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Serological and virological investigations of young children with acute respiratory syndrome associated with respiratory syncytial virus
81
DIAGN MICROBIOL INFECT DIS 1985;3:8145
NOTES
Serological and Virological Investigations of Young Children with Acute Respiratory Syndrome Associated with Respiratory Syncytial Virus Giulio Tarro, Riccardo Smeraglia, and Ciro Esposito From January 1979 to March 1979, 341 young children from the metropolitan area of Naples, Italy, were hospitalized with respiratory virus disease. Diagnosis of patients made from virus isolation and seroconversion indicate that the respiratory syncytial virus was a primary cause of this acute respiratory syndrome.
During the winter of 1979, the most severe in our area in the past 15 years, 341 children, most under 3 years of age, presented to several local metropolitan hospitals with acute infection of the branched bronchial tree. Prior data (Chanock, 1977, Martin et al., 1978) have shown that the clinical symptomatology for respiratory syncytial virus {RSV} infection appears with greatest frequency and severity in early childhood patients (McClelland et al., 1961, Ross et al., 1964). The 2- to 36-month age group appears to be at greatest risk. Severe bronchiole infections resulting in death have been observed in these patients {Chanock and Parrot, 1965; Parrot et al., 1961). RSV infection is also seen in children over 36 months, but the consequences are usually far less severe. Inflammation of upper and middle respiratory tracts, usually not requiring hospitalization, is normally the extent of the problem in this age group (Kravetz et al., 1961}. In our study, the respiratory anamnesis was compatible with the clinical picture of an acute respiratory syndrome (rhinitis, pharyngitis, laryngitis, and the most severe illnesses such as croup, tracheobronchitis, pneumonia, and bronchiolitis}. This outbreak was studied without any need to use special modern techniques and its study might be performed in any peripheral virological laboratory. Several pharyngeal swabs were taken on each patient over a given period of time. These samples were taken and immediately inoculated into cell cultures at the patient's bedside. Seventeen pulmonary samples obtained at autopsy from patients deceased in the first 2 years of life were transported to our laboratories in refrigerated containers 100 ml of Eagle's Basal Medium {BME} + 0.2% NaHCO3 + antibiotics 3 times concentrated: penicillin {200 units/ml}, streptomycin (0.2 mg]ml}, gentamicin
From the Division of Virology, D. Cotugno Hospital, and Oncologic Virology, University of Naples, Naples, Italy. Address reprint requests to: Professor Giulio Tarro, via Posillipo 286, 80123 Naples, Italy. Received August 31, 1983; revised and accepted March 13, 1984. © 1985 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Avenue, New York, NY 10017
0732-8893/85/$03.00
t~
TABLE 1. Seropositivity to RSV and Other Viruses in 189 Young Children Hospitalized with Acute Respiratory Syndrome
Patien ts admitted in: Pediatric department Intensive care Total
High positive antibody titer w i t h no rise in second serum t i t e r for RSV
Meaningful rise i n t i t e r for RSV No. of patients
Tot a l seroconversion and positive a n t i b o d y to RSV
No.
%
CF e
No.
%
CF
No.
%
164
94
58
8-256
37
23
8-256
131
80
25 189
13 a 107 b
52 57
8-256 --
12 49
48 26
16-256 --
25 156
100 83
High positive antibody titer with no rise in second serum titer for o t h e r viruses
Meaningful ri s e i n t i t e r for other viruses
"Four serum pairs were from patients that died of this disease. bin 64 of these cases, RSV was also isolated form pharyngeal swabs. ZOne seroconversion was Coxsackie B6, one was Mycoplasme pneumoniae. ~Three cases of seropositivity were Cytomegalovirus, two were Adenovirus, two were Influenza A, two were one was Coxsackie B6, and one was Parainfluenza 2. eComplement fixation units expressed as reciprocal of serum dilutions; ranges of titers.
No.
%
CF
2c
1
8-16
.
. 2
. 1
. --
Mycoplasma Pneumoniae,
No.
%
CF
12 d
7
16-64
7
--
. 12
.
one was Coxsackie B4,
O
Acute Respiratory Syndrome and RSV
83
(0.05 mg/ml)], homogenized and suspended to 10% of Minimum Eagle's Medium (MEM) with 5% chicken serum and 0,1% NaHCO3. Of each suspension sample, 0.2 ml was inoculated into two test tubes containing HEp-2 cells and two tubes containing human fetal fibroblast monolayers. HEp-2 cells grown in our laboratory using the techniques described by Tarro and Sabin (1970) and frozen in BME with 10% calf serum, 0.1% NaHCO3, and antibiotics, were brought to + 37°C and used. The cell confluent cultures were washed and replaced with BME with 2% chicken serum and 0.2% NaHCO3 and used for sample inoculum. The samples (697 pharyngeal swabs and homogenized suspensions of samples obtained at autopsy) were inoculated into cell cultures at + 37°C. The cell cultures were observed daily to plot the cytopathic effect (CPE) which became evident 2-15 days after inoculation. When CPE was seen in a sample, 3-4 serial passages were taken to ensure that the activity was due to the presence of the virus. For virus identification, the liquid culture containing the scraped infected cells plus 5% chicken serum was frozen at -80°C, thawed, and centrifuged at 1500 rpm for 10 min. The sediment was suspended in 0.2 ml of balanced salt solution (about 10% of cellular suspension) and tested by complement fixation (CF), using four units of antisera (Microbiological Associates, Baltimore, MD). For Control measures, CPE negative cell cultures were also tested using the same technique. Sera from 189 patients exhibiting symptoms of acute respiratory syndrome were studied. For each patient, at least two serum samples (the first one taken immediately upon hospitalization and the second after 15 days) were tested for antibody titration of different viruses. Of these 189 patients, 25 had been initially admitted into intensive care units for coma and 21 had survived. The CF test was performed using the technique described by the Center for Disease Control (Atlanta, GA). Four units of CF antigen, (Microbiological Associates) and two units of complement (Sclavo Laboratories, Siena, Italy), were added to the sera dilutions. Table I data show that, of 164 patients admitted to the pediatric departments, 94 cases (57%) exhibited a significant increase in RSV titer (~>4 fold) between the acute stage and convalescent stage serum samples. Another 37 patients (23%) showed definite positive results for RSV antibody--that is, elevated titers (~>4 CF units) in comparison with other viruses--in both samples, but exhibited no increase in titer concentration in the second serum. The 25 patients admitted to intensive care units showed the following serum results: 13 samples (4 from nonsurvivors) showed a significant increase in RSV titer (~> fourfold) between the serum pairs, and 12 showed a positive RSV antibody titer but no increase in titer in the second serum. The results from the total 189 sera pairs tested showed that 107 exhibited a significant increase of the second serum RSV titer (/> fourfold) over the first serum sample (56%) and 49 pairs showed a high RSV-positive titer in both samples with no measurable differences between acute and convalescent phases (26%). It is possible that the lack of antibody concentration increase was due to the stage of infection: RSV-antibody was already formed when the first sample of the patient was taken, since all the children hospitalized in this study presented the acute phase of viral involvement. Assuming that the above statement is true, it can be seen that 83% (156/189) of the serum pairs studied had a significant RSV-positive antibody titer. In addition, it was found that serum pairs from two of the patients admitted into pediatric wards for viral respiratory syndrome showed a significant increase in titer to other viruses---one patient to Coxsackie B 6 and the other to Mycoplasma pneumoniae. Moreover, twelve other patients exhibited a significant positive antibody titer without titer increase between serum pairs when tested with the following viruses: three patients, cytomegalovirus; two, adenovirus; two, influenza A; two, Mycoplasma pneumoniae; one, coxsackie B 6; one, parainfluanza'2. Nineteen of the patients admitted to pediatric departments,
84
G. Tarro et al.
TABLE 2. F r e q u e n c y of Isolation of RSV and Other Viruses by Pharyngeal Swab of 341 Young Children Hospitalized w i t h Acute Respiratory S y n d r o m e
Patients admitted in: Pediatric departments Intensive care Children who survived coma Nonsurvivors Total from both departments b
Children from whom RSV was isolated
Seronegative for RSV
Children from whom other viruses were isolated
No. of patients
No.
Percent
No.
No.
Percent
305
136
44
11
6
2
27
17
63
not done in 6
1
4
9 341
9 160
100 47
1 12
-7a
-2
aFour cases of Herpes Simplex Virus (HSV) were isolated--one of CoxsackieB6, one of Influenza A, and one of Adenovirus. ~The range of RSV serum titer among culture-positive patients, was 8-256 CF units vs. 6-32 CF units culture-negative patients.
s h o w e d no increase in a n t i b o d y formation in the sera pairs w h e n c o m p a r e d with any of the tested antigens. Table 2 presents data on pharyngeal swabs of the total 341 patients in the s t u d y group. The results illustrate that RSV was isolated by p h a r y n g e a l swabbing in 136 of the total 305 patients admitted to pediatric departments (44%). RSV was successfully isolated in 17 of the 27 patients a d m i t t e d to intensive care w h o survived c o m a and in all nine of the patients who died of coma w h i l e in intensive care. In total, 47% of all patients e x a m i n e d by pharyngeal swabbing (160/341) were found to be RSV-positive. In about 2% of the patients a d m i t t e d to pediatric departments, other viruses were isolated, including three cases of herpes s i m p l e x virus (HSV). One of the patients a d m i t t e d to intensive care w h o also survived coma was HSV-positive. Other viruses were isolated in a total of 7/306 of the patients in pediatric wards. P u l m o n a r y s a m p l e s taken at autopsy were e x a m i n e d in lieu of sera pairs and p h a r y n g e a l swabs for 17 c h i l d r e n w h o died shortly after arrival to the hospital. RSV was isolated in 11 of these samples. It s h o u l d be m e n t i o n e d here that in our region, the occurrence of RSV is very p o s s i b l y c o m p l i c a t e d b y a n u m b e r of a d d i t i o n a l factors besides the usual patient age a n d prevailing weather conditions. These i n c l u d e o v e r c r o w d e d and inadequate housing, i n a d e q u a t e heating, high levels of h u m i d i t y , a generally low socioeconomic level, a n d i n a d e q u a t e nutritional intake (Likar, 1979). The concurrent presence of isolated virus and a significant increase in serum titer was seen in 64 of the patients h o s p i t a l i z e d during our study. The results are shown in Tables 1 a n d 2. A l t h o u g h virological investigations were performed only on hosp i t a l i z e d patients a n d therefore represent a relatively small n u m b e r of cases as comp a r e d to the larger n u m b e r of outpatients, it is our belief that the e x a m i n e d cases represent a significant example, giving evidence that during the winter of 1979 in the m e t r o p o l i t a n area of Naples, there was i n d e e d an e p i d e m i c focus of RSV well p u b l i c i z e d as the " d a r k disease" (Pocchiari, 1979).
Acute Respiratory Syndrome and R S V
85
REFERENCES Chanock R (1977] Viral Infections of Humans, Epidemiology and Control. Ed., AS Evans. New York: Plenum Publishing Co. p. 189. Chanock R, Parrot R (1965) Acute respiratory disease in infancy and childhood; present understanding and prospects for prevention. Pediatr/cs 36:21. Kravetz H, Knight V, Charlock R, Morris J, Johnson K, Rikfind D, Utz J (1961) III: Production of illness and clinical observations in adult volunteers, lAMA 176:657. Likar M (1979) Are outbreaks of RSV infections depending on socio-economic conditions? International Symposium on Biology of the Future, Milan, Italy, May 18-20, Biologi Italiani, pp. 49-55. Martin AJ, Gardner PS, McQuillin J (1978) Epidemiology of respiratory syncytial viral infection among pediatric patients over a six year period in North East England. Lancet 2:1035. McClelland L, Hilleman MR, Haraparian W , Ketler A, Reilly CM, Cornfeld D, Stokes J {1961} Studies of acute respiratory illness caused by respiratory syncytial virus. II. Epidemiology and assessment importance. N Engl J Meal 264:1169. Parrot R, Vargosko A, Kim H, Cumming C, Turner H, Huebner R, Chanock R (1961} Serologic studies over a 34 month period of childhood with bronchiolitis, pneumonia and minor respiratory diseases, lAMA 176:653. Pocchiari F (1979} Report of international group of experts on children's mortality in Naples. Progress Report, Istituto Superiore della Sanita 3:13. Ross CAC, Stott EJ, McMichaelis S, Crowter IA (1964) Problems of laboratory diagnosis of respiratory syncytial virus infection in childhood. Arch fur die Gesamte Virusforschung 14:553. Tarro G, Sabin AB [1970) Virus-specific, labile, nonvirion antigen in herpes simplex virus infected cells.Proc Natl Acad Sci U S A 65:753.
DIAGN MICROBIOL INFECT DIS 1985;3:8145
NOTES
Serological and Virological Investigations of Young Children with Acute Respiratory Syndrome Associated with Respiratory Syncytial Virus Giulio Tarro, Riccardo Smeraglia, and Ciro Esposito From January 1979 to March 1979, 341 young children from the metropolitan area of Naples, Italy, were hospitalized with respiratory virus disease. Diagnosis of patients made from virus isolation and seroconversion indicate that the respiratory syncytial virus was a primary cause of this acute respiratory syndrome.
During the winter of 1979, the most severe in our area in the past 15 years, 341 children, most under 3 years of age, presented to several local metropolitan hospitals with acute infection of the branched bronchial tree. Prior data (Chanock, 1977, Martin et al., 1978) have shown that the clinical symptomatology for respiratory syncytial virus {RSV} infection appears with greatest frequency and severity in early childhood patients (McClelland et al., 1961, Ross et al., 1964). The 2- to 36-month age group appears to be at greatest risk. Severe bronchiole infections resulting in death have been observed in these patients {Chanock and Parrot, 1965; Parrot et al., 1961). RSV infection is also seen in children over 36 months, but the consequences are usually far less severe. Inflammation of upper and middle respiratory tracts, usually not requiring hospitalization, is normally the extent of the problem in this age group (Kravetz et al., 1961}. In our study, the respiratory anamnesis was compatible with the clinical picture of an acute respiratory syndrome (rhinitis, pharyngitis, laryngitis, and the most severe illnesses such as croup, tracheobronchitis, pneumonia, and bronchiolitis}. This outbreak was studied without any need to use special modern techniques and its study might be performed in any peripheral virological laboratory. Several pharyngeal swabs were taken on each patient over a given period of time. These samples were taken and immediately inoculated into cell cultures at the patient's bedside. Seventeen pulmonary samples obtained at autopsy from patients deceased in the first 2 years of life were transported to our laboratories in refrigerated containers 100 ml of Eagle's Basal Medium {BME} + 0.2% NaHCO3 + antibiotics 3 times concentrated: penicillin {200 units/ml}, streptomycin (0.2 mg]ml}, gentamicin
From the Division of Virology, D. Cotugno Hospital, and Oncologic Virology, University of Naples, Naples, Italy. Address reprint requests to: Professor Giulio Tarro, via Posillipo 286, 80123 Naples, Italy. Received August 31, 1983; revised and accepted March 13, 1984. © 1985 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Avenue, New York, NY 10017
0732-8893/85/$03.00
t~
TABLE 1. Seropositivity to RSV and Other Viruses in 189 Young Children Hospitalized with Acute Respiratory Syndrome
Patien ts admitted in: Pediatric department Intensive care Total
High positive antibody titer w i t h no rise in second serum t i t e r for RSV
Meaningful rise i n t i t e r for RSV No. of patients
Tot a l seroconversion and positive a n t i b o d y to RSV
No.
%
CF e
No.
%
CF
No.
%
164
94
58
8-256
37
23
8-256
131
80
25 189
13 a 107 b
52 57
8-256 --
12 49
48 26
16-256 --
25 156
100 83
High positive antibody titer with no rise in second serum titer for o t h e r viruses
Meaningful ri s e i n t i t e r for other viruses
"Four serum pairs were from patients that died of this disease. bin 64 of these cases, RSV was also isolated form pharyngeal swabs. ZOne seroconversion was Coxsackie B6, one was Mycoplasme pneumoniae. ~Three cases of seropositivity were Cytomegalovirus, two were Adenovirus, two were Influenza A, two were one was Coxsackie B6, and one was Parainfluenza 2. eComplement fixation units expressed as reciprocal of serum dilutions; ranges of titers.
No.
%
CF
2c
1
8-16
.
. 2
. 1
. --
Mycoplasma Pneumoniae,
No.
%
CF
12 d
7
16-64
7
--
. 12
.
one was Coxsackie B4,
O
Acute Respiratory Syndrome and RSV
83
(0.05 mg/ml)], homogenized and suspended to 10% of Minimum Eagle's Medium (MEM) with 5% chicken serum and 0,1% NaHCO3. Of each suspension sample, 0.2 ml was inoculated into two test tubes containing HEp-2 cells and two tubes containing human fetal fibroblast monolayers. HEp-2 cells grown in our laboratory using the techniques described by Tarro and Sabin (1970) and frozen in BME with 10% calf serum, 0.1% NaHCO3, and antibiotics, were brought to + 37°C and used. The cell confluent cultures were washed and replaced with BME with 2% chicken serum and 0.2% NaHCO3 and used for sample inoculum. The samples (697 pharyngeal swabs and homogenized suspensions of samples obtained at autopsy) were inoculated into cell cultures at + 37°C. The cell cultures were observed daily to plot the cytopathic effect (CPE) which became evident 2-15 days after inoculation. When CPE was seen in a sample, 3-4 serial passages were taken to ensure that the activity was due to the presence of the virus. For virus identification, the liquid culture containing the scraped infected cells plus 5% chicken serum was frozen at -80°C, thawed, and centrifuged at 1500 rpm for 10 min. The sediment was suspended in 0.2 ml of balanced salt solution (about 10% of cellular suspension) and tested by complement fixation (CF), using four units of antisera (Microbiological Associates, Baltimore, MD). For Control measures, CPE negative cell cultures were also tested using the same technique. Sera from 189 patients exhibiting symptoms of acute respiratory syndrome were studied. For each patient, at least two serum samples (the first one taken immediately upon hospitalization and the second after 15 days) were tested for antibody titration of different viruses. Of these 189 patients, 25 had been initially admitted into intensive care units for coma and 21 had survived. The CF test was performed using the technique described by the Center for Disease Control (Atlanta, GA). Four units of CF antigen, (Microbiological Associates) and two units of complement (Sclavo Laboratories, Siena, Italy), were added to the sera dilutions. Table I data show that, of 164 patients admitted to the pediatric departments, 94 cases (57%) exhibited a significant increase in RSV titer (~>4 fold) between the acute stage and convalescent stage serum samples. Another 37 patients (23%) showed definite positive results for RSV antibody--that is, elevated titers (~>4 CF units) in comparison with other viruses--in both samples, but exhibited no increase in titer concentration in the second serum. The 25 patients admitted to intensive care units showed the following serum results: 13 samples (4 from nonsurvivors) showed a significant increase in RSV titer (~> fourfold) between the serum pairs, and 12 showed a positive RSV antibody titer but no increase in titer in the second serum. The results from the total 189 sera pairs tested showed that 107 exhibited a significant increase of the second serum RSV titer (/> fourfold) over the first serum sample (56%) and 49 pairs showed a high RSV-positive titer in both samples with no measurable differences between acute and convalescent phases (26%). It is possible that the lack of antibody concentration increase was due to the stage of infection: RSV-antibody was already formed when the first sample of the patient was taken, since all the children hospitalized in this study presented the acute phase of viral involvement. Assuming that the above statement is true, it can be seen that 83% (156/189) of the serum pairs studied had a significant RSV-positive antibody titer. In addition, it was found that serum pairs from two of the patients admitted into pediatric wards for viral respiratory syndrome showed a significant increase in titer to other viruses---one patient to Coxsackie B 6 and the other to Mycoplasma pneumoniae. Moreover, twelve other patients exhibited a significant positive antibody titer without titer increase between serum pairs when tested with the following viruses: three patients, cytomegalovirus; two, adenovirus; two, influenza A; two, Mycoplasma pneumoniae; one, coxsackie B 6; one, parainfluanza'2. Nineteen of the patients admitted to pediatric departments,
84
G. Tarro et al.
TABLE 2. F r e q u e n c y of Isolation of RSV and Other Viruses by Pharyngeal Swab of 341 Young Children Hospitalized w i t h Acute Respiratory S y n d r o m e
Patients admitted in: Pediatric departments Intensive care Children who survived coma Nonsurvivors Total from both departments b
Children from whom RSV was isolated
Seronegative for RSV
Children from whom other viruses were isolated
No. of patients
No.
Percent
No.
No.
Percent
305
136
44
11
6
2
27
17
63
not done in 6
1
4
9 341
9 160
100 47
1 12
-7a
-2
aFour cases of Herpes Simplex Virus (HSV) were isolated--one of CoxsackieB6, one of Influenza A, and one of Adenovirus. ~The range of RSV serum titer among culture-positive patients, was 8-256 CF units vs. 6-32 CF units culture-negative patients.
s h o w e d no increase in a n t i b o d y formation in the sera pairs w h e n c o m p a r e d with any of the tested antigens. Table 2 presents data on pharyngeal swabs of the total 341 patients in the s t u d y group. The results illustrate that RSV was isolated by p h a r y n g e a l swabbing in 136 of the total 305 patients admitted to pediatric departments (44%). RSV was successfully isolated in 17 of the 27 patients a d m i t t e d to intensive care w h o survived c o m a and in all nine of the patients who died of coma w h i l e in intensive care. In total, 47% of all patients e x a m i n e d by pharyngeal swabbing (160/341) were found to be RSV-positive. In about 2% of the patients a d m i t t e d to pediatric departments, other viruses were isolated, including three cases of herpes s i m p l e x virus (HSV). One of the patients a d m i t t e d to intensive care w h o also survived coma was HSV-positive. Other viruses were isolated in a total of 7/306 of the patients in pediatric wards. P u l m o n a r y s a m p l e s taken at autopsy were e x a m i n e d in lieu of sera pairs and p h a r y n g e a l swabs for 17 c h i l d r e n w h o died shortly after arrival to the hospital. RSV was isolated in 11 of these samples. It s h o u l d be m e n t i o n e d here that in our region, the occurrence of RSV is very p o s s i b l y c o m p l i c a t e d b y a n u m b e r of a d d i t i o n a l factors besides the usual patient age a n d prevailing weather conditions. These i n c l u d e o v e r c r o w d e d and inadequate housing, i n a d e q u a t e heating, high levels of h u m i d i t y , a generally low socioeconomic level, a n d i n a d e q u a t e nutritional intake (Likar, 1979). The concurrent presence of isolated virus and a significant increase in serum titer was seen in 64 of the patients h o s p i t a l i z e d during our study. The results are shown in Tables 1 a n d 2. A l t h o u g h virological investigations were performed only on hosp i t a l i z e d patients a n d therefore represent a relatively small n u m b e r of cases as comp a r e d to the larger n u m b e r of outpatients, it is our belief that the e x a m i n e d cases represent a significant example, giving evidence that during the winter of 1979 in the m e t r o p o l i t a n area of Naples, there was i n d e e d an e p i d e m i c focus of RSV well p u b l i c i z e d as the " d a r k disease" (Pocchiari, 1979).
Acute Respiratory Syndrome and R S V
85
REFERENCES Chanock R (1977] Viral Infections of Humans, Epidemiology and Control. Ed., AS Evans. New York: Plenum Publishing Co. p. 189. Chanock R, Parrot R (1965) Acute respiratory disease in infancy and childhood; present understanding and prospects for prevention. Pediatr/cs 36:21. Kravetz H, Knight V, Charlock R, Morris J, Johnson K, Rikfind D, Utz J (1961) III: Production of illness and clinical observations in adult volunteers, lAMA 176:657. Likar M (1979) Are outbreaks of RSV infections depending on socio-economic conditions? International Symposium on Biology of the Future, Milan, Italy, May 18-20, Biologi Italiani, pp. 49-55. Martin AJ, Gardner PS, McQuillin J (1978) Epidemiology of respiratory syncytial viral infection among pediatric patients over a six year period in North East England. Lancet 2:1035. McClelland L, Hilleman MR, Haraparian W , Ketler A, Reilly CM, Cornfeld D, Stokes J {1961} Studies of acute respiratory illness caused by respiratory syncytial virus. II. Epidemiology and assessment importance. N Engl J Meal 264:1169. Parrot R, Vargosko A, Kim H, Cumming C, Turner H, Huebner R, Chanock R (1961} Serologic studies over a 34 month period of childhood with bronchiolitis, pneumonia and minor respiratory diseases, lAMA 176:653. Pocchiari F (1979} Report of international group of experts on children's mortality in Naples. Progress Report, Istituto Superiore della Sanita 3:13. Ross CAC, Stott EJ, McMichaelis S, Crowter IA (1964) Problems of laboratory diagnosis of respiratory syncytial virus infection in childhood. Arch fur die Gesamte Virusforschung 14:553. Tarro G, Sabin AB [1970) Virus-specific, labile, nonvirion antigen in herpes simplex virus infected cells.Proc Natl Acad Sci U S A 65:753.