INTERFERENCE BETWEEN INACTIVE AND ACTIVE
INFLUENZA VIRUSES IN THE CHICK EMBRYO
III. INHIBITOR OF \'IRUS H.AKMAGGLUTINATION IX THE CHORIOALLANTOIC MEMBRANl'
by MARGARET KDNKY^ AND A L I C K
IS
(From the Walter and Eliza Hall liistitiitp of M<MliPaI Rosparoh, Mcthoiirne).
(Accepted fm- puhlicotion 6th June W50.)
A number of inhibitors of haemagglutination by influenza viruses has been
tlo.^erihcd, e.fjf. the normal serum inhibitor (Francis, 1947), cyst mucoid
(Biirnet, McCrea and Anderson, 1947) and the inhibitor in normal allantoie
fluid (Svedmyr, 1047). Many of these have been shown to be enzymically
destroyed by influenza viruses and by the soluble enzyme, RDE, of V. cholerae.
Priedewalil el al. (1947) extracted inhibitor from various tissues, inid Bovarniek
and de Burf?h (1047) prepared an cxtraet from red blood i-ells wbieli behaved
iu solution as a virus inhibitor. Hirst (1943) described eell reeeptors in the
ferret luns;, which adsorbed iufiueuza virus, and similar rcceptoi-s have been
describetl in otbcr tissues susceptible to intluenza virus iufectiou (Stone, 1947;
Pazekas de St.Groth, 1948). These receptors, like tlie inhibitors, were fonnd to
be acted upon by viruses and RDE.
The present paper describes the characteristics of inhibitor extracted from
the chorio-allantoic membranes of chick embryos, and the reduction in inhibitory
titre occurring during the course of infliienza virus infection in the allantoie
cavity.
MATERIALS AND METHODS.
Virus Strains.
The following strains were used in the form of infected ailantoic fluid, harvested 2 days
after ailantoic inoculation of 11-day chick embryos.
ItiflnevsaA: WSE, PR8, MEL, BEL.
InflucmaB; LKE.
RDE.
An extract of the 4Z strain of V. cholerae containing the receptor-destroying enzyme
(RDE) was prepared by the method of Burnet and Stone (1947). The preparation of BDE
used in these experiments had a titre of 4,000 units/mL
Normal saline. 0-0 gm. sodium chloride in 1,000 ml, distilled water.
Citrate saline. 20 gm. sodium citnito in 1,000 ml. distillerl water.
1 Francia Haley Research Officer, Depnrtment of Experimental Medicine, Univerflity of
Melbourne.
* Rockefeller Travelling Research Fellow.
604
MARGARET EDNRY
AND A L T C K T S A A C S
Valcium-acetate-saline.
Isotonic sodium acetsite—acetic acid buffer adjusted to p H 6-2 and
containing 0-1 p.p. calcium chloride.
Borate Buffer.
Equal parts of M/20 smliiim metaboratP and M/Ij boric acid (pH 8*f)).
Fowl Bed Cells.
Blood waa obtained from the wing vein of adult white Leghorn fowla.
in normal aaline nnd made up to a 1 p.c. suspension in normal saline.
Maemapglntination
Cells were washed
Titration.
Serial twofold dilutions of virus {0'2;'» ml.) were prepared in norinal saline and an equal
volume of 1 p.e. fowl red cells was added to each dilution. The cells were nllowfd to settle at
room temperature and readings were made by observing the pattern of sedimpntcd cells. The
end-point was taken as the amonnt of virua causing partial agglutination, this amount being
one agglutinating dose (A.D.). The haemagglutinin titrea are expressed as the reciprocal of
the initial dilution of viriia at the end-point.
InhiMtnr
Titration.
Serial dilutions of inhibitor were prepared in normal saline. An equal volume of virus
containing 5 A.D. was added and the mixtures kept for 30 minutes at room temperature. A
third volume of fowl cells was added .^nd these were allowed to settle at room temperature.
Partial agglutination indicated the ond-point. Inhibitory titres are expressed as the reciprocal
of the initial dilution of inhibitor present at the end-point.
Infeetivity
Titration.
Serial ten-fold dilutions of virus were prepared in 10 p.c. horse-sernm saline. 0-0.^ ml.
volumes were inoculated allantoically into 11- or 12-day chick embryos, <i per group. After
incubation for a further 42 hours at ^rt" C. the ailantoic fluids were harvested and one drop
added to 0-25 ml. of 1 p.c. fowl cells, growth of virus being detected by haemagglutination.
The 50 p.e. infectivity end-point (ID.-^.Q) is the dilution of virus which will infect 50 p.e. of the
inoculated egga.
Preparation of Indicator Viruses,
The methods of Stone (1949a) were used to convert viruses into the inactive indicator
state, in which their haemagglutinins are particularly sensitive to inhibition by mueoid
inhibitors, anti the viruses lack cuzymic activity.
Seated LEE (E-LEF.)
Treated PRS (T-PE8) )
T
id MFT (T MF7 1 '
™. , •• .^Tn^r ,™. n-r.T ,
Treated BEL (T-BEL)
Heated WSE (H-WSE)
LEE virus heated as undiluted allantoic fluid at 56" C. for 30
minutes.
^ pai'ts of virua heated at 56° C. for 30 minutes in 2 parts of
2 p.c. citrate saline aiul 1 part of bornte buffer.
^
'
Virus heated as undiluted aUantoic fluid at 52° C. for 30 minutea.
Preparation of Interfering Virii.ies.
Viruses were inactivated for use as interfering agents by the methods described for indicator viruses except that they were heated for 60 minutes (to ensure loss of infectivity). Interfering viruses were tested for infectivity by inoculating 0-05 ml. (less than a minimal interfering dose) into 6 eggs. Only viruses which did not infect any of the eggs after 42 hours'
incubation were used as interfering agents.
INTERFERENCE IN' INFLUENZA VIRUSES. III
605
preparation of Chorio-allantoic Membrane Suspension.
11- or 12-day old chick embryos were used throughout. After rcmo%'ai of the shell over
tlie air-sac, the shell membrane and underlying chorio-altantoic tissue were cut and the contents of the egg discarded, leaving the t-hoiio allantoie membrane lining the shell. By this
method the portion of the membrane covering the viscera was not iiitluded. The membrane
was removed carefully and cleared of adhering mucinous material. A number of washings in
uormal saline removed most of the blood. The membrane was ground with alundum and suspended in 1 ml. of normal saline. After thorough mixing, the ground membrane was lightly
centrifuged and the supernatant fluid used as the standard membrane suspension.
Interference Experiments.
Groups of six 11- or 12-day embryos were inoculated allantoically each with an interfering
doac of inactive virus. If 0-5 ml. or greater volumes were injected an equal amount of allantoic fluid was first withdrawn. The eggs were incubated for one hour at 37° C. and then
inoculated agaiu with 0-05 ml. of varying dilutions of challenge virus. Controls were inoculated
with challenge virus only. The eggs were then incubated for 42 hours at 35° C. when the
allantoic fluids were harvested and titrated individually fo'r their haemagglutinin content.
EXPERIMENTAL.
Virus Inhibitor in Suspensions of Giuund Chorio-allantoic Menihruncs.
Chorio-allaiitoie membranes of 11- or 12-ilay chick embryois were tested
for the presence of inhibitor of influenza viruses. A suspension of membrane
was prepared (see Methods) and titrated for inhibitor of haemajjglutination by
LEE and MEL viruses, both heated and unheated. Table 1 shows typical
inhibitory titres.
TABLE 1.
Inhibitor in suspension of ground ehorio-allantoio menibrane.
State of virus
Active
Ueated
Inhibitory titre
LEE
MEL
18
480
80
750
Inhibitor titrated against 5 A.D. of each virus.
It can be seen that membrane suspension inhibits agglutination particularly by heated ("indicator") viruses. This behaviour is characteristic of
inhibitors prevously described.
Normal membrane suspension did not inhibit infeetivity of active virus. Dilutions 10-«,
10-7, and 10-8 of MEL virus were mixed with equal volumes of membrane suspension and with
normal saline as a control, and allowed to stand for one hour. These mixtures were inoculated
into groups of eggs and the 50 p.c. infectivity end-points were determined. Virus mixed with
membrane suspension, and control virus both showed infectivity end-points of 10-7.8,
606
MARGARET EDNEY .\ND ALICK I8AACS
Heat Stability of Membrane Inhibitor.
When undiluted membrane suspension was heated at 65° C, a heavy precipitate formed
and the inhibitory titre of the supernatant was reduced. If the suspension was first diluted
1 in 10 in normal saline, there was no significant change in inhibitory titre after heating at
65° C. for 30 minutes, or boiling for 2 minutes.
Action of BDE on Membrane Inhibitor.
The action of RDE on the inhibitor was tested as follows:
1 ml. of membrane suspension and 0-2o ml. EDE (1,000 units) were int^^ubated at 37° C.
Membrane suspension was incubated with normal saline as a control. The action of RDE was
stopped after one hour by diluting the mixture 1 in 10 in normal saline and heating at 65° C.
for 30 minntes, the eontrol being treated simiiariy. The inhibitor content of each mixture was
titrated. Table 2 shows the marked reduction in inhibitory titre produced by BDE.
TABLE '2.
Action of EDE on inhibitor in ground membrane suspension.
Inhibitory titre
Membrane incubated with
RDE (1,000 units)
Normal aaline
H-LBE
T-MEL
<10
480
<io
750
Inhibitor titrated against 5 A.D. of each virua.
Presence of intracL'Uuh/r Inhibitor.
Stone (1947) has sliowii that treatment with RDE reduces the eapaiiity of
tlie liiiinj,' of the allantoie eavity to adsorb influenza virnses. tlius prevenliu';
infection by the viruses. The virus receptors ou the cell surface demonstrated
by Stone's experiments would be expected to contribute to the inhibitory titre
obtained with ground membranes, and surface eontamination witli allantoie
fluid (Svedmyr, 1947) and egg-white (Lanni and Beai'd, 1948), both known to
contain inhibitors, might also contribute. Experiments were made to eliminate
these surfaee sources of inhibitor by careful washing in saline, followed by
the use of RDE ou the iutact excised membrane.
TABLE 3.
Action of SDK on intact membrane.
Treatment of membrane before grinding
Incubated with RDE (4,000 units') for
oneliour
Nil
Inhibitory titre of ground membrane suspension
H-LEE
TMEL
240
480
Inhibitor titrated against 5 A.D. of each virus.
320
060
INTEKFEKENCE IN INFLUENZA VIRUSES. Ill
607
The membrane wun removed, cleared of jidlicring egg-vvliite and washed thoroughly in
normal tiulinc. It was suspended in 5 ml, of a 1 in 5 dilution of RDE (4,000 units) in ciiU-iuuiatctate-naliiie and iufubated at 37° C. for one hour. The niunibrane was washed in citriitesaline (to reduce activity of tlie BDE immediately) and then ground aud suspended iu 1 ml.
of eitnite-saiine. Tlie suspension was diluted 1 in 10 in normal saline, heated at 65° C. for
30 minutes in order to inactivate the euzyme, and titrated for inhibitory activity against
H-LEE aud T-MEL, with suitable controis. Table 3 shows titros representative of many such
experiments.
Thus, treatment of tin- nu'iiiliraiu' before ^riruUnjr (i.e. surface treatment)
with 4,000 units ol' IU)K caused partial reduetiou in inhil)it(H-y titre whereas
treatment with ],U00 units of KDE after grinding eau-sed complete reduction
(see Table 2). Hence some of the inhibitor is not accessible to RDE action
before gi'lnding aud is presumably not present at the surface of the allantoic
membrane.
Action of Viruses on Menibrane Inhibitor in vitro.
A characteristic property of .soluble inhibitors is the "inhibitor gradient'*
(Stone, 1949b}, analogous to the red cell reeeptor gradient (Buniet, McCrea
and Stoue, 1946). The inhibitory activity of certaiu mncoids for dilferfent
indicator viruses is destroyed by active viruses to varying extents. Thus Stoue
showed that the inhibitory aetivity of ovomucin for various indicator strains was
reduced by virus action in the order II-WSE, H-LEE, T-MEL; e.g. LEE virus
destroyed iuhibitor for Ii-WSE und II-LEE but not for T-MEL, whereas MEL
virns destroyed iuhibitor for all three. The gradient was characteristie of a particular mucoid but varied from one mucoid to another.
TABLE 4.
Action of viruses on inhibitor in ground mevibrane suspension.
T-PB8
WSE
PR8
MKL
BEL
LEE
67
<5
<5
Inhibitory titre (percent.)
H-WSE
T-MEL
T-BEL
33
17
10
14
<o
67
'2G
<5
7
<5
84
:i4
21
l.T
<6
H-LEE
78
62
35
HO
5
Membrane siispension inciibateil for 18 hours with active viruses.
Eesiduai inhibitor titrated against 5 A.D. of indicator viruses; expressed as percentage of
titre, with that indicator, of nu'mbriuie incubated with normal saline.
The inhibitor gradient of membrane auspcnsion was determined by tlie method of Burnet
(1949) as follows. Several membranes were pooled, ground and suspended in normal saline,
1 ml. of saline per membrane. 0-25 ml. of eaeh test virus diluted to 100 A.D., was added to
1 ml. of membrane susi)enBion. A drop of penicillin (containing 200 units) was added, and the
mixture incubated for 18 hours at 37° €., with a control containing saline instead of virus.
After iucubatioii, each mixture was diluted 1 in 10 in uorinal saline and heated at 65° C. for
608
MARGARET KDXEY AND ALICK ISAACS
:iO miuutcsi to iiiactivati' virtis hacuiagglutiuiii. The iuhibitory tonteut wus titrated ngainst tha
test viruses in the iiidieator statt'. The results of a icpicstjiitativc cxi»ciiment art; shown in
Table 4, where tlie inhibitory titres following virua action are expressed as percentages of control titres.
It is seen that tlie order in whieh inhibitors were destroyed was T-1*R8,
H-WSE. T-MEL, T-BEL, H-LEE. The increasiug order of activity of th.:
viruses was WSE, PR8, MEL, BEL, LEE.
Action of Vimses on Membrane Inhibitor in vivo.
Since some viruses were found to be very active enzymicaliy ou uieinbrane
iuhibitor in vitro it was of interest to see whetlu-r membi'a)ie inhibitor was
destroyed during growth of intluenza virus in the developing chick
I'mbryo. Tlie time of disappearauce ol' iuhibitor iroui the lueiiibraue was investigated in parallel
with the time of appearance of
liaemagglutiuin.
Henle aud Henk- (1949) have
showu that when a large amouut
of PR8 virus is iujeeted iuto the £
allautoic sac, multiplication of
virus as measured by increase iu
haemagglutinin is first detected in
the chorio-allantoic membrane
after about 4 hours, aud geuerally
1 hour later iu the allautoic fluid.
In the present experiments disappearance of iuhibitor and proO
a
4
6
•
lO 12
]4
ductiou of Iiaeniagglutiniu iu tlie
TIME (ho«..J
uicjubraue were traced after inoculation with MEL virus, the Fig. 1. Iteduetion of Inhibitor in Menibrune during growth of MICL virus.
"lag" period for MEL virus, i.e.
* Inliibitury titre of membrane eusthe time before the iucreasc in *
sion ugainBt ;") A.D. of T-MEL.
liaciuafrglutiiiiu iu the allautoic
O Haemagglutinin titre of membrane
tliiid, having previously been
suspension.
found to be about I) hours (unHaemagglutinin titre of allantoic
published experiments).
fluid.
Tlie points iu brarkets rtipresent titres leas than
0-1 ml. of 1 in 10 dilution of MEL
tlie value shown.
(^8 A.D.) was inoi-ulateil allantoically
into each of a group of '21 U-day t'lnliryo:^ wtiicli were iiifiiliatcil \vitli G control embryos at
35° C. At 2 liourly iiiteivals, three inoculated eggs :nid oiu' control were removed from the
incubator, samples of alluntoic fluid taken, and the membranes removed. These were washed,
ground and suspended each iu 1 ml. of normal saline. A portion was immediately diluted 1 in
10, heated at (J5° C. for 30 minutes to inaetivate haemagglutinin. yiid titrated for inhibitor
TXTERFERKNCE TN INFLUENZA VIRUSES. TTT
609
against T-MEL. At the same time, the allantoie fluids ami uiilioiited membrane suspenaiona
were titrated for tiaemagglutiniii. Potitvols .showed that inliihitor jiri'aent in the membrane and
fluid did not affoot the Iiaemagghitiiiin titre of MEL virus iii tlii^ coiiiTntMition, since this titio
WIIH not increased by treating the samples with excess EDE
Tlie results are plotted in Fig. 1. Each point represents t!ie geometric moan of resulta
of individual titrations of three samples.
Thus MEL virus caused marked reduetiou of tho membrane inhibiiory
titre in vivo, the drop in titre Ijefrinninjr at the same time as haemap:;:liitinin
appeared in the membrane.
A possible objection to thp technique is tlmt virus might react with membrane inhibitor
released during grinding. In a number of eontrol pxpt'riment>i, in which varying amounts of
virus and ground membrane were left at room temperature for periods up to an liour, there
waa nn demonstrable drop in intiiliitory titre, i.e. virus did not aet appreciably on membrane
inhibitor in iess than an liour, at room temperature.
Memhrane InhihUnr Oiadifvl in vivo.
Tlie inhibitor *;radient iv vivo was determined by titrating: the residnal
inhibitor in membranes taken from infected embryos. Efrfrs were inoeiilated
allantoieally with lO"* TDr,,, of virns, and after 42 hours' ineubation, membranes were removed and ground. They were snspended in normal saline,
diluted 1 in 10, heated at 65° C. for 30 minntes and titrated for inhibitory
activity ajjainst five different indieator strains.
The results of these experiments were variable. The general picture
showed that the order of the inhibitor gradient was the same as in vitro, i.e.
T-PR8, H-WSE, T-MEL, T-BEL, and H-LEE. However, LEE and BEL
viriises were not as aetive in vii^o as expected from the n? vitro results. Thus
LEE virus, which was more active than MEL in vitro, showed a much lower
action in vivo even when LEE infeeted eggs were incubated for 48 hours longer
than the M'EL eggs.
Allantoic Fluid Inhibitor Gradient.
The inhibitor gra<li('iit of normal allantoic flnid was investigated at tbe
same time as that of the membrane suspension. Modification of the method
nsed for membrane inbibitor was necessary, because long incubation of allantoie fluid with most viruses caused almost complete destruction nf inhibitory
activity.
1 ml. of allantoie fluid waa incubated at 37° C. for one hour, with 0-25 ml. of virus containing 25 A.D. and with normal saline as a control. Haemagghitinhi was inactivated by
heating at 65° C. for 30 minutes and the inhibitory activity titrated.
The inhibitor gradient of allantoic fluid was found to be H-WSE, H-LEE,
T-MEL. For these three viruses the gradient of membrane suspension was
H-WSK. T-MEL. H-LEE. and that of ovomuein H-WSE, H-LEE, T-MEL
(the latter from Stone, 1949b).
MARGARET EDNEY AND ALTCK T S A A C S
610
The Effect of Interference ou the Action of Vintse.<i on Memhrane Inhibitor.
It was of interest to see whether the suppression of liaemafjglutinin production oeeurriug in interference was aeeompanied by a reduction iu the
activity of virus on membrane inhibitor. The heated virus used as an interfering ajrent is eiizymieally inactive and would uot therefore be expected to
affect the experimental results.
Interference experiments were earried out aa in Methods, uaing H-LEE as interfering
virua and MEL ns ehnllenge. After the alljintoic fluids were harvested and titrated, the membranes were removed from the pggs, and suspensions prepared and heated to destroy haemagglutinin, Trhibitor against T MEL wjis estimated: the reaults of a representative experiment are
shown in Table 5.
TABLE 5.
Lacic of inhibitor deniriiction in interference.
Inoculum
Nil—control
H-LEE—control
H-LEE + aftive MP:L
Active MEL
Mean liaemngghitinin titres of
allantoic fluids
Mean inhibitory titres of
chorio-allantoie membranes
17
450
480
420
380
17
Interfering virua—400 A.D. of H LEK.
Challenge viriis—10- IDr^o active MEL,
6 eggs per group: geometric mean titrea are shown.
Inhibitor titrated against 5 A.D. of T-MEL.
It can be seen that where interference has considerably reduced haemagglutinin produetioii no significant inhibitor destruction ean be detected.
DISCUSSION.
The properties of inhibitor in choi io-allanioie membrane suspensions are
similar to those described for other inhibitors of influenza virus haomagglutination: membrane inhibitor is heat stable, has {,n-eater inhibitory activity against
heated than unheated virus, is sensitive to the enzymic action of influenza
viruses and the receptor-destroying enzyme of V. cholerac, RDE, and shows a
characteristic inhibitor gradient. It is interesting that quite a large part of
tbe inhibitory activity of the membrane suspensions is not accessible to the
action of RDE before the membranes are ground. This suggests that part of
the inhibitor is intracellular—a suggestion which is supported by the fact that
inhibitor is accessible to the action of influenza viruses during their multiplication in vivo.
INTERFERENCE IN INFLUENZA VIRUSES. IIT
611
The enzj'mie action of viruses on membrane inbibitor in vivo might occur
during the initial stages of multiplication of virus, i.e. before haemagglutinin
is produced or at a stage when the increase in baeiuagglutiinn can l)e detected
in tlie membrane. The results favour the latter alternative, luteri'erence
inhibited liaemagglutinin production and inbibitor destruction, and inbibitory
activity in the membrane was not reduced before iin-rcase in haemagglutinin
cMiidd be detected (Fig. 1),
The inhibitor gradient of chorio-allantoic membrane suspension differs from
that of allantoic fluid. This is evidence against Svedniyr's (1948) assumption
that allantoic fluid iiihihitor represents cellular receptor substance in solution.
During speculation on the function of intracellular inhibitor in virus multiplication, the possibility of "Intracellular virus receptors" arose, de Burgh
(1948) has shown Ihat tissue particles separated frnin nornijil rat lung by
differential eentiifugation were agglutinated by influenza viruses, that the
combination of particles and virus inhibited virns haemagglntiiiation. and that
RDE ])reveuted combination of virus and tissue particles. Tntraccllular virus
receptors miglit play a part iu the interferenee phenomenon, particularly in
view of the high affinity of heated (interfering) virns for chorio-allautoic membrauc inhibitor. Tf so, quantitative differences in behaviour in interference
might l)e expected among different viruses dcpendiun' ou tlieir relative positions
in tlie chorio-allantoie membrane inhibitor gradient. This possibility is at
present under investigation.
SUMMARY,
Some properties of iu hibi tor of haemagglutinatiou by influeuza viruses
found in suspensions of grouud cliorio-allantoic membranes tiii\e been tlescribed.
Membrane inhibitor is heat-stable, lias greater inhibitory activity against
heated than unheated viruses, and is destroyed by the V. cholerae enzyme, RDE.
In the intact membrane, part of the inhibitor is inaccessible to the action
of RDK and is probahly intracellular.
Membrane inbibitor and allantoic flnid inhibitor differ from one another
in the order of their inhibitor gradients.
Influenza viiuscs act enzymically on membrane inhibitor both in vitro after
normal membrane has been ground and in vivo duriug growth of the viruses in
the developing chick embryo.
During interference, when production of virus hapniatrglntinin is prevented, no reduction in inhibitor can be demonstrated.
612
MARGARET EDNEY AND ALICK ISAACS
REFERENCES.
Bovarnick, M. and de Burgh, P. M. (1947) : Scienee, 105, p. 550.
de Burgfa, P. M, (1948): personal communication to Prof. F. M. Burnet.
Burnet. F. M. (1949) : Austral. J. exp. Bid., 27, p. 575.
Burnet, F. M., McCrea, J. F. and Anderson, S. G. (1947) : Nature, 160, p. 404.
Burnet, F, M., MeCtea, J. F. and Stone, J. 1). (1946) : Brit. J. eitp. Path., 27, p. 228.
Bnrnet, F. M. and Stone, J, D. (1947) : Austral, J. exp. Biol., 25, p. 227.
Fazekaa de St.Groth, S. (1948): /btd., 26, p. 29.
Francis, T. (1947) : J, exp. Med., 85, p, 1.
Friedewald, W. F,, Miller, E. S, and Wliatley, L. E. (1947): J. exp. Med., 86, p. 65,
Henle, W. and Henle, G. (1949): Ihid., 90, p. 23.
Hirst, G, K. (1943) : Ibid., 78, p. 99.
Isaacs, A. and Edney, M. (1950a and b) : Austral. J. exp. Biol., 28, p. 219 and 2^1.
Lanni, F. and Beard, J. W. (1948) : Proc, Soc. exp, Biol., 68, p. 312.
Stone, J. D. (1947) : Nature, 159, p. 780
Stone, J. D. (li)49a) : Austral. J. exp. Biol,, 27, p, 337.
Stone, J, I>. (1949b) : Ihid., 27, p, 557.
Svedmyr, A. (1947) : Ark. Kom. Min. Geol., 248, p. 1.
Svedmyr, A, (1948): Brit, J. exp. Path,, 29, p. 295.