imwjnologic studies on pichindevirus · 2014-06-18 · abstract '..---.. infection by pichinde...
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IMWJNOLOGIC STUDIES ON PICHINDEVIRUS
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IMMUNOLOGIC STUDIES ON PICHINDE VIRUS INFECTION IN CELL
CULT'URE AND IN SYRIAN HAMSTERS
J/
By
MICHAEL JOSEPH BUCHMEIER, B.S., M:S.
A TheSis
Submitted to the School of Graduate Studies
in Partial Fulfilment of the Requirements
for the Degree
Doctor of Philosophy
McMaster University
June 1976
i
(
r @) MICHAEL JOSEPH BUCHMEIER 1977
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DOCTOR OF PHILOSOPHY (1976)(Medical Sciences)
McMASTER UNIVERSITYHamilton,cOntario
TITLE: Immunologic Studies on Pichinde,Virus Infectionin Cell Culture and in Syrian Hamsters
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AUTHOR: Michael Joseph Buchmeier,.B.S.
M.S.
SUPERVISOR: Professor W.E. Rawls
NUMBER OF PAGES: xvii, 210
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(Washington Sta~eUniversity)
(Washington ~t~teUniversity)
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ABSTRACT' ..
..---Infection by Pichinde virus, a member of the
arenavirus group, was studied in vivo in Syrian hamsters,-- .
and in vitro in cell culture. Emphasis in the in ~
studies was placed on the mechanism of resistance' to
~irus infection in adult hamsters. Two hamster str~ns
were found to"differ in their susceptibility to lethal
Pichinde virus.. infection. LVG/Lak hamsters were found,
to be roO%.susceptib-le to low doses of Pichinde virus
dur~ng ~he first 0 days of life, but after 8 days·of.
~ife, mortality was uncommon. Peak serum virus titers
in animals infected at 3 days of life were 4 logs
greater than in animals infected at 12 days. MHA/Lak
)
\
hamsters, in contrast, were found Ito be susceptible•
.' to lethal virus infection both as newborns and as
adults. Peak serum virus titers of greater than' 108
PFU/ml were observed 8 days after infecti~n of adult
MHA hamsters compared with l~ss than 10 3 PFU/ml in
their LVG counterparts. Cultured primary kidney' cells
and peritoneal macrophages from either hamster strain
supported Pichinde Virus. rePlic~tion equall~ rll in vitro.
Antibody levels me~sured by complement-fixation test wereI--~\
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similar at 13, 21, and 30 days after infection.,. ..
I
Cyclophosphamide immunosuppression, administered 3, .
days after infection, rendered adult LVG animals
susceptible while slightly increasing the mortality, ,
/among MHA hamsters. These fin~lings suggellt thatr ,
immunologic factors maturing early "in life in' LVG
hamsters and deficient in MHA hamsters limit Pichinde. ~~~----
virus infection. The relationship of theBeob~ervations
with previously reported arenavi;rus diseases is
discussed ~
, The antigenic structure,of Pichindevirus was
I ' examined. Lysates of BHKn cells" infected with
Pichinde virus and harvested 48-96 hours af~er infection
contained virus-specific antigens detectable by comple
~ent-fixation (CF) test. Immunodiffusion anaiysis
of the lysates dJmonstrated two antigens which,differed
'",
in their properties of heat and proteolytic• ,<
res~stance. The antigen accounting for the
enzyme.major,
.,(
proport~onof the CF a'ntigen
pronase resistent protein of
activity was a heat stable,I
.)
20~30,000 molecular we~ght·
estimated by gel' Hlt"I'ation. The minor antigen was heat,'/ '
..W .
labile and s~sceptiqle to proteolysis. Antiserum pre-. (.pared aga~nst part~ally purified major antigeQ, yielded" '.
~ -v~ Ja band of identity when tested,against antiserum
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.-.... ,
. rprepared against virus "cores" obtained by NP-40
solubilization ~of purif.ied Pichinde virus.' Disruption1 •
of purified virus bytreatm~nt with 1\ NP-40 and SO\ ~
ug/ml RNase liberated 2 soluble antigens which,
identified wi~h ~hose.demonstrated in lysates of,
,-
'infected cells.
1~own'to'contain
The liberated an~igenic activity was•
3 of the 4 polypeptides found in the
.- virion. These. findings suggested-that the antigens
detectable by CF and immunodiffusion were components.
of the virion core. ~ajor antigen derived from infected
cells was purified by rate-zonal sedimenta~ion, iso
electric focusing and gel filtration. Two low molecular
weight polypeptides were demonstrable in the purified
antigen.
Since'multiple segments of RNA exist in the-Pichinde virion, it was of interest to determine,
I. •whether ant~gen synthesis and virus production could
be dissociated in the infected cells. In Vero cells
infected by Pichinde virus, antigen on the cell surface,
and production of infectious virus shut down 48-96
hours after infection, whereas antigen detectable in
the cytoplasm of infected cel}s appeared stable fo; over"5 days. In BHK 21 ~ells, actinomycin D (AD) at dosage
levels of 1-4 ug/ml reduced virus yields by greater than
95% while reducing antigen detectable by CF by only 30%.
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The quantity of a~tigen\ .
. \prodl!ced was independent
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of AD dosage within the range t~sted. Both the major
and the minor antigens were identified in lysates
of AD treated cells. The observed decrease in infectious
virus production could not be ·attributed to increased.cell associated virus or to greater production of
•defecti~e interferring particles. Surface antigen,
,demonstrable by immunofluorescence. and by antibody
binding assay was .found to accumulate
of infected cel~s incu;ated with AD.
on the membrane
These findings
!
suggest that Pichinde virus replication in ~D treated
BHK 2l cells is blocked at the level of virus maturation •
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ACKNOWLEDGEMENTS
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I would like to thank Dr. 'Rawls for suPPo€t,
guidance, and encouragement in these studies, and ther
,-entire Rawls family, for friendship both in and out of
the lab. Thanks are also extended to Ms. Carol McMillan,
Ms. Elaine Jaggard and Dr. RamaraoGa~gavalli for
asshtance in the performa~e ~f some of the experiments
described. The contribution of Dr. Jack Gauldie~in
supplying helpful discussion and suggestions regardIng
protein purification is greatly\appreciated.
I would also like to thank Dr. Frederick
Murphy, in Atlanta, for evaluating the histopathology
of Pichinde virus infection in hamsters, and Ms. Terry
DeCola for preparing this manuscript. ,~
':lery special thanks are offered to Nancy
and to Josh ~or love, patience, understanding and moral
support.
This work described in this study was supported
by grants from the Natiunal Cancer Institute of Canada,
and the Medical Research Council.
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Introduction
TABLE OF CONTENTS
I I.
II."-
III.
IV.
V.
VI.
VII.
Definition.and Characteristics of ' 1th~ Arenaviruses.
Biophysical Properties.· 4
Biochemical Prpperties - Nucleic Acids. . 5
Biochemical Properties - Proteins. ' 10
Persis~ent Infections by Arenaviruses. 13
Acute Disease Following Arenavirus Infection. 16
Immunosuppressive Effect of Arenavirus. 21.Infection.
VIII. ~urpose of the Study. 24
~Materials and Methods
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I.
, II.
Cell Culture and Virus Growth.
A. Cell Culture.l' ,
B. Virus Source and Propogation.. '" ..C. Soluble Antigen Extraction.
Immunological Methods.
A.' Antisera./
B. Complement Fixation Tests.
C. Immunodiffusion.
D. Immunofluorescent Staining ..
E. Antibody Binding Assay.
viii
25
25
26
28
29
29
33
33
34
36
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Results 46
46I. ' Susceptibility of Unnatural Hosts toInfeotion by Pichinde Virus.
A. 'Immunization of Laboratory Animals with 46Piohinde Virus.
,B. Pathogenesis of Pichinde'Virus Infeotion 56in Hl\msters'.
1. Susceptibility of LVG and MHA Hamsters 56to Lethal Infection by Pichinde Virus.
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2. Virus ~iters in Organs after Infection 63of LVG and MHA Hamsters.
3. Primary Cell Cultures from both Strains 67Support Virus Replication Equally well.
"
4. The Effeot of CyclophosphamkD Immuno- 72suppression on Resistance to Infection.
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'['AI.ILI: 01' CONTENTS (oondnut>cl-) . c
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5• Anti'1Viral Anti:bocaou in LVO lindMUA Hllmottlru Aftt>f' P:Lo)1i,ncl{l Vi.rutl
·Infootion. . ,
6. Suoooptibifity to othQl' HnmntorStrain" to Piohindn Virun Infnction.
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7. IIintopntholop.y of Acute Jij,chincln,. Virutl Infec~ion iri the Hdmntnl';
\C. Infeotion of Humtlntl by" Pichindt'l V:Lrun~
•Antigon Synthooi' llnd Exprouoion on Col,lCulturo,
A. Virun-~ocific Antigon ExprofJuionCorroliltod wJ.t'h Now Virun Production.> in Infectod Voro Cell 0,
B. ViruD Replioation llnd Antigon Productionin Cello Tretltod with Aotinomycin D.
C. POlype~e com~oui~iO; of Cellu Inoubated in tho nrouonce and Aboonce ofAotinomycin D.
72
'IG
79,
ell
88
96
III. Bioohemical Studiflo of tho Antigenullof Pichi~do 101;
Virus. .
A. Soluble Antigona Detoctable in Lynatoo ofCells Infootod by Piohinde Viruo.
B. Some' BiopnY!Jica~er~iC!l!l of the Mtlj'orAntigon.
C. Purification of the Major CF Antigon fromInfected BrK2 i Colla.
D. Relationship of the Major Antigen withStructural Componento of the Viruo.
x,.
1'05
109
112
121
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TABLE OF CONTENTS (continued)
Page
E. Association of Antigens with the Virion 137as Detected by Immunodiffusion.
F. Relationship of Antigenic Activity. with . 141Substructures ?f Pichinde Virus.
G. Comparison of the Polypeptide Composition i48of Purified Major Antigen Derived fromInfected Cells with ~he Virion 101ypeptides.
H. Purification of Viral Polyp~ptides by 155Isoelectric Focusing.
Discussion
't.
-1.67
. \.."
Summary and Conclusion.
Appendix I
Report from Dr. Frederick Murphy - Histopathologyof Pichinde Virus Infection in Hamsters.
References
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xi
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-. ',",.~: ,~
198
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i,,LIST OF FIGURES AND CHARTS
Pag~
31IFlow Chart of the Methods Used toObtain Antiserum against the HeatStabie Major Antigen. "
2. Complement-fixing Antibody Produced by 48a Rabbit after a Single 'IntravenousInjection with Purified Pichinde,Virus.
,Figure 1.
3. Complement-fixing Antibody Titers in 53Adult LVG Hamsters after Infection withPichirde Virus. Q
"
4.
5.
Immunodiffusion Reaction Between Serumfrom Pichinde Virus-immune Guinea Pigs'and Soluble Antigen Prepared from Virusiri~ected and C?ntrol BHK21 Cells.
Mortality among LVG Hamsters after PichindeVirus Infection at Various Ages.
55
58
6. Mortality Among MHAHamsters after Iptraperitoneal Infection with Various Dosesof Pichinde Virus.
60
7 .
8 •
9.
10.
11.
Viremia in MHA and LVG Hamsters afterPichinde Virus Infection.
Growth of Pichinde Virus in PrimaryKidney Cell Cultures Established From3 Day Old and Adult MHA and LVG Hamsters.
Growth of Pichinde Virus in LVG and MHAHamster Macrophages Cultured In Vitro.
The'Effect of Cyclophosphamide Immunosuppression on Mortality in Adult LVGand MHA,Hamsters.
Complement-fixing Antibody :Titers, toPichinde Virus in Laboratory Workersin Relation to the Time of PossibleExposure.
65
69
71
74
83
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'.LIST OF FIGURES AND CHARTS (continued)
12. Expressio.n of~Cytoplasmic and SurfaceAntigen in Relation to' Infectious .Viru~ Production after )ichinde.VirusInfection in V~o Cells.
13. Immunodi~fusion Test Showing AntigensProduced in BHK21 Cells Incubatedwithan~ without A~tinomycin·D.
l~. Immunofluorescence Staining of theSurface of Cells Infected with PichindeVirus.
87
95
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16.
17.
18.
Quantitation of Surface Antigens in 98Actinomycin.D Treated Cells Using 125ILabelled Antibody.
Virus-specific Polypeptides Present in 101BHK21 Cells Infected with Pichinde Virusand Incubated with or without Actinomycin
:~creased Particle Production in Cells ~Infected with Pichinde Virus and Inc~ated
in the Presence of Actinomycin D.
Immunodiffusion Te~ Showing the Effect of 107Hefting on the Antigens Detected in LysatesoflBHK21 Cells Infected with PichindeVirus.
..~
..19. Estimation of the Molecular Weight of the III
CF Antigen of Pichinde Virus by GelFiltration in G-200 Sephadex.
20. Sedimentation of Soluble Antigen in a 5-20% 114Sucrose Density Gradient.
21. Isoelectric Focusi~g of Cell-derivedPichinde Virus-specific and ControlAntigen.
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LIST OF FIGURES A~D CHARTS (Continue:,
'\.,
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22. Biogel P-200 Gel'Filtration of Pichinde 120Virus-specific CF Ant~gen.
23. Indirect Immunofluorescent Stain of 124Pichinde Virus-infected Vero Cells byAntiserum Against Partially Purified,Heat Stable CF Antigen.. ,
24. Direct Immunofluorescent Stain of·~eroCells Infected with Pichinde Virususing Immune Hamster Globulin Conjugated with FITC.
25. Flow Chart of the, Methods. Used to Obtain 128Antisera.Directed Against Components ofNP-40 Disrupted Pichinde Virus.
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26.' Immunodiffusion Test Showing Identity. between Antigens Detected in Lysates ofInfected Cells by Immune Serum and Antisera agairi~t NP-40 Obtained Virus "Cores"and Partially Purified CF Antigen., . .
27. Indirect Immunofluorescent Stain ofPichinde Virus-infected Ver~ Cells byAntiserum against Intact Virus.
28. Indirect Immunofluorescent Stain ofPichinde Virus-infected Vero Cells by
~_Antiae~um~against the NP-40 InsolubleVirion "Core" Fraction.
130
132
134
29. Indirect Immunofluorescent Stain of "136Pichinde Virus-infected Vero Cells byAntiserum against the NP-40 SolubleVirion Component.
30. Immunodiffusion Reaction Showing Identity 140of Antigens Detected in Lysates of In-fected BHK21.Cells with Antigens Detectablein Disrupted Pichinde Virus.
xiv
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