SQME CHARACTERISTICS OF THE
HEMAGGLUTIMATBNG ACTNITY
OF iNFECTEOUS BRONCHETES VlRUS

Thesis in: tho Dam cf Ph. D.
MlCHIGAN STATE UNWERSITY
Robert I.» Munn
i960

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IYfis is to cerfifg that the
thesis enthled
Some Characteristics of the

Hemagglutinatiny Activi

CV
of Infectious Bro

tchitis Virus

presented b9
Robert Lee Huidoon

has been accepted towards fulfilhnent

of the requirements {or

I); . '7. l . .
..1 0. degree 1n In cr()ot()logy

and Public Heath

hIajor professor

Dam Februarv 2 JJOO
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SOME CHARACTERISTICS OF THE HEMAGGIUTINATING ACTIVITY

OF INFECTIOUS BRONCHITIS VIRUS

By

ROBERT LEE MULDOON

A THESIS

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and Applied
Science in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1960

 

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This thesis is

respectfully dedicated

to

MY FAMILY

 

 

APFWRACT

mhe common method for titration of infectivity of
infectious bronchitis virus and its antibody uses the
embryonating chicken egg as the indicator. Hemagelutina~
tion by this virus can be demonstrated after it has been
modified by tryDSin.

The present study deals with certain properties of
the trypsin modified hemagglutinin of infectious bronchitis
virus with the primary purpose of development of a hemagglu-
tination~inhibition,test.

The hemagglutination titers obtained after 30
minutes incubation with trypsin at 56 C are comparable to
those obtained after three hours incubation at 37 C. ggga
white trypsin inhibitor is added after incubation. The
maximum hemagglutination titer is obtained when one percent
trypsin and one percent eggwhite trypsin inhibitor is used.

Trypsin modified virus can be stored at ~65 C for
three w-eks without loss of hemagglutinating activity.
Chicken erythrocytes could not be stored in Alsever’s
solution for longer than one week and be used in the
hemagglutination test.

The only reactive erythrocytes for hemaqglutination
of IBV are those from chickens and turkeys. Cells from

chickens three weeks or older are reactive, but maximum

11

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“w-....~ em

I *‘m'*' h....__ .

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activity is observed from birds five weeks or older.

The hemagglutinin prior to trypsinization of
infectious bronchitis virus is stable at 56 C for at least
six and one-half hours, whereas infectivity is lost within
two hours. Antigenicity as detected by serum neutralization
tests is retained for two hours at 56 C. The infectivity of
the virus decreases significantly after incubation with
trypsin at 37 C.

Analysis of viral multiplication and hemagglutinating
activity reveals that the hemagglutination titer is maximal
at 72 hours post inoculation whereas infectivity is highest
at 24 hours. No decrease of infectivity is observed after
reaction of the trypsinized virus with chicken erythrocytes.
Filtrates from a 50 millimicron filter are infectious, but
hemagglutinating activity is not demonstrated.

The activity of the receptor destroying enzyme of
Vibrio cholerae is of the same magnitude when tested with
influenza strain PR8 or infectious bronchitis virus 17-40.

Extracellular fluid from virus infected chicken
embryo kidney cell culture gives the same hemagglutination
titer as non-infected fluid. Hemadsorption is not observed
prior to or after trypsinization.

The development of a usable hemagglutination-inhi-
bition test probably depends on the removal of inhibitors
of hemagglutination which are present in negative and anti—

infectious bronchitis virus sera. Heat or filtration does

111

-wn. ¢

 

 

 

 

 

not remove inhibitors. “rypsin, periodate, butanola ether,3

zymosan or the receptor destroying enzyme of Vibrig,oholcrae
does not influence the action of serum inhihitorfl when
trypsin treated serum was subseouently reacted with periodic

acid, all inhibitors are destroyed.

iv

4!

 

 

 

 

EC KNO WLEII} I“; ENG" S

Deep appreciation is expressed to Doctor Charles H,
Cunningham, Professor of Microbiology and Public Health,
for his guidance throughout this thesis.

The author also wishes to acknowledge the interest
shown by Doctor Lloyd C. Ferguson, Dean of the College of
Science and Arts; Doctor Richard U. Byerrum. Assistant
Provost; Doctor James L. Dye,9 Assistant Professor of
Chemistry; Doctor Ralph C. Eelding, Associate Professor
of Microbiology and Public Health; and Doctor James L.
Fairly, Associate Professor of Chemistry.

The author is also indebted to his fellow colleagues
for their help and criticisms, with special appreciation
for the help tendered by Mrs. Martha P. spring.

This study was supported by the Agricultural

Experiment Station of N chigan State University.

14 ‘-....~-.-mn-.‘t._-._...—-1
,
I

 

P A

L .‘L, ‘ LE OF CONT FCNT 5;

J

I

Page
Introduction . . . . . . . . . . 1
Literature Review. . . . . . . . 2

Material and Methods . . . . . . 15

Results. . . . . . . . . . . . . 23
Discussion . . . . . . . . . . . 82
summary. . . . . . . . . . . . . 96

Bibliography . . . . . . . . . . 99

vi

 

Table

II

III

IV

VI

VII

VIII

IX

 

LIST OF TAPLEd

The Hemagglutination Titer of IBV 18-11
Modified by Trypsin at 37 C . . . . . . . .

The Hemagglutination Titer of IBV 40-23
Modified by Trypsin at 37 C . . . . . . . .

The Effect of Time and Temperature on the
Induction of Hemagglutination of IBV 17-37,
40-19, and 41-8 by Trypsin . . . . . . . .

Comparison of Time-Temperature Relation
and Concentration of Trypsin-Eggwhite
Trypsin Inhibitor Used for the Induction
of HA of IBV. . . . . . . . . . . . . . . .

The Effect of Time and Temperature on the
HA Titer of Trypsin Modified and Untreated
IBV no- 2 3 c I o c o o o c o o o o I o c l o

The Effect of Age and Infection on the
Reactivity of Chicken Red Blood Cells with
the Hemagglutinin of IBV 41 . . . . . . . .

The Effect of Age and Infection on the
Reactivity of Chicken Red Blood Cells with
the Hemagglutinin of IBV 41-3 . . . . . . .

Thermostability of the Hemagglutinin of
T20 Different Egg Passages of IBV 41 at
5 C. O D I O O O I O O C c C I o O O O O 9

Thermostability of the Hemagglutinin
and Infectivity of IBV 17-37 at 56 C. . . .

Thermostability of the Hemagglutinin
and Infectivity of IBV 16-43 at 56 C. . . .

Thermostability of the Hemagglutinin
and Infectivity of IBV 3—15 at 56 C . . . .

Thermostability of the fiemagglutinin
and Infectivity of IBV 40-23 at 56 C. . . .

vii

Page

24

24

27

31

33

36

39

41

43

43

44

44

 

 

“q“. _ '
-‘_1—,_‘ ._ _- ‘l‘. ah-

XXVI

Thermostability of the Hemagglutinin
and Infectivity of IBV 40-23 and
“1'9 31: 56 C o o o o c o c a o . o o o o o

The Effect of Trypsin at 37 C on the
Infectivity of IEV 41-7. . . . . . . . . .

The Effect of Trypsin at 37 C on the
Infectivity of IBV 40-23 . . . . . . . 0 .

The Effect of Trypsin at 37 C on the
Infectivity of IBV 3-16 . . . . . . . . .

The Effect of Trypsin at 37 C on the
InfeCtIVIty of IBV 3‘16 0 o I o o I o c I

The Effect of Trypsin at 37 C on the
Infect1v1ty 0f IBV “1’9 - a o o o o o o I

Infectivity and Hemagglutinatin§ Activity
of IBV 41-6 (Undiluted Inoculum . . . . .
Infectivity and Hemagglutinating Activity
of IBV 41-6 (Inoculum Diluted 10‘ ), , , .

Infectivity and Hemagglutinating Activity
of IBV 40—23 (Undiluted Inoculum) . . . .

Infectivity and Hemagglutinating Activity
of IBV 17—40 (Undiluted Inoculum) . . . .

Infectivity Titers of IBV 40-23 After
Hemagglutination . . . . . . . . . . . . .

The Effect of Ultrafiltration Upon
Separation of the Infective Particle
and the Hemagglutinin of IBV 40-22 . . . .

The Effect of Ultrafiltration Upon
separation of the Infective Darticle
and the Hemagglutinin of IBV 40-22 . . . .

The Effect of Modified 1BV on the
kroduction of Neutralizing Antibodies
Neutralization Indices of Pre-

infection Sera . . . . . . . . . . . . . .

The Effect of Modified IBV on the
Production of Neutralizing Antibodies
Neutralization Indices of Post—

infection dera . . . . . . . . . . . . . .

viii

 

Page

57

57

60

60

64

b5

70

I')1

73

76

77

 

Table Page

KXVIII The Effect of Freon on Hemagglutination
Of IBV “0‘23 9 o o o o o o I 0 o o O o o O 79

XXIX Titration of RDE Using PR8 and IBV 17-40 . 80

ix

 

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Figure

10

ll

12

13

LIST OF FIGURES

The Hemagglutination Titer of IBV 18-11
and 40-23 Modified by Trypsin at 37 C. . .

The Effect of Time and Temperature on the
Induction of Hemagglutination of IBV 17-37
by Trypsin o O O O 0 I o c O c o c O o l 0

The Effect of Time and Temperature on the
Induction of Hemagglutination of IBV 40-19
by Trypsin o o o o o c o o o o o o o o o o

The Effect of Time and Temperature on the
Induction of Hemagglutination of IBV 41-8
by Trypsin . . . . . . . . . . . . . . . .

The Effect of Age and Infection on the
Reactivity of Chicken Red Blood Cells with
the Hemagglutinin of IBV 41. . . . . . . .

The Effect of Age and Infection on the
Reactivity of Chicken Red Blood Cells with
the Hemagglutinin of IBV 41- 3. . . . .

Thermostability of the Hemagglutinin and
Infectivity of IBV 17- 37 at 56 C . . . . .

Thermostability of the Hemagglutinin and
Infectivity of IBV 16-43 at 56 C . . . . .

Thermostability of the Hemagglutinin and
Infectivity of IBV 3-15 at 56 C. . . . . .

Thermostability of the Hemagglutinin and
Infectivity of IBV 400- 23 at 56 C . . . . .

The Effect of Trypsin at 37 C on the
InfeCtIVICY of IBV 41-7. 9 o o o o c o O O

The Effect of Trypsin at 37 C on the
Infectivity of IBV 40-23 . . . . . . . . .

The Effect of Trypsin at 37 C on the
InfeCtIVIty Of IBV 3‘16. 0 o o o o I o 0 O

X

 

Page

25

28

29

30

37

4O

46

47

 

Figure Page

14 The Effect of Trypsin at 37 C on the

InfeCtIV1ty Of IBV 3'16. 0 o o o o o o c o 58
15 Infectivity and Hemagglutinating Activity
16 Infectivity and Hemagglutinating Activity

of IBV 41-6 (Inoculum Diluted 10-3)_ , , . 62
17 Infectivity and Hemagglutinating Activity

of IBV 40-23 (Undiluted Inoculum). . . . . 66
18 Infectivity and Hemagglutinating Activity

of IBV 17-40 (Undiluted Inoculum). . . . . 67

xi

 

INTRODUCTION

Infectious bronchitis virus and its neutralizing
antibody are generally assayed using embryonating chicken
eggs as the indicator host of viral infectivity.

Infectious bronchitis virus will not agglutinate
erythrocytes, but when the virus is treated with trypsin
hemagglutination occurs.

The present studies are concerned with the charac-
teristics of some of the properties of the agglutinating
agent with the primary purpose being the possible develop-

ment of a hemagglutination—inhibition test.

 

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LITERATURE REVIEW

The virus of infectious bronchitis of chickens is
classified as Tarpeia pull; by van Rooyen119. This disease,
first described by Schalk and Ham“2 in 1931, is limited
to chickens.

Infectious bronchitis virus (IBV) as determined by
electronmicroscopy is a sphere ranging from 60 to 100 milli-
microns91-92. Filamentous projections are present in Speci-
mens prepared in saline or in distilled water, but they are
more prominent in the former. These filaments have not been
detected in the infected chorioallantoic membrane where the
elementary bodies have a mean diameter of 200 millimicrons31.

The infected tracheal mucosa of the chicken undergoes
cyclic changes of acute, reparative, and immune phases for
18 to 21 days7o,

The embryonating chicken egg is used as the indica-
tor host for assay of infectivity of IBV and its antibody.
Serial passage of IBV in the embryonated egg results in an
increase of virulence for the embryo and a loss of antigeni-
city for the chicken5o30.

Viral activity in eggs can be detected by macroscopic
as well as microscopic alterations. Pathognomonic lesions
0f the embryo are caused by early egg passage virus.

"Curling" of the embryo consists of a wry neck and deformed
2

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feet compressed over the head. Embryos may be dwarfed as

much as one half size with a firm, ball like shape5'30!33'78.
Another indication of viral infectivity is the presence of
urates in the mesonephros78.

Some microscopic changes in virus infected cells are
edema of the chorioallantoic membrane; hepatic hemorrhage,
necrosis, and abcess formation; and interstitial nephritis
and necrosis. Inclusion bodies have not been observed78.

Embryo mortality is not a constant finding in the
26

early egg passages of IBV. Cunningham and Jones observed
the greatest direct correlation between embryo mortality and
number of egg passage when eggs were inoculated via the allan—
toic cavity. Although this virus may be cultivated using
several routes of inoculation, the allantoic cavity is the
route of choice because of simplicity of operation and

amount of infected material recovered.

binghIOh found that egg propagated IBV has a di-
phasic characteristic and a bimodal rate of inactivation at
56 C which is indicative of a three-halves order reaction.
At room temperature, virus infected allantoic fluid remains
viable for five to seven daysae.

Inactivation of this virus is achieved within three
minutes by certain chemicals, of which some are, one percent
phenol, one percent metaphen, 90, 70, 40, and 25 percent
ethanol and one percent formalin27.

Infectious bronchitis virus is more stable at 4 C

 

._..’~ At-.-

 

 

 

 

in an acid medium than in an alkaline medium for 60 days.

However, the reverse is true from 60 to 170 days with an
optimum pH of 7.80. The isoelectric point is probably
about pH 4.0528.

Buthalalg determined the density of IBV to be
approximately 1.15 with minor variations depending on the
solvent. He was unable to concentrate the virus by centri-
fugation at 40,000 g for two hours.

Infectious bronchitis virus does not possess hemagglu—
tinating activity detectable by the usual methodsSu'és.
Corbo and Cunningham25 have shown thar virus-infected allan-
toic fluid, to which trypsin and eggwhite trypsin inhibitor

(ETI) have been added, contained an active hemagglutinating

agent.
Hemagglutination
Erythrocytes may be agglutinated by extracts of
plant86 and animal tissues105, the higher fungi35, bacteria86

rickettsiae and pluropneumonia-like organiSms35 as well as
viruse857. Hemagglutination (HA) by viruses offers a tool
for analysis of the virus-cell interaction and definition
of some biochemical properties of the virus. It may also
serve as a possible basis for the classification of viruses.
Viral hemagglutination, and the inhibition of this

reaction by specific antibody, was first reported by

 

 

 

 

 

 

 

 

 

5

Hirst57 in l9hl, Working independently, McClelland and
Hare79 also reported on viral HA, hemagglutination-inhibition
(HI), and the species of cells that may be used to charac-
terize the reaction.

Viruses which participate in HA have been classified
into four groups; 1) myxoviruses, 2) arthropod-borne viruses,
3) pox viruses, and 4) viruses which cannot be grouped into
the first three categories63.

The myxovirusesu

are those of influenza, Newcastle
disease, fowl plague, and mumps. Basic research on the
mechanism of HA of this group has been done with the PR8

strain of influenza as the prototype. The members of this
group possess the ability of splitting neuraminic acid
derivatives from mucOproteins which are present in body

fluids and on the surface of erythrocytes63. The hemagglutinin
is part of the virus particle58’59.

The sequence of reaction is adsorption of the virus
onto the receptor sites on the cell, agglutination, and
finally eluticn of the virus from the cell after destruction
of the receptors.

At the time of adsorption of the virus to the cell,
the reaction is reversed by decreasing the salt concentration
0f the diluentllz. Adsorption may occur over a wide virus-—
cell ratio. The temperature may range from 4 C to 37 C.

Physical data indicate that the initial attachment is ionic

whereas chemical data suggest that the attachment occurs

 

 

 

 

 

 

 

9
through the hydroxyl group on the substrate“0.
15

Adsorption can be prevented or limited“0 by control
of the ionic concentration, presumably by reducing the
mutual replusion of the virus and of the cell. The HA titer
of PR8 is constant within the range of pH 6.0 to 8.083. The
HA of Newcastle disease is optimal within the range of pH
5.8 to 6.2, although titers were obtained from pH of 5.0 to
9.097.

The second event in the sequence is agglutination

68 10?

of the cells. Isaccs and Edney , and Schlesinger and Karr
suggest that the hemagglutinin of the myxoviruses is poly-
valent, and forms a lattice of cells.

The final process is the elution of the virus from
the cell. Elution is dependent upon the presence of cations
of which calcium is the most efficientlfi. Sodium hexameta—
phosphate adversely affects the degree of elution probably
by negation of the effect of the calcium ion89. Active
virus can destroy the major portion of the receptors on a
cell without becoming detached from it12'53’110. bagik98
Suggested that the virus is fixed in one location and causes
a rippling disturbance on the remainder of the cell surface.
Burnet12 proposed that the virus migrates over the cell
Surface and in so doing destroys the receptors. Regardless
0f the mechanism, viruses can agglutinate fresh cells
repeatedly without loss of activity. Cells which were acted

upon by the virus were not reagglutinable by the same virus.

 

 

 

 

Hirst57 had previously supgested that the HA mechanism was
enzymatic in nature. Cells after as lutination with a given
virus may or may not be agglutinated by another virus. For
example, cells which have been acted upon by mumps will not
be agglutinated by influenza strain MEL. Cells agglutinated
by M:L will be agglutinated by mumps. such a series consti-
tutes a "receptor gradient" as described by fiurnetl7, and
adds supplemental support to Hirst's enzyme hypothesis.

The final proof of an enzyme reaction is the isolation
and purification of the enzyme and substrate. As a corollary,
the cleavage products should be identified and the kinetics
of the reaction studied. The components of the virus respon-
sible for infectivity, HA, and elution have not been
separated by physical meanssl‘117. Hoyle67 was able to dis-
integrate influenza particles with ether with the subsequent
release of two fractions, one of which carried HA activity.

Inhibitors of HA activity have been found in many
laiological materials one of which is urine. Tamm and Hors—
fald}lu were able to purify this inhibitor. When an electro-
Innoretically homogenous mucoid was incubated with purified
influenza virus type B, enzymatic cleavage occurredu7.

Klenk71 identified the end product of this reaction as

N-acetylneuraminic acid.

Inhibitors

There are three major inhibitors of viral hemagglu-

 

 

 

A]

 

 

 

 

tination. The first is specific antiserum which is produced

in response to the hemagglutinin proper or to the infective
virus particle. It is stable at 62 C for 30 minutes, and is
not destroyed by the receptor destroying enzyme (RDE) of
Vibrio cholerae, or moderate concentrations of periodateég.

The second inhibitor is the thermolabile Chu inhi-
bitorgo, present in serum, which will at times reduce viral
infectivity. It is precipitated with gamma globulin, and is
destroyed by RDE3 only if the inhibitor preparation is
crudezz. Inactivation occurs in the presence of trypsinllé,
but the action of periodate is debatable22'6o.

The third inhibitor of HA activity is a mucopoly-

saccharide found in many normal biological materials, such

as serumuz, red blood cellsuB, allantoic fluidlle, and
human urinellB. The initial work was done with the serum
mucoid.

Stone108 observed that when influenza virus was

heated at 56 C, the ability of the virus to elute from cells
was destroyed. This was named an indicator virus. Certain
strains required specific conditions before conversion
occurred. Indicator virus may be produced by the action of
trypsinlog. Incubation with one mg per ml of potassium
periodate will yield an indicator virus, but HA activity
is destroyed when eight mg per ml of periodate is used39.
Francis“2 observed that the HI titers obtained with

indicator viruses have no correlation with Specific antibody

 

 

 

 

as determined by neutralization tests. Indicator virus has

no effect on the inhibitor, which is destroyed by an active

virus9’6o.
Burnet16 found that this serum inhibitor precipitated
with the globulin fraction and is heat stable. Hirst6O noted

a marked reduction in inhibitory titer of normal rabbit serum
after incubation with trypsin. The non-specific inhibitor

of HA of chicken erythrocytes by mumps and Newcastle disease
is eliminated by treating the serum with trypsin and per-

121. The inhibitor of influenza virus in allantoic

55

iodate
fluid can be destroyed by trypsin and periodate77. Lanni
and Beard74 destroyed the eggwhite inhibitor with dilute

periodate. Inhibitors of some arthropod-borne viruses can

be removed by filtration of the serum21. Normal serum may

be dialysedlz2 or treated with sodium citrate29

to remove
the inhibitor of certain myxoviruses.

Burnet18, and Friedewald gt g;.n3 showed that the
inhibitor described by Francis is similar to the receptor
substance of intact erythrocytes to which the virus attaches
to produce HA.

Capsular material frOm pneumococci23 and a toxin from
(Ilostzidium welchii81 exhibit the same effect as the viral
enzyme in that red cells are rendered inagclutinable. Anderr
son gfl.3
destroyed by RDE from 1‘ cholera‘ It is unknown whether

this latter enzyme preparation is homogeneousla.

demonstrated that the "Francis inhibitor” is

 

 

—--———-:-'r~ ‘I

 

 

fig“

 

 

10

The activity of RDE is optimal at pH 6.2, and calcium
is required32. The activity is destroyed after 30 minutes at
52.5 C in the absence of calcium, but is stable for 60 minutes
in the presence of M/lOO calciumz. RDE is destroyed by

10'109. The electro-

crystalline trypsin at pH 7.2 to 8.5
phoretic mobility of the cell is decreased after the action
of BBB2 or more active virus9o, indicating that a new surface
antigen may be produced or uncovered by the action of the

RDEl3. All mucoid inhibitors which inhibit the HA of indi—
E10,62,109‘ If

the mucoid is in solution, complete destruction occursll.

cator viruses are rendered less active by RD

If RDE is administered to mice prior to infection by in-
fluenza virus, the susceptible tissue is resistant to infecr
tion, probably due to the stripping off of receptor site on
the cells107.

The action of active virus and RDE on mucoid inhibi-
tors is the same in all cases but two. According to Tamm
and Tyrrellll5, BBB has no effect on the inhibitor in normal
allantoic fluid and human urine which affect the HA activity
of murine encephalomyelitis GD VII and certain influenza
strains. By starch zone electrophoresis, it has been shown
that it is chemically distinct from the LEE influenza B in-
hibitor. The inhibitor of Newcastle disease virus, in
allantoic fluid, is not destroyed by this enzyme.

Although all inhibitors which have been studied

extensively are mucopolysaccharide, certain differences

 

11

are observed. The degree of inhibition of HA by active

virus varies with the source of the inhibitora. If an in-

hibitor which had been degraded by active virus was subse-

quently tested with an indicator virus, a receptor gradient

was established showing characteristic differenceslog. The

action of periodate differs with various inhibitors9'37i

In an attempt to analyse the composition of the

substrate for the viral enzyme, inhibitors from various

sources were isolated and studied. The urinary inhibitor

purified by Tamm and Horsfall113 has a molecular weight of

7 X 106. It is soluble in water but insoluble in 0.85

N NaCl, and contains 20 percent carbohydrate by weight.

Hexoses, hexosamines, and 2—carboxy~pyrrole appear in equal

amount83. It is destroyed by active influenza virus and the
receptor destroying enzyme of 1. choleraela. The product of
‘the reaction of highly purified influenza B virus on electro-
lakuoretically homogenous urine mucoprotein or ovamucir.47
reesembles N-acetylglucosamine. On further analysis, Klenk71
icientified the end product of the reaction as N-acetylneura-
minic acid.

Green and Wooley52 reported that HA of PR8 strain

Of‘ influenza was inhibited by the presence of an extract
Otrtained by heating chicken red blood cells at 100 C for
fisve minutes. Friedewald gt al.u3, using a Waring blendor,
extracted an inhibitor from chicken cells. This inhibitor

was rapidly inactivated at 65 C. An extract obtained when

 

12

dry ether was used was stable for two hours at 100 C66,

Other extractions were performed using ether, chloroform and
acetone in sequence85. McCrea82 idolated an inhibitor using
a serial extraction method of pentane and ethanol followed
by cyclic freeze-drying. Trypsin had no effect on this in—
hibitor, but 0.005 M periodic acid destroyed it. Hirstél
found that 0.001 M periodate could completely destroy the
receptors of a 1.5 percent suspension of cells.

sialic acid, the group name for ON-diacetylneuraminic
acid, N—acetylneuraminic acid, N—glycolylneuraminic acid,
and other acylated neuramic acids6 had been found in all

inhibitory mucoproteinsuet87. Klenk and Lempfrid72 were

able to isolate N-acetylneuraminic acid from human erythro—

cytes after reaction with RDE. Using treatment with certain
taases, GottschalkfiO isolated N-acetylneuraminic acid linked
l<etosidically to N-acetylgalactosamine from bovine sub-
nuaxillary gland mucoproteinug. Enzymatic action of influenza
vaiflll hydrolyse the disaccharide into these two components.
Neuraminidase is the name given to this supposed v1ral
erizyme. The action of BBB yields the same products as the
Viral enzymeso,

The second group of viruses which have been classi-
fixed on the basis of the HA mechanism are the arthropod-
bornae viruses, which are the typical encephalitic viruses,
Tire virus-cell reaction requires exacting conditions for

demonstration of HA activity. West Nile virus will only

 

 

 

 

 

react with cells from chicks not older than 12 hours and

from sheep at pH 7.595. The hemagglutinin is thought to be
associated with the virus particle96. There is no enzyme
found as with the myxoviruses, and RDE has no effect on the
cellular receptor site.

All known inhibitors of HA by this group of viruses
can be destroyed by ether, chloroform, benzene, acetone95,
or Sietz filtrationZI. The inhibitors are stable at 100 C
and not affected by potassium periodate95.

The third group of viruses includes those of
psittacosis and the pox diseases. They are characterized
by a hemagglutinin distinct from the viral particle. Vacci-
nia has two hemagglutinins which are separable by centrifu—
gationnu. In extracts from the chorioallantoic membrane,
there is no differentiation in activity. The agent first
sedimented in centrifugation is not destroyed at 70 C,
vvhereas the other is destroyed at 56 C within #5 minuteszn.
EBOth hemagglutinins are phospholipid, and are inactivated

toy lecithinase types A and C105.

6

The hemagglutinin of mouse meningopneumonitis5 and

ffieline pneumoniaué viruses is smaller than the infective
particle.

The fourth group of viruses includes those of ence-
ptualomyocarditis, which agglutinate only sheep cells, the
prueumonia virus of the mice which agglutinates only mouse

cells and the polyoma virus of mice. With these viruses,

 

«3". . '

 

 

 

14

the hemagglutinin is believed to be associated with the

viral particle, but there is no enzymatic destruction of

receptor sites. There is a question whether this group is

distinct from the arthropod—borne viruses.
The GD VII strain of Theiler's murine encephalomye-

litis virus agglutinates human group 0 cells at u C, but not

at 37 C73. After treatment with trypsin, HA activity can be

08“ Periodate and RDE have no effect

demonstrated at 22
on the cell receptors36. After 30 minutes at 56 C, there
was a 99 percent reduction of HA aotivity73.

Corbo and Cunninghamzs demonstrated that trypsin can
The rate and degree of adsorption of the

Elution

induce HA of IBV.

hemagglutinin varied directly with temperature.
varied indirectly with temperature. The HA activity was
inhibited by negative and anti-IBV serum, ovomucoid, and
riormal and virus—infected allantoic fluid. It was concluded
tliat the hemagglutinin was probably present in two fractions:
<311e in close association with the virus particle, and the

second, which is in the majority, was distinct from the viral

particle.

W—c» _— ._—‘—"_ ".

 

 

 

MATERIAL AND METHODS

Virus

The strains of IBV used were obtained from the North
Central Repository at Michigan State University, and were
identified as follows:

Repository Source
Code .
3 M. S. Hofstad, (104). Iowa State College, Ames,

Iowa. 1947. .

16 Q. Hipolito, (66), Universidade Rural de Minas
Gerais, Escola Superior de Veterinaria, Brazil.
1955.

17 F. R. Beaudette, (B-13-25), New Jersey Agricul-
tural EXperiment Station.

18 C. S. Roberts, (1005-7), Alabama Department of
Agriculture.

no G. H. Cunningham, (586), Michigan State Univer-

sity. 1956.

41 R. Van Roekel, University of Massachusetts.
1941. (29lst Bird Passage).

uz F. R. Beaudette, (B 588), Rutgers University,
New Brunswick, New Jersey. 1935. Egg Adapted.
The strains of IBV will be identified by the Rape.
SituDry Code Number and the number of egg passage; e.g.
3-CL6 indicates strain 3 in the 16th egg passage.
Embryonating chicken eggs, nine-to-eleven days old,
were used for the cultivation of all strains of IBV. The

15

 

16

standard inoculum was 0.1 ml per egg. Strain 42, which is
completely egg adapted, kills all embryos within approxi-
mately 36 hours. Strains in earlier egg passages do not
consistently kill embryos. The allantoic fluid was usually
harvested 72 hours after infection. pooled and stored at
-30 C in screw cap vials. Bacteriological sterility tests
were performed. Strains 41 and 42 from chicken embryo kidney
cell cultures, prepared by M. Spring, were also studied for
induction of HA.

At the time of use, the virus was thawed at room
temperature, and centrifuged at 3,000 rpm for 10 minutes.

The supernatant fluid was tested for studies of HA.

Reagents

A one percent suspension of Bacto trypsin (1:250)
111 distilled water was incubated at room temperature for
JHS minutes, and then passed through a Seitz EK filter. The
f‘fl.ltrate was dispensed into screw cap vials, and stored at
~230 C. The final concentration of this enzyme suspension is
\AIiknown due to the amount of adsorption on the filter.

A one percent solution of eggwhite trypsin inhibitor
(EIDI) in distilled water was prepared and stored at -30 C.
True solution was not filtered due to loss of activity.

The receptor destroying enzyme is obtained in

lyophilized form from Behringwerke, Marburg-Lahn, Germany.

 

 

 

 

 

17

It is reconstituted in distilled water and diluted in
calcium borate buffered saline. To 12 X 75 mm test tubes,
0.25 ml of one percent chicken red blood cells is added to
eoual volumes of diluted enzyme, and incubated at 37 C for
30 minutes. Ten HA units of test virus is added to the RDE
red blood cell mixture, and incubated at 37 C for 30 minutes.
The end point is the lowest dilution showing partial agglu-

tination.

Diluents

Bacto hemagglutination buffer was the commonly
employed diluent in the HA test. This buffer consists of
766 ml of M/l5 NaZHPOh and 233 m1 of m/15 Kszpou solution.
'Phe final pH is 7.3.

The diluent used for infectivity titrations was

131.fco Nutrient broth.

Earle's solution was used to prepare dilutions of
tlie virus infected allantoic fluid for HA and infectivity
tfl.trations in the experiments using an ultrafilter because
tine action of Bacto HA buffer on infectivity was unknown.

The ingredients of Earle's solution are:

Grams/liter , Grams/liter
NaCl 6.80 NaHzPOu'Hgo 0.1“
KCl 0.40 Ncho3 2.20
C3012 0.20 Glucose 1.00

MgSOu-7H20 0.20

 

18

The CaC12 was autoclaved separately to prevent its
precipitation from the solution.
Calcium borate buffered saline was used for the

dilution of RDE. The ingredients are:

Grams/liter
CaClz 1.000
NaCl 9.000
H3B03 1.203

Nazsuc7oioazo 0.052

The final pH should be 7.1 to 7.3.

Erythrocytes

Erythrocytes, obtained by cardiac puncture from
Single Comb White Leghorn chickens, were the standard cells
for the HA test.

ErYthrocytes from certain fowl, for the determina-
tion of cell spectrum, were collected at the Gull Lake Bird
Sanctuary. Cells from turkeys and mammals were obtained at
the College of Veterinary Medicine, Michigan state Univer-
sity.

The blood was collected in test tubes containing
1 ml of a two percent sodium citrate solution for each six
m1 of blood. The cells were washed three times by centri-
fugation at 1,800 rpm in 0.85 percent saline. After the
final washing, the saline was removed, the packed cells were

stored at h C, and used within five days.

 

19

For storage of erythrocytes, Alsever's solution was
used which consists of the following:
2.05 grams of dextrose
0.80 grams of sodium citrate
0.42 grams of sodium chloride
0.055 grams of citric acid
100.00 ml of distilled water
The ingredients were mixed, the pH was adjusted to
6.1, the solution was passed through a beitz EK filter,

placed in a sterile flask, and stored at u C.

Methods

The procedure, as described by Corbo and Cunninghasz,
for the HA test was the addition of one m1 of one percent
trypsin to two ml of virus infected allantoic fluid, and
incubated for three hours at 37 C in a water bath. Upon
removal, one m1 of ETI was added.

Serial two fold dilutions of the treated virus were
prepared in buffer. To 12 X 75 mm tubes was added 0.25 ml
each of the buffer, diluted virus and a 0.5 percent suspen‘
sion of chicken erythrocytes. The control contained 0.5 ml
of buffer and 0.25 ml of cells. The tubes were shaken and in—
cubated for 50 minutes at room temperature.

The end point was the highest dilution of virus
which showed complete hemagglutination. The titer, expressed
as hemagglutination units, was the reciprocal of the highest

dilution of virus, before the addition of buffer and cells.

2O
Hemagglutinafion-Inhibition (HI) Test

Alpha Procedure:

This test was prepared in the same manner as the
HA test except that 0.25 ml of serum, diluted 1:5, was
substituted for 0.25 ml of buffer. The control tube con—
tained 0.5 ml of serum and 0.25 ml of cells.

The serum titer was the reciprocal of the lowest
dilution of virus in which hemagglutination was completely
inhibited.

The HI titer of the serum was computed from the

following formula:

1133§—311§3 X dilution of serum = HI titer
serum titer '

Peta Procedure:

Constant amounts of virus, 10 HA units, were employed
with decreasing concentration of serum ranging from 1:5
through 1:2560. The proportion of reagents were the same
as used in the Alpha procedure.

The serum titer was the reciprocal of the highest
dilution of the serum in which hemaaglutinetion was completely
inhibited. The HI titer of the serum was computed from the

following formula:

Serum titer X number of HA units = HI titer

 

 

21

here

All donors were reared in isolation quarters, and
sera were assayed for antibody by the serum neutralization
test. Various treatments of sera were performed before
dilution to be used in the HI test.

For the production of anti-IE serum, mature roosters
were inoculated intratracheally with 0.2 m1 of virus infected
allantoic fluid, and the serum was collected after six weeks.
In one experiment. untreated, trypsin modified, or heat

inactivated IBV, was inoculated intravenously or intratra-

cheally.

Viral Infectivity

For determination of viral infectivity, serial ten
fold dilutions of the virus were prepared in nutrient broth,
or Earle's solution, using separate pipettes per dilution.
Five eggs were employed per dilution, and each was inoculated
with 0.1 m1 via the allantoic cavity. The eggs were incubated
at 99 - 99.5 F for five days following inoculation. Embryo

mortality during the first 18 hours was considered to be due

txa non-Specific causes, and these were not included in the

final results.

Mortality, curling and dwarfing, and urate deoosits
111 the mesonephros were the criteria of infectivity and were

used in computing the titer, which was expressed as the

 

 

 

 

 

22

50 percent infectivity and point, ID50, according to the
method of Reed and Muench93.

Serum Neutralization Tests

Dilutions of viruses were prepared in the same
manner as described for infectivity titrations. Two rows
of tubes were set up parallel to the dilution tubes. One
row contained 0.3 m1 of serum per tube. The second row
contained 0.3 m1 of nutrient broth per tube. To each was
added 0.3 ml of the corresponding virus dilution. The
ingredients were incubated at room temperature for 30
minutes. Serum mixtures were inoculated prior to the con-
trol to exclude the possibility of thermal environmental
effects on the virus. Inoculation and incubation of eggs
were the same as used in viral infectivity titrations.

The 50 percent end point was used to evaluate all
titrations. The ID50 neutralization index (IDSONI) was the
difference between the reciprocal of the virus and the

serum titers.

 

 

 

results
trypsin
sisted

volumes
incutat
tervals
eggwhit

for ser.

after f1
actiVi ty
Sampling

was 89 ,

[5.

02/0 31‘ the

 

Ellantoi 3 f

   

 

RESUDTS

Using Corbo's procedure which gave reproducable
results, the time of appearance of HA activity following
trypsinization of IBV was studied. This procedure con-
sisted of adding one volume of one percent trypsin to two
volumes of IBV infected allantoic fluid. The mixture was
incubated at 37 C and samples were removed at various in-
tervals. To this mixture was added one ml of one percent
eggwhite trypsin inhibitor. The material was then ready
for serial dilution.

The HA activity of IBV 40-23 was first detected
after five minutes incubation, whereas with IBV 18-11,
activity appeared in 15 minutes. This was the first
sampling period for this virus. With both viruses the titer
was 80, and progressed to a maximum titer of 10,240 and
6&0 at the respective sampling time (Table I and II; Figure
1).

It was immediately apparent that the incubation
time proposed for this technique was inconvenient. Projects
were proposed involving series of HA determinations which
should be completed in one day. Trypsinized IBV infected
allantoic fluid could not be stored at -30 C or b C with
any degree of stability. Attempts were made to shorten

the incubation period without loss of accuracy.

23

 

 

 

 

 

24

TABLE I

THE HEMAGGLUTINATION TITER 0F IBV 18-11
MODIFIED BY TRYPSIN AT 37 C

Time HA Titer
(Minutes)

0 0
15 8o
30 160
#5 320
60 320
90 320

120 6b0

150 640

180 640
TABLE II

THE HEMAGGLUTINATION TITER OF IBV 40-23
MODIFIED BY TRYPSIN AT 37 C

Time HA Titer
(Minutes)

0 0
5 so
10 160
15 320
30 320
u5 320
60 640
90 320
120 1280
150 10,2»0
180 1280
240 2560

Fl
.4. NW A i c .
WV A: 1 1 1 WW kw
’ I l , 6
AU 0 3c 1
7— 1

 

 

 

 

FIGURE 1

THE HEMAGGLUTINATION TITER OF IBV 18-11 AND UO—BB

MODIFIED BY TRYPSIN AT 37 C

 

20,080-

10,2uo

5120

2560

1280

320

HA Titer

16d

80

40

2d

10

 

 

 

O

is 50 05 60

I I l
90 120
Time - Minutes

 

 

 

PrOced. 1,. e ,

 

with tr?

at one-'
after :
those a

and b).

worthv

acueous
The 1683
maximal ‘L‘
Valeria)

trypsin 5

(7f “7ij n

  
   
  

Fun

and. Ex.

“‘31“th we. a

J

(03516 no

 

 

26

Cultures of IBV 17-40, 40-23, and 41-8 were incubated
with trypsin at 24, 37, and 56 C; and samples were removed
at one-half, one, two, and three hours. The HA titers
after one-half hour incubation at 56 C were comparable to
those after three hours at 37 C (Table III; Figures 2, 3,
and 4). Upon further incubation with trypsin, the HA
activity of 17-37 and 40-19 decreases slightly, levels off
and remains constant until the termination of the test. The
sudden loss of activity of IBV 41-8 is not explainable at
this time. It may be a strain difference in HA activity
worthy of further study.

The concentration of the filtrate from a one percent
aqueous suspension of trypsin is probably not constant.

The least amount of trypsin necessary to produce consistant
maximal HA activity should be known to reduce extraneous
material. A phosphate buffer of pH 8.2, the optimal for
trypsin solubility, was prepared to which varying amounts
of trypsin were added.

Further studies of possible modifications of Corbo's
procedure were made using different concentrations of trypsin
and ETI. The HA titers after incubation at 56 C for 30
minutes were compared to those at 37 C for three hours
(Table Iv).

The results clearly indicate that the only modifi—
cation of Corbo's procedure which can be made without loss

of precision is one of temperature and time. When trypsin

 

 

 

Virus

17-37

40-1 9

> ”\AVAA
R
v“

Virus

17-37

40-19

41-8

TABLE III

THE EFFECT OF TIME AND TEMPERATURE

ON THE INDUCTION 0F HEMAGGLUTINATION

0F IBV 17-37. 40-19, AND 41-8 BY TRIPSIN

Temperature

24
37

2b
37
56

24

37
56

30 Min.
2560
5120
2560

640
2560
5120

10
10
80

HA Tit
Time

1 Hr.
10,2uo

5120
20,u80

2560

1280
5120

10

er

2 Hrs.
2560
2560
5120

1280
5120
1280

40
80

 

2?

2560
1280
640

80

 

 

 

FIGURE 2

THE EFFECT‘OF TIME AND TEMPERATURE

on THE INDUCTION or HEMAGGLUTINATION or IBV 17-37 BY TRYPSIN
20,u8

 

10.2%
SIZCL \
256q+ 37 C
1280.

6nd

32CL

HA Titer

16C}

#0-

20

 

 

 

Time - Minutes

1

#005? <2

{4

/\

 

 

 

29
FIGURE 3

THE EFFECT OF TIME AND TEMPERATURE
ON THE INDUCTION OF HEMAGGLUTINATION OF IBV 00-19 BY TRYPSIN

 

10,2uoi
512d
2560
1280
640
320

160

HA Titer

80

#0

20

10

 

 

 

l 1 J I i 1 1 i L n I

o 30 60 120 150

 

Time - Minutes

 

 

 

 

HA 'T‘iter

 

 

THE EFFECT OF TIME AND TEMPERATURE

FIGURE h

 

30

ON THE INDUCTION OF HEMAGGLUTINATION OF IBV U1-8 BY TRYPSIN

HA Titer

160

80

40

20

10

 

—

 

”—560

J J l

60

i
120

Time - Minutes

 

 

31

Hem mac.
9 Mao.

 

o owH om 0:0
0 one ow comm
o 00H oH owma
Hem ma Hem ma. Hem ea. Ham ma
9 ma. 9 ma 9 ma. 9 ma

..n«: on god 0 mm

Louda <m

>LH m0 <m m0 ZOHBODOZH

mmh

own m-H:

comm mm-on

omma o:-sa
3%....” Wm

.mum n now 0 mn

mshfi>

QO QNHD mOBHmHmZH anANmB MBHmBOUmIZHemNmB mo

ZOHBdeZmUZOU 32¢ ZOHB¢Jflm mmbfiimmmzuecnsz mo ZQonmmEOO

>H mqmde

*-'~*v.o—_—~-

 

 

 

32

and ETI are used in concentrations of less than one percent,
the HA titers show marked reduction. Trypsin is necessary
for HA activity and the concentration must be greater than
0.01 percent before the activity becomes detectable. The
phosphate buffer is not inhibitory to either the trypsin
action or the virus reaction with the erythrocytes.

The procedure used in the remainder of this work
for the induction of HA activity of IBV is the addition of
one volume of a one percent aqueous solution of trypsin to
two volumes of virus infected allantoic fluid followed by
incubation at 56 C for 30 minutes. Upon removal, one
volume of one percent ETI is added, and the mixture is
diluted as before.

Although the time of incubation of virus with
trypsin can be reduced to 30 minutes, it would be more
practical if a quantity of virus could be trypsinized and
stored without loss of activity. Studies were performed
on treated virus stored at -65 and —30 C and untreated virus
at —30 C. The ETI was not added until the treated virus
was thawed for use.

samples at -65 C were stable for three weeks, where-
as at -30 C titrations showed reduction of HA activity over
the same period. Untreated IBV 00-23 remained stable for
three weeks.

The data (Table V) must be considered on a relative

basis for each sampling period because it was necessary to

 

 

 

 

 

THE

T‘ime in
weeks

N

“ox?”

TABLE V

 

THE EFFECT OF TIME AND TEMPERATURE ON THE HA TITER

OF TRIPSIN MODIFIED AND UNTREATED IBV QO~23

Time in
weeks

mGWNl-J

—65 C
Trypsin
Modified

1280

2560

1280

80

2560

640

HA Titer
-30 C
Trypsin
Modified
2560
320
6uo
40
320

640

-30 c
Untreated
1280
1280
1280
160
160
640

 

 

draw fr
the fOu

1n reac‘

solution
cells ar
4C. Ce.

reduced 2

reactiv-t
reactivit
I-
and of 131
With tryp:
were coll:
old. Arte

were divic‘

tamed in

infection 1

  
 

cellect an
hare a Sufi

were also
f

\
fiver and 1‘

‘/
J
511 weeks, /

I

f
325 not 096

I
I

/
z

 

3h

draw fresh cells each week. The discrepancy observed at
the fourth and seventh week may be due only to variations
in reactivity of cells.

Chicken cells which are collected in Alsever's
solution can be used for testing HA activity of IBV. The
cells are unsuitable for use after one week of storage at
4 C. Cells colected in saline and stored at b C will show
reduced HA titers after five days.

Cells from individual chickens vary in their
reactivity with the trypsin modified IBV. With some donors,
reactivity is decreased after prolonged bleeding schedules.

Investigations were made of the influence of age
and of infection of the donor on the reactivity of cells
with trypsin modified IBV. Starting with 30 chicks, cells
were collected when the chicks were one-day and one-week
old. After the cells were collected at one week, the chicks
were divided into two groups of 10 each. One group received
0.1 ml of IBV 41—8 intratracheally. The remainder was main-
tained in a separate room as a control group. For the pre—
1nfection and post-infection periods, it was necessary to
<:ollect and pool the blood from several chicks in order to
liave a sufficient quantity of cells for the HA test. Cells
inere also collected when the chicks were two, three, four,
five, and seven weeks old, or one, two, three, four, and
81:: weeks, respectively, after infection. Hemagglutination

was not demonstrable with cells from chicks two weeks old

 

and only a minimal reaction was detected when they were

four weeks old. At the fifth and seventh week, significant
HA titers were obtained but there was no clear differential
evidence of the influence of cells from infected or control
chicks (Table VI; Figure 5).

To test further the influence of age and infection,
a similar group of chicks was used. Cells were collected
when the chicks were one, eight, and ten days old. On the
tenth day, IBV 41-3 was inoculated intratracheally. Cells
were also collected at weekly intervals from two to twelve
weeks after hatching. No test was performed at the eleventh
week.

On the eighth day, and at the 12th week, sera were
collected and pooled for respective neutralization tests.
The pre-infection IDSONI was 100°34. The ID50NI for the
control birds was 100'6 at the 12th week, whereas the ID50NI
for infected birds was 102°5. The low NI of the infected
birds may be a reflection of either a low antibody level
1n.the decline phase from the maximum which is generally
from the sixth to tenth week after infection, or the response
<>f immature birds.

Hemagglutinating activity could not be detected
Ivith cells from chicks less than three weeks old. This
:strain of IBV gave titers of 640 when cells from mature
birwis were used, but such activity was not demonstrable

with the cells from the birds in this experiment.

 

 

0F CHI

A
Test

7 We

’ At Ont.

my

OF'CHICKEN RED BLOOD CELLS WITH THE HEMAGGLUTININ OF IBV #1

i

TABLE VI

THE EFFECT OF AGE AND INFECTION ON THE REACTIVTTY

Age of
Test Bird

1 Day
1 Week*
2 Week

3 Week

4 Week

5 Week

7 Week

At one week of a
with IBV Strain

Infected
Control

Infected
Control

Infected
Control

Infected
Control

Infected
Control

Virus
for RA

41-9
41-9
41-8

41-8

41-8

ui—e

01—8

HA Titer

U'IO CO C O

10

320
320

160
1280

fie, some of the chickens were infected
1'90

 

36

 

 

NIH"
151.3

0? CHICK

HA "‘1 ter

0

 

FIGURE 5

THE EFFECT‘OF‘AGE AND INFECTION ON THE REACTIVITY

0F CHICKEN RED BLOOD CELLS WITH THE HEMAGGIUCININ OF IBV 41

 

 

640

320

160

HA Titer

80

40

20

10

 

 

   

 

 

t
L A
/
/’
/
/
p /
/
./ Control
/
’ I
I
Infected;
L- I
I
- / l
/ I
/ .
/

l l 1 I I _ I.
1 3 4 5 #65 7

Time — Weeks

 

 

 

experime:
from infi
control ‘:
in .1}; act
group of
CI
turkeys ca
Cells fro:
guinea pi;
could not
were not r
".‘h
strains of
Procedure I
taming t'z-‘I
At 30 minu‘
one percen‘
in a Water
one m1 of Q
finding, tn
ITable VIII
To

and HA ac?‘
"I
I

One 31 Of [I

-I

I

I

/I

I

 

Fluctuation of HA activity occurred throughout the
experiment (Table VII; Figure 6). The HA titers using cells
from infected birds paralleled those using cells from the
control birds. It would seem most likely that variations
in HA activity were due to the cells of this particular
group of chicks because IBV 41 was used in both experiments.

Chickens three weeks old or older, poults, and
‘turkeys can be used as donors of cells for the HA test.
(Zeljs from the cow, horse, sheep, pig, rabbit, hamster,

gxzinea pig, blue goose, mallard, mute swan, and snow goose
ccnild not be used. Cells from lB-day—old chicken embryos
were not reactive.

The thermostability of the hemagglutinin of several
si:rains of IBV at 56 C was studied using the trypsinization
IDINocedure described by Corb025. Tubes, 12 X 75 mm, con-
tzaining two m1 of virus, were placed in a 56 C water bath.
.A1: 30 minute intervals, one tube was removed and one ml of
ozierpercent trypsin was added. After re-incubation at 37 C
iri a water bath for three hours, the tube was removed and
0116! ml of one percent ETI was added. Contrary to Corbo's
filldlng, the HA activity was stable for 180 minutes
(Table VIII).

To compare the thermostability of viral infectivity
axml HA activity, five ml of virus was added to an appro-
priate number of 13 X 100 mm tubes. At each test interval,

One ml of fluid was removed, cooled, and frozen for future

38

 

 

,. can».

TABLE VII

THE EFFECT OF AGE AND INFECTION ON THE REACTIVITY

OF' CHICKEN RED BLOOD CELLS WITH THE HEMAGGLUTININ 0F IBV 4123

A

ge of

Test Bird

1
8
10

10

12

Day
Days’
Days**
Weeks
Weeks
Weeks
Weeks
Weeks
Weeks
Weeks
Weeks

Weeks

Weeks***

Infected
Control

Infected
Control

Infected
Control

Infected
Control

Infected
Control

Infected
Control

Infected
Control

Infected
Control

Infected
Control

Infected
Control

HA Titer

N

N
VIC 00 mo “0 \AU‘ 00 O O

N

com
00

O
20

160
80

80
20

Serum Neutralization Index equals 100’3

Some of the birds were infected intratracheally
with IBV 41-3

Serum Neu
equals 10
Serum Neu
equals 10

Eralization Index of infected chickens

Erglization Index of control chickens

 

39

 

 

 

40

FIGURE 6

THE EFFECT OF AGE AND INFECTION ON THE REACTIVITY
()1? CHICKEN RED BLOOD CELLS WITH THE HEMAGGLUTININ OF IBV 41-3

 

   

HA Titer

 
 
 
 

Infectedq,
80-

40_

   

\
,/\

Control‘

 

20b

10;

 

Time - Weeks

:5.

lit—mama

 

 

Time
IHinut ea

 

TABLE VIII

THERNOSTABILITY OF THE HEMAGGLUTININ OF
TWO DIFFERENT HE} PASSN}ES 0F IBV 41 AT 56 C

41-7 41—9
Time HA Titer Time HA Titer
(Minutes) (37 C for (Minutes) (37 C for
3 Hours) 3 Hours)
0 1280 O 5120
30 640 30 2560
60 640 60 5120
90 1280
120 1280 120 5120
150 640

180 640 180 5120

41

 

infec‘ivi.

for deter‘
I

The rent!

 

infectiviI

incubaticI

 

the trypsf

I
minutes :I
I

 

are recorI
TI
Idtreas t?

60 to

\f)

C r

I:
41‘9 war»;-
time port:
determine
for 3Q mi
Independé
“0 SiFfiif

at 56 C I

 

42

infectivity assay. At the same time, two ml was removed
for determination of HA activity by the Corbo procedure.
The remaining two ml was treated with trypsin at 56 C for
30 minutes. At zero time, one sample was titrated for
infectivity, while two others were trypsinized at the two
incubation temperatures and respective times. Although
the trypsin modified virus was incubated at 56 C for 30
minutes or at 37 C for three hours, the respective titers
are recorded for zero time.

The hemagglutinin was thermostable for 180 minutes,
whereas the virus was innocuous for chicken embryos within
60 to 90 minutes (Tables IX to DU; Figures 7 to 9).

In an extension of this experiment, IBV 40-23 and
41—9 were incubated at 56 C for nine hours, during which
time portions were removed at hourly intervals for HA
determination. Trypsinization was performed only at 56 C
for 30 minutes, because the thermostability of IBV is
independent of the trypsinization procedure. There was

no significant decrease of the HA activity after nine hours
at 56 C (Table XII and XIII; Figure 10).

To compare the effect of trypsin on the infectivity
Of IBV 40-23 and 41-7, two samples of each virus, one con-
taining trypsin as used in the HA test and one without
trYpsin were incubated at 37 C for three hours.

The addition of trypsin to virus resulted in a

threefold dilution. To correct for this the log of the

 

 

vs
712

Time
(Minute:

TECH;

 

 

43

TABLE IX

THERMOSTABILTTY OF THE HEMAGGLUTININ AND INFECTIVITI OF
IBV 17-37 AT 56 C

Time HA Titer HA Titer Infectivity
(Minutes) (37 c for (56 c for Titer
3 Hours) 30 Minutes)
0 1280 1280 7.2

15 3.5

30 640 320 2.2

1+5 0.8

60 640 320 0.1

90 640 6&0 0.0
120 640 1280 ,
180 640 euo

TABLE X

THERMOSTABILITY OF THE HEMAGGLUTININ AND INFECTIVITY OF
IBV 16-“3 RT 56 C

Time HA Titer HA Titer Infectivity
( Minutes) (37 C for (56 C for Titer
3 Hours) 30 Minutes)
0 2560 2560 5.5

15 0.8

30 2560 2560 0.8

45 0.1

60 5120 2560 0.0

120 1280 1280

180 5120 5120

 

 

 

 

 

TABLE XI

THERMOSTABILTTY OF THE HEMAGGLUTININ AND INFECTIVITY 0F

Time
(Minutes)

THERMOSTABILTTY’OF THE HEMAGGIUTININ AND INFECTIVITY OF

Time
(Minutes)

IBV 3-15 AT 56 c

HA Titer HA Titer

(37 C for (56 C for

3 Hours) 30 Minutes)
2560 2560
2560 2560
1280 1280
1280 1280
1280 1280

TABLE XII

IBV 40-23 AT 56 6

HA Titer
(56 C for

30 Minutes)

2560

5120

5120
1280
5120
2560
6#0
640
640
640
6M0

Infectivity
Titer

Infectivity

Titer

OHWONwF

 

nu

03.456789

mime
(Hour

 

 

 

 

TABLE XIII

THERMOSTABILTTY OF THE HEMAGGIDTININ
OF IBV 40-23 AND 41-9 AT 56 C

Time HA Titers (56 C for 30 Minutes)
(Hours)
40.2 3 41-9
6&0 160

\oooxio‘mmo
E
M)
N
O

 

46
FIGURE 7

THERMOSTABILITY OF THE HEMAGGLUTININ AND INFECTIVITY
OF IBV 17-37 AT 56 C

 

7.0

 

 

 

 

5.0
h
0
.p
*1
as
Q «400
H :E:
w
0
A
‘ /,.i\ HA Activity
300 K// \\\\
// \\N
//
2.0
1.q
Infectivity
I J l L I x A i J
0 60 120 l 0

Time — Minutes

FIGURE 8

THERMOSTABILITY OF THE HEMAGGLUTININ AND INFECTIVITY
OF IBV 16-43 AT 56 C

 

ID50
Log HA Titer

/
/
\-_ /
\ .
‘

‘ I HA Activity

/‘
\

Infectivity

 

60 | l J l

 

A l
120

L
180
Time - Minutes

 

u?

 

 

FIGURE 9

 

48

THERMOSTABILITY OF THE HEMAGGLUTININ AND INFECTIVITY

0F IBV 3-15 AT 56 C

 

7.d»

3-!

0

4.:

c: E:

\r\

Q<
P4 :5
as

0

5—1

 

Infectivity

 

 

Time - Minutes

HA Activity

 

 

1+9
FIGURE 10

THERMOSTABILITY OF THE HEMAGGLUTININ AND INFVCTIVITY
OF IBV HO-Zj AT 56 C

 

 

 

 

 

"‘_“"_-‘\
\\\ ,r’*\\
‘\\r,,/' \\\
\
\e_ _______
HA Activity
\
\
1.05
Infectivity
i l l I | _,_,gil i‘_L)
0 l 2 3 4 5 6 7

Time — Hours

 

 

 

 

50

dilution factor (3), 100°5, was added to the ID50 values.

The reduction of titer of the control samples due
t<> heat inactivation alone must also be taken into consi-
<1eeiration and added to the ID50 obtained with the trypsinized
virus. The corrected values are considered to be a more
£1<>curate reflection of any change due to the action of
trypsin alone. After three hours, there was a 100°9
Iceaduction in viral infectivity of the trypsinized sample
ass; compared to the control. With IBV uo—23, the titer of
'trle control was 100'3 lower than that of the trypsinized
saanufle at the same period. The initial titer of the con-
‘t:r<31 was 100'6 lower than the corrected value of the tryp—
sinized sample. Whether this is due to the disruption of
‘viiral aggregates or an error in technique is unknown.
(anrnparison of uncorrected values shows the titer of the
Ccarntrol sample to be 10009 lower than the titer of the
1::‘3psin treated sample (Table XIV and XV; Figure 11 and 12).

As an extension of this experiment, IBV 3—16 was
1:1'eated in a similar manner except that the testing period
‘75155 increased to 20 hours. After three hours there was
a. reduction of 101'3 in the titer of the trypsinized sample.
After eight hours, the titers of the trypsinized sample
arm: the control sample were approximately the same. ‘The
Virus was non-infectious at the 20th hour (Table XVI;
Figure 13).

A second sample of IBV 3-16 was tested. After four

 

 

 

 

 

TABLE XIV

THE EFFECT OF TRIPSIN AT 37 C ON THE INFECTIVITY

0F IBV 01-?
'Time Infectivity Titer
(Hams)
Trypsin Correction Correction Control
Modified Value for Value for
Dilution Heat
, 0 6.6 7.1 7.0
K 1 6.6 7.1 6.2
2 6.2 6.7 7.0 6.?
3 5.3 5.8 5.8 6.7
TABLE XV
THE EFFECT OF TRIPSIN AT 37 0 ON THE INFECTIVITY
0F IBV h0—23
Time Infectivity Titer
(Hours)
Trypsin Correction Correction Control
Modified Value for Value for
Dilution Heat
0.0 6.8 7.3 6.?
100 50“ 5.9 6.1
' 1.5 6.4
f 2.0 4.6 5.0 5.2
t 3.0 [+05 “-9 5.7 504
L

 

 

TABLE XVI

THE EFFEET OF TRYPSIN AT 37 C ON THE INFECTIVTTY

OF IBV 3-16
Time Infectivity Titer
(Hours)
Trypsin Correction Correction Control
Modified Value for Value for
Dilution Heat
0.0 7.6 8.1 8.1+
1.0 7.3 7.8 7.8
1.5 8.2
200 608 703 70“
3-0 5.7 6.2 6.5 7.8
8.0 [‘03 408 601 605
20.0 0.0 0.0

 

FIGURE 11 53

THE EFFECT OF TRYDSIN AT 37 C ON THE INFECTIVITY

 

 

 

 

 

 

 

0F IBV #1-7

- I

7. K\\ ”Gontl'OI
6.d-

50dL .

M \§

Trypsing

a: z
e W

300' ('

2.0L 3

i

3

1.0L _

i;

3

i

0 1 2 —~-‘- 3*

Time - Hours

 

 

 

ID50

FIGURE 12

THE EFFECT OF TRIPSIN AT 37 C ON THE INFECTIVITY
OF IBV 40-23

 

54

 

M. . __,.‘_~...

 

   

 

 

 
 
  

   

   

 

8.0-
l
7.0
——————— “°\
\
\\\
\\\ Corrected
6.0- \.\ Trypsin
\\ [J
\
\
Control
5.0L
Tl“
r sin
4.0- yp
3.d—
2.d+
1.qu
1 - ___.l_
0 1 2L *‘Tj

Time - Hours

ID50

N Lo 4:- Ln
0 O 0 0

win"... P. w»... -- .1an .. “Mg“..- . .P... “—9....”—

#1

FIGURE 13

THE EFFECT OF TRYPSIN AT 37 C ON THE INFECTIVITY
OF IBV 3-16

 

 

   
 
 

(

K.

.9.

Time - Hours

“\.\ -L— Control

   
 
 

Corrected
Trypsin

”1' Trypsin

m)—

 

 

hourS, the titer of the trypsinized sample was 100-6 lower

than the control. Again the initial titer of the control
was 10007 lower than the corrected value of the trypsinized
sample. Analysis of uncorrected values reveals that the
titer of the trypsin treated sample was 101-5 lower than
the control. At the eighth and 15th hour, the corrected
titers of both samples were equal. Infectivity was
observed after 23 hours of incubation, but an end point
was not determined (Table XVII; Figure ,1“)-

IBV 14-1-9 was tested at zero, 12, 16, and 20 hours.
Infectivity was observed at the 20th hour with the titer
Of the trypsinized sample being greater than that of the
cBonn-cl (Table XVIII).

To determine the correlation between viral multi-
plication as detected by infectivity and HA activity
following inoculation of embryonating chicken eggs, 60
eggs were inoculated, respectively, with 0.1 ml of un-
diluted and a 10'3 dilution of 113V 41-6. Immediately
after inoculation and at four, eight, and 12 hours, and
at subsequent 12 hour intervals through 72 hours, five
eggs containing living embryos were selected at random
and chilled for 12 hours at 1+ C. The allantoic fluid
from these embryos was then collected and pooled, and
infectivity and HA titrations were performed. The re-
maining fluid was stored at -30 C for tests to be done

one week later to determine the effect of freezing and

 

 

 

TABLE XVII

THE EFFECT OF TRIPSIN AT 37 C ON THE INFECTIVITI

OF IBV 3-16
Time Infectivity Titer
(Hours)
Trypsin Correction Correction Control
Modified Value for Value for
Dilution Heat
0 7o? 802 703
2 7.2 7.7 7.8 7.
4 5.5 6.0 6.4 7.0
8 4.8 5.3 6.1 6.2
12 4.5 5.0 6.7 4.“
15 3.2 3.7 4.1 4.0
23 >0.0 >0.0
TABLE XVIII

THE EFFECT OF TRIPSIN AT 37 C ON THE INFECTIVITY

0F IBV 41-9
Time Infectivity Titer
(Hours)
Trypsin Correction Control
Modified Value for
Dilution
0 0 8.8 o
12 . 4.7

a 3

a 2 >
16 2.8 3.3 >‘

3 0 3.5

 

 

 

 

)l C [If
a . i i 1 t .. . t, t L ((1
7 6 . _.. I. t)‘: m? _ , /(/‘ (I
c. A . . .. . z i ( /
3 . 0
AC .
Ohm:
ll“-

ID50

FIGURE 14

THE EFFECT OF TRYPoIN AT 37 C ON INFECTIVITY

 

OF IBV 3-16

 

 

 

  
 

 

 

6 0 1Corrected
. Trypsin
51-
“.0»
3.0-
Trypsin
2 s Ob-
1.0-
L 1 l l Jn I I I l 1 n _L_J_L l 4L
0 2 h 6 8 10 12 15

Time - Hours

 

 

 

 

 

 

 

 

the f
mum of
infect
106‘? a

throng?

was 101.

reaches?

107.“ a

A

C

’L“

U ('EI’ Wa A
b

/
/

J

J
y/

i

a)

was {Ollr

t(

J

J

 

thawing on HA activity of the fluid as compared to the

fluid which had not been frozen.

Infectivity titrations of IBV z4.1—6 showed the
typical lag, log, decline and stationary phases of viral
multiplication (Table XIX and XX; Figure 15 and 16)..

With the undiluted inoculum, the 1050, 10u°0, at
the fourth hour, represented a 102°” decrease from the
11350 106°“ of the inoculum. The log phase reached a maxi-
mm of 11350 108'9 at the 24th hour, or a 101'“9 increase in
infectivity. Following this, there was a 102'2 decline to
106‘? at the 48th hour followed by the stationary phase
through the 72nd hour.

Hemagglutinating activity of the frozen sample
could first be detected at zero time when the titer was 10.
QTIIere was a progressive increase to a titer of 640 at the
24th hour. With the exception of the titer of 320 obtained
at the 48th hour, the titer continued to increase to 1280
at the 72nd hour.

The IDSO of the 10"3 diluted inoculum of IBV til-6
Was 101°2 at the fourth hour. The maximum ID50, 107°8, was
reached at the 24th hour, or an increase of 106-6. This
Was followed by minor fluctuations in a general decrease to
107‘“ at the 72nd hour.

Only with the frozen sample could hemagglutinating
aetivity be detected four hours after inoculation when the

titer was 10. The titer remained constant at 80 from the

 

 

 

 

Ori g1

\jm[\\_\r\\-

Time
{Hours

0MEinal C

 

60

TABLE XIX

INFECTIVITY AND HEMAGGLUTINATING ACTIVITY
OF IBV 41-6

Undiluted Inoculum

'Time Infectivity HA Titer
(Iiours) Titer Before Freezing After Freezing

Original Titer 6.4 320
0 5.0 40 10

4 4.0 10 40

8 o.# 20 no

12 5.0 160 320
2# 8.9 160 640
36 'l. 8 (:40 640

#8 6.7 2‘60 320
60 6.8 2‘60 1280

72 6.8 040 1280

TABLE XX

INFECTIVITY AND HEMAGGLUTINATING ACTIVITY
OF IBV 41-6

Inoculum Diluted 10"3

Time infectivity
(Hours) Titer HA Titer
Before Freezing After Freezing

02-1 ginal Titer 6.6 o
4 1.2 0 10
8 4.1 160 80
12 6.8 160 80
24 7.8 80 no
36 7.3 320 80
#8 7.6 320 320
60 6.8 2560 640
72 7.“ 320 1280

 

-
v

 

f...- I A fillet
v
’
.
rn ’ iv

Loaut,<: id;
Ohm-Q H

A.

1

J . vjk

 

 

11m...“ ._ _

._ ~-..__¢. -w

A... 6,)...» w: ”‘MF‘

 

61
FIGURE 15

INFECTIVITY AND HEMAGGLUTINATING ACTIVITY

 

OF IBV hl-6
Undiluted Inoculum
9.0-
8.0L
7.0-

Infectivity

 

\

ad-

 

 

 

3-.
3. 0
<3 {3"
as"
H g
gm
3'0}- //.»—Q_H—'
2" ~ -‘~—\ /
’_/" “\.\ /‘/ HA Titer
/" \f
/
2.0» /
/
/—J
/
1.0!
l i l 1 1..-... _. .1,
0 12 24 36 48 60 72

Time - Hours

 

 

,fi, w”

r..

a.
LGLrF.<I K04
OwQH

A ,

(I. 'i

 

 

 

16

AND HEMAGCLUmINATING

FIG UP. 73
lNFECTTVITY
CF IBV 31-6

Inoculum Diluted 10"3

8.0L

 

/’

 

 

 

g...____...___.__ ____ __ F..-

ACTIVITY

 

 

 

I fectivity
6,P-
5.0L
a
m
+9
'4
526*
Q\¢g1~¥.0p
H 21':
a
o
.1
3.0b ’/_/ "
/“ HA T fer
J /
l /
2.0a ,/
/,/r
I \. ,,/’
I N,“
I
I
l
!
1.0t 7
/
f,
1: ,_i_4..__——-J————«—-——~ ‘ ' l. 7
o _“flafl—-12 24 36 “8 60

Time - Hours

)\

D)

 

 

 

63

8th through the 36th hour and then increased to 1280 at the
72nd hour. This general trend in HA activity was paralleled
rather closely with the unfrozen samples.

Using IBV 40-23 and 17-UO with the same procedure
as previously described, infectivity tests were performed
with unfrozen allantoic fluid whereas HA activity was
determined only with samples frozen for one week. The test
periods ranged from 12 through 101 hours after inoculation
(Table XXI and XXII; Figure 17 and 18).

The maximum infectivity titer of IBV 40—23 was
108-3 at the thh hour, which represented a 102'1 increase
from the original inoculum of 106°“. The titer decreased
to 6.3 at the 72nd hour and remained constant until the
termination of the experiment. The HA activity was not
detected until the 72nd hour when the titer was 160. At
the lolst hour, the titer was 1280.

The titer of IBV 17-40 remained at 107°2 through
the thh hour, decreased to 106.5 at the b8th hour, and
then remained rather constant to the 101st hour. HA
activity was first detected at the 48th hour with a titer
of 160 and to a titer of 2560 at the 72nd hour. At the
101st hour, HA activity was not detected.

With only the frozen samples, the HA activity of
£111 strains reached a maximum at 60 hours or later. The
fluaximum infectivity of all strains was reached at 24 hours

eaccept for IBV 17-40. The titer of this virus remained

 

64

TABLE XXI

INFECTIVTTY AND HEMAGGLUTINATING ACTIVITY
OF IBV 40-23

Undiluted Inoculum

Time Infectivity Titer HA Titer
(Hours) After Freezing
Original
Inoculum 6.2 320
12 6.8 0
2b, 8.3 0
48 7.2 O
72 6.3 160
101 6.2 1280

 

 

Ori
INC:

72
101

 

65

TABLE XXII

INFECTIVITY AND HEMAGGLUTINATING ACTIVITY
OF IBV 17-00

Undiluted Inoculum

Time Infectivity HA T1tep i
(Hours) Titer After Freezing i
Original t
Inoculum 7.2 320
12 7.2 0
24 7.2 O
”8 6.5 160
72 ' 6.5 2560

101 6.2 0

 

 

{ (fl)
“5‘ )ill.ll/$H|{IIL 0
1‘. “90 pl“ 7. ‘IM
LmvuaH. 431 E04
OWQH

 

 

 

 

9a04'

 

 

66

FIGURE 17
INFECTIVITY AND HEMAGGLUTINAmING ACTIVITY
OF IBV 00-23

Undiluted_Inoculum

__..__.. H...~r_._—-_—_.—-—.-__'—._.--.--—— -h-a - — ———~.-~~—-_¢—~__———._.——-——-o— - Q-om—-----_.-.4w.1

‘m

 

 

 

 

 

. Infectivity
1 :1
3-4 ; 0
0 1
+3 (’0’
d I 0
(DE! E
‘0 i
a <t: ;
H II: n
d)
b.
3 ‘0’ g
i
BOQL /I
/ )
,/
/
Jr / ’1
// / HA Titer
2.6- /
/
/
/
/
LQL /
\ /
\ / l
\ /’
\ / l
L___-_ \L__+__ _____l"/,_ i__i__, ,_i a--_7|7 l i__,.__l__i__J
O 12 2“ 08 72 101

Time - Hours

 

 

 

3
M9 1,“
Lou «L. tax: MOJ
OwQH

2.91

18

FIGURE
INFECTIVITY AND HEMAGGLUTINATING ACTIVITY
OF IBV 17-40

Und 1 1237 851-3999919n3-

8.0f—— m'

 

I“‘\~‘\\‘“‘-a

infectivity

\ /‘ \ HA Titer

-1- “~-.--i_L.-_ -_ i _. ,

72

m- .L—A4--__.__-_..L_...-_....-J. -- - _-.a .‘ L4,“--- _

Time - Hours

‘-—-—-—- —~.——-—-~____.—__.._. ”d_‘..-u-

 

 

’—_--i-_4—-"-

 

 

 

68

constant through the ZAth hour and then decreased. Although
there is no evidence of correlation between infectivity and
HA activity, practical application can be made of these
data. Harvests of allantoic fluid at 20 hours post inocula-
tion yield high infectivity titers but little or no HA
activity. To obtain high yields of HA activity, the allan-
toic fluid should be harvested after 60 hours.

In an attempt to determine the role of the infective
viral particle in the HA test, a sample of virus infected
allantoic fluid, which had been treated with trypsin at 37 C
for three hours, was serially diluted in Earle's solution
under sterile conditions. A modification of the standard
HA test was used. Instead of the conventional 0.25 ml
volume of ingredients, 0.5 ml portions of the appr0priate
virus dilutions and cell suspensions were mixed in order to
have sufficient material for infectivity tests. For the
control, 0.5 ml of Earle's solution was substituted for the
cell suSpension . All tubes were incubated at room tempera-
ture for 45 minutes. At the end of this time, hemagglutina-
tion was complete in the 1:80 dilution of the virus; and ne»
gative in the 1:160 dilution of virus. After centrifugation,

the cell free supernatant fluid from the virus dilutions of

1:40, 1:80, and 1:160 were used for titrations of infectivity.-

If the infective particle was adsorbed to the cells,
it would be expected that a reduction of infectivity of the

supernatant fluid would be deteCted as compared to the

 

.-_“‘-‘—_—*_—‘———-

 

 

69

control in which cells were not present. With the 1:40
dilution of the virus, the infectivity titer of both samples
was the same. with the lz80 and the 1:160 dilutions, there
were 100'2 and 100°3 reductions of infectivity, respectively,
with the cell-containing samples as compared to the control.
These differences are not considered to be of sufficient
magnitude to permet any conclusions, but do suggest the
possibility that the infective viral particle does not have
a direct function in the hemagglutination of IBV under the

conditions of the test (Table XXIII).

 

Ultrafiltration experiments were performed to
determine whether the hemagglutinin of IBV 40-22 could be
separated from the infective particle. Virus infected
allantoic fluid which had been frozen and thawed was clari-
fied by centrifugation at u,500 rpm for 20 minutes at u C.
The supernatant fluid was passed through a millipore filter
having a #50 millimicron porosity and the filtrate was then
passed through a filter having a 50 millimicron porosity.

When each filtration process was completed, the
filter pad was removed from the holder, broken into several
pieces, and placed in a screw cap vial containing Earle's
solution. The vial was shaken vigorously to resuspend any
residual virus on the pad. The filtrates and residues from
the pad were tested for infectivity and HA activity (Table
.KKIV). Earle's solution containing a filter treated in the

same manner as above had been shown to be non-toxic to

 

   

70

TABLE XXIII

INFECTIVITY'TITERS OF IBV #0-23 AFTER HEMAGGLUTINATION

Dilution Titer
of Virus
1:40 Cell free supernatant
£11116. 2 o 5
Control 2.5
1:80 Cell free supernatant
fluid 2.5
Control 207
1:160 Cell free supernatant
fluid 1.7
Control 2.0

Undiluted Virus before
Trypsinization 6.8

Undiluted untreated virus
incubated with red blood
cells 7.5

 

 

 

 

 

Pin
mill

Viru
tilt

 

71

TABLE XXIV

THE EFFECT OF ULTRAFILTRATION UPON SEPARATION
OF THE INFECTIVE PARTICLE AND THE HEMAGGLUTININ
OF IBV 00-22

Volume HA Infectivity
of sample Titer Titer
(m1)
Virus clarified by
centrifugation at 0,500
rpm 15.0 2560 5.8
Filtrate from 450
millimicron filter 10.1 1280 4.3

Virus retained on
filter of #50 millimicron
10.0 80 0.0

Filtrate from 50
millimicron filter 9.5 0 1.0

Virus retained on
filter of 50 millimicron
10.0 320 3.0

 

. ..

.01.

l. .. 2...

0.. qr!“ .r“
. 0

LL P l «L

4‘

l . VI 1 . Yfi . a ' f
”—m o... x.» . 42v ‘4} Her“ M. ”1‘ .... uAHo OIIJII, r. .I I I I I I

as 24 n .F M. D. T. .u r” a. a... . . J

. { Jul . : . .5

a .2. c s a e u . _ u. l c 5

f e . hu H... rd 3 h M. .. n c .3. C. 1.. r .

oi: “w. b . . D a 5L w. .. Q . Ck 1

r A. . u.” no h .. 4

AK‘ if V a v H

val pan

JIIII!

 

 

 

 

 

72

emDTTOS.

The virus used for this experiment had an infectivity
titer of 105°8 and an HA titer of 2560 prior to filtration.
The filtrate from the 450 millimicron filter had an infecti-
vity titer of 10u*3 and an HA titer of 1280. Apparently no
virus was retained by the pad since it was innocuous after
being suspended in Earle's solution, but the HA titer.was 80.
The filtrate from the 50 millimicron filter had an infectivity
titer of 101'0, but without demonstrable HA activity. The

residue from the pad had an infectivity titer of 103-“ and

\

an HA titer of 320.

A second sample of IBV 00-22 was similarly treated
two weeks later. Before the pad was removed from the
holder, the filter apparatus was inverted and Earle's solu-
tion was introduced into the delivery tube. The apparatus
was rotated gently to Wash any virus which might have been
passed through the filter, but remained on the underside of
time pad and on the filter apparatus. The apparatus was then
rueturned to normal position and the washing collected. In-
fwectivity and HA determinations were made on the filtrates,
wasfluings, and residues (Table XXV).

The original infectivity and HA titer of the second
samzale was 105°C, and 2560 respectively. The filtrate from
the 450 millimicron filter was 10‘h6, whereas the HA titer
was 1160. The infectivity titer of the washing was 102°8

and izhe HA titer was 80. The residue of the pad had an

 

 

TABLE m

THE EFFECT OF ULTRAFIBTRATION UPON SEPARATION

OF THE INFECTIVE PARTICLE AND THE HEMAGGLUTININ

0F IBV #0-22
Amount of Amount of
material final
in m1 material
in m1
Virus clarified
by centrifugation
at h,500 rpm 20 20.0
Filtrate from #50
millimicron filter
15 14.2
Washing from 450
millimicron filter
2 2.0
Virus retained on
450 millimicron
filter 14.2
I?iltrate from 50
inillimicron filter
11 8.0
Washing from 50
millimicron filter
h “.0
Virus retained on
50 millimicron
filter 8.0

HA Titer

2560

160

80

2560

Infectivity
Titer

2.3

0.3

  

 

I.
.5-
_t
r

i
. ,f/////, ,f" i ’f~:
I 7) _
I' An,
' - I’M” " ' 0’
v I Q ,
. 7".

  

, . t p t ‘. e:,4 ‘ _ ._ _ . p. Y . _
infectivity titer of lb with no ooservaole nA actiVity.

The filtrate of the 50 millimicron filter was 100'3 with no
detectable HA activity. Infectivity and HA activity was not
demonstrated in the washing. The residue had an infectivity
titer of 101“0 and an HA titer of 2560.

While the data from both experiments cannot be
analysed objectively and correlated on a definite numerical
basis, certain differences between viral infectivity and HA
activity can be demonstrated with respect to an apparent
role of each.

It is apparent that the majority of the virus, as
represented by infectivity tests, passes rather readily
through a 450 millimicron filter, but is retained by a 50
millimicron filter. In both experiments, the filtrates
from the 50 millimicron filter had no demonstrable HA
aactivity, and a low degree of infectivity. Both HA and in-

feectivity were readily demonstrated with the filter which
amass resuSpended in Earle's solution. While this data cannot
be: used for definite conclusion, they do suggest that HA
.a<31:1vity of IBV induced by trypsinization of the virus may
be? independent of the infective viral particle.

The immunological response of chickens was studied

‘Vifitlu normal, trypsin-treated and heat-inactivated IBV.

Sera from four chickens receiving two inocula of

a C3.1 ml via the trachea four weeks apart with IBV hl-9,

Wklixzh had been trypsinized at 37 C for three hours, had an

 

 

 

 

IDEONI of at least 107'8 at the sixth week using untreated

IBV 41-9 for the neutralization test. Sera from chickens
receiving the same inoculum intravenously and weekly for the
next five weeks showed an IDEONI of 107'3 at the sixth week.

Virus inactivated at 56 C for two hours without
trypsinization, and which was non-infectious for chicken
embryos, was administered intravenously according to the
schedule for chickens receiving trysinized virus by this
route. The IDSONI was at least 107*8 at the termination of
the experiment.

Untreated virus was administered according to the
schedule for intratracheal inoculations of trypsinized
virus, and the IDEONI was 10790 at the sixth week.

The average ID50NI for control chickens was 100-7
at the beginning and end of the experiment (Table XXVI).

The N.I. of the serum produced against intravenously
ariministered trypsinized virus is 100°5 lower than the N.I.
01’ serum produced against intratracheally administered
tz'srpsinized IBV. The N.I. of the serum produced against
heeeat inactivated virus is 100‘8 higher than the N.I. of the
Searwam produced against an untreated virus inoculated intra-

tI'acheally. Although there is a difference between the
1511“ter serum N.I. and the N.I. of the former sera, the
Slain'iificance of this value is uncertain. IBV hl—9, trypsin-
1Zed at 37 C for three hours or heated at 56 C for 30 minutes,
r‘etains its antigenicity (Table XXVII).

 

/
'20

TABLE XXVI

THE EFFECT OF MODIFIED IBV ON THE PRODUCTION
OF NEUTRALIZING ANTIBODIES

Neutralization Indices of Pre-infection Sera

Serum Serum Titer Virus Titer N.I.
of IBV hl-lo "
6.3
Serum B 4.8 1.5
Serum C 5.2 1.1

Serum D 6.1 0.2

 

TABLE XXVII

THE EFFECT OF MODIFIED IBV ON THE PRODUCTION
OF NEUTRALIZING ANTIBODIES

Neutralization Indices of Post Infection Sera

Serum Serum Titer Virus Titer N.I.
IDSO ID50
Trypsinized
Virus
Intravenously O. 5 7 .81} 7 . 31+
Trypsinized
Virus
Intratracheally 0.0 7.84
Heat Inactivated
Int ravenously O. 0 7 . 81+
Untreated Virus
7.01

Int rat racheally O. 83

R Control 7.43 0.’+1

77

 

IBV Al and uz were propagated in chicken embryo

kidney cell culture with Hank's balanced salt solution. 0.5

percent lactoalbumin hydrolysate, and two percent bovine
At 48 hours when cytopathogenic effect was distinct,

serum.

the extracellular fluid was harvested. The untreated virus

infected cell culture medium showed no HA. When infected

cell culture fluid was processed exactly as infected allantoic

fluid, the HA titer for both strains of virus was 160. The

fluid from uninfected cell cultures treated in the same

fashion was 40.
Several tests were performed to determine if hemad-

sorption of IBV would occur in a manner similar to influenza
when the red blood cells are added to infected cell cultures.
To chicken embryo cell cultures inoculated 24 or 48 hours

‘previously with IBV 41, 0.2 ml of a 0.h percent suspension

caf red blood cells was added to the nutrient medium and in-

otabated for one minute. The same procedure was followed

at’ter removal of the nutrient medium. Hemadsorption was not

observed.
In other tests, the nutrient medium was removed and

ei.tflner 0.25 or one percent trypsin solution was added for
VEaI"1ous intervals ranging from 30 seconds to five minutes.

TIIEB cells were washed with saline, and 0.2 ml of a 0.4 percent

SuSpension of red blood cells were added. Hemadsorption was

n£>tz observed. After five minutes in contact with trypsin,

tYue cells sloughed from the wall of the tube.

 

Trypsr

 

Freon .
Preon t

Freon t

 

 

Periodate was tested to determine its effect on the

HA of IBV. To two volume of infected allantoic fluid was
added one volume of sodium periodate, 0.1 to 0.001 M. After
incubation at 37 C for one, two, and three hours, the re-
action of periodate was stopped by the addition of one
volume of a one percent glycerol-saline mixture. HA could
not be detected by this method.

Heat alone will not induce HA.

Freon and n-heptane, one volume, was added to two
volumes of virus infected allantoic fluid before or after

trypsinization of IBV 40—23 at 37 C for three hours.

TABLE XXVIII

THE EFFECT OF FREON UN HEMAGGLUTINATION 0F IBV 40-23

Treatment HA Titer
Trypsinized Virus 5120
.F1~eon treatment before trypsinization 5120
Freon treatment after trypsinization 0
.Fxseeon treatment alone 0

Freon alone did not induce HA and was without effect
When used before trypsinization, but negated HA activity
When used after trypsinization of the virus.

The receptor destroying enzyme of l. cholezae

 

essenti
Several

minutes

 

 

80

destroys the cellular receptor sites to which the myxoviruses
adsorb. The standard virus for titration of R05 is the PR8
strain of influenza. The RDE is serially diluted in calcium
borate buffer. After incubation with a one percent suspension
of cells for 30 minutes at 37 C, ten units of virus is added
and the mixture reincubated for 30 minutes. The end point

is the lowest dilution of RDE in which HA is inhibited.

TABLE XXIX

TITRATION 0F RDE USING PR8 AND IBV 17—40

Time lnhibition of HA Titer of
(Minutes) PR8 17-00
30 no 20
1&5 8O 80
6O 80 80

The magnitude of inhibition of HA by RDE was
essentially the same with either PR8 or IBV 17-40 at the
seaxreral test intervals. The only difference was at 30
nultlutes where the titer was 40 for PR8 and 20 for IBV.
F'71-"0mthese data, it would seem that the cell receptor site
\ltifl.lized by IBV are similar to those used by influenza.

Definite separation of naturally occuring non—specific
31161 specific inhibitors of the hemagglutination test has not

been accomplished for the utilization of trypsin modified

 

3?
-.

IBV in.the assay of antibody.

The following treatments of normal and anti~IEV
serum did not influence the inhibitory properties of the
sera:

l. 56 C for 30 minutes

2. 62 C for 20 minutes

3. filtration through a Seitz BK filter

A. one percent trypsin and five drops of toluene for
three hours at 37 C followed by 100 C for two minutes

5. toluene alone plus 62 C for 20 minutes

6. trypsin in concentrations of 0.8 or 16 ma/ml, at
37 C or 56 C for 30 minutes to three hours

7. sodium or potassium periodate, 0.9 to 0.01 M at 4 C
overnight, 37 C for three hours, or 56 C for 30
minutes

8. zymosan in concentrations of one, two, five, ten or
20 mg/ml had no effect on the inhibitor, indicating
that properdin was not the inhibitory agent

9. equal volumes of RDE and sera

A phosphate buffer, pH 8.2, containing 8 mg of
trypsin 1 m1 and M/90 potassium periodate removed the in-
lllbitory properties of both negative and anti-IPV sera,

Tdithout such treatment the inhibitory property of both sera
.1£3 the same. This treatment offers perhaps the most fruitful
approach for detection of specific antibody as it is the only

<Drue in which removal of the inhibitor has been demonstrated.

 

 

arthro;
men 1:
Chicken
V1 ty t1
remains

gradual

 

 

DISCUSSION

The mechanism of the induction of hemagglutination
by IBV must be determined if an hemagglutination~inhibition
test is to be developed using trypsin modified virus-infected
allantoic fluid. Trypsin is a relatively specific enzyme
which can act as a peptidase or an esterase, when an L-
arginine or L-lysine residue contributes to the carboxyl
grouplzo. Attention must then be directed to the location
of the substrate upon which trypsin reacts.

The hemagglutinin of the myxoviruses-Ss'59 and the
arthPOpOd-borne96 viruses is the virus particle itself.
When influenza strain PR8 is inoculated into embryonating
chicken eggs via the allantoic cavity, the maximum infecti-
vity titer is reached at the ninth hour post inoculation,
remains fairly constant until the 20th hour, followed by a
gradual loss of activity. The HA titer gradually rose to
a maximum titer at the 20th hour and remained constant until
the termination of the experiment 40 hours after inocula-
tion106.

Mumps virus inoculated via the allantoic cavity
(would first be detected u8 hours after inoculation. Hemagglue
txinating activity was first detected after 96 hours. Maximum
.111fectivity was observed at the 120th hour post inoculation,

Whereas HA activity is maximum at the 11mm hour. The 24

82

 

- ‘l

 

T.
{to

e.“

V.‘ _

’v-

a.» v

.‘4‘b
I. <00

Cv
~54.

 

 

83

hour difference between the observance of maximum infectivity
and maximum HA activity is considered to be of no signifi-
cance. It was concluded that log 10‘“3 E150 or higher was
necessary for HA to be detectedu5. A figure of about
log 106 ID50 is given for one HA unit of influenza virusBe,
Growth curves of the egg-adapted strain of IBV in
eggs inoculated via the allantoic cavity shows a maximum
titer at 12 hours post inoculation. Early egg passage virus
enters the 10g phase in six hours and a maximum concentration
is attained within 24 to 30 hourséb’. The Showa—machi strain
of IBV in the 40th egg passage reaches a maximum infectivity
titer at approximately 16 hours after inoculationloo.
Infectious bronchitis virus 17-40, 40-23, and 41-6
inoculated into embryonating chicken eggs via the allantoic
cavity, showed maximum infectivity 24 hours post inoculation.
The infectivity titer decreased during the next 24 to 48
hours, after which the activity remained fairly constant to
the lOlst hour. The hemagglutinin of the trypsin modified
IBV 41-6 decreases in activity 24 hours post inoculation.
At this time, the HA activity of 17-40 and 40-23 was not
detectable. At the time when the infective particle entered
'the stationary phase, the HA activity was detected.
If the HA activity of IBV is an intrinsic property
CDf' the infectious particle, there should be an increase in
1311 titer corresponding to a rise in the infectivity titer.

éiizice such a relationship is not observed, it is concluded

 

84

that another mechanism is operative.

Further evidence indicates that the hemagglutinin
is not the infective particle. Chickens were inoculated
intravenously with.trypsin modified virus, and intratra-
cheally with untreated and treated virus. When neutraliza-
tion indices were calculated, the differences were not great
enough to conclude that trypsin so modified the infective
viral particle that a specific antibody was produced.

There are two phenomena reported in which the
hemagglutinin was associated with the virus particle with
no relation between the HA titer and the infectivity titer.
Under certain conditions of inoculation of influenza virus,
the allantoic fluid will yield high titers of noninfectious
hemagglutinins as well as normal infectious particles which
will participate in HA118. The noninfectious particles
lack a complete complement of ribonucleic acidl, It has
been suggested that the infected cell produced more protein
than nucleic acid and thus noninfectious as well as infectious
particles are produced67.

The other possibility would be the presence of an
irwubitor of HA activity which would mask any correlation
Ioetween infectivity and hemagglutinating activity. An
iinhibitor of HA of influenza virus was found to exist in the
allantoic fluidlll. The virus in the active state is
<zarpable of destroying the mucoid inhibitor and HA will re-

Stzlt. Indicator viruses are not capable of destroying the

 

85

mucoid, but HA will occur when the inhibitor is destroyed
by trypsin, periodate, or RDE.

The lack of correlation between the HA activity of
IBV and the infectivity cannot be explained by von Magnus'
description of the incomplete form of influenza. Rather,
the reduction or disappearance of hemagglutinating activity
would indicate that more nucleic acid is produced than is
protein, or a necessary antigen is not being incorporated
into the viral particle. If this were so, then the HA
activity would be intimately associated with the infective
particle at the time of appearance.

Data were collected which indicated that the infecti-
vity titer of IBV after hemagglutination is of the same
magnitude as that which had not been reacted with cells.
This would indicate that the infective particle does not
significantly contribute to HA.

If a mucoid inhibitor were masking any correlation
between infectivity and HA, it may be destroyed by either
active virus or trypsin . It is unknown whether IBV can
ciestroy the mucoid inhibitor. However, since this virus is
sstored in a frozen state or handled for short periods of
thne at temperatures not exceeding 5 C, the enzymatic

activity of the viral particle is not expected to be very
great. The conditions employed for the trypsinization of
tk11.s virus to induce HA favor the formation of an indicator

‘V11~us, which of its nature cannot destroy the inhibitor.

 

86

Nevertheless, trypsin should completely or partially
inactivate the inhibitor which would result in detection

of HA. Since trypsin induces HA in later harvests of in—
fected allantoic fluid, it seems that the answer is not the
presence of an inhibitor unless conditions change that re-
sult in the destruction of the inhibitor. One condition
may be longer exposure to IBV. Thus, this possibility may
not be neglected.

Another mechanism which may be operative is that
the hemagglutinin of IBV is distinct from the infective
particle, similar to the viruses of the pox and psittacosis
group. If this were the case, the growth curve and HA curve
for IBV need not show a direct relationship. In support of
this hypothesis is the fact that viral infectivity, but not
HA activity, can be detected after passage through a 50 milli—
filter. Also lending support to this hypothesis is the fact

that trypsinized infective particles are not absorbed to red
blood cells.

It is concluded from the results of infectivity and
11A determinations, ultrafiltration, and infectivity after HA
‘that the HA activity of IBV infected allantoic fluid is a
pnroperty of an antigen distinct from the virus particle. Of
‘tkie various agents tested, trypsin is the only one which
svinl consistently cause induction of HA by IBV suspended in

a llantoic r1u1d25.

Trypsin reacts with a normal constituent of allantoic

 

8?

fluid to cause HA. When ETI is added, the non-specific HA

is eliminated. The ETI does not effect the HA of virus in-
fected allantoic fluid. It seems possible that trypsin is
reacting with a “specific" and "non-specific" agent to induce
HA. Hemagglutination is evident only with the "specific"
agent after the addition of ETI. The equations for such a
reaction may be:

1. (Substrate) + (Trypsin): HA+ (ETD-rho reaction

2. (Substrate) +(Trypsin)-—>HA+ (ETI)—>HA

3. (Trypsin)+-(ETI)€Z$(Trypsin-ETI)

Therefore, ETI is necessary for the specificity of
the test, but the exact role is unknown. It may simply be
that all equations are reversible except number 2. It can
be seen from the above reactions invloving ETI that either
the non-specific substrate is absent in virus infected
allantoic fluid or, if present, does not play a significant
role when the Specific substrate is present.

In the consideration of this mechanism, the ETI
probably is competitive in nature, and all reactions are
(dependent on the concentration of inhibitor, enzyme, and
:substrate. This fact is clearly seen from the data obtained
LISing various amounts of trypsin and ETI. Maximal activity

1&3 detected when both reagents are at a one percent concentra-
tion. Activity is not detected at 0.01 percent concentration.
1Truat higher titers are observed when the concentration of

trJr'psin is ten times greater than ETI may be a reflection

 

88

of the non-specific hemagglutinating agent. When the
concentration of ETI is ten timesgreater than trypsin, the
lack of detectable HA activity may be a reflection of the
dependence of the reaction on concentration.

A means to elucidate the action of trypsin may be the
employment of various antisera. Whether trypsin acts as a
proteolytic enzyme or adsorbs onto an antigen distinct from
the viral particle to induce HA, the specificity of the
homologous antibody may be so altered as to show a difference
in titer when compared to antibody produced against normal
virus.

If chickens were inoculated intravenously with
trypsin modified virus, antibodies homologous to the antigen
should be produced. Trypsin modified virus and its specific
antiserum in a HI test may yield different results as that
when treated virus is employed with antiserum produced
against untreated virus. A difference was observed in the
serum neutralization test but was not regarded as significant.

The non-specific inhibitor of the HA of IBV present
1n.normal and immune serum would have to first be removed.
ESince ether extraction and butanol separation failed to
remove the inhibitor, it is concluded that the inhibitor is

probably not lipoidal in nature. Heat and/or trypsin had
11c> effect on the inhibitor. Trypsin and potassium periodate
irrueatment of negative and anti-IBV sera results in the

IUSggation of any effect of inhibitors or specific antibody.

 

 

 

 

89

Perhaps by varying the concentrations of the reagent, the
inhibitors may be removed without the destruction of the
specific antibody. The possibility that the inhibitor is
an enzyme which negates the action of trypsin or destroys
the erythrocyte receptor site cannot be dismissed.

Further investigation may possibly determine the mode
of action of trypsin either in two ways. If trypsin acted
as a proteolytic enzyme, a new substrate should be produced

by destruction of a surface moiety. The trypsin treated

infected allantoic fluid could be subjected to various
enzymes and if destruction of HA occurred with a certain I
enzyme reaction, the substrate would be partly defined.
The receptor site to which the myxcviruses attach
during hemagglutination is destroyed by the action of BBB.
This enzyme also destroys the mucoid inhibitor of HA of this
group of viruses.
The receptor destroying enzyme destroys the receptor
site on the erythrocyte to which the hemagglutinin of IBV
adsorbs in the process of HA, indicating that the same
receptor site is possibly used by the myxoviruses and IBV.
fiDE activity is destroyed by trypsin and the IBV sample
ennployed in this test may have active trypsin present. The
activity of the RDE on the red blood cell is completed
'beefore the trypsin is added and the conclusion is valid.

N€2‘Vertheless, equal volumes of RDE and serum will not remove

the inhibitor of HI.

 

90

A property which has been frequently employed in
characterization of a virus is the thermostability of the

infective particle and the hemagglutinin.
The hemagglutinin of the influenza strain PR8, which

is the prototype of the myxoviruses, does not follow a first

order rate of inactivation at 61 C. A straight line is

obtained when l/J log C is plotted against time which is a

three halves order reaction. After ten minutes at this

temperature, the HA activity was almost completely de-

stroyed75.
The thermostability of the hemagglutinin of the LEE

strain of influenza type B, and the Weiss and PR8 strain of

influenza type A was studied at 61.5 C. The Weiss strain

was almost completely inactivated in one and one half hours,

and the PR8 in six hours. Hox-yever, the LEE strain showed no

Significant loss of titer in eight hours at this temperature,
1 ndicating the great variation in stability among these

Strains99.
The hemagglutinins of influenza, PR8, and LEE are

Stable at 55 C for one hour, but a significant reduction of

ti ter occurred within 15 minutes at 60 C59.
Variations in the thermostability of hemagglutinins

have been observed among various strains of Newcastle disease

V1 P1135“.
Infectious bronchitis virus subjected to 56 C gave

reSults similar to those obtained with the LEE strain of

 

91

influenza B. There were no differences among the various

strains of IBV. A11 strains tested were stable for at

least three hours , and some were tested at nine hours with

no reduction of HA titer. However, there is some evidence

presented which indicates a variation of the hemagglutinating
activity of various strains of IBV when subjected to trypsin

at 56 C. Further study must be done to validate these pre-

liminary findings.
The hemagglutinin of influenza virus is more heat

stable than the infective viral particlesg. The rate of

inactivation of a virus may proceed according to the kinetics

of a first order reaction, as in the case of the PR8 strain

of influenza76. Inactivation rates of other viruses, such

as murine polio7 and human poliolz3, show deviations from

the normal curve, suggest a more complex reaction than

described by first order kinetics7. Infectious bronchitis

virus follows a bimodal rate of inactivation which can be

Studied as a three halves order reactionlol‘. The data

collected in the present thermostability study confirms this

'3 Onclusion. ‘
Viral infectivity, but not antigenicity, was

destroyed within 90 minutes at 56 C. The neutralization

index of serum from chickens inoculated intravenously with
v11‘us heated at 56 C for 120 minutes was comparable to the
1rldex of serum from chickens inoculated intratracheally with

untreated virus. The HA activity was not destroyed after

 

92

nine hours at this temperature. These differences emphasize

the variety of chemical composition of the various antigens.

Probably, the infectivity of IBV is associated with the

nucleic acid portion of the viral particle. This property

of nucleic acid was first demonstrated with tobacco mosaic

[*1 The antigenicity of IBV may be associated with

Virus 0
the viral particle and perhaps proteinecus. The antigenicity

of the fowl plaque virus resides on the protein coat of the

virus particle101. The thermostability of this neutralizing

antigen may be of some consequence in the preparation and

storage of vaccines.
The effect of trypsin on viral infectivity has been

Studied with a number of different viruses. .Nith certain

viruses as feline pneumonitis and the CD VII strain of murine

encephalitis, the infectivity titer will increase due to the

dissociation of aggregates73. This enzyme has been used in

the preparation of virus for electromicroscopy by destruction
The LEE strain of influenza, and

o f amorphous mater ial9u.
After three hours

pOtato virus X are inactivated by trypsin.
at 37 C, the infectivity titers of trypsinized IBV 141-7 and

3—16 were significantly lower than that of the controls.

The titer of the trypsinized sample of IBV 40-23 was approxi-

mately the same as the control. The mechanism of inactive—

t10n is presumably one of digestion of protective proteineous

material. It may either be the destruction of an antigen

necessary for replication of the particle, or the unfolding

 

of a peptide which would render the nucleic acid more

susceptible to heat.

After 12 hours of incubation, the trypsinized sample
of IBV 3—16 has a greater titer than the untreated control.
The same has occurred at 20 hours with IBV hl-9. This may
be an artifact or it may be that the enzyme has been de—
natured by heat and is now acting as a protective colloid.
After 20 hours at 37 C with trypsin, one sample of IBV 3-16,
and IBV 41-9 still retained some infectivity in both the
trypsinized and untreated sample.

Disruption of viral aggregates may have occurred
with some samples as the titer of trypsinized IBV 3-16 was
ID50 100°7 greater than the control. In some cases no
effect was observed. The question concerning the disruption
of viral aggregates in allantoic fluid could be studied by
means of a sonic oscillator.

In studying enzymatic digestion, the concentration
of extraneous material must be considered. The lag period
observed in the infectivity titrations of IBV hl-7 and 3-16
may be due to the destruction of soluble material which were
in higher concentrations than the infective particle, and
only after the unaltered enzyme was released from this sub-
strate did it react with the virus particle. This lag may
be due to the digestion of an inhibitor coating on the in-
fective particle much like that which has been proposed for

influenzazo. It has been shown that trypsinized virus

 

 

 

 

9U

administered intravenously will stimulate the production of
antibodies to the same level as untreated virus given intra-
tracheally.

Any system involving the agglutination of red blood
cells is hampered by the variation found in the reagents
used. One of the variations which must be considered is the
reactivity of the erythrocyte. HA titers of vaccinia varied
considerably depending on the species of fowl cells employed.
Cells from only 50 percent of donors tested were reactive in
the HA tests and this proportion is dependent on the age of
the donor animallu'zu. Another factor which will influence
the HA titer is the length of storage time of drawn cells
at 4 C.

Such variables are found when the HA activity of
IBV is determined. Erythrocytes from different chickens
will not react with a given sample to give the same titer.
Erythrocytes collected in Alsever's solution could be used
to determine HA activity of trypsin treated allantoic fluid,
but they were unsuitable for use after one week of incubation
at 4 C.

In a preliminary experiment chickens were hatched,
and reared in isolation. Some birds were infected with IBV.
Reactivity of erythrocytes is apparent in birds which were
three weeks or older. The activity of the cells from
infected birds was the same as those from birds free of IBV.

In a second experiment using different birds, it was again

 

found that HA activity could not be demonstrated until the

birds were three weeks old. The data of this experiment

paralleled the previous results which indicate that previous
exposure to the virus does not increase the reactivity of the
erythrocytes. Nevertheless, the data shows an alteration of
the receptor site three weeks after hatching, which indicates

the site is absent, blocked, or non-reactive. Since exposure
to virus has no effect on the development of the receptor
site, all variations in reactivity were merely reflections

of the intrinsic variation of the erythrocytes.

 

SUMMARY

Hemagglutination titers of various cultures of infectious
bronchitis virus after treatment with one percent trypsin

at 56 C for 30 minutes are comparable to those at 37 C

for three hours. Eggwhite trypsin inhibitor is added

after incubation. The former procedure has been

selected for the standard incubation period for the

trypsin modified hemagglutination test of infectious

bronchitis virus. I
The hemagglutinin of infectious bronchitis virus can

be demonstrated only with erythrocytes from chickens
and turkeys. I

Erythrocytes from chickens less than three weeks old

are not reactive to the hemagglutinin of infectious
bronchitis virus.

Reactivity of erythrocytes varies among donors. Frequent
bleeding may result in the loss of reactivity of the
erythrocytes.

Trypsin modified infectious bronchitis virus is stabile
for hemagglutination for three weeks at -65 C, but
fluctuations in titer occur during the next four weeks.
After six and one-half hours at 56 C, prior to trypsini—
zation, infectious bronchitis virus retains its ability

to agglutinate erythrocytes.

10.

11.

12.

 

97

After two hours at 56 C, the virus is non-infectious
for chicken embryos; but it is antigenic for chickens
as shown by antibody response measured by serum
neutralization tests.

There is a reduction of viral infectivity when
incubated with trypsin at 37 C.

With infectious bronchitis virus 17-uo, 40-23, and
41-6 the maximum viral infectivity for embryos and

the hemagglutination titer of infected allantoic fluid

is attained at 24 and 72 hours, reSpectively, after

\

inoculation.

Infected allantoic fluid after hemagglutination shows

no difference in infectivity as compared to similarily
treated controls which were not reacted with chicken
erythrocytes.

Embryo infectivity and hemagglutinating activity of
infectious bronchitis virus l7-b0 and 20—22 can be
demonstrated in filtrates from NSC millimicron Milli-
pore filters. Filtrates from a 50 millimicron filter
are infectious for chicken embryos but do not possess
hemagglutinating activity.

The same endpoint is attained with infectious bronchitis
virus 17-40 and influenza virus strain PR8 when 10
hemagglutinating units of each are reacted with chicken
erythrocytes previously treated with receptor destroying

enzyme of Vibrio cholerae.

13.

14.

 

98

Hemadsorption does not occur with infectious bronchitis
virus 41 cultivated in and producing cytopathogenic
effects in chicken embryo kidney cell cultures.
Extracellular fluids from infectious bronchitis virus
41 and 42 cultivated in chicken embryo kidney cell
cultures show a hemagglutination titer of 160 as
compared to 80 for uninoculated cells.

Various methods for the removal or destruction of the
non-specific inhibitor of hemagglutination present

in normal serum proved ineffective.

3.

 

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