Hsiung, G. D.; Pinheiro, F.; Gabrielson, M. O. The diagnostic laboratory: functions and problems based on three years’ experience 339

The virus diagnostic laboratory Functions and problems based on three years’ experience*

Hsiung, G. D. Pinheiro F°, F. Gabrielson, M. O.

The development of tissue culture techniques during the past 15 years has facilitated the isolation of a variety of viral agents responsible for in man. Today a modern virus laboratory can be of assistance to the clinician in the diagnosis and management of his patient and to the epidemiologist in the prevention and control of disease. Although virus isolation and identification is not a rapid procedure and specific therapy for most viral is still not available, knowledge of the viral agents responsible for various clinical syndromes and their prevalence in a community at a given time can enable the physician to sharpen his diagnostic acumen in differentiating viral and bacteriological illnesses. This will in turn reduce the unnecessary, and sometimes harmful use of antibiotic or chemotherapeutic agents in . In this communication we shall describe the application of newer methods for virus isolation and identification used in the Virus Diagnostic Laboratory of The Yale-New Haven Medical Center and discuss the significance of the results obtained over a three-year period. Correlation of the clinical diagnosis with Laboratory findings and the epidemiological aspects of viral disease in the community will also be considered.

* Publicado originalmente em The Yale Journal of Biology and Medicine, v. 36, n. 1, August, 1963. 340 Memórias do Instituto Evandro Chagas: Virologia

THE

Since the discovery by Enders, Weller and Robbins1 that could be propagated in cultures of non-nervous tissues, cell cultures have been used extensively in the studies of enteroviruses2. New members of this virus family are constantly being recognized. There are approximately 60 types, with many more pending final classification. The major problem with this group has not been virus isolation but identification. As a general routine, identification of enteroviruses is based upon . Suggestions made by several investigators3,4 using anti-serum pools have facilitated the separation procedure of this group of , but neutralization tests in tissue culture are still time-consuming and laborious. Obviously a simple diagnostic tool is urgently needed for the differentiation of these viruses, especially in a clinical laboratory.

Fig.1 - Schematic diagram showing grouping of enteroviruses. Plaque patterns were obtained in rhesus bottle cultures seven to ten days after inoculation. HA = hemagglutination with human O erythrocytes. Hsiung, G. D.; Pinheiro, F.; Gabrielson, M. O. The virus diagnostic laboratory: functions and problems based on three years’ experience 341

Rapid diagnosis of Previous studies5,7 have shown that in addition to the distinctive plaque patterns of various enteroviruses, different groups of these viruses have specific host cell ranges, so that differential cell cultures may be used in grouping them in a manner analogous to the use of selective media in a bacteriology laboratory. Use of these techniques can shorten the time for routine diagnosis. As shown in Figure 1, preliminary grouping of the isolate can be made once a positive (CPE) or virus plaque is observed. Final identification is then established by serological means. Using this scheme, a presumptive diagnosis can usually be made as soon as the agent is detected, usually within five to ten days (Table 1). Final serological identification may still take weeks or months depending upon the properties of the isolates. For example ECHO-22 isolated from patient D.I. proved difficult to identify. Since the virus produced no signs of disease in infant mice but produced small irregular plaques in both Rhesus and patas monkey kidney cultures (agar overlay), it was placed in the ECHO virus group B according to Figure 1. Attempts to subculture the plaque virus into fluid media were without success, since no CPE was observed in either Rhesus or patas monkey or human kidney cells. It has been noted previously7,8 that ECHO-22 and 23 give higher virus titers when measured by the plaque method than by CPE method, but the prototype does produce CPE. Since the new isolate did not produce distinct CPE in any of the culture systems tested, it was not until a neutralization test was carried out by the plaque reduction method that identification was completed. Thus although a presumptive diagnosis of an ECHO virus infection was made within ten days, it required several months to determine the type involved. ECHO-6, 8, and 11 (listed in Table 1) represent single isolations of each virus type during the past three years. Final identification in each case was troublesome, especially when strain 342 Memórias do Instituto Evandro Chagas: Virologia

variation occurred as in the case of ECHO-11, which did not agglutinate human O erythrocytes. This was in contrast to the report of Rosen9 that all 200 strains of ECHO-11 tested yielded high titers of hemagglutinin. Isolation and identification of ECHO-8 virus from a patient with raised questions as to the etiologic significance of the agent, because this disease has commonly been associated with Coxsackie B viruses. Nevertheless, serological study confirmed that the patient was actually infected with ECHO-8 and not with a Coxsackie B virus10.

Fig. 2 – Mixed infection. Bottle at left shows one large poliovirus plaque and numerous small ECHO virus plaques. Bottle at right shows small irregular plaques of ECHO virus type 4. Hsiung, G. D.; Pinheiro, F.; Gabrielson, M. O. The virus diagnostic laboratory: functions and problems based on three years’ experience 343 4 5 Virus isolation ECHO-4 ECHO-22 iden- tification Final 7 25 ECHO-4 Days after specimens received Presump- tive diagnosis clear 5 28 ECHO-8 Fig. 2) Virological studies Virological rregular, rregular, clear 5 9 3 ge, clear 6 10 Polio 1 dium hazy boundaries 7 31B Cox. And small (see Not done hazySmall, irregular, 10 6 270 60 ECHO-11* Rhesus monkey cell Medium hazy 5 23 Cox. B Small, irregular, hazy Small, irregular, – Not done 5 18A9 Cox CPE Tissue cultures systems Tissue Monkey cell Rhesus Patas Hep-2 meningitis – + + clear types: large, Two 10 85 Polio 1 and hospitalized patients illness FUO§ + – – Aseptic meningitisBornholm disease + + – – – –done Not i Small, 10 30 ECHO-6* Clinical diagnosis Hemagglutination positive with human O erythrocytes. † Paralytic. § of unknown origin.

Table 1 – Time required for presumptive and final identification of enteroviruses isolated from 1 – Time Table * J.B. Aseptic D.I. Upper-respiratory – – – L.G. P.J. Poliomyelitis† + + + Lar J.I.aC. D.O.A.‡L.M. FUO§ + + ++ + + Large, Me Z.Fr. S.E. B.J.W.J. Poliomyelitis† Poliomyelitis† + + ++ – + L.R. FUO§ + – – ** Hemagglutination negative with human O erythrocytes. ‡ Dead on arrival. Patient 344 Memórias do Instituto Evandro Chagas: Virologia

-5

0 0 0 10

-4

0 0 ++ 10

+

-3

0 + ++ 10

+

-2

0 ++ 10

-1 ++ ++ 0 ++ 1

.

+ +++ Undl

-5 0 0 0 0 0 0 0 10

-4 0 0 0 0 0 0 0 10

-3 0 10

CPE in cultures after inoculation of stool sample dilutions dilutions sample of stool inoculation after cultures CPE in -2 0 10

-1 ++ 1 Rhesus monkey kidney cell cell kidney monkey Rhesus cells kidney Human

.

ECHO virus from stool sample Undl

. s r

1 0 0 0 0 0 0 0 1 0 0 0 0 2 0 0 0 0 3 ± 0 0 4 ± 0 0 + 5 ++ 6 + 0 0 0 + 6 7 ay noc

afte i D Table 2 – Comparison of sensitivity Rhesus (Rh) and human (Hk) cell cultures in the isolation Table Hsiung, G. D.; Pinheiro, F.; Gabrielson, M. O. The virus diagnostic laboratory: functions and problems based on three years’ experience 345

Numerous factors may influence the isolation of any viral agent; among these, the presence of sufficient amounts of viable virus particles in the specimen to be tested and the use of the most susceptible host system are of great importance. Table 2 illustrates this point in the case of an ECHO virus isolated from a stool specimen. In this instance, human kidney was the more sensitive cell system, and virus isolation would have been delayed several days had the Rhesus monkey kidney (MK) cultures alone been used. On the other hand a positive isolation was obtained in 24 hours in the highly sensitive human kidney cell system. The latter result was obtained even with a minimum amount (10-5) of sample. Mixed As shown in Table 1, mixed infection occurred in case J. B. from whom both Poliovirus type 1 and ECHO-4 virus were isolated (Fig. 2). The presence of two agents was determined by means of the plaque technique which indicated at once that more than one agent was present. Since this patient had received type 1 live Poliovirus vaccine one month prior to his current illness, it was not surprising that a small amount of Poliovirus, i.e. one plaque per ml was recovered from the stool sample. More than 100 plaque-forming units (PFU) of ECHO-4 virus were present in the same specimen, and this agent was interpreted as the most likely cause of the from which the patient suffered. Since an ECHO-4 epidemic was in progress at the time there was epidemiological support for this diagnosis as well. Problems in the isolation of Poliovirus Concurrent with the use of Poliovirus vaccines in recent years the number of isolations of wild Poliovirus in hospitalized patients has decreased. Only four paralytic cases were admitted in 1960,–three due to type 3 and one to type 1. However, isolations of Poliovirus during or after individual vaccination, or during periods in which community- wide programs have been carried out have presented problems. Mixed infections may occur, as mentioned above. It must also be determined 346 Memórias do Instituto Evandro Chagas: Virologia

whether the isolated Poliovirus is the vaccine strain of avirulent nature or a wild virulent strain. Even with extensive studies on each isolated Poliovirus strain, the interpretation in terms of association with clinical disease is often impossible. The isolation of Poliovirus type 3 from case B.F. illustrates some of the difficulties encountered. This six month old child died suddenly after a nonspecific febrile illness of several days duration. Clotted blood, brain, cerebrospinal fluid, spinal cord and colon contents were submitted for virus studies. Among all specimens tested, only one virus plaque, subsequently identified as Poliovirus type 3, appeared in one of the bottle cultures inoculated with the colon contents sample. Subsequently we were notified that the child had been vaccinated with live Poliovirus vaccine type 3 one month prior to death. There was no evidence at autopsy to suggest that the Poliovirus isolated had any relation to the child’s disease. Furthermore, the small quantity of virus recovered, and the history of vaccine ingestion some week’s earlier, indicated that the isolation of Poliovirus was only an incidental finding in this case. However, the sensitivity of the current tissue culture method for the isolation of enterovirus, especially , was well demonstrated. Two seasonal outbreaks of aseptic meningitis The association of ECHO and Coxsackie viruses with aseptic meningitis has been reported frequently in recent years. Among the ECHO viruses, types 4, 6 and 9 have been most frequently associated with epidemics. ECHO-6 and ECHO-9 have been studied in great detail in the New Haven area in association with aseptic meningitis in 1956 11,12 and 1957-58 . In the summers of 1961 and 1962 Coxsackie B4 and ECHO-4 respectively were isolated from patients clinically ill with aseptic meningitis. Figure 3 shows the distribution of the two groups of viruses in the summers of the two consecutive years. In both, the peak of viral activity was in the month of August, as is usual with enterovirus infections. Strains isolated in 1961 were predominantly Coxsackie B4

(30 of 43) with a few Coxsackie B5 (12 of 43). ECHO-4 was not isolated Hsiung, G. D.; Pinheiro, F.; Gabrielson, M. O. The virus diagnostic laboratory: functions and problems based on three years’ experience 347

during 1961 but among the 46 non-polio enteroviruses isolated in the summer of 1962, 38 were ECHO-4 and six were Coxsackie B3

Fig. 3 – Distribution of Coxsackie B and ECHO-4 viruses in New Haven, 1961 and 1962.

Comparison of types of specimens in the isolation of Coxsackie B and ECHO-4 viruses The data in Table 3, in agreement with previous observations, indicate that stool samples are the best source for isolation of enteroviruses, with rectal swabs and throat swabs following, in that order. Cerebrospinal fluid specimens showed a striking difference in per cent positive for ECHO-4 (73.3%) and Coxsackie B (23%), although both viruses were found in association with central nervous system disease. Isolation of Poliovirus and other enteroviruses from the blood, largely during the incubation period, has been reported 13,14 previously. Table 3 indicates that both Coxsackie B4 and ECHO-4 viruses were isolated in the present study from serum specimens collected during the early phase of illness. In one instance the

Coxsackie B4 virus titer in the serum was as high as 730 plaque-forming 348 Memórias do Instituto Evandro Chagas: Virologia

units per ml. In the other three patients only a few virus plaques were detected. In general, virus disappears from the blood as antibody develops; but it is possible that for a short time virus may be present in the blood but cannot be detected because it is bound to antibody. In 15,16 other studies it has been found that Poliovirus and Coxsackie B4 can be dissociated from neutralizing antibody by acid treatment. Attempts to dissociate possible virus-antibody complexes in the blood of patients in the present study were without success. Table 3 – Comparison of types of specimens yielding enteroviruses

Virus isolated Coxsackie B ECHO-4 Total No No Per cent Total No No Per cent Specimens tested Positive Positive tested Positive Positive Blood (serum) 23 2 8.7 28 2* 7.1 CSF 17 4* 23 15 11 73.3 Throat swabs 35 18 52 30 13 43.3 Nasal-pharyngeal 6 2 33 15 3 20.0 swabs Rectal swabs 28 24 86 18 13 72.2 Stool 19 19 100 11 10 91.0 Pericardial fluid 1 1* * Virus obtained by the plaque technique only.

Few Coxsackie B viruses have been isolated from fecal specimens obtained from the sporadic cases of and tested during the course of this study. However, Coxsackie B4 virus was recovered from pericardial fluid aspirated immediately after death of a patient (J.E.) in February 1963. This 13 year old girl entered the hospital in a moribund state and died of acute myocarditis within a few days. Tests on throat and rectal swabs obtained before death failed to yield any viral agent, but a few virus plaques appeared in bottle cultures inoculated with pericardial fluid obtained immediately post mortem. Other specimens including heart muscle, liver, brain, kidney and colon contents obtained at autopsy were all negative. Pathological lesions in the heart muscle, liver, and pancreas were Hsiung, G. D.; Pinheiro, F.; Gabrielson, M. O. The virus diagnostic laboratory: functions and problems based on three years’ experience 349

compatible with a infection. A serum specimen collected before death had an antibody titer of 1:160 against Coxsackie

B4. The failure to detect virus in rectal and throat swabs (ante mortem) and the minimal amount present in pericardial fluid are interpreted as being related to the time in the course of the infection when specimens were collected; the child had been ill for two weeks before admission and already had significant amounts of antibody which would interfere with virus isolation. Antibody responses to enterovirus infection Antibody rises in patients following enterovirus infections can often be of diagnostic value, although not infrequently maximum titers have been reached at the time of admission and no further rise can be detected. As illustrated in Table 4 good responses were shown in the present study in patients with Coxsackie B virus infections. Fourfold or greater antibody rises were demonstrated in sera collected from four to 47 days apart. Studies on paired sera from seven patients with aseptic meningitis admitted to the hospital during this same period but from whom no virus was isolated, showed significant antibody rises in only two cases. However, of the seven patients tested, four exhibited high initial antibody level to Coxsackie B4 suggesting that this agent may have been responsible for their disease. Table 4 – Enterovirus infection and antibody response in patients with aseptic meningitis No showing fourfold or greater Per cent with Total no Virus isolated neutralizing antibody rise to antibody tested homologous virus response

Coxsackie B4 54 80

Coxsackie B5 6 6 100 None 7 6* 85 ECHO-4 13 5 38

st * Four of these patients had high antibody titers to Coxsackie B4 in their 1 serum samples; the other two showed rise to Coxsackie B . 5 Problems were encountered in measuring antibody to ECHO-4 virus. Similar difficulties were reported by Barron and Karzon 350 Memórias do Instituto Evandro Chagas: Virologia

in the study of an ECHO-4 epidemic in Buffalo17. In the present study using the plaque reduction neutralization technique, five pairs of sera were found to have significant antibody rises to ECHO-4 virus, but such rises were not detected when conventional CPE methods in tube cultures were used. The ECHO-4 virus strain used for antibody tests in the present investigation was isolated from the cerebro-spinal fluid of patient W.A. No comparison of its antigenicity was made with that of the DuToit strain of ECHO-4 which seems to be the most satisfactory ECHO-4 strain for serologic tests17. THE RESPIRATORY VIRUS GROUP The known agents commonly involved in respiratory tract infections are the and Parainfluenza groups of Myxoviruses, Adenoviruses, Reoviruses, Respiratory Syncytial viruses, Rhinoviruses and occasionally certain enteroviruses. Conversely a single virus family can produce a variety of syndromes from the to pneumonia.

Fig. 4 – Correlation of respiratory syndromes and virus isolation (total 124 cases) Hsiung, G. D.; Pinheiro, F.; Gabrielson, M. O. The virus diagnostic laboratory: functions and problems based on three years’ experience 351

Correlation of clinical illness and virus isolation During the period from July 1960 to March 1963 specimens received from 2,004 patients yielded a total of 125 virus isolations from individuals with a variety of respiratory syndromes. As shown in Figure 4 the Parainfluenza viruses of all three types were the most frequently isolated agents from cases of pneumonia and croup. Respiratory syncytial (RS) virus, reported to be associated with bronchiolitis by Chanock, et all.,18 was apparently not active in New Haven during the period of the present study. Coxsackie B and ECHO-4 viruses were isolated from cases of pneumonia during the period in which each virus type was active in the community. Association of ECHO-11 and ECHO-22 with respiratory illness has been noted previously by others8,10. Parainfluenza viruses The hemadsorption technique originally described by Vogel and Shelokov20 has greatly facilitated the isolation of the Parainfluenza virus group. However, difficulties have been encountered from time to time with an endogenous agent, SV5, a simian virus commonly found in normal monkey kidney cell cultures21. Since the latter cell system is the most sensitive method for the isolation of the

Parainfluenza viruses, and since SV5 is serologically identical to a human 22 isolate of Parainfluenza virus, the DA virus, an isolation of the DA-SV5 group of viruses from human specimens in monkey cell cultures, requires considerable interpretation. Isolation of Parainfluenza virus type 2 from infants with sudden unexplained death occurred on three occasions during the past three years.23 The significance of this finding and its importance in respiratory illness of infancy requires further elucidation. Outbreak of Parainfluenza type 1 in the winter of 1962-63 In the winter of 1962-63 Parainfluenza virus type 1 was isolated from a total of 29 patients, ranging in age from one month to 43 years. Slightly over half (19/29) of these patients presented with 352 Memórias do Instituto Evandro Chagas: Virologia

illnesses involving the respiratory tract. Croup and bronchiolitis predominated in the group under three years of age; pneumonia, influenza-like syndromes and upper respiratory tract infections in the older children and young adults. The other ten patients from whom isolations were made had non-specific febrile illnesses (4/29), gastrointestinal symptoms (2/29), or other conditions having no obvious relationship to the virus isolated. Antibody responses and serodiagnosis of Parainfluenza virus infection Heterotypic antibody responses in humans following Parainfluenza virus infections have been observed by various investigators. In a previous paper Hsiung, et all.24 reported the interrelationships between the Parainfluenza, DA and viruses and suggested that specific etiologic diagnosis often cannot be made on the basis of serology alone, without virus isolation. Table 5 shows a few examples of homotypic and heterotypic antibody responses in patients infected with Parainfluenza virus type 1. It was noted that there was some antigenic variation in Parainfluenza type 1 virus in the three different years judging by their antibody responses. For example, patients K.B. and O.P. (1961) showed no antibody rises to Para-1 virus isolated in 1960 (NH60), but a greater than fourfold rise was obtained when the 1961 strain (NH61) from patient O.P. was used as antigen. On the other hand, several pairs of sera from patients from whom Para-1 was isolated during the 1962-63 epidemic showed antibody rises to both the current strain (NH62) and the 1960 strain (NH60). However, sera from patients in 1960 (L.E. and J.N.) did not show significant rises to the 1962 strain (NH62) although they did show at least fourfold rises to the 1960 strain (NH60). It would appear that the 1962 strain (NH62) has a broader antigenic spectrum than the strains of the two previous years. Such an antigenic variation of Parainfluenza virus types has not been noted previously. Hsiung, G. D.; Pinheiro, F.; Gabrielson, M. O. The virus diagnostic laboratory: functions and problems based on three years’ experience 353

– – – – 40 40 40 160 160 40 40 20 20 80 80 20 40 80 40 80 40 40 40 DA Mumps 40// 40// –

/ Para-3

Para-2

Para-1 (NH62)

Para-1 (NH61) 160 20 <80 80 160 40 – – – – 40/ 160// – 160// 80/ – 80 320// 640 – – – 20

/ / / / / /

Para-1 / Heterotypic response. response. Heterotypic / response. Homotypic //

11/28/62 11/28/62 – – 40 2/14/63 – – – – – 80

yrs yrs 10/27/60 –* – 80 80 yrs 2 2 / / 1 1 Age Age obtained (NH60) 5 mos 1/13/63 – – – – – 20

* L.E. 4 K.B. 19 yrs 2/15/62 40 M.C. 80 10 mos – 80 1280

serum Date Patient Data 4/12/61 80/ 3/7/62 80 640// 1/4/63 titers antibody HI of Reciprocal – – 80 1280 – 160 3/1/63 160/ 1/20/63 320/ 1961 K.U.* <1:20. titer HI * Novirus ** was isolated. J.N. 2 yrs 10/30/60 – <80 20 20 4/19/61 R.S. >1280/ 21 yrs 12/1/62 20 20 40 1960 1962 O.P. 2 21 yrs D.B. 2/16/62 20 20 R.M. 4 yrs 2/27/62 – 20 12/28/62 640// 40 160 12/18/62 – – 1/8/63 80/ 320/ 40 160 – – 640 Table 5 – Antibody responses following infection with Parainfluenza virus type 1 Table 354 Memórias do Instituto Evandro Chagas: Virologia

Adenoviruses The original isolation of Adenovirus was made from human adenoid tissue in cultures which underwent spontaneous degeneration. Most subsequent isolations have been obtained from patients with respiratory illness. A total of 16 strains of Adenovirus has been isolated in our laboratory during the three-year period 1960-63. Of the 16 isolates, all but two were associated with respiratory illness (Fig. 4). Fourteen strains were recovered from children under three years of age, ten of them from infants three weeks to 12 month old. At present there are 28 known serotypes of human Adenoviruses25. Since all share a common complement-fixing antigen, it is easy to identify an agent as belonging to the Adenovirus group. However, the designation of the type involved presents a considerable exercise in the laboratory. This has been simplified recently by Rosen26 who has reported that these 28 types can be placed into four subgroups on the basis of their agglutinating properties when tested with Rhesus monkey and rat erythrocytes. Adenovirus types 12 and 18 have been reported recently to be capable of producing tumors in new-born hamsters27,28. Since these are the 1st human viruses that have produced tumors in experimental animals, the isolation of Adenoviruses in young infants may take on a new significance in the future. MISCELLANEOUS VIRUS GROUP Herpes simplex This virus has a wide host cell range, growing well in the Hep-2 cell line and in human amnion or kidney cells, but less well in Rhesus monkey cell cultures. However, easy isolation of this virus requires freshly obtained specimens and the use of the most sensitive cell system. In our laboratory human amnion cells have proved to be the most susceptible cell system for (Table 6). As noted earlier, a highly sensitive cell system accelerates isolation and Hsiung, G. D.; Pinheiro, F.; Gabrielson, M. O. The virus diagnostic laboratory: functions and problems based on three years’ experience 355

diagnosis. The chorio-allantoic membrane of embryonated hen’s eggs has also been used for isolation of this virus. The latter method is a helpful and practical one for differentiating between Herpes simplex and viruses of the adenovirus, vaccinia or pox virus group, since the pocks produced by Herpes simplex virus on the chorio-allantoic membrane are quite typical. Measles* Isolation of Measles virus is not easily accomplished, even from optimum specimens, i.e. throat washings and blood collected from the patient either before or within the first 24 hours after the appearance of rash29. Since clinical diagnosis of the disease has a high degree of accuracy, laboratory tests are rarely required to establish the diagnosis, but are often useful to determine susceptibility. Currently, serological tests,** either complement-fixation or hemagglutination-inhibition using monkey erythrocytes, can be used to determine whether an exposed person is immune, since antibodies to Measles virus persist for many years after infection. Mumps As with Measles, attempts to isolate from hospitalized patients have not been generally successful, especially during the late phase of the disease. Serological tests by complement- fixation and hemagglutination-inhibition are available for demonstrating antibody rises during the course of illness. However, since Mumps antibody rise has been observed in patients infected with Parainfluenza viruses24 (see preceding section Parainfluenza), the practice of diagnosing Mumps on the basis of antibody rise alone might be questioned.

* The authors are grateful to dr. F. L. Black, department of Epidemiology and Public Health, Yale University School of Medicine, for determination of measles antibody. **The authors are indebted to dr. J. R. Henderson, department of Epidemiology and Public Health, Yale University School of Medicine, for the virus isolation and serological studies of this case. 356 Memórias do Instituto Evandro Chagas: Virologia

+ -4 ++ 10 ++

+ -3 ++ 10 ++

+ + -2 10 ++ ++

+ -1 10 +++ +++

l. +

undi

-4 0 +++ 0 +++ 0 + 10

-3 0 0 0 10

+ -2 + ++ 10

+

-1 Hep-2 cells cells amnion Human ++ 10 +++

l. + ++ + +++ +++ undi

4 s

- 0 0 0 0 0 0 0 10

-3 0 0 0 0 0 0 0 10

2

- 0 0 0 0 0 0 0 10

1

- 10 l.

Virus Virus dilution undi 0 0 0 0 Inoculated 0 0 0 0 0 0 0 0 0 0 0 4 6 9 1 3 7 0 e cell kidney monkey Rhesus in cultures CPE Dat 9/1 9/1 9/2 9/1 9/3 10/4 9/2 9/2 Table 6 - Isolation of a strain Herpes simplex virus in diferent cell cultures Table Hsiung, G. D.; Pinheiro, F.; Gabrielson, M. O. The virus diagnostic laboratory: functions and problems based on three years’ experience 357

Fig. 5 – Virus types isolated by months, 1960-1961, 1961-1962, and 1962-1963. 358 Memórias do Instituto Evandro Chagas: Virologia

Arthropod-borne viruses* Viruses belonging to this group have not been isolated from naturally-occurring human infections in Connecticut during the past few years. However, West Nile virus was recovered from one patient who acquired a laboratory infection in May, 1961*. Clinically this patient showed classical symptoms of West Nile fever, and the virus was isolated from the blood 24 hours after onset. Subsequently antibody tests confirmed that the patient had been infected with the West Nile virus. EPIDEMIOLOGICAL ASPECTS OF VIRAL INFECTION The foregoing discussion has been concerned with individual virus groups and their isolation from patients. However, data obtained from these studies give some indication of seasonal trends in certain virus infections. The cases studied were hospitalized ones i.e., those most severely ill, but it is probable that many milder infections were prevalent among individuals in the community at the same time. All samples were treated in the same manner, regardless of season, but isolations of virus types were distinctly different, as shown in Fig. 5 and 6. Myxovirus isolations were at their peak in the winter, enterovirus isolations in the summer. Furthermore there was a marked variation from year to year in the prevalence of virus types, not necessarily reflected by the types of clinical illnesses. For example aseptic meningitis was the predominant syndrome seen in both the summers of 1961 and 1962, but virus isolations indicated that two different groups of enteroviruses (Coxsackie and ECHO) were involved. Of the Myxoviruses, Parainfluenza virus type 1 was active in the New Haven area in the winters of 1960-61 and 1962-63, but only four isolations of this virus type were made in the winter of 1961-62. Remarkably similar results were obtained by Canadian investigators30, 31 during

* The authors are indebted to dr. J. R. Henderson, Department of Epidemiology and Public Health, Yale University School of Medicine, for the virus isolation and serological studies of this case. Hsiung, G. D.; Pinheiro, F.; Gabrielson, M. O. The virus diagnostic laboratory: functions and problems based on three years’ experience 359

the same time period. Between November 1960, and April 1961 Parainfluenza virus type 1 was isolated from 73 of 155 children in Toronto. Predominance of this virus in the Washington, D. C. area has also been observed in the winter of 1962-6332 The distribution of the Parainfluenza virus types in different geographical areas in different years may thus follow a specific pattern similar to that of the Influenza viruses.

Fig. 6 - Seasonal incidence of enterovirus and myxovirus infections in hospitalized patients, July 1960 to March 1963.

COMMENT A modern virus diagnostic laboratory can be a valuable asset to clinicians, pathologists, epidemiologists and virologists. Facilities can be made available for virus isolation, serological studies and observations on the over-all patterns of virus activity in a community. If a virus diagnostic laboratory is to provide comprehensive service, however, it must be of a reasonable size and have adequate equipment and well trained personnel to keep pace with the increasing diversity of techniques available. 360 Memórias do Instituto Evandro Chagas: Virologia

Once a virus isolation has been made a number of problems may still arise. Endogenous viral agents present in many of the primary animal cell culture systems used must be distinguished from pathogenic agents in the patients’ specimens. Difficulties may be encountered in the recognition of new agents and the identification of known agents which may vary in their behavior from the prototype strain. Finally the relationship between the virus isolated and the patient’s disease requires careful interpretation. REFERENCES 1. Enders, J. F., Weller, T. H., and Robbins, F. C.: Cultivation of Lansing Strain of poliomyelitis virus in cultures of various human embryonic tissues. Science, 1949,109, 85.

2. Committee on the Enteroviruses, National Foundation for Infantile Paralysis: The enteroviruses. Amer. J. publ. Hlth, 1957, 47, 1556.

3. Lim, K. A. and Benyesh-Melnick, M.: Typing of viruses by combinations of antiserum pools. Application to typing of enteroviruses (Coxsackie and ECHO). J. Immunol., 1960, 84, 309.

4. Schmidt, N. J., Guenther, R. W., and Lennette, E. H.: Typing of ECHO virus isolates by immune serum pools. The “intersecting serum scheme.” J. Immunol., 1961, 87, 623.

5. Hsiung, G. D. and Melnick, J. L.: Morphologic characteristics of plaques produced on monkey kidney monolayer cultures by enteric viruses (poliomyelitis, Coxsackie, and ECHO groups). J. Immunol., 1957, 78, 128.

6. Hsiung, G. D. and Melnick, J. L.: Comparative susceptibility of kidney cells from different monkey species to enteric viruses (poliomyelitis, Coxsackie, and ECHO groups). J. Immunol., 1957, 78, 137.

7. Hsiung, G. D.: Further studies on characterization and grouping of ECHO viruses. Ann. N. Y. Acad. Sci., 1962, 101, 413. Hsiung, G. D.; Pinheiro, F.; Gabrielson, M. O. The virus diagnostic laboratory: functions and problems based on three years’ experience 361

8. Wigand, R. and Sabin, A. B.: Properties of ECHO types 22, 23 and 24 viruses. Arch. ges. Virusforsch., 1961, 11, 224.

9. Rosen, L.: Hemagglutination and complement-fixation. In, Perspectives in Pediatric Virology. Columbus, O., Ross Conference on Pediatric Research, 1959, 53.

10. Kantor, F. S. and Hsiung, G. D.: Pleurodynia associated with ECHO virus type 8. New Engl. J. Med., 1962, 266, 661.

11. Davis, D. C. and Melnick, J. L.: Association of ECHO virus type 6 with aseptic meningitis. Proc. Soc. exp. Biol. (N. Y.), 1956, 92, 839.

12. Dubin, L. and Horstmann, D. M.: The epidemiology of aseptic meningitis and related non-specific disease in Connecticut, 1957. Virological and clinical studies. Yale J. Biol. Med., 1958, 30, 429.

13. Horstmann, D. M., McCollum, R. W., and Mascola, A. D.: Viremia in human poliomyelitis. J. exp. Med., 1954, 99, 355.

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ACKNOWLEDGEMENT The authors wish to express their appreciation to dr. E.M. Wakeman, resident in Pediatrics, to the other members of the resident staffs of the departments of Pediatrics and Internal Medicine, and to dr. John S. Hathaway, director, Department of University Health, for their collaboration in collecting specimens and for their constant interest. We also wish to acknowledge the excellent technical assistance of mrs. Grace Tucker, mrs. Maria Zweng, mr. John Gennari and mrs. Maria Baisier. 364 Memórias do Instituto Evandro Chagas: Virologia