key: cord-1026415-bj5nff9o
authors: Kapikian, Albert Z.; James, Harvey D.; Kelly, Sara J.; Dees, Jane H.; Turner, Horace C.; McIntosh, Kenneth; Kim, Hyun Wha; Parrott, Robert H.; Vincent, Monroe M.; Chanock, Robert M.
title: Isolation from Man of “Avian Infectious Bronchitis Virus-like” Viruses (Coronaviruses) similar to 229E Virus, with Some Epidemiological Observations
date: 1969-03-03
journal: J Infect Dis
DOI: 10.1093/infdis/119.3.282
sha: a481bc9b06d52f6974b47b72a46f3ddff1f545bc
doc_id: 1026415
cord_uid: bj5nff9o

nan

isolated only in human embryonic tracheal organ cultures [7] . The B814 virus, the 229£ virus, and the NIH a.c. viruses were all shown to possess a similar morphology which resembled that of the avian infectious bronchitis virus (IBV) but was distinct from that of the myxo-or paramyxoviruses [7-9V Subsequently, it was found that mouse hepatitis virus (MHV) shared the common morphologic features of these viruses [10] [11] [12] [13] .

As a group, the "IBV-like" viruses of man are fastidious in their host-cell requirements:

neither the B814 nor the 6 NIH a.c. viruses could be adapted to grow in a monolayer tissue culture system; 229E virus was isolated in such tissue culture, but only with difficulty. Virus 229E was isolated originally after a second blind passage in human embryonic kidney (HEK) cells; attempts to isolate this virus in human diploid cell strain (HDCS) WI38 cultures after 4 blind passages were unsuccessful although it was adapted subsequently to grow in these cells [6] . Virus 229E was capable of producing respiratory illness in volunteers after 1 passage in human embryonic tracheal organ cultures [11] .

In an attempt to study the incidence of 229E virus infection in a population of civilian adults, complement fixation (CF) tests were performed on paired sera obtained during a previously reported cross-sectional study of upper respiratory illness which spanned 2 years (1962) (1963) (1964) [2] . Five of 256 patients developed serologic (CF) evidence of 229E virus infection. At the same time, various experimental cell cultures were under evaluation for their sensitivity to rhinoviruses and strain 229E. We therefore took advantage of this opportunity by attempting to isolate virus strains resembling 229E from the original specimens of these 5 patients in such cell cultures. In addition, specimens were obtained from patients with upper respiratory tract illnesses who participated in a more recent (1965) (1966) (1967) cross-sectional study. 2 With the use of semi-continuous human embryonic intestine (HEI) cell cultures, a virus similar to 229E virus was isolated from 3 of the 5 patients with serologic evidence of infection and from 6 patients in the more recent study whose serologic response to 229E virus was not known at the time of the attempts at isolation. The recovery of these 9 virus strains represents the only reported isolation of viruses similar to 229E virus from natural infections since the original description of this agent. A description of these virus strains and certain observations concerning their epidemiology form the basis of this report.

Human embryonic intestine (HE/) tissue culture. HEI tissue culture tubes (MA 177) were purchased from Microbiological Associates. The intestine was obtained from a 3 1 /2lb stillborn male infant with an estimated gestational age of 34 weeks. This fibroblast cell strain could not be grown consistently beyond the twentieth passage; passages 13-17 were used for isolation of the 229E related viruses. Growth medium consisted of Eagle's minimum essential medium (MEM) in Earle's balanced salt solution (BSS), supplemented with 0.1 mmole each of 7 "nonessential" amino acids, I mmole sodium pyruvate, 10% inactivated (56 C for 30 min) fetal calf serum, 100 units of penicillin, and 100 f.lg of streptomycin per milliliter; maintenance medium consisted of equal parts of Eagle's MEM in Earle's BSS and Medium 199 in Hanks' BSS supplemented with 2 % inactivated calf serum and antibiotics as above [14] .

Source of specimens and virus isolation procedures. Specimens were obtained on or before the fourth day of illness from employees of the NIH who had acute upper respiratory tract ill- nesses. In the 1962-1964 study, 0.2 ml of freshly collected nasopharyngeal wash fluid (veal infusion broth with 0.5% bovine albumin) was inoculated into 2 roller tubes each of Hep-2, rhesus monkey kidney (MK), and HDCS WI26 or WI38 [2] . In the 1965-1967 study of NIH employees, 0.85% NaCI was used as nasal wash fluid, and this fluid was immediately diluted approximately 'h in veal infusion broth with 0.5% bovine albumin. These specimens were inoculated within I hr of collection into 2 roller tubes each of Hep-2, MK, WI26 and/or WI38, human aorta (AT-39), and HEK cultures [15] . The remaining nasal fluid was stored at -60 C. Tissue cultures were obtained from commercial sources, maintained as previously described, incubated at 33-34 C on drums rotating at 12 revolutions per hour, and observed for cytopathic effect (CPE) twice weekly [16] . MK cultures were tested for hemadsorption at 5-7-day intervals, and a single blind subpassage of the HEK culture harvests was made at 21 days. Acute phase sera were obtained at the time the washings were collected and convalescent sera about 3 weeks later. Five specimens from the 1962-1964 study and all specimens from the more recent study (1965) (1966) (1967) were also inoculated as above into HEI tissue culture; although some specimens were inoculated into HEI cultures within 1 hr of collection, most had been stored at -60 C prior to inoculation.

Organ cultures. Human embryonic tracheal organ cultures were prepared and maintained by a modification of the method of Hoorn and Tyrrell as described previously [7, 17, 18] .

Electron microscopy. One to 5 ml of tissue culture or O.C. fluid was clarified by lowspeed centrifugation at 2,000 rpm for 10-15 min in the PR-2 International centrifuge, and then centrifuged at 111,000 X g for 60 or 90 min in the SW-39 rotor of a Spinco model L ultracentrifuge. Pellets were resuspended in 0.1-0.2 ml of 1% ammonium acetate, negatively stained with 2% phosphotungstic acid (PTA) at pH 5.0 or 7.0, and spread on formvar-coated copper grids [7] . All electron micrographs were taken with a Siemens Elmiskop 1A at magnifications of X 40,000-80,000.

Viruses and sera. Dr Chloroform sensitivity, 5-iodo-2-deoxyuridine sensitunty, and acid lability tests. These tests were performed as previously described [16, [19] [20] [21] [22] . The 229E-related viruses isolated in HEI cells were adapted to HDCS WI38, and tests were performed in tube cultures of HDCS WI38.

Infectivity titrations. HEI or WI38 cultures were used in infectivity titrations. Tenfold dilutions of virus were made in Hanks' BSS containing 0.5% gelatin, 100 units of penicillin per milliliter, and 100 /-Lg of streptomycin per milliliter. Two-tenths milliliter of the appropriate virus dilution was inoculated into each of 2-4 tube cultures; the cultures were examined for the appearance of CPE 3 times weekly for approximately 2 weeks. Infectivity titers were based on CPE and calculated by the method of Reed and Muench [23] .

Inoculation of suckling mice. Swiss mice of the CD-l strain were obtained from Charles River Mouse Farms, Incorporated, Wilmington, Massachusetts. Retired breeders from this mouse colony were tested and found to be free of CF antibody to MHV, strain A59 [24] . One litter each of suckling mice, 0-3 days old, was inoculated intracerebrally with 0.01 ml of tissue culture harvests of viruses 489, 511, 515, and 844 and observed for 21 days. If a mouse developed illness, it was sacrificed and brain suspension was passaged to additional suckling mice intracerebrally and intraperitoneally; in addition, such suspensions were inoculated into HDCS WI38 cultures.

Complement fixation (CF) tests. 229E virus was inoculated into 32-oz bottles of WI38 cultures and allowed to adsorb for 1 hr. Fluids harvested after 2 freeze-thaw cycles, about 48 or 72 hr after inoculation, were employed as CF antigen. CF tests with appropriate controls were performed as previously described [7] . CF tests involving large numbers of paired sera could not for practical reasons be completed in a single day of testing. Therefore, such CF results reported in this study were from tests performed several days in succession.

Neutralization tests. Equal volumes of virus and fourfold dilutions of inactivated serum (56 C for 30 min) were incubated at room temperature for 2 hr. Two-tenths milliliter of the mixture was inoculated into each of 2 WI38 cultures. These were examined for CPE at a time when a simultaneous titration indicated that approximately 32-320 TCD so of virus were present. The cultures were examined at 2-3-day intervals for approximately 2 weeks, since CPE with the 229E-related viruses did not appear before the fifth or sixth day. Serum neutralizing end points were calculated according to the method of Reed and Muench and were expressed as initial serum dilutions [23] .

Isolation and growth in tissue culture. The 3 nasal wash specimens which yielded 229Erelated viruses from the 1962~1964 study were obtained within a 7-week period, March 2 to April 13, 1964 , while the 6 virus-positive nasal wash specimens from the 1965-1967 study were obtained during the first 3 months of 1967 (table  1) . The specimens were inoculated into roller tube cultures as stated in the Methods section. None of the inoculations into rhesus MK, Hep-2 HDCS WI26 or WI38, AT -39, or HEK cultures g~ve CPE. However, all 9 specimens produced CPE on initial passage in HEI tissue culture. The CPE was characterized by a gradual elongation of the cells throughout the monolayer beginning on the fifth or sixth day after inoculation; specific foci were not evident. Gradually, small granular round cells appeared throughout the monolayer. The cell sheet was rarely destroyed completely. At times uninoculated HEI tissue cultures appeared somewhat "stringy," and for this reason recognition of virus-specific CPE was occasionally difficult, especially at the beginning of the study. Electron microscopic and filtration studies. HEI culture harvests were clarified and concentrated by ultracentrifugation and examined by electron microscopy. Figure I shows representative particles seen in the harvests of isolate 489. Morphologically similar particles were seen in harvests of each of the other 8 isolates. A comparison of the isolates with strain 229E (figure 2), and avian IBV (figure 3), revealed their remarkable similarity. The particles usually appeared round or elliptical, but many were somewhat pleomorphic. A characteristic feature of the particles was the widely spaced club-or pear-shaped surface projections, which were narrow at the base, about 10 m,u wide at the outer edge, and approximately 20 mu in length. The mean of the largest diameter (including projections) of 67 samples of the 9 isolates was 152 m,u ± 6 mu (i.e., ±2 X standard error [SE] of mean) with a range of 104-250 me; the mean of the shortest diameter of these 67 samples was 107 m,u ± 4 m,u (i.e., ±2 X SE of mean) with a range of 76-160 mu. Details of the morphology of this group of viruses have been described elsewhere [7] [8] [9] [10] 25] . In filtration studies, in which Swinnex-25 filter units attached to 20-ml vacuum tubes were employed, virus strain 844 passed 285 through a 450-m,u, 220-m,u, and 100-m,u but not a 50-m,u or 10-m,u Millipore filter [26, 27] .

Attempts to grow agents in organ culture (D.C.). Attempts were made to cultivate these agents in human embryonic tracheal a.c. and to detect them by electron microscopy using methods described previously [7] . Three passages of the original specimens obtained from 5 while another control virus, herpes simplex (a known chloroform-sensitive virus) was inactivated by chloroform. Acid lability. Each of the 9 virus isolates exhibited at least a 100-fold reduction in infectivity titer after exposure to pH 2.7 for 3 hr at 25 C (table 4 ). The table also shows that rhinovirus IA (a known acid-labile virus) demonstrated at least a 100-fold reduction in infec- Clinical findings. Each of the 9 virus-positive patients was cultured either on the day of onset (1 of 9) or the day after onset of symptoms. Their average age was 33 years, with a range of 21-54 years. The most common symptoms recorded were coryza (in all 9), nasal congestion (in 8), sneezing (in 7), and sore throat (in 5). Less common were headache (in 4), cough (in 3), muscle or general aches (in 3), 'and chills and feverishness (in 2). The chief complaint in 8 of the 9 patients was coryza or nasal congestion; 1 patient's chief complaint was sneezing. None of the patients had an abnormal temperature elevation on the day of examination.

Epidemiologic observations. Sera obtained from pediatric patients with acute respiratory illnesses from several populations were tested by the CF technique for evidence of 229E infection. Only 1 of 892 paired sera obtained from October, 1962, through August, 1965, from infants and children admitted to Children's Hospital, Washington, D.C., for predominantly lower respiratory tract illness, exhibited serologic evidence of 229E infection. In a similar study of 222 infants and young children admitted to Children's Hospital from December, 1966, through April, 1967, none of these patients developed an antibody rise for 229E virus. In addi-Number of individuals tivrty titer, while the infectivity of poliovirus type I (a known acid-resistant virus) was unaffected after exposure to pH 2.7 for 3 hr.

Pathogenicity in mice. Virus strains 489, 511, 515, and 844 were inoculated into suckling mice as described in Materials and Methods. No illness was observed in mice inoculated with strains 489, 515, and 844; 4 of 8 mice inoculated with virus 511 died on the fourth day after inoculation. However, brain suspensions passaged intracerebrally and intraperitoneally to additional suckling mice failed to produce illness; in addition, such suspensions inoculated into WI38 cultures failed to produce CPE.

Serologic studies. 10-32 TCD so of each of the 9 isolates were neutralized by a 1: 160-1 : 1280 dilution of 229E guinea pig antiserum which had a homologous titer of 1: 320-1 : 1280 against 32-100 TCDso• This would indicate that each of the 9 isolates was similar, if not identical, to 229E VIrUS. Table 5 shows that 8 of the 9 patients from whom the 229E-related viruses were isolated had a significant CF antibody response to 229E virus. In neutralization tests, 3 of the 9 virus-positive patients exhibited significant increases in antibody to both strain 229E and their own isolate; [7] . Two of the 6 NIH O.C. strains (OC38 and OC43) were subsequently adapted to suckling mouse brain, but none of the 6 strains grew in monolayer tissue culture [29] . The 9 strains recovered in this study Isolation or"IBV-like" Viruses were found to be similar, if not identical, to strain 229E by one-way neutralization tests and, in addition, to possess similar morphologic and biophysical properties.

The 9 229E-related strains were recovered on initial passage in semi-continuous HEI tissue cultures; they could not be isolated on initial passage in conventional tissue cultures. In addition, since the technique of O.C. passage followed by electron microscopic examination of pooled, concentrated O.C. harvests had proved to be a sensitive system for the recovery of NIH O.C. viruses, this technique was applied both to several clinical specimens yielding agents resembling strain 229E and to several tissue-culture-adapted strains as well. In all cases, attempts to detect virus by electron microscopy in O.C. harvests failed, although titration of such harvests showed in 1 case that low-grade replication of virus probably occurred. It appears, therefore, that HEI tissue culture is preferable to O.C. for isolation of certain strains of "IBV-like" viruses such as these 229E-related agents. It should be noted, however, that the 6 "IBV-like" strains recovered in O.C. in this laboratory could not be adapted to grow in HEI tissue culture [K. Mcl ntosh, unpublished studies]. Therefore, for isolation of ""IBV-like" viruses, it would appear to be necessary to employ both HEI tissue culture and O.C. However, additional ways to facilitate the growth of "IBV-like" viruses such as 229E and others must be found since HEI cultures, although more sensitive than any conventional tissue culture or O.C. system, were much less sensitive than the measurement of CF antibody rises for detection of 229E infection.

The serologic survey of 229E virus infection in various population groups revealed that infection with this agent was rare in infants and children with lower respiratory tract illnesses. In the 1962-1964 study of upper respiratory tract illness among adults, 5 (2%) of 256 individuals had serologic evidence of 229E infection. It was noteworthy that these 5 infections occurred from January through April, 1964, when rhinovirus and other known respiratory virus infections were uncommon but upper respiratory tract illnesses were prevalent; during this period 229E virus infection was associated with 5 (10%) of the 50 illnesses sampled. In the 1965-1967 study of upper respiratory tract illness in adults, 23 (7%) of 317 patients dev.eloped serologic evidence of 289 229E infection. It was striking that 21 of the 23 infections occurred during a 5-month period (December, 1966 -April, 1967 when infections with rhinoviruses and other cytopathic or hemadsorbing viruses were uncommon while upper respiratory tract illness remained prevalent. During this 5-month period, 21 (24%) of 89 patients studied developed serologic (CF)· evidence of 229E infection. It was previously reported that 5 NIH O.C. viruses were recoveredin O.C. from specimens obtained from 9 patients during a 3month period of the previous year (December, 1965 -February, 1966 [7] . In addition, in a serologic survey using as antigen 2 serologically identical NIH O.C. viruses (strains OC38 and OC43) originally recovered in O.C., and subsequently adapted to grow in the brain of suckling mice, 18 (31%) of 59 patients (including the 5 patients above who yielded NIH O.C. viruses) developed serologic evidence of infection with these viruses during this 3-month period (December, 1965 -February, 1966 [29] ; only 1 of the 59 patients developed a CF antibody rise to 229E virus during this period. It was of interest that, in these CF antibody surveys, patients with serologic evidence of 229E infection rarely developed concurrent CF antibody rises to the OC38 and OC43 mouse brain antigens [30] .

These observations suggest that "Iflv-like" viruses may be etiologic agents of a portion of respiratory illnesses which occur during the winter season, when the prevalence of rhinovirus and other known respiratory virus infections is often low, but that of upper respiratory tract illnesses quite high [2] [3] [4] .

The occurrence of a typical common-cold-like illness in the 9 patients from whom 229E-like viruses were isolated is consistent with the clinical findings in volunteer studies with B814 and 229E viruses [5, 11] .

Nine virus strains resembling "IBV-Iike" virus 229E of Hamre were recovered in human fetal intestine fibroblast cultures from nasopharyngeal washings of adults with acute upper respiratory tract disease. Eight of the 9 virus-positive individuals developed serologic (CF) evidence of 229E virus infection. The virus isolates exhibited properties typical of the "IBV-like" virus group: distinctive morphology, resistance to 5-IUDR, and chloroform sensitivity. Measurement of CF antibody response was found to be approximately twice as sensitive as virus recovery for detection of infection. Standard monolayer tissue cultures as well as human fetal tracheal a.c. were ineffective for recovery and recognition of the 229E related isolates. A seroepidemiologic survey indicated that 229E virus infection was rare in children with lower respiratory tract disease. However, such infection occurred in 10%-24% of adults with upper respiratory tract illnesses during 2 of 4 winters-a season when rhinovirus infection was uncommon but respiratory disease morbidity was high.

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