herpes simplex virus. Group A virus type 16 has been recovered from dermal lesions, pharyngeal secretions, and feces. Echoviruses. Echoviruses have been associated also with a wide variety of skin lesions, mainly maculopapular, occasionally vesicular, sometimes rubelliform or petechial in nature. Of the 31 echoviruses, at least 16 have been associated with rashes. Types 9 and 16 have a fairly consistent association; others, such as types 2, 4, 6, and 11, have less consistent association with cutaneous lesions. At this time only type ] 1 has been reportedly recovered from cutaneous lesions. on 14 March 218

4 756 WENNER A.J.CP. Vol. 57 Injections of the Respiratory Tract. Enteroviruses often involve the pharynx as part of a broader clinical expression. Infections such as the common cold have been described for Group A types 21 (Coe) and 24 coxsackieviruses. The Group B viruses, e.g., types 3, 4, and 5, may be encountered in brief febrile illnesses consisting of rhinorrhea, pharyngitis, and conjunctivitis. Group B types 1 and 4 have been recovered from children with bronchitis and bronchiolitis, and several of these viruses (A 9, A 16, A 2lJ B 4, and B s ) have been associated with pneumonia during infancy. Various echoviruses may be associated (Table 2) with upper respiratory infections. The Alimentary Tract. The causative role of enteroviruses in gastroenteritis essentially remains unresolved. Coxsackieviruses do not appear to be etiologically important. echoviruses are linked rather solidly with episodic diarrhea. Types 14 and 18 may cause diarrhea in nursery infants. Type 11 may produce steatorrhea. On the other hand, these viruses may spread silently among nursery cohorts, or they may be found as often among well as among sick children. Immunity Type-specific humoral antibodies develop during the infective period. Neutralizing antibodies appear within a fortnight after colonization of the alimentary tract. Such antibodies may be found either at the onset of illness (rash or meningitis, or both) or shortly thereafter. Following their appearance they persist, with but slow decay over the ensuing years. Complement-fixing (CF) and hemagglutination-inhibitor (HI) antibodies generally lag behind neutralizing antibodies in development, but only for a few days. Echovirus type 6 neutralizing and HI antibodies reach peak levels early in the second week of illness. In some serotypes there is a decrease in antibody titer over the ensuing weeks with low levels only detectable for the next 3 or 4 years. Specific CF antibodies may not be detectable during the same time interval. There are variations on the theme for different enteroviruses; an example is cited for echovirus type 16, in which both CF and neutralizing antibodies were detected, albeit at low levels, for 3 to 6 yr. after infection. Antibodies acquired prior to or shortly after conception cross the placenta and enter the circulation of the unborn infant. This select newborn population is probably protected from the generalized disease associated with Group B coxsackieviruses. However, it is abundantly clear that passively transferred gamma G may not preclude infection of the alimentary tract during the early months of infancy. Examples of recovery of enteroviruses from the pharynxes and feces of seropositive persons can be cited. On the other hand, seronegative individuals who acquire infection may not develop disease. The modulation of infection (as opposed to disease) is related to a number of separate events a quenching of primary virus multiplication, elimination or sequestration of virus, and a postulated state of clinical refractoriness about which very little is actually known. Once enteroviruses reach secondary target organs, however, humoral antibody appears to be relatively unimportant, and induction of other factors (sequestration in macrophages, interferon, and possibly cellular resistance) occurs in response to the processes of cellular injury. At this point repair usually leads to full recovery, and only exceptionally is tissue injury so extensive that a fatal outcome is anticipated. Recovery, Identification, and Association with Disease. 17 ' 24 > 2 The recovery of viruses from sick patients provides a direct and cogent means of revealing the nature of infection. Enterovirus isolation depends on the use of sensitive indicator systems on 14 March 218

5 June 1972 THE ENTEROVIRUSES 757 Table 3. Tissue Cultures Used for Detection of Enteroviruses* Class of Enterovirus Poliovirus Coxsackievirus Echovirus Kind of Culture Group A Group B Human Origin 1. Primary Amnion Kidney Thyroid 2. Continuous HeLa HEp-2 * A A Monkey Origin 1. Primary Kidney Rhesus Cynomolgus Patas * See text regarding problems relating to growth of Group A coxsackieviruses in tissue cultures. At grows readily in monkey kidney cells; An, 13, 15, 16, 18, 2, and 21 may be isolated directly in human cell cultures. A21 grows in HeLa, HEp-2, and KB cells, but not on monkey kidney cells. Enteroviruses may be grouped according to plaque morphology and host susceptibility of enterovirus serotypes (Hsiung, GD, Ann. N. Y. Acad. Sci. 11: , 1962.) All except types 1, 19, and 22 Group A coxsackieviruses have been adapted to grow in human amnion cells. wherein a cytopathic effect (CPE) is identifiable. Many enteroviruses are cytopathogenic in selected tissue culture systems. 17 For maximum recovery of indigenous viruses several different culture systems are often used (Table 3). Unfortunately, some Group A coxsackieviruses on primary passage do not produce detectable CPE. Infant mouse inoculation, with subsequent expression of disease, in association with characteristic pathologic lesions may be necessary for their discovery. Even so, failure may follow, for strains of low virulence for mice are known; such Group A coxsackieviruses are difficult to recover and especially difficult to identify. As was noted earlier, enteroviruses may be recovered from a variety of clinical samples particularly from feces and oropharyngeal secretions. They have been recovered from blood, cerebrospinal fluid, pericardial effusions, and vesicular cutaneous fluids during the early active period of infection. At autopsy several Group B coxsackieviruses have been isolated from cardiac muscle, brain, spinal cord, liver, and several other organs. They are rarely recovered from saliva or the nasopharynx; a few have been recovered from the urine. In general, enteroviruses grow best in primate epithelial cells. All three types of polio- and Group B coxsackieviruses grow well in monkey kidney cells, as well as in a variety of human cell lines. Among the Group A coxsackieviruses, types 7, 9, 14, and 16 produce CPE on monkey kidney cells; types 11, 13, 15, 16, 18, 2, and 21 are more readily cytopathic on human cell lines. Primary cultures of human kidney and thyroid cells and continuous cultures of WI-38 cells are particularly useful. Other useful culture systems include HeLa, HEp-2 lines, and primary amnionic cell cultures. With repeated passages in tissue culture some Group A viruses (e.g., A 21 ) may acquire pathogenicity for suckling mice. Enteroviruses produce a characteristic cellular degeneration observable microscopically. The CPE is often characterized by rapid (> 36 hr.) and extensive destruc- on 14 March 218

6 758 WENNER A.J.CP. Vol. 57 tion of the cell monolayer. Infected cells round up, shrink, become refractile, show nuclear pyknosis, and fall away from the glass surface of the culture vessel. In stained preparations it is possible to delineate a series of morphologic events. With echovirus type 9 the earliest changes occur at the cytoplasmic rim of the nucleus as a zone of increased basophils. Eosinophilic granulation of the nucleus intervenes. Slightly later the nucleus is distorted, ridged, and plicated. A homogeneous pole mass increasing in size accumulates in a juxtanuclear position and increases in size, displacing a pyknotic nucleus and cytoplasmic residue to the cell periphery. These findings may be reinforced by fluorescence and electron micrographic studies. Most enteroviruses conform to the type of CPE noted above. Exceptions relate to a few. Attentuated polioviruses may be separated from wild-type virus by varying conditions of cellular milieu and temperatures of incubation. Decreasing the bicarbonate concentration of the medium favored the growth of attentuated (d) strains; increasing the temperature of cultivation to 4 C. favored the growth of wild-type polioviruses (T rct/4 strains). Other useful "markers" are known. differences also relate to echovirus types 22 and 23, in which cell degeneration under usual conditions of growth is discontinuous, and the cells are more or less refractory until after several passages in tissue cultures. Enteroviruses may be cultivated in cells under agar, and produce focal islands of cell destruction commonly called "plaques," because they cannot take up the vital stain, neutral red. Certain viruses which are not cytopathic under a fluid medium may produce plaques under agar. Nutritional factors affect the susceptibility of cells. Even the type of agar may play a crucial role. Certain echoviruses (types 2, 3, 5, and 6) have failed to produce plaques under Noble agar. Inhibitors present in the agar may be removed by treatment with DEAE dextran, among other compounds. Alternatively, the use of methylcellulose as an overlay avoids the inhibitor effect. MgCl 2 or CaCl 2 (25 to 4 mm) added to nutrient media enhances plaque formation. Various pitfalls interrupt work with tissue cultures. Monkeys harbor viruses of their own, and these may propagate in primary kidney cultures. Their presence is revealed by either distinctive cytopathology (e.g., SV 4 ) or the absorption of erythrocytes (e.g., SV 5 ) onto the cultured cell sheet. times adventitious viruses present in culture fluids may be quenched by heat stabilization with 1 M MgCl 2, by treatment with ether (enteroviruses are resistant to ether), by the use of.2 mm A1C1 3 in growth medium or by incorporation of selected antisera (e.g., SV 5 ) in the growth medium. Moreover, continuous lines of cells may become contaminated with Mycoplasma and these may interfere with enteroviral CPE. Certain animal sera used in growth and maintenance media may contain viral inhibitors, a problem fortunately not often encountered with enteroviruses. All laboratories have periodically encountered nonspecific cell destruction due to faulty substrate and dirty glassware, among other tangible factors. Virus Typing and Antibody Measurements Antigenic Properties. Polioviruses types 1 and 2 share common antigens; type 2 poliovirus may be neutralized by early sera from patients infected by type 1 virus, and vice versa. Pronounced intratypic variation (antigenic heterogeneity) may be encountered among both coxsackie- and enteroviruses. With some enteroviruses antigenic variants are encountered among "prime" strains. The prime strain is poorly neutralized by prototypic antisera, but antiserum on 14 March 218

7 June 1972 THE ENTEROVIRUSES 759 raised against the prime strain neutralizes the prime and prototypic viruses equally well. Prime strains have been reported for echovirus types 1, 4, 5, 6, 8, 11, and 3, and for coxsackievirus types A 2 and A 24. Such strains are difficult to recognize by tube neutralization, and relationships may be hard to find even by CF or plaque reduction tests. Difficulties in demonstrating neutralization of certain viruses may be clue to virus aggregates; deaggregation by ultrafiltration renders the dispersed viruses neutralizable. Recently, Schmidt and Lennette 2 " 22 applied immunodiffusion in delineating antigenic relationships between intratypic strains of widely diverging antigenic structure. Concentrated antigen is necessary; nonspecific reactants are absorbed from antisera. Distinctive antigenic relationships by cross-reacting precipitins disclosed relationships among diverse strains of A 2i coxsackieviruses. Additionally, some cross-relationships for heterologous serotypes have been noted. Minor cross-neutralization with prototypic monkey antisera has been reported for coxsackieviruses A 3 and A 8, and A n and A 15, A] 3, and A, 8, and for echoviruses 1 and 8, 1 and 12, and 6 and 3. The reported relationship between echoviruses 1 and 12 appears to be due to contamination of an echovirus type 12 stock by type 1 virus. 8 enteroviruses agglutinate human "O" type erythrocytes. 7 ' 1S Agglutinating serotypes are listed in Table 4. The relative simplicity of the procedure suggests its use as a first step in identification. However, it should be noted that all wild-type strains within a serotype may not agglutinate erythrocytes at least not on initial isolation. Moreover, with passage, agglutinable strains may lose their agglutinability. Other factors affecting hemagglutination are variabilities among erythrocytes, thermolability of agglutinins, ph of diluent, and temperature of incubation. Coxsackie A 7 aggluti- Table 4. Enteroviruses Known to Hemagglutinate* Group A Coxsackievirus Agglutinating Serotypes Group B 7,2,21,24 1,3,5,6 Echovirus 3,6, 7, 11, 12, 13, 19, 2, 21, 24, 25, 29, 3,33 * For some serotypes only a few agglutinating strains are known, whereas for others almost all strains have the property. Other serotypes, as yet not classified and possibly new, agglutinate human erythrocytes. nates those chicken cells agglutinable by vaccinia virus. Many enteroviruses provide reliable CF antigens for routine diagnosis, and with the use of appropriate antisera type relationships between most strains can be established. ' 17 The CF test is less sensitive than serum neutralization for revealing prime relationships, and thereby has a certain supplemental advantage. The method, in our laboratory, but not in others, is beset with a number of difficulties, principally the failure to obtain potent antigen for some serotypes, the anticomplementary effect of antigens, and nonspecific reactions occurring principally with sera containing antibodies engendered by proteins in the viral milieu. By standardization of test procedures and attentiveness to proper antigens and antisera, these handicaps are largely surmountable. Applied immunofluorescent technics 3 ' 2 for rapid diagnosis of enterovirus infections have not found routine use in most laboratories, despite encouraging reports appearing in the past 8 yr. The burden, like that of preparing CF antigens, is largely one of conjugating many prototypic sera and the application thereof to diverse strains encountered in sporadic illness. Nevertheless, the method has great value in rapid identification during episodic outbreaks. Absorbed sera have been used to on 14 March 218