(SANITIZED)THREE PAPERS ON BIOLOGICAL STANDARDIZATION(SANITIZED)

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• Collection: General CIA Records
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• Publication Date: October 29, 1963
• Content Type: REPORT

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COUNTRY
USSR/Czechoslovakia/Yugoslavia
REPORT
SUBJECT
Three Papers
Biological Standardization
on
DATE DISTR.
29 Oct. 62
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REFERENCES
DATE OF
INFO.
PLACE &
DATE ACQ
HIS Is UNEVALUATED INFORMATION
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three papers
on
Biological Standardization
Titles and authors of the papers are as follows:
a) "The Ilse of Primary Human Amnion Cell Cultures for the
_Diagnosis of Poliovirus Infection" -M am, J Vesen'ak-
Elden, S Ralcic, D Blatnik, MMatjasie, S Smerdel, E Soos,
and B212.1; ;Institute of Health, Department of Epidemiology,
Virus Laboratory, Ljubljana, Yugoslavia, and School of Public
Health "A Stamper", Medical Faculty, Zagreb, Yugoslavia.
"Utilization of a New Diploid Cell Strain Derived from Human
Embryo Lung Tissue for the Cultivation of Enteroviruses and
Measles-Virus" - VI Hozinski, VB Seybil, LB Cypkin, NS
Panteleevn, and CM Mazurova; Institute of Poliauyelitis and
Virus Encephalitis, Academy of Medical Sciences, USSR, Moscow.
c) "Cultivation of Vaccinia Virus in Human Diploid Cell Strains"
J Trlifaiova, V Strizova, and F Stastny; Institute of Epidemi-
cology and Microbiology end Institute of Sera and Vaccines,
Praha, Czechoslovakia...... UNCLASSIFIED.
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From the National Institute of Health, Dept. of Epidemiology,
Virus LaboratOryT Ljubljana, Yugoslavia, and the School of
Public Health "A, 6tampar", Medical Faculty, Zagreb, Yugoslavia
THE USE OF PRIMARY HUMAN AMNION CELL CULTURES FOR THE DIAGNOSIS
OF POLIOVIRUS INFECTION
M, JUNG, JELKA VESENJAK-HIRJAN, S. KALoIe, D. BLATNIKt
ZDENKA HEIGL, M. MATJAIO, S. SMERDEL, E. 600, and B. SNOJ
This study was supported by grants obtained from the
Boris Kidri6 Foundation, Ljubljana, and the Institute of
Social Insurance, Ljubljana, Yugoslavia.
The Slovenian Foundation for Poliomyelitis in Ljubljana
provided financial support for collection and laboratory
investigation of specimens from healthy children,
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INTRODUCTION
Multiplication of the three types of poliovirus in
primary human amnion (PHA) cell cultures has been reported
(Zitcer, Fogh and Dunnebackel 1955). It has been also
demonstrated, that PHA cell cultures were sensitive for
cytopathic action of Adenovirus types 1-8, Coxsackie B
viruses, herpes simplex, vaccinia, and measles virus
(Wilt, Stanfield and Leindl, 1956; Takemoto and Lerner,
1957; Milovanovie, Enders and Mirus, 1957). These cultures
have been also used for virus isolation attempts. ComparU-
tive infectivity titrations in monkey kidney, PHA, and HeLa
cell cultures (Lehmann-Grube, 1961) have shown that PHA cell
cultures supported growth of ECHO virus types 1, 2, 3, 5-130
15, 19, 20, 21 and 24, and of types 4, 14, 16, 17, 18, 22
and 23 in. a variable degree, It is of considerable interest,
that even types 9, 11, 13, 15 and 18 of Coxsackie A group of
viruses could be propagated in these cells with satisfactory
infectivity titers, Other types of Coxsackie A viruses could
be also cultivated after adaptation (Krech, 1961; Lenahan
and Wenner, 1961),
The communications, which have been cited here, provide
informations on the usefulness of PHA cell cultures for the
propagation of many viral agents causing cytopathic effects
in tissue culture. There is, however, little evidence on
the large scale use of these cells for primary isolation
of viruses from clinical specimens. In this communication,
we shall present the results of the use of PHA cells for
the diagnosis of poliovirus infection. In addition, succes-
full primary isolations of viruses other than poliovirus
will be reported.
MATERIALS AND METHODS
Pathological specimens were obtained from clinical
patients in Slovenia during 1958-1961. Materials were also
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collected from patients in Croatia, where poliomyelitis
occured in epidemic form in the summer of 1960; during the
year 563 cases were reported. In 1961 a vaccination campaigne
with Sabin poliovirus vaccine was initiated in Slovenia,
while in Croatia the Koprowski type vaccine was employed;
in Slovenia the Salk type poliovaccine was also used on a
large scale since 1957.
Only a few cases of poliomyelitis-like diseases were
reported during 1961-1962 in both countries, but the number
of cases with aseptic meningitis remained practically
unchanged; a part of these patients was also investigated,
and the results included in this report. The participating
Virus laboratories were at the National Institute of Health
in Ljubljana, Slovenia, and the School of Public Health "A.
kampar" of the Medical Faculty in Zagreb, Croatia. Laboratory
methods of testing pathological specimens were similar in both
laboratories. In many instances two or more faecal samples
were examined in each od the cases under study except during
1960 epidemic, when it was apparent that this would
overburden available facilities.
Virus isolations were accomplished in PHA cell cultures,
and in HeLa and Detroit-6 cells in some cases. Tissue cultures
were prepared according to the methods previously described
(Jung, Strah, Blatnik and Vozelj, 1959; Jung, Blatnik, Strauch,
MatjagiC, Tovornik, Kegu, Drnov6ek, Kralj and Zadnik, 1960).
Test tube cultures were examined daily for 10-14 days for
evidence of cytopathogenicity. Two blind passages were always
made with specimens from clinical patients; no blind passages
were performed with rectal swabs obtained from healthy children.
The cytopathogenic agents, which were isolated in tissue
culture were preliminary grouped by testing the tissue
affinity and pathogenicity for suckling mice, as shown in
Table 1. Final identification was made in neutralization test
with 100 TCID50 of the virus, in 0,1 ml, against a limited
number of Polio - ECHO - and Coxsackie antisera pools which
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were available; they were diluted as to contain at least 20
units of each antibody per 0,1 ml, Neutralization tests were
incubated for 3 hours at 37C, and overnight in the cold-room.
The virus-serum mixtures, in 0,2 ml amounts were then inoculated
into each of two PHA cell cultures, If neutralization of
cytopathogenicity occured, the virus was tested against
components of the pool. The poliovirus typing antisera were
obtained from Parke, Davis and Co., the Swiss Serumand Vaccine
Institute, and from dr. U, Krech, Hilterfingen/Thun. The ECHO
antisera types 1, 2, 3, 4, 5, 6, 7, 8, Coxsackie A 9 and 231
and Coxsackie B 1, 2, 3, 4 and 5 were obtained from dr. U. Krech.
In 1962, ECHO antisera for types 16 and 18 were also available.
Adenoviruses were included into the group by testing the
tissue culture supernate for the presence of complement-
fixing antigen against a known antiserum. No type identifica-
tion was made.
For virus titrations serial tenfold dilutions were
prepared in the maintenance medium, and 0,1 ml inoculated into
each of 2, 3, 4, or 6 PHA cell cultures, according to the
experiment, Cell culture tubes for determining the reproductive
capacity of polioviruses at different temperatures were kept in
specially constructed water baths, maintained at 36?C and 400C;
internal Beckmann type thermometers were used to check the
temperature, which varied by 0,01500. Infectivity titers
were calculated according to the Karber method (Kgrber, 1931).
Complement-fixation with non-inactivated poliovirus
antigens was done with sera from majority of patients;
neutralization tests were also performed in many cases.
RESULTS
Results of enterovirus isolations in Slovenia, during
1958-1959, from patients with paralytic poliomyelitis and
aseptic meningitis in PHA, HeLa and Detroit-6 cell cultures
are shown in Table 2. No significant difference was observed
in various tissue culture systems, as to poliovirus isolation,
although four strains of unidentified enteroviruses could be
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detected only in PHA cells. During this period, 39 patients
were reported to have paralytic poliomyelitis.
76 cases of paralytic poliomyelitis occured in Slovenia
during 1960-1961; materials from 61 patients were tested in the
Virus laboratory. Seasonal incidence and vaccination history of
these patients are presented in Table 3. Results .of poliovirus
isolation, in various age groups, and of immunologic typing of
polioviruses are evident from Tables 4 and 5. In. total, clinical
diagnosis of paralytic poliomyelitis could be confirmed by virus
isolation, in 88,5 per cent of cases. It is of considerable
interest, that 40,7 per cent of total poliovirus isolates were
immunologically related to the type 3; this percentage was even
higher in the older children and adults.
During the same period rectal swabs from 1493 healthy
children were collected, and tested in _PHA cell cultures for
the presence of cytopathogenic viruses. Poliovirus strains
isolated from this study group, as compared with total number
of enterovirus isolates are shown in Table 6. It is evident,
that the incidence of poliovirus infection in healthy children
was significantly higher in November-December 1960, and February
1961. This is in good egreement with seasonal incidence of
paralytic poliomyelitis cases, as presented in Table 3.
Results of virus isolations from healthy children, and
clinical patients with paralytic poliomyelitis and aseptic
meningitis in Slovenia, during 1960-1961, are evident from
Table 7; materials from only a part of aseptic meningitis cases
were examined. As shown in the table, 54 strains of polioviruses
could be isolated in PHA cell cultures from patients with
paralytic poliomyelitis, and 2 strains from aseptic meningitis
cases". In contrast, 7 non,.-poliovirus strains were isolated from
non-paralytic patients. These results co-relate well with
poliovirus isolations from healthy children, since only types
1 and 3 could be identified by immunologic typing in. both study
groups, except a single strain of type 2 poliovirus. It is also
evident, that 95 strains, cytopathogenic for primary cultures,
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were isolated; most of these strains probably belong to the
ECHO virus group, since they were non-pathogenic for suckling
mice, did not react with Polio and Coxsackie B antisera, and
could not be grouped as adenoviruses.
As it was mentionned before, the Republic of Croatia
experienced its largest epidemic of poliomyelitis in the
history with 563 cases reported during 1960. This epidemic
was virologically investigated, and some of the results
published elsewhere (Jung, Vesenjak-Hirjaa, Lulio, Matja516,
Blatnik, Spalatin and Fryda-Kaurimsky, 1961). In the first
group, materials from 64 patients were tested serologically,
and in tissue culture. Results of virus isolations in. PHA cells
are presented in Table 8. Cytopathogenic viruses were isolated
from faecal material or CNS tissue in 47 out of 60 patients
with paralytic forms of the disease, and from 3 out of 4
non-paralytic patients. The type 1 poliovirus was found in. 36
cases. The sensitivity of PHA cell cultures, as evaluated by
the time of appearance of cytopathogenic effect,. is evident
from Table 9; 93 per cent of polioviruses were detected within
the first 10 days of incubation at 37?C.
Stools from a second group of 218 patients were tested
some months later in PHA and Detroit-6 cell cultures, and 122
strains of cytopathic agents were isolated of which 62 strains
in PHA cells, out of 76 specimens, and 60 strains in Detroit-6
cultures, out of 142 specimens. Immunologic typing with
poliovirus antisera showed 58 strains to be type 1, 3 type 2,
and 8 strains to be type poliovirus.
Comparative infectivity titrations of poliovirus strains
in various tissue culture systems have also been performed.
Table 10 presents infectivity titers of some standard attenua-
ted and virulent strains of polioviruses in PHA and monkey
kidney cells, incubated at 36oC and 40oC, and in HeLa cells,
incubated at 36?C. Comparable results were obtained with PHA
and monkey kidney cells, although results with HeLa cells were
less satisfactory. Tables 11 and 12 show infectivity titers
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of type 1 poliovirus strains in various tissue culture systems
incubated at 36?C and 40?C. It is evident, that PHA and monkey
lsidney cells gave comparable results. In HeLa cells the infec-
tivity titers were less satisfactoty, and in some cases they
were completely resistant to cytopathic effects of polioviruses.
A small number of type 3 polioviruses were also tested, and the
results are given in Table 13. The reproductive ,capacity of type
3 strains was not significantly reduced at 40oC. It must be
noted, however, that most of the strains were isolated very late
after the poliovaccine was fed,
The effect of temperature 40?C was examined in some cases,
by additional incubation at 36?C for 72 hours. As evident from
table 14, with majority of strains the infectivity titers were.
identical or the difference was minimal, not exceeding 1,0 log.
As shown previously, cytopathic agents, other than polio-
virus, could be isolated in PHA cell cultures. Infectivity
titers of some ECHO viruses are presented in. Table 15.
Satisfactory titers were obtained with virus types 1(8), 6, 9,
and 16. In Table 16 the results of primary non-polio enterovirus
isolations, from patients with aseptic meningitis in Croatia
and Slovenia, during 1962, have been. summarized. In total, 52
strains were isolated, of which 6 ECHO viruses out of 26 strains
which were identified. It is of considerable importance0-that
no polioviruses were found in both countries during 1962,
DISCUSSION
Freshly explanted human amnion cultures consist of normal
cells with a basic diploid DNA content, and with a relatively,
slow mitotic process (Leuchtenberger, Boyer and Strain, 1959).
The abundance and availability of placental membranes enables
a large scale production of these cultures. On the basis of
trypsinization of over 600 membranes, it can be stated, that
sucessfully grown cultures are free of spontaneously occuring
cytopathic viruses; this fact is of obvious importance when
cultures are used for primary virus isolations. The PHA
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cells can be easily maintained for several weeks with relatively
infrequent changes of the nutritient medium. The cytopathic
effects of viruses can be easily Observed under low power
magnification of the microscope. Details of cytopathology in.
PHA cells have been reported elsewhere (Jung, Strauch, Blatnik,
Kresnik, Tovornik, eleznik and Matja6i6; 1960; Kresnik and
Jung, 1961).
As a results of investigation of 197 patients with paralytic
-poliomyelitis in. Slovenia and Croatia, it can be concluded that
PHA cell cultures represent a reliable tool for the diagnosis
of poliovirus infection. In addition, Simultaneous virus
isolation, attempts were made in PHA, HeLa, and Detroit-6 cells.
In. Slovenia, during 1960-1961, the clinical diagnosis was
confirmed by poliovirUs isolation. in. 88,5 per cent of cases
under study, ranging from 76,9 per cent in adults, up to 95,0
Der cent in children aged 0 ? 4. During the 1960 epidemic in
Croatia, viruses were isolated in. PHA cells in. 47 out of 60,and
in. 62 out of 76 materials, from paralytic poliomyelitis patients,
thgt is in. 78 and 81 per cent, respectively.. These results have
been in accordance with previous experience. Virological studies
of epidemic poliomyelitis, during 1956, in .Chicago and Cook
rlounty were made with materials from 664 patients, which were
tested for virus isolation in monkey kidney cell cultures; in
total, Viruses were found in 71 per cent of cases, of which 65
per cent were polioviruses (Nathanson, Thrupp, Hall, Langmuir,
Cornell, Forester, Church, Hall, Hildebrand, Shaughnessy and
Morrissey, 1959). Enterovirus isolations, including Polio, ECHO,
and Coxsackie viruses, which were performed in the same tissue
culture system during 1958 epidemic in Detroit, were positive in
73 per cent of paralytic poliomyelitis patients (Brown, Lent
and Agate, 1960). Virus isolations in, monkey kidney cell
cultures during 1959 poliomyelitis epidemic in, Des Moines and
Polk County, Iowa, gave positive results for polioviruses in, 56
out of 67 paralytic patients (83 per cent) from which stools
were available for laboratory investigation.; all the isolates
belonged to the type 1 poliovirus. The recovery of enteroviruses
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from stools of non-paralytic patients was 34,4 per cent; in
this group Coxsackie B 2, ECHO 7, and unidentified cytopatho-
genic agents were _also found (Chin, Marine, Hall, Gravelle
and Speers, 1961),
These results co-relate well with our experience altho h
PHA cell cultures were used through the study.
Comparative infectivity titrations with virulent and
attenuated poliovirus strains were performed in PHA, monkey
kidney, and HeLa cells. PHA and monkey kidney cell systems were
equally susceptible to the virus strains under study, while
HeLa Cells gave results, which were less satisfactory. PHA
cells could also be sucessfully used for_performing infectivity
titrations in cultures incubated at 400C,
PHA cells were used for primary isolations of polioviruses
(Lahelle, 1957; Chadwick, Welsh and Lennette, 1959), ECHO
viruses type 6 and 9 (Lahelle, 1957a; Krech and Wulff, 1957;
McLean and Melnick, 1957)? adenoviruses (Takemoto and Lerner,
1957), and measles virus (Ruckle and Rogers, 1957), In our
experience, the following viruses were isolated from patients
with paralytic poliomyelitis and aseptic meningitis : poliovirus
types 1-3, Coxsackie B virus types 1, 3, and 5, Coxsackie A 9
and 23 (ECHO 9), and some unidentified group A strains, and. _
ECHO virus types 1(8), 2, 3, 6, 7, 9 (Coxsackie A23), and 16.
It is also very likely, that many of the viruses, which were not
typed and/or identified, belong to ECHO viruses, since only a
limited number of ECHO antisera types were available to us. This
is especially the case with 95 strains of cytopathogenic agents,
presented in. table 7, which were isolated from healthy children,
and which were cytopathic for PHA cultures only. In previous
experience, adenoviruses, herpes simplex, and vaccinia viruses
were also isolated in this cell system (Jung and Matja6i6, 1962),
SUMMARY
The sensitivity of PHA cell cultures for primary isolation
of polioviruses has been investigated with materials of over
200 patients with paralytic poliomyelitis. The clinical
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diagnosis could be confirmed by virus isolation in Slovenia,
during 1960-1961, in 88,5 per cent of cases, ranging from 76,9
per cent in adults, up to 95,0 per cent in children aged 0 -4,
Investigation of materials from the 1960 poliomyelitis epidemic
in Croatia, performed by two laboratories, gave positive
. results in 78 and 81 per cent of cases under study, respectively.
In Slovenia, only paralytic cases were found in paralytic cases,
while in Croatia other enteroviruses could also be demonstrated.
Results of comparative virus isolation attempts in PHA, HeLa and
Detroit-6 cells were similar, as to poliovirus isolation. t PHA
cells were more sensitive for primary isolation of non-polio
enteroviruses, Infectivity titrations of virulent and attenuated
polioviruses in PHA and monkey kidney cells gave equally good
results, while HeLa cells were less satisfactory.
PHA cell cultures were susceptible for primary isolation
of Coxsackie B virus types 1, 3, and 5, Coxsackie A 9 and 23
(ECHO 9), and some unidentified group A strains, and ECHO virus
types 1(8), 2,3, 6, 7, 9 (Coxsackie A 23), and 16. More than a
hundred of non-polio enteroviruses, of which most detected in
healthy children, were not identified. In previous experience
of this laboratory adenoviruses, herpes simplex, and vaccinia
viruses could also be isolated in these cells,
substitute
,
PHA cells seem to be a useful and economical . ?
for monkey kidney cells, since successful primary isolations of
many cytopathic enteroviruses could be obtained.
REFERENCES
1./ Brown,G.C.,Lenz,W.R.land Agate,G.H.: J.A.M.A.,1960472,807.-
2./ Chadwick,D.L.,Welsh,H.H.,and Lennette,E.H.: J. Lab. Clin.
Med.,1959,54,409.-
3./ Chin,T.D.Y.,Marine,W,M.,Hall,E.C.,and Gravelle,C.R.,and
Speers?J.F.: Amer. J. Hyg.?1961,74,67.-
4./ Jung,M.,Strah,M.,Blatnik,D.land Vozelj,M.: Zdrav. vestnik,
1959,28,45.-
5./ Jung,M,?B1atnik,D.,Strauch,L.,Matja6i6,M?Tovornik,D.,Kegu,
M.?Drnov5ek,V.,Kralj,Z.,and Zadnik,V.: Zdrav.vestnik,29,205.-.
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6./ Jung,M?Strauch,L.,Blatni4D.,Kresnik0V.,Tovornik,D?
Zeleznik,Z.,and Matja6i6,M.: Proc. Int. Symp, Microbiol,
Stand.1, Opatija, Yugoslavia, 19602.p. 297.-
7./ Jung,M.,Vesenjak-Hirjan,J.,Lulio,V.,MatjagiCtM.,Blatnik,D.,
_ Spalatin,J.0and Fyda-Kaurimsky,2.: Lij. vjes?1961,..81,587,-
8./ Jung, M., ,and Matja6i6,M.: Unpublished informations.-
9./ Kgrber,G.: Arch. ges. Path. Pharm.,1931,162,480.-
10./ Krech,U.: Personal communication,1961.-
11.1 Krech,U,land Wulff,H.: Schweiz. Zschr. allg. Pathol.,1957,
20,651.-
12./ Kresnik,V.,and Jung,M.:-Acta med._Iugosl.,1961,12,446.-
13,1 Lahelle,O.: Acta pathol.microbiol.Scand.,1957,40,436 -
14,/ Lahelle,O.: J. Hyg.,1957a155,475.-
15,/ Lehmann-Grube,F.: Arch. ges. Virusforsch.,1961,11,276.-.
16./ Lenahan,M.F.land Wenner,H.A.: Proc, Soo, Exp. Biol, Med.,
- 1961,107,511.-
17./ Leuchtenberger,C.,Boyar,G,C.,and Strain J.J.: Ann. N.Y.
Acad. Sci.T1959,81,73.- _
18./ McLean,D.M.pand Melnick,J.L.: Proc, Soc. Exp. Biol. Med.,
- 1959,2?,656.-
19./ MilovanoviC,M.V.,Enders,J.F.,and Mitus,A.: Proc. Soc. Exp.
- Biol. 1Ied.,1957,95,120?-
20./ Nathanson,N.,Thrupp,L.D.,Hall,WM.J.,LangmuirIA.D.,Cornell,R.
G,,Forester,H.E.,Church,R.E.,Hall,J.B.,Hildebrand,M.,Shaugh-
nessy,H.J.,and MorrisseyrR.: Amer. J. Hyg.,1959,70,107,-
21./ Ruckle,G,,and Rogers,K.D.: J. Immunol.,1957,78,341.,
22./ Takemoto,K.K.,and Lerner,A.M.: Proc. Soc. Exp. Biol. Med.,
? 1957,94,179.-
23./ Wilt,J.C.,Stanfield,F.J.,and Leidl,L Canad. J. Pub. Health,
? 1956,47,433.-
24./ ZitcerE.L,Pogh,L, and DunnebackeT.H.? Science,1955,
122,30.-
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TABLE 1. PRELIMINARY GROUPING OF VIRUS ISOLATES BY SEARS OF TISSUE
AFFINITY AND SUCKLING-MICE PATHOGENICITY TESTS
Viruses
Cytopathogenic effect in cultures of
PHA Bele ?) . Embryonic
pig kidney
Pathogenicity
for suckling-
mice
.
Polioviruaes
+
4.
.
0
0 (a)
Comackie B
.
+
+
Coxaackie A
0 (%)
0
0
+
ECHO viruses
+ (?)
0
0
.0
Adenoviruses
+
+
0 (")
0
(?) HeLa cells strain Zurich. The cell strain has undergone 160
passages in this laboratory during the past five years.
(a) Some type 2 strains may be pathogenic.
(%) Types 9 and 23 are positive. Strains of types 11, 13, 15 and 18
may also be cytopathogenio.
(?) Some types could not be propagated in these cells.
(") Some types may cause cytopathogenic changes.
TABLE 2. RESULTS OF ENTEROVIRUS ISOLATION IN SLOVENIA DURING 1958 - 1959
FROM PATIENTS WITH PARALYTIC POLIOMYELITIS AND ASEPTIC
MENINGITIS IN VARIOUS TISSUE CULTURE SYSTEMS
Virus ',trains
PARALYTIC POLIOMYELITIS
ASEPTIC MENINGITIS
PHA (?)
Bela
Detroit-6
PHA
HeLa
Detroit-6
Poliovirue type 1
17
16
16
1
1
1
- type 2
type 3
6
6
6
1
1
11
1
Unidentified (A)
2
2
TOTAL
26
23
23
4
1
2
(?) Primary human amnion cell cultures.
(8) Cytopathic activity of the V*12110 strains could not be neutralized by
immune antisera pools against Polio 1-3, ECHO 1-14, Coxsackie A 9 and
23, Coxsackie B 1-5, and Adeno 1-7.
TAELE 3. SEASONAL INCIDENCE AND VACCINATION HISTORY OP PARALYTIC
POLIONYELITIS PATIENTS 16 SLOVERIA DURING 1960 - 1961
Date
Ha. of
caeca
reported
VACCIKATHIL(?)
NON -VACC
RATED 1
1,657-07-116-;
ca.?
reported
of
0.a
confirmed
ITEr.-Ff--
cane,
reported
-No. of
ease,
confirmed
P 11 v1ruaeo
1 2
3 Total
1960/1
2
3
4
5
6
7
0
9
10
11
12
1961/1
2
3
;
6-9
1
2
2
4
2
14
17
16
6
4
3
1
1
2
7
5
1
1
3
3
1
2
2
3
2
1 2/2 12
3/5 10
2 5/5 11
1/1 5
1
1
1
1
3 1
6
4
2
1/2
1 2/2
1/1
1/2
4 0/9
2 0/0
4 0/9
3 5/5
2 ;%;
4 1
1 1/1
0/1
1
2
1
1
1/1 1%1 / 2
1 1/1 I
1
Total
76
20
(1O
4 14/1c 56
21
10 40/45
(6) Children were vaccine ed with the Salk vaocine produced in 1957-1959
by Parka, Davis 6 Co., and in 960 by the InOtitute of hygiene of
Serb I.
(N) MomlnatOr Mean' the number of patients, from which polioviruaee could
be teolated in PHA cell cultur a; denominator meant, the number of
patients, from ehleh pethologi al epecimene were eent for
laboratory invceti6at100. ?
TABLE 4. RESULTS OF POLIOVIRUS ISOLATION IN VARIOUS AGE GROUPS OF
PATIENTS WITH PARALYTIC POLIO!
--- ----
DURING 1960 - 1961
Age group
(years)
POLIOVIRUS ISOLATION IN TISSUE CULTURE (?)!Not
Positive (8) Negative Per cent positive examined
(?)
0 - 4 '
19
1 95,0 5
5 , 9
15
2 88,2
10 - 19
10
1 90,9 2i
20+
10
3 76,9 5
I
TOTAL
54
i
7 88,5 I 15
I
I
(?) Primary human amnion cell cultures were used for virus isolation
attempts. Complement-fixation and neutralization test were
performed with sera from majority of the patients.
(&) Virua strains were identified by neutralization tests with the
type-specific poliovirus antisera.
(?) Patients were reported as to have paralytic poliomyelitis;
pathological specimens were not sent to the laboratory.
???
TABLE 5. POLIOVIRUS TYPES IN VARIOUS AGE GROUPS OP PATIENTS WITH
PARALYTIC POLIMEMITIS IN SLOVENIA DURING 1960 - 1961
Age group
(years)
POLIOVIRUS STRAINS4
Type 1 Per cent Type 2 Per cent Type 3 Per cent
Total
poliovirus
0 - 4
12
63,1
1
5,2
.6
31,6
19
5 - 9
11
73,3
4
26,6
15
10 - 19
4
40,0
6
60,0
10
20 +
4
40,0
6
60,0
10
TABLE 6. POLIOVIRUS STRAINS ISOLATED IN PRI !ARY HUMAN AMNION CELL CULTURES
FROM HEALTHY CHILDREN IN SLOVENIA DURING 1960 - 1961
Materials
collected
No. of I Positive
speci- (?)
mans (Per cent)
Negative
POLIOvIRUSES
.gO.-o-f -Pei'
strains
isolated
cent poliovirolis
from
-
no. of
enterovirus.
no. of
specimens
May-June 1960
.483
70
(14,5)
4134
0,83
5,7
100v.-Dec. 1960 512
90
(17,5)
420
36
7,0
40,0
February 1961 498
23
(4,6)
475
19
3,8
82,6
TOTAI 1 1493
4133
(12,2)
1310
59
3,9
32,2
(?) Total number of enteroviruses isolated in PHA cell cultures.
TAM 7. RESULTS OP ENTEROVIRUS ISOLATION PROM HEALTHY CHILDREN AND CLnICAL
PATIENTS WITH PARALYTIC POLIOMYELITIS AND ASEPTIC MENINGITIS IN
SLOVENIA DURING 1960 - 1961
Materiels
collected
Virus strain0 cytopathogenic for primary
(5) and continuo. (&) cell culturee
Virue strains
cytopethogenic
2.1t1"1.rrY
Polio
1 21 3
Cozaackie
B type 3
Adeno
Unidentified
(?)
Healthy childrea
Day -June,1960
2 1 2
16
6
1
43 (e)
NOV-::Dec. 1960
15 1213
2
49 (c)
-
February 1961
9- 110
. i--
3. (;)
TOTAL
26 133
17
93
95
Clinical patients
1
1.I.1960-31.V.1961
1
Paralytic caeee 31
1 22
Non-parelytic 2
1
2
5 (-)
TOTAL 33
1122
2
5
(?) Primary human amnion cell cultures.
(a) Helga cello, strain Zurich.
(?) Cytopathogenic activity of the virus etraina could not be neutralised by
immune antisera poola againet Polio 1-3, ECHO 1-8, Coxoackie A 9 and 23,
and Goxeookie B 1-5.
(...) Immunologic identification not completed.
InCluding one strain of ECHO virue type 1(8).
(-) Including one strain of ECHO virue type 2, 3 and 1(8).
TABLE 8. RESULTS OF VIRUS ISOLATION IN PRIMARY M.IN.AH 6161I00 CELL CULTURES
FROM PATIENTS WITH PARALYTIC POLIOMYELITIS IN CROATIA DURING 1960
Virus isolation
Paralytic cases
Non-paralytic cam;
Total
Poliovirue type 1
33
3
36
type 2
type)
3
3
2
2
ECHO virus type 1(8) 2
2
type 7
3
3
Coxsackie A 23
1
1
Adenovirum
2
2
Unidentified (9)
1
1
Negative
13
1
14
(9) Cytopathic activity of the virus strains could not be neutralized by
immune antisera pools against Polio 1-3, ECHO 1-8, Coxsackie A 9 and
23, and Coxsackie B 1-5.
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1
TABLE 9.TIME OF APPEARANCE OF MICROSCOPICALLY VISIBLE CYTOPATHIC ACTIVITY
OF VIRUS STRAINS ISOLATED IN PRIMARY HUMAN AMNION CELL CULTURES
Virus strains
DAYS OF OBSERVATION (9
Total
1 2
3
4
5
6
7
8
9
10
11
12
15
17
26
Poliovirus 1
1 10
5
13
2
1
4
2
2
1
43
2
[
1
4
3
1
1
1
3
ECHO
.. 4
1
....._
_

?
5
. . __
Adenovirus
2
2
Coxsackie A23
1
_
1
Unidentified(&)
1
1
(9) Cell cultures have been examined for 10 days; two blind passages
were made if isolation attempt was negative.
(&) See table 8.
TABLE 11. ISPECTIVITY TITRATIONS OP TYPE 1 POLIOVIRUS STRAIES IF PRIMARY
RUYAN AMNION CELL CULTURES INCUBATED AT 36?C AND 40?C, AND IN
UCLA CELL CULTURES INCUBATED AT 36?C
LaterialDays
0)
afte r
vacci-
nation
106107CID50 (0)
,,,,,,I.TCID5.36?C
TCID5.40?C
PHA
PHA
HeLa
36?C
40?C
36?C
6572 RS
14
5,75
51,50
5,75
64,25
6573 RS
14
5,50
-11.50
5.75
kl,00
6588 RS
14
6.00
1,50
5,75
4,00
6601 RS
14
1.50
5.25
4,75
660900
14
_6.25
6,75.
1,50
4.00
5,25
6620 RS
14
5.75
1,75
5,75
4,00
6624 RS
14
6,00
1,50
5.75
4.50
RS
14
6,00
1,75
neg. (7)
4.25
_Jkak
6397 HS
14
6,25
1,25
neg. (?)
5.00
6640 RS
14
6.25
1,00
5.50
5,25
5080 V
27 (9)
5,00
3,00
5,00
2,00
5086 P
31 (%)
5,50
14,50
5.50
(?) Second tissue culture peseege wee need. Virus stroll), were leoleted
In PHA cell cultures from rectal Swabs of children 14 doge after the,
were fed the babin type 1 poll virue vaccine. P . faeces
(4.) Infectivity titer, ?screamed log TCID,.. per 0,1 al.
(7) In Hole cell, no CPE wee desonstrated. '- although 1,000.000 30I0,0
Of the virus was used as inoculum.
(%) Paralytic poliomyelitie caeca from 1960 epidemic in Croatia.
TABLE 13. INFECTIVITY TITRATIONS OF TYPE 3 POLIOVIRUS STRAINS IN PRIMARY
HUMAN AMNION AND MONKEY KIDNEY CELL CULTURES INCUBATED AT
36?C AND 40?C
Material
(g)
Clinical
diagnosis
Days
is;g1.
3
vaooine
1og10TC1D50 (1)
1081oTCID5038?C
PHA
MK
TCID5040?C
36oc
40?C
36?C
40?C
PHA
MX
5829 F
5829aF
Aeeptio
meningitis
/
6,50
4,75
1,50
2,60
7,00
4,75
0,75
2,75
5,00
2,75
6,25
2,00
6171 P
Entero-
colitis
/
4,75
3,50
4,25
2,75
1,25
1,50
6180 F
Pertussis
25
5,75
4,00
5,00
4,50
1,75
0,50
6472 RS
Healthy
86
5,00
3,25
5,50
3.25
1,75
2,25
6564 RS
Healthy
86
5,00
2,50
5,75
4,75
2,50
1,00
6690 RS
Healthy
86
5,00
2,00
13,50
0,75
3,00
92,75
(9) Second tissue culture passage was used.
(&) Infectivity titers expressed as log TCID50 per 0,1 ml.
TABLE 15, INFECTIVITY TITERS OF SOLE ECHO VIRUS STRAL.S
ISOLATED IN PRIMARY HUMAN AMNION CELL CULTURES
Virus type
Virus strain (9)
lcg10TCID50 (&)
ECHO 1(8)
I
.5
g_33A _
---10065
__ _
_ Z,.!0_ ._
7.0
10091
J.
7,0
7,0
ECHO 6
---131:_
- -I-
--
9831
f 7,5
10744
ECHO 9
7295
7,0
7_206
ECHO 169634-
---i0440-
_.__4.5_.___
-1
5 5
,- - - ---
5,0
(9) Second tissue culture passage.
(A) Infectivity titers expressed as log TCID50 per 0,1 ml.
I 1
. TABLE 10. INFECTIVITY TITRATIONS OP STANDARD ATTENUATED All) VIRULENT
POLIOVIRUS STRAINS IN PRIMARY HUMAN AMNION, MONKEY KIDNEY,
AND HEIA CELL CULTURES INCUBATED AT 36?C AND 40?C
Virus
strains
1Viru-
Bence
1
l_1f81. TCID5. (9)
- ----
---MK -
38?C ---I
o
log,.TCID5o36 C
pHA
6? ' 40?C
3C
1
---
licLa
40?-6- 136?C
1
' TCID ,40?C
1f5'
PRA

la(
Sabin
type 1
' , 0
I
1 7,0/2(9.)
2,50/2
6,83/1
I
160,50/1 8,50/2 4,50/2 1
?6,33/1
Sabin
-;KT1n2
type 3
.
0
1 6,83/2
1,50/2
1 5,33/2
, 0
1 6,61/3 2,17/3
6,50/1
1,50/13,50/1 4,44/3
5,00/1
Chat
type 1
Brunhilde
-
Itgl 1.105f
_tylp.D 2 I______
E543:494
type 3 I
0
,
I 7,17/2 1,84/2
I
5,50/1 5,33/2
.
11,09/4 6,50/4
7,10/1
____A
6,50/1 6,50/2
1 ----]
5,50/114,27/1
1 1
0,59/4
0,60/1
.
.
1 7,50/2
6,83/2
-
--
6,50/1
0,67/2
1,07/3 ,
i 5,79/3
4,72/3
1,00/1
Infectivity titers expressed es log TCID50 per 0,1 ml.
(&) Denominator means the number of titrations performed; nomimator
represente the median value of repeated experiments.
TABLE 12. INFECTIVITY TITRATIONS OF TYPE 1 POLIOVIRUS STRAINS IN PRIMARY
HUMAN AMNION CELL CULTURES AND MONKEY KIDNEY CELLS INCUBATED AT
36?C Al)]) 40?C
Material
(9)
log10T0ID50 (&)
logi. TCID5036?C
PHA
36?C
40?C
36?C
mx
40?C
TCID5?40?C
PHA HE
6588.
6,00
1 50
7 25
1,25
4,50
6601
6,25
1,50
6,25
0,50
4,75
.6,00
45,75
6609
6,75
1,50
5,25
60,50
5.25
54,75
6620
5.25
1,75
1,50
7,25
5,75
60,50
60,50
4,00
4.50
96,75
95,25
6624
6,00
6396
6,00
1,75
5,50
0,75
4,25
4,75
6397
6,25
1,25
5,50
60,50
5,00
5,00
6640
6,25
1,00
6,50
1,50
5,25
5,00
Virus strains were isolated in PHA cell cultures from rectal swabs of
children 14 days after they were fed the Sabin type 1 poliovirus
vaccine.
(&) Infectivity titers expressed as log TCID50 per 0,1 ml.
TABLE 14. INFECTIVITY TITHATIONS OP VINUIENT AND ATTENUATED POLIOVIRUS
STRAD. IN PRILARY HUNAN ABNION AND MONKEY EIDNEY cal
CULIEIRES INCUBATED AT 3600 AND 40?C. APPFIt 7 DAYS THE mammas
AT 40?C %BRE ADDITIONALLY INCUBATED AT 36?C POR 72 HOURS
Poliovirus
stream
.
Type
100507CID50 (?)
P H A
:0?C
36(40)?C
36?C
4000
36(40)?C
36?C
Chet
1 7,17
2.17
2,17
Sabin
1abin3
2 (5)6,83/2
1,50/2
1,50/2
6.50
1,50
2.75
0551-1105
E543-4934
2
3
7.50/2
6,83/2
6.03/2
6,50
5.50
5.50
6588
6601
1
1
6,00
6,25
1.50
1,50
1,50
1,50
6375
6391
2
2
5.75,2,25
6.00
44,50
2.75
04.50
6467
6468
2
2
6.25
6.50
2.75
2.25
3,00
2.50
6492
6493
2
2
7.25
6.25
2.25
2.50
2.25
2.75
6508
6523
2
2
5.25
6.75
1.50
2.73
2.75
3,00
6529
6)39
2
2
6.27
5.75
1,00
1,75
2,00
2.75
5,329
6171
3
3
1:n
'3:7g
1:n
6179
6180
3
3
6.25
5.75
4.25
4.50
4.50
4,50
5.00
4.50
4.50
6181
6542
3
3
5.75
4.75
04,50
2,00
14,50
2,00
4,75
2,75
2,75
(?) Infectivity t tare expressed es log TCIDa?, per 0,1 ml.
(2) Denominator mesas the number of titration)) perforned, nominator
seen, the median value of repeated experimente.
TABLE 16. PRIMARY ENTEROVIRUS ISOLATIONS IN PRIMARY HUMAN AMNION CELL
CULTURES FROM PATIENTS WITH ASEPTIC MENINGITIS IN CROATIA
AND SLOVENIA DURING 1962
ECHO viruses
Coxsackie B
Coxsackie A
Unidentified
Not
37(-q 6
16
1 3
5
9
not typed
0)
typed
Croatia
112 14
1
2
1 7
Slovenia
3
1
2 1 I
11
7 11
Total
3 1
2
12 14
2
2
1
7 ' 18
(9) Cytopathic activity of the virus strains could not be neutralized by
immune antisera pools against Polio 1-3, ECHO 1-8, Coxsackie A 9 and
23, and Coxsackie B 1-5.
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STAT
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VI/8
CULTIVATION OF VACCINIA VIRUS IN HUMAN DIPLOID
CELL STRAINS
J.Trlifajova, V.Stiova, F.Stastn3,
Institute of Epidemiology., and Microbiology and
institute of Sera ancl:Vaccines, Prague, C.S.S.R.
Numerous tissue cultures of the most diverse origin are
sensitive to the virus of vaccinia. In view of the presumed
suitability of human diploid cell strains for the preparation
of antiviral vaccines it was interesting to ascertain if these
cells are sensitive to the vaccinia virus and what height of
titre the virus attains in them as compared with other cell
systems.
Material and Methods
Diploid cell 'strains WI-26 and WI-38 obtained from
Dr Hayflick of the WiStar Institute in Philadelphia, were used in
the experiments. The V71-26 strain was used in its 24th to 43rd
passage, the WI-38 strain in its 21st to 31st passage. Eagle's
medium with 1096of calf serum and1/3:bf0.5%.-LAH in Hank's
solution was used as :t1,(- growth medium. The maintenance medium
after inoculation of the tissues Was incomplete Parker medium:199
in a modification of the Serum and :Vaccine Institute, Prague
Mikrobiol.:Imunol.; 2, 11, 1960/, -10;th 2% of ?calf serum.
The-test-tube:cultures were set-up-from a..cell-suspension
containing 100,000 cells per ml and test-tube.
Parallel experiments with,primary:tissue.cultures from
Macaccus rhesus kidney /OL/ and from dog.kiany-/PL/ were carried
out. Modified Parker medium. .199 /as above/ with. different
quantities of calf serum was employed as growth medium. The density
of. the seeding suspension was 10.0,000 cells per. ml and test tube
for the monkey-kidney primary cultures and. approximately 200,000
cells_ per ml and test ?tube for the dog' kidney primary-cultures.
Modified Parker tedium 199 With. 2%-of calf serum was used as
?maintenance.mecliut.in both :types of: tissue culture.
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?
VI/8 2
Vaccinia virus strains E-4 /ID50/0.2 ml for the CAM equalled
-43
10 ?/ and 20 E-2, ovovaccine /1D50/0.2 ml for the rabbit equlled
10-3.7/ obtained from the Serum and Vaccine Institute were used
for inoculating the cells.
Virus was titrated fron the cytopathogenic effect, pronounc
though focal destruction being taken as the endpoint. Four to
five test tubes were used for each virus dilution. The tissue
cultures were observed for 10 days following inoculation. Dteween
the passages the virus was stored on dry ice or in a Revco ice-box
at -60.)C.
The TCID50 was calculatedHby the Reed-Luench method.
Results
The vaccinia virus produces a pronounced cytopathogcnic effect
in human diploid cell strains. The destruction of the tissue
consists in a ,clustering of the cells and in a retraction and
disintegration of the cytoplasm, which bridges by narrow strips
the vacancies in the tissue joining the, cell clusters. The clusters
consist of deeply stainable pathologicallychanged nuclei and
cytoplas,1 debris. A hi-,1er virus dilution causes enly a focal
cytopathegonic effect.
In the WI-26 diploid strain. five .passages of the vaccinia
strain E-4 and four passages of the 20 E-2 ovovaccinun'Arariolae
strain were performed /Table 1/.
.Table ?1
7:1;xpe- Material Virus Log.TCID50/0.1 ml
riT:ient Inoculated Tissue Lt1lUt1fl let 2nd. 3rd 4th 5th -pcIssaF;e
No
Strain WI-26 concent. -6.5 -4.7 -4.2 -4.2 -4.5
E-4 31st-
36th
passage
Ovovaccin. JI-26 -6.3 -5.6
vaiolae 37th-
20 E-20 43rd
passage
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VI/8
3
In the JI-3.8 diplAd cell strain twice four pasages of the
ovuvaccinun variUlae 20. E-2 strain were performed /Table 2/.
Table -2
2x1De' 'Material Virus
riment Tissue
Inoculated dilution 1st 2nd 3rd 4th passage-
roG.
50
??????????????1?????????????????
1 20 E-2 ? .41-38: 10-2 35-, .6.4..t5.5
19th-
23rd
passage
20 E-2 WD-38. 10-2
27th-
31st
passage
7.0
-5-6 -5.2 -5.5 -6.0
The virus titre did not increase by passaging virus diluted
10-1, 10-3, 10-4 /log.TCID50/0.1 ml unier these c nditions
equalled -5.5 and -6.0/.
An approximately equivalent sensitivity of thc. aa-26 and the
WI-38 strains to the vaccinia virus was C.e=strated by the
cultivation and titration cf the virus under identical c:Aiditicns
in both strains /Experinunts No 2 in Tables 1 and 2/.
Furthermore , comparative titrations of the vaccinia virus in
the diplr,id cell strains and in primary tissue cultures iron
monkey and dog kidney were carried out /Tables 3 and 4/.
Table 3
Expe-
riment
No
Material
inoculated
Virus
dilution
Tissue
Log. TCID50/0.1 .m1
1st passage
2nd passage
1
20 E-2
-1
10
CL
-4.6
-5.1
2
20 E-2
10-2
WI-38
-4.5
-6.5
30th p.
OL
-5.3
-5.2
dI-26
-5.5
25th p.
OL
-6.2
NI-26
-5.6
29th p.
OL
-4.6
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VI/8 4
.The above comparison of titre of the vaccinia virus in first
passages in diploid cell strains and in primary monkey kidney
cultures shows that the sensitivity to the virus of both cell
systems is more or less the same.
Table 4
e- Material Virus
riment Tissue
inoculated dilution
No
Log.TCID50/0.1 ml
1st 2nd 3rd 4th passage
-2
E20 10 PL -4.8 -5.4 -5.0 -4.2
2 E WI-26
4
29th D. -5.6
PL -5.3
No substantial differences between the titres of vaccinia
virus obtained in the first passages in human diploid cell strains
and primary dog kidney cultures was noted.
It was found in two experiments that the titre of vaccinia
virus cultivated in the human dipinid strains '.4I-26 and ,vI-38 is
not changed by storage on dry ice for three to eight months.
Discussion
The above results show that human diploid cell strains are
highly sensitive to vaccinia virus. Should these tissues prove
of use for the production ()f antivriral vaccines, the possibility
of utilizing them for the prparation of an anti-variola vaccine
could, be considered.
We are continuing our c:laparative studies on the sensitivity
og human diploid cell strains and ether systems and on the
stability of virus in long-term cultivation and storage.
Surirjaa
A hight sensitivity to the vaccinia virus was observed in
human diploid cell strains and was compared with the sensitivity
of primary monkey kidney and dog kidney cultures.
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s...) I In I
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V/6
UTILIZATION OF A NEW DIPLOID CELL. STRAIN DERIVED
FROM HUMAN :EMBRYO LUNG TISSUE FOR THE CULTIVATION
OF ENTEROVIRUSES AND MEASLES-VIRUS
Hozinski,V.I., Seybil.V.B. Cypkin,L.B. Panteleeva,N.S.
Mazurova,C.M.
Institute of Poliomyelitis and Virus Encephalitis,
Academy of Medical Sciences of USSR, Moskva, SSSR
Diploid cell strains from the very beginning adapt to the
composition of the nutrient medium, and the success of their
maintenance in passages is, apparently, to a considerable degree
dependent on the identity of cultivation conditions .and the
conditions of primary culture. A serious drawback in the use of
primary Hayflick-Moorhead diploid strains is their marked
adaptability to the amino acid complex manufactured by the
Microbiological Associates Company. The experience of many
researchers, our own included, has been that the replacement of
that complex by amino acids of a different origin results in the
death of the strain.
An attempt has been made to obtain our own diploid strains
on available media. After trials with a number of media w,s
chose a mixture of 70 parts of "Igla" medium /of amino acids
of the California Corporation for Biochemical Research and of
Czechoslovak-made Chemerol, vitamins produced by. NBC/ and 30
parts of 0.5% solution of lactalblmine hydrolysate in Hanks
medium+. The "Igla" medium contained 10% of calf serum
Using Hayflick and Moorhead techniques /1,2/, we obtained
five cellular strains from the lung tissue of human embryos.
Our paper presents the materials concerning research into one of
them, which was passed throng-!1 48 subcultivation passages
/strain LT-16A
+Thnaks to person-21 communication from Dr J.Trlifajova /I.E.M.
Prague/ we were aware of the successful use of lactalbumine
hydrolysate, to supplement 7hu "Igla medium /prepared from amino
acid and vitamins by Microbiological Associates Co/ in the
culture of original Hayflick and Moorhead strains
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The object of the first part of our research was to show
the correspondence of the charcateristics?of our strain with the
features of original Hayflick and Moorhead strains. The principal
of these characteristics were in our opinion the dynamism and
morphology of growth, intensity of cellular metabolism, karyo-
typic picture, and absence of oncogenicity in the experiment0
ism owf_graih_ of LT-16 strain. The strain passed
through three stages of development: formation /primary culture,
1-2 passages/1 active growth /up to 43-45 passages/, and ageing.
In the stage of active growth, as early as 24 hours after
transplanatatien of the culture, the protruding cells formed a
net, merging into a monolayer after 48 hours of cultivation.
After 74-98 hours, an at least twofold cellular layer was
observable, oriented in intercrossing directions. Subcultivation
passages were made twice weekly, with the growth surface being
doubled. By the end of each subcultivation period /.i.e. after
3-4 days of growth/ the cell countin a one-litre matrix flask
reached 18-24 millions. There was a decline in the intensity of
growth during the stage of ageing and after 48 passages mitotic
activity was discontinued,
Morpholo'77, of LT-16 strain. In the primary culture the cells
had a predominantly epithelic form /Fig.1,a/. Three or four
passages later they attained a fibroblastic form, took parallel
positions and were characterized by monomorphism /Fig,l,b/.
Cells mainained in passage for 10-12 days formed a multilayer
membrane at the end of this period /Fig.1,b/. In the stage o:7
ageing the cells assumed a loose arrangement, at times amorphous.
Parellel with protruding elements a considerable number of cells
was observable with oversize hyperchromic nuclei sometimes
amorphous, and with voluminous cytoplasm /Figl /. Fluorescent
-
microscopic examination revealed a considerable RNE.- content in
cells of LT-16 strain. The differentation of cells was effected
by alterations in RNK content.
Intensity of .cellular metabjism, expressed trough acidifica-
tion of the nurient rs.ldium9 was considerably enhanced in sub-
cultivations and exceeded thct commonly observable in cell
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1/6 3
Cultures of monkey kidney or inoculated lines, LT-16 cells
preferably grew in media with a comparably low RN index.
auamt. Cells of LT-16 strain had the female diploid
Chromosome arrangement, common in man. Fig.2,a shows a cell in
metaphase. The chromosomes of the same cell, arranged in groups
:416cording to Denver classification, are shown in Fig. 2,E
,t1C conspicuous quality alterations were observed in the idiogram.
.06Unts acrried through with 200 cells undergoing metaphase
TeVealed 8 /4%/ heteroploid cells in the 12th passage, 6 /0:7 in
.01.0 21st Passage, and 2 /1%,/ in the 34th passage, The decrease
iit:number of heteroploid cells in the process of cultivation
suggests the diploid stability Of the LT-16 strain.
H OncogenicitV. In the inoculation of hamster cheek Pouches
Cells of LT-16 strain caused no increasing swelling. Between the
4th'and: 8th day following inoculation, very small /millet-grain-
sized', infiltrations were observed, which were re-Sorbed after a
few days. Histological research revealed that these infiltrations
represented an ordinary inflammatory reaction developed at the
site of induction of LT-16 cells /Fig.3/.
The charactetistics outlined allow, in our opinion, to
consider LT-16 strains to be close in their qualities to
Hayflick and Moorhead strains. Our task was to study the sensiti-
vity of the new diploid strain to cerrain viruses.
It is known that somewhat lower titres of enteroviruses are
obtained in diploid cell cultures. This is explained by the
adaptability of laboratory virus strains to monkey cells /2/,
We checked titres of attenuated Sabin polioviruses on
various levels of passage in LT-16 culture. It was revealed that
during adaptation to the new culture the poliovirues P-r-c-Ti no -
less intensively than in the cells of monkey kidney /Table 1/.
ECHO viruses of types 1-19, 201 21, 26, 27 as well as
Coxackie A9 were cytopathogenic for LT-16 strain, but in titres
less than 1 log10 in monkey kidney cultures /the possibility of,
adaptation has not been checked/. Coxackie B1_5 viruses caused
no cytopatogenic alterations in our cell culture
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Table 1
Titres of Polioviruses in LT-I6
Culture
Virus adapted in
tissue culture
Titres after C13, /log TCD50/m1
in tissue culture of'
in LT-16 culture:types
monkey kidney:
I II types I
III 1 I II III
Monkey kidney 7.0 6.5 7.0 5.8
LT-16 /1st passage/ 7.0 6.5 8.0 6.6
LT-16 /4th passage/ 7.0 6.5 6.2
LT-16 /7th passage/ 740
LT-16 /9th passage/ 7.4 7.0 8.0
?
The strains of measles viruses, Edmonston and Leningrad-Li
were successfully adapted in LT-16 culture, caU6ing cytopathogen-
ic alterations. In the process, the virulent vn14ant of Edmonston
strain and the vaccinia strain Lenitgra1-4 daubed the formation
of oversize multinuclear cells; whereas the dytopathogenic effect
of the attenuated variant of Ed.Monston strain was limited to the
ocdurretce of a general cellular degeneration and dilution of
celltir layer /Fig.41 a, , /. Fig.5 shows the dynamism of
growth of virulent Edmonston strain by titres it LT-16 culture.
tetingrad-4 strain was reproduced in this culttire by approxima-
tely identical titres.
Coiori
1. It the process of adaptation of polioliiruses to a new strain
of LT-16 diploid cells, the virUses were grown in this culture
up to titres comparable with those commonly obtained in the
cell allture of monkey kidtey.
2. The virulent variant of the strain of Edmonston measles virus
and the la.ccinal measles virus Leningrad-4 gate rise to the
formation of symplasts in LT-16 cell culture; upon infection
by the attenuated variant of Edmonston strain, a degeneration
of cells of LT-16 culture, with no formation of symplasts,
was observed.
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V/6
References
5
1. Hayflick,L. and Moorhead?P.S., The Serial Cultivation of
Human Diploid Cell Strains, Exper.Cell.Res., 1961, Vol.251
N 3, 585-621.
2. Sayflick,L., Plotkin,S.A., Norton,T.W.
Pv,eparation of Poliovirus Vaccines in
Cen Strain. AmerJ.Hyg., 1962, 22:2,
3. Report to the Director General, World
Scientific Group on the Human Diploid
July 1962 /WHO/PA/ 140, 62.
and Koprowski,H.,
a Human Fetal Diploid
20-258
Health Organization,
Cell. Geneva, 16-18
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STAT
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