key: cord-0007334-61e9obb3 authors: Jackson, George Gee; Muldoon, Robert Lee title: Viroses Causing Common Respiratory Infections in Man. III. Respiratory Syncytial Viroses and Coronavimses date: 1973-11-03 journal: J Infect Dis DOI: 10.1093/infdis/128.5.674 sha: 236cb70fc132c252c445471f94b913f30173f3b4 doc_id: 7334 cord_uid: 61e9obb3 nan 1. Size. RS virus was estimated, from sucrose density gradient centrifugation studies, to be 90-120 nm in diameter [2] ; viral particles in infected cells measured 65 nm by electron microscopy. Particles negatively stained with phosphotungstic acid measured 120-300 nm [23] . Some viral particles were as large as 860 nm [25] . Structure. In ultrathin sections of infected tissue culture, electron microscopy revealed viruslike particles in vacuoles or invaginations within the cytoplasm [14] , but complete particles were not found in the cell cytoplasm [21] . The enveloping membrane has been described as fringed and the nucleoprotein strand as having a herring-bone appearance, with a mean diameter of 13 CF antibody formed by infants during an RS infection has a higher antigen requirement than antibody present in sera of adults or than that found as maternal antibody [113] . Neutralizing secretory antibody occurs in one-half or more of patients with lower respiratory disease and in 10%-20 % of those with milder infections [97, 116] . b. Secondary. A rise in CF and neutralizing antibody was observed after reinfection in the presence of pre-existing serum antibody [18] . The CF antibody response in adults was always weak compared with that in children [65] . a. Infection of tissue culture. Isolates were made from 0.1-0.2 ml of specimen inoculated onto a monolayer consisting of 100,000-300,000 HEp-2 cells. Eagle's basal medium, containing 5 %inactivated chicken serum, is a suitable maintenance medium that should be changed every three to four days. Stationary incubation at 36 C is satisfactory. Inoculation of a culture of Chang liver cells, four to six days old, maintained with a medium of eight parts Eagle's basal medium, two parts inactivated horse serum, and 0.2 parts L-glutamine is satisfactory. The medium is changed every four days. Virus can be propagated in KB cells in a medium consisting of Eagle's basal medium with 2 %chicken serum. Preferred cell line. The most sensitive cell cultures are HEp-2, monkey kidney, human amnion, and human kidney, in decreasing order [5, 44] . i. The cytopathic effect begins with small syncytial areas randomly distributed early in infection. Within one to four days, the entire cell sheet may be involved, with syncytial areas enlarging and becoming more numerous [2] . The time of appearance of CPE depends, within limits, on the number of serial passages of the virus [2] but is nearly always demonstrable within five days. ii. Cytology. Eosinophilic cytoplasmic inclusions are commonly found in infected cells, especially in the syncytia [15] . These inclusions are devoid of DNA, RNA, virions, and demonstrated specific antigens. Chromosomal abnormalities are not observed. In infected Vero cells, dense intracytoplasmic inclusions have diameters ranging from 90 to 130 nm [115] . iii. Plaque formation. Small macroscopic plaques developed after incubation for seven to nine days in HEp-2 cells overlayed with agar [30] . Four conditional-lethal, temperature-sensitive mutants of RS have been isolated and shown to be genetically stable. One of the mutants produced a typical, nonsyncytial plaques [93]. iv. Hemadsorption. None demonstrated. No data available. One-day-old mice inoculated intracerebrally or intraperitoneally [1, 2] , weanling hamsters, rabbits, guinea pigs, mice, weighing 10 g, and young adult rats inoculated via the previously mentioned routes were refractory to infection [1] . A. Natural Infection 1. Clinical. Infection with RS virus has been observed every year since its recognition. It occurs in rather sharp epidemics, recurring at intervals of nine to 14 months, usually in the fall or spring. Illness may be gradual or abrupt after an incubation period of three to five days. The symptoms associated with infection in children are cough (97 %), fever (93 %), rhinitis (57 %), pharyngitis (47%), malaise (38 %), vomiting (30 %), anorexia (27%), lymphadenopathy (22%), otitis media (17%), conjunctivitis (13 %), and abdominal pain (7 %) [13, 29, 49] . In an analysis of symptoms produced by infections with RS and by influenza A viruses, the two diseases could not be differentiated on a clinical basis [112] . In infants, as many as 60% The first isolations of virus were made one to two days before the onset of symptoms. Isolates were more frequent and positive over a longer period from subjects with illness than from those who were infected but without symptoms. 3. Immunity. All adults tested possessed detectable levels of neutralizing antibody to RS virus before challenge, but the titer of naturally acquired antibody had no significant effect on subsequent RS infection of volunteers and was poorly correlated with development of mild clinical illnesses. Resistance to infection and illness appeared to be related to the level of nasal antibody but not to the level of serum antibody [121] . Infection elicited an increase in the titer of serum antibody by CF and neutralization. Immunity upon rechallenge was not tested. Illnesses from infection are more severe in children than in adults [8, 10] . The CF test, with 8 units of antigen, will detect 90 %of infections among individuals older than six months and is as sensitive as neutralization. Below six months, the CF test detects only 20 %of infections. The neutralization test is more sensitive than CF when serum from infants is used, but rises in neutralizing antibody have been detected in only half of the virus-positive infections in this age group. The use of unheated serum or the addition of antibody-free fresh serum increases the sensitivity of tests for neutralizing antibody [42, 60]. Antiserum was produced in guinea pigs by three weekly ip inoculations of 1 ml of infected tissue culture harvest [2] . Rabbits given three weekly iv inoculations of 1 ml each followed by two weekly im inoculations of 1 ml of infected tissue fluid combined with 2 ml of a mixture of Mycobacterium butyricum, paraffin oil, and arlacel, produced CF and neutralizing antibody [2] . In adult rabbits, an alternative procedure is to give three injections at two-week intervals, the first two consisting of 8 ml of virus and adjuvant given im, and the third injection of virus alone administered iv [5] . Respiratory syncytial virus is considered to be a paramyxovirus on the basis of its size, appearance by electron microscopy, and sensitivity to ether; it differs from other paramyxoviruses in that it has no known hemagglutinin. Although minor antigenic variants have been found, they are not well discriminated by antibody in human sera. Because there has been no sequential drift in the antigenic character of the prevalent strain, RS virus is considered to be a single type. Epidemiologically, respiratory syncytial virus is very important, because it causes annual epidemics of acute respiratory diseases affecting infants, children, and adults. Infection spreads rapidly from person to person and characteristically occurs as a discrete outbreak of acute respiratory illness in the winter or early spring. In infants and children, especially during the first six months of life, respiratory syncytial virus is the most important cause of bronchiolitis and a major cause of pneumonia. Serum antibody acquired by transplacental passage does not provide immunity against infection and might possibly augment the local respiratory disease by an immunopathologic process. The virus may replicate in the middle ear, but its role in otitis is unproven. Croup is an infrequent manifestation. Pneumonia, as determined radiographically, is frequent, usually bilateral, and multilobar; it may be lobar with secondary bacterial infection. The pathologic lesion is one of necrosis of the epithelial mucosa of the trachea and bronchi and an interstitial inflammation. In adults, infection with RS virus usually causes upper respiratory symptoms; however, because of the prevalence of infection, it is an important cause of exacerbations of bronchitis, pneumonia, and "flu," requiring the hospitalization of adults. In some years, virus infection has given rise to an increase in secondary pneumonia due to Diplococcus pneumoniae. Infection is followed by an increase in serum CF and neutralizing antibody; also by secretory neutralizing antibody in the nasopharyngeal and tracheal secretions. Primary infection does not establish complete immunity, and reinfection is common at all ages. Inactivated vaccines of the type and potency previously produced have been detrimental because they have failed to prevent infections and they have induced a more severe disease with exaggerated pneumonia. Attenuated live virus vaccines have not yet been successful, nor has any effective chemotherapy been developed. Takano A virus isolated from wild cottontail rabbits was shown to possess chemical, physical, and biologic characteristics of the paramyxovirus family. Although formation of syncytia was characteristic of its growth in tissue cultures, no antigenic relationship was detected by CF or neutralization tests with any known member of the paramyxovirus family [1] . Isolates shown to be antigenically related to human RS virus were recovered from cattle with bronchopneumonia [2] . Cytologic examination of BHK2 1 cells infected with bovine RS virus revealed intranuclear and intracytoplasmic viral components [4] . An A type particle with a diameter of 65 nm has been described in the cytoplasm of such cells [3] . The viral envelope was added as the virion passed through the cytoplasmic membrane in a budding process [4] . A. Physical Properties CHARACTERIZATION 1. Size. By gradocol filtration, the size of the virion was calculated to be 89 nm [2] . By electron microscopy, the usual diameter was measured as 80-160 nm [5] . 2. Structure. The virions are pleomorphic. Most particles are covered with projections (spikes) more densely packed than those seen on influenza viruses. These spikes are attached to the virion-by narrow stalks with a thickening (90-110 A) at the distal end [5, 11] . The internal component is a hollow, threadlike structure with a diameter of 70 nm and a definite structural pattern [21] . 3. Heat stability. Infectivity was destroyed at 56 C within 10 min. There was no loss of titer after 2 hr at 37 C or 10 days at 4 C. Rates of thermal inactivation are dependent on the amount of particle aggregation. Aggregation is dependent on the concentration of serum in the medium [ A. Group Antigen Common CF antigens have been observed in known members of the coronavirus family, except in avian bronchitis virus. Serologic data are still incomplete, but some observed interrelations are shown in table 1. Table 1 . Antigenic mosaic of coronavirus as determined by neutralization (N) and CF tests with animal serum [20, 25] . Antigens produced in tissue culture or mouse brain CF antigens have been prepared from harvests of infected tissue culture and mouse brain, but attempts to prepare antigens from organ cultures have not been successful [2] . Strains OC38 and OC43 cross-react, as shown by neutralization tests in mice or monkey-kidney cell culture. Strains 229E and LP cross-react in neutralization tests but not to identical titers, indicating a close but not completely similar antigenic mosaic. A one-way cross exists between 229£ and OC32; antisera against the latter and B814 do not neutralize other coronaviruses. Avian bronchitis virus reacts only with homologous virus (see table I ). In immunodiffusion tests, the number of detectable lines of precipitation varies from one (strain B814) to four (strain OC43). This may be a result of the procedure used for production of antibody [25] . C. Antigenicity I. Animals. In animals, specific antibody is elicited by the initial series of inoculations of cell-culture harvests. In neutralization tests with animal serum, no antigenic relationship has been detected between strains 229E and OC38-43 [20] . a. Primary response. Initial infection results in an increase of specific neutralizing antibody. About 50% of volunteers challenged with the OC strains of coronaviruses developed CF rises to MHV [20] . b. Secondary Response. The antigenic primacy of the initially infecting strain, heterologous interrelationships, and anamnestic responses are still to be worked out. [16] . Coronaviruses of avian origin have phenotypic differences in the susceptible avian host cell range [4] and grow to a limited degree in nonavian tissues [32] . Murine coronavirus has grown adequately only in tissues from mice [20] . Coronavirus of rats has Isolation Propagation grown in only one of several cell lines studied [24] . Coronaviruses of swine grow only in tissues of porcine origin [37] . a. Infection of tissue culture. For growth of explants, a medium of 2 ml of Eagle's medium with 0.2 % (wt/vol) bovine plasma albumin and incubation at 33 C in a humidified atmosphere containing 5 %(vol/vol) CO 2 in air is satisfactory [5] . A pH of 7.0 is required for growth since inactivation is accelerated at pH 7.7 and 6.7 [25] . i. Preferred cell line. Human tracheal organ cultures. ii. Growth cycle. After 1 hr at 33 C, only 18%of L132 cells wereinfected with strains 229E. Virus structures were detected 6-8 hr later [17] .· Infection of WI-38 cells with strain 229E resulted in a reorganization of the cytoplasm, as determined by electron microscopy. New viral structures were observed 6-12 hr after infection. Clusters of virus were observed in intracytoplasmic vacuoles, called cisternae, 24-36 hr after challenge. Strain OC43 in WI-38 matures in intracytoplasmic vesicles similar to those observed with strain 229E.. Budding, such as that described for strain 229E was not observed [30] , and the budding process described for coronavirus is into cytoplasmic vesicles rather than from the plasma membrane, as has been observed with myxoviruses [6, 8, 28] . iii i. CPE. The CPE that gradually developed in human diploid cells gave them a stringy appearance. Some intracytoplasmic vacuoles were observed [2] . Strain B814 caused no CPE and could be detected only by electron microscopy or by interference with Echovirus type 11 [1] . ii. Cytology. No inclusions have been observed [2] . Fluorescent antibody has been used for identification of viral antigen [15] . Morphology, as observed by electron microscopy, is characteristic [5, 37] . The use of specific antisera, in combination with electron microscopy, can facilitate recognition of the virus in culture harvests [38] . iii. Plaques. At 33 C, with a methyl cellulose overlay, plaques were formed in WI-38 cell cultures [8] . Plaques can be produced in L132 cells infected with the 229E strain [17] . [4, 12, 24] . After four to five passages in tissue culture, strains OC38 and OC43 were administered intracranially to suckling mice. On the first passage in mice, illness, characterized by tremors, rigidity, and lethargy, was observed on days 11-15 after challenge. By the fourth passage in mice, these viruses were lethal for mice within 48-60 hr after challenge [9] . There is marked host specificity of different strains. A. Natural Infection 1. Clinical. The use of explants of human embryonic nasal epithelium or trachea has resulted in the isolation of coronaviruses. Serology has shown them to be associated with acute respiratory diseases of man. The exact importance of these viruses with accompanying epidemiologic data is unavailable because of the variability of strains and the difficult techniques required to establish diagnosis of the infection. In most studies, coronaviruses usually caused infections in the period from January through March [18, 33, 34] . Only about one-half of naturally occurring infections cause clinical illness [26] . Spread appears to be preferentially within families. In one study, secondary cases occurred in 17 of 26 families [22] . Serum neutralizing antibody does not influence the occurrence of reinfection [33] . Acquisition of infection with avian bronchitis virus from chickens is suggested by studies in poultry handlers [13] . Nonsusceptible Cells Infection in Man sponses occurred in 10%-20 % of these and other volunteers infected with coronaviruses [29] . Strain 229E was recovered from sick as well as healthy volunteers in each of the four challenge passages [7] . Volunteers challenged with the OC or B816 strain often showed CF rises to strains 229E and LP [20] . 3. Immunity. Incomplete. c. Prevention 1. Vaccine. None. None. Diagnosis is based on electron microscopic examination of cell explants or, with some strains, detection of CPE in monolayers after serial blind passage. B. Serology Rises in the titer of CF antibody against strain 229E and HAl antibody against strain OC43 are the most practical tests. Neutralization is the serologic test of choice for specific identification. Commercially unavailable. Prevention Laboratory Diagnosis Coronaviruses are a distinct group of viruses with common morphology and various degrees of antigenic similarity. The number of different coronaviruses that infect man and their exact interrelations are still unknown. Their etiologic role in respiratory diseases has been established. Also, they are associated with hepatitis in mice and avian bronchitis. Data suggest that coronaviruses cause Comment 3 %-4 % of acute respiratory illnesses in humans. The clinical syndrome is usually that of a common cold. Asymptomatic infections also occur, possibly because of partial immunity to reinfection. Coronavirus infections are most prevalent in the winter months and may occur in epidemic fashion, with the same strain being geographically widespread. Recurrent epidemics of the same type do not seem to occur in sequential years. Preliminary serologic investigations indicate that certain strains may infect children preferentially, whereas others are prevalent in adults. An alternative explanation of the findings is the emergence of new epidemic strains with disappearance of older strains for a period. Transmission of the virus within families is frequent, and acquisition may be possible from poultry and other sources. No effective vaccines or chemotherapy have been developed. Recovery of cytopathogenic agent from chimpanzees with coryza Recovery from infants with respiratory illnesses of a virus related to chimpanzee coryza agent (CCA). I. Isolation, properties,and characterization Recovery from infants with respiratory illness of virus related to chimpanzee coryza agent (CCA). II. Epidemiologic aspects of infiltration in infants and young children Association of the chimpanzee coryza agent with acute respiratory disease in children Growth characteristic of chimpanzee coryza agent in tissue cultures Role of respiratory syncytial virus in bronchiolitis, pneumonia, and minor respiratory disease. I. Virus recovery and other observations during 1960 outbreak Studies of acute respiratory illness caused by respiratory syncytial virus. I. Laboratory findings in 109 cases Respiratory syncytial virus infection in adult volunteers. II. Correlation of illness and clinical observations An outbreak of febrile illness and pneumonia associated with respiratory syncytial virus infection Respiratory syncytial virus infection in adult volunteers. III. Prediction of illness and clinical observations Studies on acute respiratory syncytial virus. 2. epidemiology and assessment of importance Role of respiratory syncytial virus in bronchiolitis, pneumonia, and pharyngitis with bronchitis in children. II. Serologic studies over a 34 month period Studies on acute respiratory illnesses caused by respiratory syncytial virus. 3. Clinical and laboratory findings Morphology and development of respiratory syncytial virus in cell culture Growth and serologic characteristics of respiratory syncytial virus Respiratory syncytial virus Experimental cytial virus antigens by agar gel diffusion and immunoelectrophoresis Interferon and respiratory syncytial virus Speculation on pathogenesis in death from respiratory syncytial virus infection The late detection of respiratory syncytial virus in cells of respiratory tract by immunofluorescence Double infection with RS virus and influenza Virus infections in children. Clinical comparison of overlapping outbreaks of influenza A2-Hong Kong-68 and respiratory syncytial virus infections Differentiation of actively and passively acquired complementfixing antibodies in infants with respiratory syncytial virus infection The use of cough-nasal swabs in the rapid diagnosis of respiratory syncytial virus infection by the fluorescent antibody technique Morphogenesis of respiratory syncytial virus in a green monkey kidney cell line (Vero) Respiratory syncytial virus neutralizing activity in nasopharyngeal secretions RSV infections and infant deaths Respiratory syncytial virus tissue culture immunofluorescence as a laboratory aid Rapid diagnosis of respiratory syncytial virus infection in children by the immunofluorescent technique Experimental respiratory syncytial virus infection of adults. Possible mechanisms of resistance to infection and illness Recovery of a new syncytium virus from a cottontail rabbit A respiratory syncytial virus of bovine origin Cultivation of a novel type of common cold virus in organ culture A new virus isolated from the human respiratory tract A new virus cultivated only in organ culture of human ciliated epithelium Immunofluorescence of avian infectious bronchitis virus in primary chicken embryo kidney, liver, lung, and fibroblast cell cultures The morphology of three previously uncharacterized human respiratory viruses that grow in organ culture Morphogenesis of avian infectious bronchitis virus and a related human virus (strain 229E) Effects of a "new" human respiratory virus in volunteers Growth and intracellular development of a new respiratory virus Growth in suckling mouse brain of "IBV-like" viruses from patients with upper respiratory tract disease Recovery in tracheal organ cultures of novel viruses from patients with respiratory disease Direct electron microscopy of organ culture for detection and characterization of viruses Neutralization of infectious bronchitis virus by human serum Cultivation of "difficult" viruses from patients with common colds Intracellular avian infectious bronchitis virus: detection by fluorescent antibody techniques in nonavian kidney cell cultures Sensitivity of L132 cells to some "new" respiratory viruses The propagation of "coronaviruses" in tissue culture Isolation from man of "avian infectious bronchitis virus-like" viruses (coronaviruses) similar to 229E virus with some epidemiological observations Some characteristics of hemagglutinin of certain strains of "IBV-like" virus Antigenic relationships among the coronaviruses of man and between human and animal coronaviruses Symposium on the biology of large RNA viruses Community-wide outbreak of infection with 229E-like coronavirus in Tecumseh, Michigan Seroepidemiologic studies of coronavirus infection in adults and children Rat coronavirus (RCV); a prevalent naturally occurring pneumotropic virus of rats Coronaviruses of man Seroepidemiologic survey of coronavirus (strain 0C43) related infections in a children's population Presence of neutralizing antibody against the 229E strain of coronavirus in the sera of residents of Sendai Electron microscopic studies of coronavirus Coronavirus antibody titres in sera of healthy adults and experimentally infected volunteers Intracellular development and mechanism of hemadsorption of a human coronavirus OC43 Studies with human coronaviruses. II. Some properties of strains 229E and Jackson and Muldoon OC43 Replication of avian infectious bronchitis virus in African green monkey kidney cell line Vero Virologic studies of acute respiratory disease in young adults. V. Coronavirus 229E infections during six years of surveillance Coronavirus infections in working adults: eight year study with 229E and OC43 Protein composition of coronavirus OC43 Hemadsorption by coronavirus strain OC43 Characteristics of a coronavirus (strain 67N) of pigs Detection of coronavirus strain 692 by immune electron microscopy Of five subjects from whom coronaviruses were isolated, all developed a serologic response [3] .b. Serologic. Measurement of the CF antibody response was found to be twice as sensitive an index of infection as virus isolation. The CF antibody tends to be transitory, whereas titers of neutralizing antibody remain elevated for a longer period of time [13] . The observed prevalence of infection varies widely in different years [29, 33] .During a spring outbreak of respiratory disease in 1967, the infection rate in a community was 34 % [22] . From observations in a children's home, it was estimated (based on serology) that 19% of respiratory illness in a single season was caused by coronavirus strain OC43 [26] . Among groups of adults during two of four winters when there were high rates of respiratory disease and infrequent virus recovery, infection with 229B occurred in 10%-24% of those with upper respiratory tract disease [18] .In three separate studies, each covering a seven-or eight-year period, coronavirus OC43 accounted for 3 % of 1,328 illnesses observed in children [26] ; coronaviruses 229B and OC43 accounted for 4 % of colds in an 'adult industrial population [34] , and 15%-35 % of students were infected during three seasons of high prevalence [33] .A serologic survey of adults and children showed that infection in infants below one year of age was infrequent. Infection with OC38 and/ or OC43 occurred principally in the preschool years, whereas infection with 229B occurred later [23] . Data from sera collected in 1966indicate that 30 % of adults and 15%-20 % of children had neutralizing antibody specific for strain 229E [7] . In a study of sera collected since 1967, 50 %-80 % of the population were found to have neutralizing antibody against 229E at a dilution of 1:20, as measured by plaque reduction in L132 cells. In CF tests (serum diluted 1: 20) between 50 %and 98 %of those tested had antibody [25] . Sera collected from 139 adults between January 1969 and October 1970 showed that 8.6% had a neutralizing antibody titer 2:: 1: 8 against strain 229B [27] . 1. Challenge. In an early study, harvest medium from human embryonic trachea containing strain B814 was used as inoculum. Illness was observed in five of 11 volunteers [1] . After tissue and organ-culture passage, strain 229E was passed four times in volunteers and produced illness in each passage [7] . Later, six coronaviruses isolated from persons with illness were given to volunteers. All six produced colds. Heterologous antibody re-