INFORMATION TO USERS
This reproduction was made from a copy of a manuscript sent to us for publication and microfilming. While the most advanced technology has been used to pho tograph and reproduce this manuscript, the quality of the reproduction is heavily dependent upon the quality of the material submitted. Pages in any manuscript may have indistinct print. In all cases the best available copy has been filmed.
The following explanation of techniques is provided to help clarify notations which may appear on this reproduction.
1. Manuscripts may not always be complete. When it is not possible to obtain missing pages, a note appears to indicate this.
2. When copyrighted materials are removed from the manuscript, a note ap pears to indicate this.
3. Oversize materials (maps, drawings, and charts) are photographed by sec tioning the original, beginning at the upper left hand comer and continu ing from left to right in equal sections with small overlaps. Each oversize page is also filmed as one exposure and is available, for an additional charge, as a standéird 35mm slide or in black and white paper format. *
4. Most photographs reproduce acceptably on positive microfilm or micro fiche but lack clarity on xerographic copies made from the microfilm. For an additional charge, all photographs are available in black and white standard 35mm slide format.*
*For more information about black and white slides or enlarged paper reproductions, please contact the Dissertations Customer Services Department.
Dissertation UMI Information Service University Microfilms International A Bell & Howell Information Com pany 300 N. Zeeb Road, Ann Arbor, Michigan 48106
8618741
Baumgartner, Wolfgang K.
MECHANISMS OF IN VITRO PERSISTENCE OF TWO CANINE PARAMYXOVIRUSES AND IN VIVO NEUROPATHOGENICITY OF CANINE PARAINFLUENZA VIRUS
The Ohio State University Ph.D. 1986
University Microfilms
Intern Stionel SOO N. Zeeb Road, Ann Arbor, Ml 48106
PLEASE NOTE:
In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V
1. Glossy photographs or pages.v /
2. Colored illustrations, paper or ______print
3. Photographs with dark background
4. Illustrations are poor copy______
5. Pages with black marks, not original copy.
6. Print shows through as there is text on both sides of page,
7. Indistinct, broken or small print on several______pages
8. Print exceeds margin requirements______
9. Tightly bound copy with print lost in______spine
10. Computer printout pages with indistinct print,
11. Page(s)______lacking when material received, and not available from school or author.
12. Page(s)______seem to be missing in numbering only as text follows.
13. Two pages num bered . Text follows.
14. Curling and wrinkled pages______
15. Dissertation contains pages with print at a slant, filmed as received______
16. Other______
University Microfilms international
MECHANISMS OF IN VITRO PERSISTENCE OF TWD CANINE
PARAMYXOVIRUSES AND IN VIVO NEUROPATHOGENICITY
OF CANINE PARAINFLUENZA VIRUS
Dissertation
Presented in Partial Etilfillment of the Requiranents for
The Degree Doctor of Philosophy in the Graduate
School of the Ohio State IMiversity
By
Wolfgang K. BaumgSrtner, Dr. itied. vet.
*****
The Ohio State University
1986
Dissertation Conmittee: Approved by Steven Krakcwka James R. Blakeslee Charles C. Capen Steven E. Vfeisbrode Adviser Department of Veterinary Pathobiology Copyright by
Wolfgang K. Baumgârtnery Dr. med. vet.
1986 Dedication
To nty vd-fe, Angelika, and iry son, Lars.
11 iOaSiCWLEDGayENTS
The author wishes to ej^ress his sincerest appreciation to iry
adviser. Dr. S. Krakcvto, for his guidance, encouraganents, enthus
iasm, and patience during this project. I wish also to eatress iry
gratitude to Dr. C. Capen, Chairman of the Department of Patho
biology, for his encouragement and support for my second visit to The
Ohio State University.
The author would like to es^ress profound appreciation to Dr. J.
Blakeslee and Dr. M. Axthelm for their collegiate support and advice.
I thank Ms. S. Ringler, N.J. Austin and J. Dubena, vÆo most
skillfully assisted me in ny in vitro and in vivo ejqjeriments.
I would like to e3q>ress m y thanks to Ms. E. Handley and 0.
Kindig for their technical assistance in preparing the electron- microscopic specimens.
I thank Ms. V. Stump and R. I^an for their secretarial assis
tance in -typing this dissertation.
Last, but not least, I would like to express my appreciation and
thanks to my colleague and friend. Dr. T. Rosol, for providing a
constructive, competitive office environment.
A final acknowledgement goes to The State of Ohio Canine
Research fund grant 2 RDI NS-14821, NIH, PHS and the Deutschen
Forschungsgemeinschaft for sipport of these studies.
iii VITA
July 20, 1952 ...... B o m - Offenbach, West Germany
1 9 7 1 ...... Abitur, Leibnizschule, Offenbach, West Germany
1978 ...... Approbation, JLO-Giessen
1979-1981 ...... Predoctoral fellow, DAAD, Department of Veterinary Pathobiology, The Ohio State University
1981 ...... Doktor med. vet.. Institute Vet. Path., Giessen
1982 ...... Private practice
1983-1985 ...... Postdoctoral fellow, DFG, Department of Veterinary Pathobiology, The Ohio State University
1986-Present ...... Postdoctoral fellow, DFG, Institut für VeterinSr Pathologie der Justus Liebig Universitât, Giessen
PUBLICATICNS
Baumgârtner, W., Krakowka, S.: A Sirtple Method for the Histochemical Demonstration of Viral Inclusion Bodies in Cell îfonolayers in Micro titration Plate. Stain Technology (in press) 1986.
Baumgârtner, W., Zajac, A., Hull, B.L., Andrews, F., Garry, F.: Paralaphostrongylosis in Llamas. J. Am. Vet. Med. Assoc. 187:1243- 1245, 1985.
IV Baumgârtner, W. : Canine Parainfluenza Virus. In: Pathology of Viral Disease, R.G. Olsen, S. Krakoto, eds. CPC Press, Vol. II, Ch. 4, 1985.
Baumgârtner, W., Posselt, H.J. : Kutane Altemariosis bei Hunden mit unspezifischen Dermatitiden. Kleintierpraxis 28:353-358, 1983.
Baumgârtner, W., Posselt, H.J. : Ostrogeninduzierte hamorrhagische Diathese bei einer Hundin. Kleintierpraxis 27:419-420, 1982.
Baumgârtner, W.K., Krakovdca, S., Kbestner, A., Evermann, J . : Ultra- structural Evaluation of Acute Encephalitis and Hydrocephalus in Dogs Caused by Canine Parainfluenza Virus. Vet. Pathol. 19:305-314, 1982.
Baumgârtner, W.K., Krakcuflto, S., Kbestner, A., Evermann, J.: Acute Encephalitis and Hydrocephalus in Dogs Caused by Canine Parainfluenza Virus. Vet. Pathol. 19:79-92, 1982.
Evermann, J., Krakowka, S., McKeiman, A.J., Baumgârtner, W.: In Vitro Identification and Characterization of a Virus Isolated from a Dog with Neurological Dysfunction. Infect. Irtittun. 31:1177-1183, 1981.
Abstracts:
Baumgârtner, W.K., Krakcudca, S., Koestner, A., Evermann, T.: Acute Encephalitis and Hydrocephalus in Dogs Caused by Canine Parainfluenza Virus. Annual Review of Hydrocephalus, 1983. S. Mabumoto, K. Satdheny, eds., Nevion Publishing Co., 1983, pp 7-8.
FIELDS OF STUDY
Major Field: Neuropathology, Ccmparatice Pathology, Pathogenesis of Viral Diseases TABLE OF CCNTENTS
DEDICATIOSr...... ii
ACKNCWLEDGEMENTS...... iii
VITA ...... iv
LIST OF T A B L E S ...... X
LIST OF F I G U R E S ...... xii
ABBREVIATICNS ...... xix
CHAPTER Page
I. A SIMPLE METHOD FOR THE HISTOCHEMICAL DEMONSTRATION OF VIRAL INCLUSION BODIES IN CRT.T. MONOLAYERS IN MICROTITRATION P L A T E S ...... 3
Introduction ...... 3 Materials and Methods ...... 5 Results and Discussion ...... 6 Summary ...... 8 R e f e r e n c e s ...... 11
II. CANINE PARAINFLUENZA VIRUS: EVOLUTION OF TO VITRO PERSISTENCE AND PRffiUCTION OF INTERFERING VIRAL PARTICLES ...... 12
Introduction ...... 12 Materials and Methods ...... 15 R e s u l t s ...... 19 D i s c u s s i o n ...... 24 S u m m a r y ...... 28 Refeirences ...... 40
VI TABLE OF OOMTEMTS (Continued)
Page
CHAPTER
III. VIRAL PEE^ISTEMCE IN VERO CRT.Tfi DUALLY INFECTED WITH IWD PARAMYXOVIRUSES: A MORPHOLOGICAL AND IMMUNOELECTRON MICROSCOPIC INVESTIGATION...... 45
Introduction...... 45 Materials and Ifethods...... 48 R e s u l t s ...... 52 D i s c u s s i o n ...... 56 S u m m a r y ...... 59 R e f e r e n c e s ...... 87
IV. PERSISTENT INFECTICN OF VERO rRT.Tfi BY PARAMYXOVIRUSES ...... 91
Introduction...... 91 Materials and tfethods...... 93 R e s u l t s ...... 96 D i s c u s s i o n ...... 99 S u m m a r y ...... 103 R e f e r e n c e s ...... 116
V. NEUROPATHOLOGY OF CANINE PARAINFLUENZA VIRUS AFTER INTRACEREBRAL INFECTICN IN FERRETS ...... 120
Introduction ...... 120 Materials and Methods ...... 122 R e s u l t s ...... 125 D i s c u s s i o n ...... 128 S u m m a r y ...... 131 R e f e r e n c e s ...... 147
vix TABLE OF CCNTENTS (Continued)
Page
CHAPTER
VI. CANINE PARAINFLUmZA VIRUS ...... 151
Introduction and H i story ...... 151 Characterization of Canine Parainfluenza (CPI) V i r u s ...... 152 Pathogenesis of CPI Virus Infection In V i t r o ...... 152 S e r o l o g y ...... 153 Clinical and Pathological Findings in Dogs with CPI Virus Infection ...... 154 Treatment and Prevention ...... 156 R e f e r e n c e s ...... 167
BIBLIOGRAPHY...... 170
Vlll LIST OF TABLES
Page
CHAPTER II
Table
2.1 Virus yield in lytic and persistently infected cell l i n e s ...... 30
2.2 Superinfection of persistently infected cells with lytic CPI(+) and R252-CDV virus ...... 31
2.3 Hanolcgous interference with replication of lytic CPI(+) virus ty CPI(+) or CPI(-) progeny virus of persistently infected c e l l s ...... 32
2.4 Effect of ultraviolet (UV)-irradiation on inhibitory material fran persistently infected cells ...... 33
CHAPTER III
3.1 Summarized effects of 3 different preembedding treatments on glutaraldehyde-fixed cells on cellular integrity and labeled viral antigen presentation...... 60
CHAPTER IV
Table
4.1 Virus yield in lytic and persistently infected cell l i n e s ...... 104
4.2 Super infection of persistently infected cells with the lytic CPI(+) and CDV v i r u s ...... 105
XX LIST OF TABLES (Continued)
Page
CHAPTER IV
Table
4.3 Interference with replication of lytic CDV virus by CDV and CPI-CDV progeny virus of persistently infected cells ...... 106
4.4 Interference with replication of lytic CPI(+) virus isy CPI-CDV progeny virus of persistently infected cells ...... 107
CHAPTER V
5.1 The time-dependent occurrence of micro scopic lesions and viral antigen in ferrets intracerebrally inoculated with canine parainfluenza virus ...... 132 LIST OF FIGURES
Page
CHAPTER I
Figure
1.1 Vero cells persistently infected with a non-giant and non-plaque producing CPI virus. A) Immuno fluorescence demonstrating cytoplasmic inclusion bodies (XlOO). B) Bouin ' s-formalin-Giemsa fixation and stain shows well-demarcated cytoplasmic inclusion bodies (arrows) (XlOO). C) Formalin-Giemsa fixation and stain; no inclusion bodies visible (XlOO) D) Methanol- Giemsa fixation and stain; inclusion bodies are less demarcated from the cytoplasm (XlOO). . 9
CHAPTER II
2.1 Equilibrium density gradient separation of CPI(+) in a potassium tartrate gradient. Ten-fold virus dilutions of 650 yl fractions shows peak activity witdi a buoyant density of 1.184 g/ml. CPE was characterized by syncytial giant cell formation. Fractions witdi a buoyant density below 1.12 g/ml showed no syncytial giant cell formation but numerous cytoplasmic inclusion bodies (numbers represent buoyant densities in g/ml) ...... 34
2.2 Equilibrium density centurifugation of lytiic CPI(+) virus (a) and CPI(+) progeny virus and from persistently infected cells, passge level 64 (b). Infected confl^gnt monolayers were labeled witdi 20 yCi S- methionine per ml. Radioactivity of each fraction was determined. Lytic virus (a) has a buoyant density of 1.185 g/ml; in con-trast, at passage level 64 the virus population has a lower buoyant density with 1.136 g/ml (b). (Numbers represent buoyant densities in g/ml) ...... 36
xi LIST OF FIGURES (Continued)
Page
CHAPTER II
Figure
2.3 Equilibrium density centrifugation of lytic CPI(-) virus (a) and CPI(-) progeny virus fron persistently infected cells, passage level,42 (b). Virions were labeled with S-methionine as described under materials and methods. Lytic CPI(-) virus (a) and progeny virus fron passage 42 (b) shew a similar distribution of the virus population, vàiich is very distinct from CPI (+). (Numbers represent buoyant densities in g/ml) ...... 38
CHAPTER III
3.1 Vero cells persistently infected with CDV virus after saponin -treatment. Membrane, budding viral particles (arrovAead), and cyteplasmic -viral antigen (arrow) are labeled (anti-CDV 1:200). Note loss of cytoplasmic organelles and disruption of mitochondria, N = nucleus, M = mitochondria (osmic acid only). Original magnification X 1450 ...... 61
3.2 Vero cell lytically infected with CPI virus, after GBS treatment. Sutmembraneous viral antigen and budding viral particles are labeled (arrows) N = nucleus (osmic acid only). Original magnification, X 4800 ...... 63
Xll LIST OF FIGURES (Continued)
Page
CHAPTER III
Figure
3.3 Vero cells persistently infected with CPI virus, after fMSBS treatment. Cyto plasmic viral antigens are labeled (arrows) N = nucleus (anti-CPI 1:100) (osmic acid only). Original magnifi cation, X 17,700 ...... 65
3.4 Vero cell persistently infected with CPI-CDV virus, passage 27. Both smooth (sNC) CPI and fuzzy (fNC) NC are present in the cytoplasm (osmic acid, uranyl acetate and lead citrate counterstain). Original magnification, X 17,000 67
3.5 Vero cells persistently infected with CPI-CDV virus, passage 27. Cytoplasmic accumulation of both smooth (sNC) CPI virus and fuzzy (fNC) CDC nucleocapsids. Several fibrillary nuclear bodies are present in the nucleus (osmic acid, uranium acetate and lead citrate). Original magnification, X 10,800 ...... 69
3.6 Vero cells persistently infected with CPI virus, passage 27. Borderline between smooth (sNC) CPI virus and fuzzy (fNC) CDV nucleocapsids (osmic acid, uranium acetate and lead citrate). Original magnification, X 123,000 71
3.7 Vero cells persistently infected with CPI virus, passage 27. Multilobulated nuclei in ^mcy- tium giant cells with prominent fibrogranular nuclear bodies (vhite arroviiead), smooth (arrcxAead) and fuzzy (arrows) nucleocapsids (osmic acid, uranium acetate and lead citrate). Original magnification, X 10,800 93
xiii LIST OF FIGURES (Continued)
Page
CHAPTER III
Figure
3.8 Vero cell persistently infected with CPI-CDV. Saponin treatment, p40, labeled cytoplasmic CDV antigen (arrow) and unlabeled CPI NC (arrcxdiead). (Stained with osmic acid and heavy metals.) Original magnification, X 13,400 ...... 75
3.9 Vero cell persistently infected with CPI-CDV, passage 40, saponin treatment. Perinuclear positively labeled CDV NCs (arrow) (anti- CDV 1:200) with adjacent diffusely distributed unlabeled smooth CPI NCs (arrovihead), N = nucleus. Original magnification, X 69,000 77
3.10 Vero cell persistently infected with CPI-CDV, p75. Labeled cytoplasmic CDV NC and budding viral particles (arrows) (anti- CDV 1:200) adjacent to unlabeled CPI NCs (arrovdiead) M-GBS method (osmic acid and uranyl acetate only). Original magnifi cation, X 9,300 79
3.11 Vero cell persistently infected with CPI-CDV (GBS method) (p63) ). Labeled cytoplasmic CPI antigen (anti-CPI 1:100) (arrows) and unlabeled CDV antigen (large arrc^Aeads) in adjoining cells (stained with osmic acid and heavy metal counterstain) N = nucleus, small arrowheads - plasma membrane. Original magnification, X 13,500 ...... 81
XIV LIST OF FIGÜEES (Continued)
Page
CHAPTER III
Figure
3.12 Segment of a Vero cell persistently infected with CPI-CDV, p75 (M-GBS treatment). CPI antigen is labeled with anti-CPI (1:200) (arrov^eads) and the adjacent, fuzzy CDV NC remains unlabeled (arrows) (stained with osmic acid and heavy metal counterstain). Original magnification, X 90,000 ...... 83
3.13 Vero cell persistently infected with CPI-CDV, p68. Sufcmanbranous positively labeled CPI antigens (anti-CPI, 1:70) (arrows) and unlabeled perinuclear fuzzy CDV NCs (arrow head) . N = nucleus, GBS method (stained with osmic acid and heavy metal counter stain) . Original magnification, X 11,960 . . . 85
CHAPTER IV
4.1 Coitparison of monolayer morphology of aged, persistently infected 3 week old, monolayers from (A) mock infected Vero cells, (B) CPI infected, and (C) CPI-CDV infected cells (D) CDV infected cells (high power magnifi cation, phase contrast) ...... 108
4.2 Coiparison of monolayer morphology of aged, 3 week old monolayers fron (A) uninfected Vero cells, and (B) persistent CDV infection. Vero cells persistently infected with CDV shew numerous small multilayer foci (high dry magnification) ...... 110
XV LIST OF FIGURES (Continued)
Page
CHAPTER IV
Figure
4.3 Equilibrium density centrifugation of lytic CDV (A) and CDV progeny virus from persis tently infected cell lines from passage level p51 (B). Infected^confluent monolayers were labeled with 20 yCi S-^nethionin/ml. Lytic virus has a buoyant density of 1.175 g/ml; in contrast, at passage level 51 the virus population has a lower buoyant densiiy with 1.165 g/ml (numbers represent buoyant densities in g/ml) ...... 112
4.4 Equilibrium density centrifugation of progeny viruses from Vero cells doubly infected with CPI-CDV from passage level 63 (numbers represent buoyant densities in g/ml) ...... 114
CHAPTER V
5.1 Ventricular lining cells of the fourth ventricle PID 8. Focal loss of ependymal cells and moderate subependymal, supraependymal and mild perivascular infiltration, H&E ( X 2 5 0 ) ...... 133
5.2 Fourth ventricle frcm same ferret as in Fig. 1 with focal loss of ependymal cells and mononuclear infiltration, V = fourth ventricle, H&E (X250) 135
5.3 Mesencephalon PID 8. Focal axonal degener ation with dilation of myelin sheaths and gitter cell infiltration, H&E (X250) ...... 137
XVI LIST OF FIGURES (Continued)
Page
CHAPTER V
Figure
5.4 Cervical spinal cord canal, PID 11. Mild to moderate mononuclear infiltration of the Virchow Robin spaces. Moderate diffuse paraventricular gliosis and intra- ventricular accumulation of mononuclear cells, 1. cervical spinal cord canal, H&E (X 100) 139
5.5 Lnraunoperoxidase labeling of CPI viral antigen with polyglonal anti-CPI (1:75). Ependymal cells of the fourth ventricle PID 4. Reaction product is present in cytoplasm of ependymal cells, 1 = fourth ventricle (X 250) ...... 141
5.6 Immunoperoxidase labeling of CPI viral antigen with polyglonal anti-CPI serum (1:75). Cervical spinal cord canal (A) of ferret on PID 4. Reaction product is present in cytoplasm of ependymal cells and cellular processes of subependymal cells (X 400) ...... 143
5.7 Immunoperoxidase labeling of CPI viral antigen with polyglonal anti-CPI serum (1:75). Section of fourth ventricle and choroid plexus of same ferret as Fig. 5.5. Reaction produce is exclusively in cytoplasm of ependymal cells (arrows) but not in the choroid plexus cells, 1 = choroid plexus, 2 = fourth ventricle (X 100) ...... 145
XVll LIST OF FIGURES (Continued)
Page
CHAPTER VI
Figure
6.1 Multinucleated syncytial giant cells with intracytoplasmic viral inclusion bodies (arrow) in a Vero cell monolayer infected 36 hr previously with CPI v i r u s ...... 157
6.2 Vero cell monolayers with persistent CPI virus infection. Viral antigen is demonstrated by indirect immunofluorescence methods ...... 159
6.3 Interstitial pneumonia and atelectasia (PID 10) induced by CPI virus infection in a gnotobiotic puppy inoculated intra cerebrally at 7 days of a g e ...... 161
6.4 Acute encephalanalacia (arrows) and meningo encephalitis induced in a dog inoculated with CPI virus (PID 10) 163
6.5 Severe internal hydrocephalus that developed 6 months after intracerebral infection with CPI virus ...... 165
XVlll ABBREVIATICNS
CDV-R252 Vero cell adapted strain of canine distatper virus
CPI Canine parainfluenza virus
G P K + ) Syncytial giant cell producing CPI virus
CPI(-) Non-syncytial giant cell producing CPI virus
CDV/CPI (+) Double infection with CDV virus and CPI virus
Parent virus Refers to lytic stock virus
Progeny virus Refers to virions released from persistently infected cells
DI particles Defective interfering particles i.e. Intracerebral inoculation ts Temperature-sensitive mutants
TCID5Q 50% tissue culture infectivity dose
M.O.I. Multiplicity of infectivity
Passage
xrx MECHANISMS OF IN VITRO PERSISTENCE OF ŒWO CANINE PARAMXXOVIRDSES AND IN VIVO NEUROPATHOGENICITY OF CANINE PARAINFLUENZA VIRUS
By
Wolfgang K. Barmgârtner, Dr. med. vet. The Ohio State University, 1986 Professor Steven Krakodca, Adviser
Canine Parainfluenza Virus (CPI) and Canine Disteitper Virus
(CDV) are both uj^)er respiratory viral pathogens of dogs. In
addition, CDV infection is accompanied by systemic infection
including gastrointestinal and/or nervous manifestations. Recently,
a CPI virus was isolated from the cerebrospinal fluid of a dog with
transient posterior paresis. The neuropathogenic potential of this
isolate is still incompletely characterized. The mechanism of in vitro persistence of CPI and CDV are controversial and largely unknown. The objectives of this study were to investigate compara
tive mechanisms of in vitro persistence of CDV and/or CPI virus and
to determine the neuropathogeniciiy of CPI virus in ferrets. The development of the Bouin's Formalin Gieitisa fixation-staining tech
nique was necessary to investigate the various viral strains, vhich
lacked promdnent cytopathic effects. CPI virus persistence was characterized by the appearance of low density interfering viral particles. Furthermore, virulent virus reisolated from experi mentally infected dogs differed significantly from the original 2 inoculum. This reisolate showed interference with the original stock virus, lack of giant syncytial cells, immediate in vitro persistence and a heterogenous virus population as determined hy buoyant density studies. In contrast, CDV persistence generated a virus population with a slightly lower buoyant density than the stock virus. Inter fering viral particles or tenperature-sensitive mutants were not observed. Similar results were obtained with the dually infected
CPI-CDV cell line. A morphological and imnunoelectron microscopic study of this cell line showed the simultaneous infection of numerous single or syncytial giant cells with both viruses. CPI virus produced smooth nucleocapsids (NC) with a diffuse or multifocal cytoplasmic distribution. In contrast, CDV infection produced fuzzy
NC with a multifocal perinuclear distribution. Both NCs occupied large portions of the cytoplasm at the early passage levels and decreased in size with progressing persistence. Both NCs were well demarcated fran each other. Seven week old ferrets were intra- cerebrally infected with CPI virus. Virus replication was most praninent on PID 4 with viral antigen present in the ependymal and subependymal cells of the fourth ventricle and cervical spinal cord canal. Ependymal cell necrosis and lyirphohistocytic subependymal and paraventricular perivascular infiltration was observed 8 and 22 days p.i. No residual lesions were present in ferrets sacrificed 60, 90 and 150 days p.i. CHAPTER I
A SIMPLE METHOD FOR THE HISTOCHEMICAL DEMONSTRATIF
VIRAL INCLUSIF BODIES IN CRT.T. MONOLAYERS
IN MICROTITRATIF PLATES
INTRFUCTIF
There are a number of methods for scoring virus neutralization tests (VNT) and virus titrations (VTT) for parait^oviruses in micro- titration plates (Mayr et al., 1977; Confer et al., 1975; ^^>pel and
Robson, 1973). Generally, virus infections in monolayers are detected plaque formation, hemadsorption (Had), antigen production demonstrated by irmunocytochemistry, cytopathic effects (CPE) or combinations thereof. Although these techniques give satisfactory results for most paramyxoviruses, the procedures involved cannot be used for all viral strains. In our laboratory, we are working with various canine parainfluenza (CPI) and canine distemper virus (CDV) substrains, many of which show poorly developed or non-existent plaque-forming capability, lack Had, and do not form syncytial giant cells. Performance of either VTT or VNT with these strains can only be performed imnunofluorescence assay, a time consuming, laborious and reagent wasteful procedure. The present paper describes a
3 4 technique vAich sirrplifies the VTT and VNT for these problem virus strains. 5
MA3ERIALS AND MEUHCDS
Both VTT and VNT with CPI and CDV viruses were performed in
96-v^ll microtitration plates (Costar) as previously described
(BaumgMrtner et al., 1981). Five days after incubation at 37°C, 5%
CO2, the growth medium is discarded and the wells are filled with
Bouin's fixative for 15 minutes at room teaiperature. After removing excess Bouin's, all wells are filled with 10% (v/v) buffered formalin solution and fixation is allowed to proceed overnight at roan temper ature. After decanting the formalin, wells are filled with Giemsa stain (4% v/v Giemsa-stock and double-distilled water) for 3-4 hours, rinsed with several changes of tap water, air-dried, and examined by lOX light microscopy. Duplicate plates were examined after fixation in methanol, formalin and Bouin's solutions followed by 4% (v/v)
Giemsa stain. 6
RESULTS AND DISCUSSION
Plates fixed and stained with the Benin's fontialin-Giemsa method
adequately preserved cellular morphology and shewed numerous, well
demarcated viral cytoplasmic inclusions. The discoloration of both
the cells and the inclusion bodies varied from bright yellow through
green-brown to dark blue (Fig. 1.1b). The residual yellow color of
the picric acid of the Bouin's fluid along with the added Giemsa
stain, produces the variation in staining of the monolayers. A
thorough wash under tap water prior to Giemsa staining diminishes the
color variation and the cells and inclusion bodies are stained blue.
However, we found color variation helpful in the differential
staining of cells and inclusion bodies.
Monolayers fixed in methanol for 20 minutes, air dried and then
stained as above were less well preserved in cellular detail. The
cytoplasm was slightly eosinophilic or basophilic. The majority of methanol fixed viral inclusions were homogeneous and eosinophilic but were not clearly separated from the cytoplasm by a halo (Fig. l.ld).
In contrast, viral inclusions in formalin-fixed monolayers stained with 4% Giemsa, were not visible (Fig. 1.1c). Extensive rinsing of
Bouin's fixed monolayers followed ty staining with 4% Giema, resulted
in severe precipitation of the stain on the slide. Picric acid is miscable with ethanol, formaldehyde, and acetic acid. After fixa tion, picric acid is readily removed by alcohol or water wash solutions (Sheehan and Hrapchack, 1980). The latter are difficult to perform in microtitration plates. 7
Bonin's fixation of cellular monolayers on micro-slides followed by multiple changes in graded ethanol and hanatoxylin-eosin (H&E) stain is a ccnmonly used and reliable fixation and staining proced ure. Using this procedure, eosinophilic viral inclusions with a pronounced halo in well preserved fixed cells are readily achieved.
However, the multiple fixation and staining steps make this technique unsuitable for obtaining uniform results in microtitration plates.
The Bouin's formalin-Giemsa technique described here showed results very similar to the latter, yet is much sinpler to perform and results in both inter- and intra-plate uniformity. ffethanol fixation, followed by polychromatic stains such as Giemsa, is a convenient method of preparing stained monolayers (Freshney R.J.,
1983) and is widely used for VTT and VNT (Mayr et , 1977).
However, the paucity of well demarcated cytoplasmic inclusions with certain viruses makes methanol fixa-tion less suitable for VNT and VIT assays.
Thus, formalin, Bouin's and/or methanol fixation techniques alone proved unreliable for demonstrating viral cytoplasmic in clusions wi-fchin infected cells in microtitration plates. Bouin's formalin-Giemsa fixation and staining method described here should facilitate screening of cellular monolayers for subtoxic e2q>ression of viral material in microtitration plates. 8
SUMMARY
The present paper describes a Bouin's-formalin-Giemsa fixation
and staining procedure for demonstrating viral cytoplasmic inclusion
bodies in cellular monolayers in microtitration plates. The method
is stçerior to formalin, methanol or Bouin's fixation-staining methods alone and carpares favorably to inmunocytochemical techniques
for sensitivity. Fig. 1.1 Vero cells persistently infected with a non-giant and
non-plaque producing CPI virus. A) Immunofluorescence
demonstrating cytoplasmic inclusion bodies (XlOO).
B) Bouin ' s-formalin-Giemsa fixation and stain shews
well-demarcated cytoplasmic inclusion bodies (arrows)
(XlOO). C) Formalin-Giemsa fixation and stain; no
inclusion bodies visible (XlOO). D) Methanol-Giemsa
fixation and stain; inclusion bodies are less
demarcated fran the cytoplasm (XlOO). 10
Fig. 1.1 11
REFERENCES
Appel, M. and Robson, D.S. (1973) A Microneutralization Test for
Canine Disteirper Virus. Am. J. Vet. Res. Vol. 34:1459-1463.
Baumgârtner, W., Metzler, A.E., Krakowka, S. and Koestner, A. (1981)
In Vitro Identification and Characterization of a Virus Isolated
fran a Dog with Neurological Dysfunction. Infect. Immun.
31:1177-1183.
Confer, A.W., Kahn, D.E.K., Koestner, A. and Krakowka, S. (1975)
Biological Properties of a Canine Distenper Virus Isolate
Associated with Demyelinating Encephaloiyelitis. Infect. Immun.
11:835-844.
Freshney, R.J. (1983) Culture of Animal Cells. A Manual of Basic
Technique. Alan R. Liss, Inc. New York.
Mayr, A., Bachraann, P.A., Bibrack, B. and Wittmann, G. (1977)
Virologische Anbeitsmethoden, Band II, Gustav-Fischer-Verlag.
Stuttgart, New York.
Sheehan, D.C., Hrapchack, B.B. (1980) Theory and practice of histo-
technology. Sec. edition. C.V. Mosby Carpany. St. Louis,
Toronto, London. CHAPTER II
CANINE PARAINFLUENZA VIRUS: EVOLUTION OF DI VITRO PERSISTENCE
AND PRODUCTION OF INTERFERING VIRAL PARTICLES
INTRDDUCTICN
Canine parainfluenza virus (CPI) causes txacheo-bronchitis in
its natural host (Rosenberg et , 1971). Recently, a neutrotropic
strain was isolated from the cerebrospinal fluid (CSF) of a dog with
temporary posterior paralysis (Baumgartner et , 1981; Evermann et
al., 1981). Experimentally, this neurotropic CPI virus caused
encephalitis with subsequent internal hydrocephalus \Æien inoculated
into gnotobiotic dogs (Baumgartner et , 1982). Similar changes
have been observed in experimentally infected ferrets (Baumgartner,
unpublished data, 1986) . The CPI virus is closely related to simian
virus 5 (SVg) (BaumgSrtner et , 1981). The latter has been
recently isolated from human bone marrow cells of multiple sclerosis
patients and fran leukocytes of a patient with subacute sclerosing
panencephalitis (Goswami et al., 1984; Mitchell et al., 1978; Robbins
et al., 1981). The significance of SVg in these human diseases
ranains undefined (Choppin, 1981; Huddlestone et al., 1979).
Little is known about the evolution of in vivo and iji vitro persistence by parainfluenza viruses, in spite of their widespread
12 13
presence in primary kidney cell lines (Choppin, 1964; Hsinng, 1973;
Robbins and Rapp, 1982; Zakstelskaya ^ , 1976). The literature
regarding in vitro persistent viral infection has been recently
reviewed (Huang, 1973; Kaaden and van Dawen, 1983; Perrault, 1981).
Several factors or combinations of factors were found to play roles
in establishment and maintenance of viral persistence (Friedman and
Ramseur, 1979). The influence of the host cell has been demonstrated
by the use of cultures from various species and by differences in
viral susceptibility depending on the age of the cells (Ccirpans et
al., 1964; Johnson et al., 1972; Wild and Dudgre, 1978). Other
factors vdiich play a role in viral persistence are generation of
defective interfering (DI) viral particles, temperature sensitive mutants and interferon (Armen et al., 1977; Huang, 1973; Fortner et
al., 1975; Youngner and Preble, 1980). The intriguing idea of incor
poration of catplementary paramyxovirus DMA in the host cell DNA
proposed by Zhadnov et (1975) as a mechanism of persistence has
not been confirmed by others (Friedman and Ramseur, 1979). Produc
tion of Dl-particles, originally called the von Magnus phenomenon, was first described with influenza virus (Huang, 1973). Generally,
Dl-particles are characterized by enrichment, defectiveness and
interference. Enrichment is shown ty preferential replication over
the standard virus. Defectiveness indicates that Dl-particles need a helper (eg, ccnplete virus) for replication. Interference is detected by reduced yield of standard virus after simultaneous
infection with Dl-particles. The Dl-particles of vesicular
stomatitis virus (VSV) represent one of the best studied systems 14
(Peinrault, 1981). Conparative studies between standard VSV and
VSV-DI showed that VSV-DI have protein profiles identical to parent
VSV yet are smaller in size. The defect appears to be in the viral
genome since DI exhibit deletions of genetic information (Huant,
1973; Lazzarini, 1981; Perrault, 1981).
During the course of pathogenesis studies in dogs with the CPI virus isolated by Evermann et (1981) above, virus reisolation was performed from infected canine brain tissues. Surprisingly, the recovered CPI isolate after only one iri vivo passage, differed
substantially in cytopathic effects (CPE) from that observed with the parental CPI virus. Further characterization of this difference along with the production of Dl-particles and evolution of in vitro persistent infection by both viruses form the basis of this report. 15
MATE3ŒAIS AND METHCDS
Viruses. A syncytial giant cell and plaque-forming canine
parainfluenza virus designated CPI (+), originally isolated fran the
CSF of a dog with neurological dysfunction, was kindly provided by J.
Evermann (Department of Veterinary Clinical Medicine and Surgery and
Diagnostic Laboratory, Washington State University, Pullman WA) (11).
A non-giant cell, non-plaque-forming CPI strain designed CPI(-) was
reisolated fran gnotobiotic dogs experirtentally infected with CPI(+)
virus (BaumgSrtner et , 1982). The canine distenper virus
(R252-CDV) has been described previously (Confer et , 1975).
cell cultures and virus titrations. African green monkey kidney
(Vero) cells were maintained as previously described (BaumgSrtner et
al., 1981). Virus titrations were performed in a microtiter system
by adding serial 10-fold dilutions of viral suspension to Vero cells 4 (1.5 X 10 cells/ well) with incubation for 7 days. Plates were
fixed and stained ty the Bouin ' s-formalin-Gionsa method as described
(BaumgSrtner et , Stain Technol., accepted for publication).
Virus titrations expressed as TCID^q were calculated by the formula
of Spearman-KSiijer (12).
Establishment of persistently infected cells. Vero cells were
infected with virus stocks, CPI(+) or CPI(-) at a multiplicity of
infection (M.O.I.) of 1.0 and maintained in the same flask until
confluency. Medium was changed every three days. Surviving virus-
infected cells were maintained until confluence and then passed thereafter by seeding 1.5 x 10^ cells onto 25 on^ flasks (pi). 16
Monolayers undergoing additional lytic crises were maintained in the same flask until they reached confluency again. If not otherwise stated, cells were passed at weekly intervals hy trypsinization.
Li<ÿit microscopy and immunofluorescence (IF). To evaluate viral
CPE, 4.0 X 10^ persistently infected cells per well were propagated on glass microscope slides (Lab-Tek Products, Westmont, IL). After 3 days, cell monolayers were fixed in Bouin's fixative for 10 min, dehydrated and stained with hematoxylin and eosin (H&E). Indirect immunofluorescence (IF) on acetone-fixed monolayer replicates was performed as described (BaumgSrtner et , 1981).
Hemadsorption (Had). Had was performed as described previously
(BaumgSrtner et , 1981). The number of Had-positive cells was determined by counting the number of Had positive cells/300 cells.
Interference assay. Srg)ematants from persistently infected cultures were tested for interfering activiiy with 200 TCID^q of lytic (CPI(+) virus in 200 yl MEME. Equal volumes (0.2 ml) of lytic virus plus interfering material were mixed and inoculated onto a 25 cm^ flask of subconfluent Vero cells. After incubation at 37°C for
72 hrs, the cells were scraped into the medium, virus was harvested after 3 freeze-thaw cycles and assayed for titratable virus by
10-fold dilution. Controls included mixtures of lytic virus with lytic virus and/or growth medium.
Ultraviolet (UV) irradiation of Dl-particles. Inactivation of
Dl-particles was carried out in Petri dishes with 1 ml virus suspen sion. The solutions were e^çosed to a 15 watt germicidal lairp
(General Electric) at a distance of 20 cm for 45 min with a dose of 17
250 watt/cm^. The UV energies were determined with a Black-Bay UV
intensity meter (Ultraviolet Products, Inc., San Gabriel, CA).
Virus purification and equilibrium density gradient centrifuga tion. Persistently or lytically infected Vero cells were labeled for
24 hrs with 80 yCi L-methionine in 25 cm^ flasks (NEN Research
Products, Dupont, specific activi-t^ 1022 yCi/iunol). Persistently infected cells were labeled 2-3 days after passage; lytically infected cells were labeled v^en CPE ccmnenced. Growth medium was replaced by methionine-deficient medium supplemented with dialyzed 35 PCS; 80 yCi S L-methionine was added 2 hrs later. Virus was harvested by 3 cycles of freezing and thawing. Cellular debris was removed 1^ centrifugation for 10 min at 2,200 X £ and the resultant virus suspensions were mixed with 10% polyethylene glycol 6000 (50%
PEG stock solution in O.IM Tris buffer pH 7.6) 1:4 (v/v), 4®C 12 hrs under constant stirring. Aggregated virus particles were collected by centrifugation (700 £, 30 min) and the pellet was resuspended in
700 y; NTE (O.IM NaCl, O.OIM Tris, O.OOIM EDTA acid in dd-water pH
7.2) buffer for 20 min at 4°C, centrifuged and resuspended two more times.
The eluates were further purified by a zonal centrifugation in
40% (w/w) potassium tartrate in NTE and 15% sucrose (w/w) in NTE for
2 hr at 21,000 rpn (SW41 Rotor, Beckman, Inc). The virus-containing band at the interface was collected with a syringe and sediraented to equilibrium on a 0%-50% potassium tartrate gradient in NTE buffer
(w/w) for 16 hr at 21,000 rpm. Tubes were punctured with a needle at the bottom and 14 drop fractions (650 yl) were collected. One 18 hundred yl of each fraction were weighed to determine buoyant density. Radioactivity of each fraction was determined by adding 10 yl of each fraction to 5 ml Aguasol-2 (New England Nuclear) and 500 yl distilled water, and counted by liquid scintillation. 19
RESULTS
CPI(+) virus. Subconfluent monolayers were infected with CPI(+)
virus (10^*^^ TdD^p/ml). Within 48-72 hr, 95% of the cell monolayer
shewed cytopathic effect (CPE) with syncytial giant cell formation
with subsequent cytolysis and detachment. Surviving cells formed a
sparse monolayer 1 to 2 weeks later. Numerous lytic crises occurred
during the following 29 passages. During this period, pronounced
syncytial giant cell formation was present, however cellular detach
ment was less praninent. After p29, the infected cells vrere
indistinguishable fran uninfected Vero cells. The yield of progeny
virus was significantly reduced after the establishment of persis
tence (Table 2.1). By pi2 and p38, 90% of the infectious virus was
cell-free (not shown). The CPE produced by progeny titrated virus
was similar to CPI(+) parent virus.
CPI(-) virus. Subconfluent monolayers were infected with CPI(-)
virus (10^*^ TCIDgQ/ml). Forty-ei^t hr later, a mild strand-forming
CPE developed accanpanied by occasional small syncytial giant cells.
Cellular confluence was reached within one week. During the follow
ing 7 passages no light microscopic changes were noted except for an
occasional small syncytial giant cell. At p8 a severe Ij.’ti c crisis
occurred. The surviving cells formed a sparse monolayer 2-3 weeks
later. During the following 23 passages, several more crises
occurred. Large syncytial giant cells similar to the CPE of CPI(+)
were a praninent feature during these crises. After p31 the infected monolayer was indistinguishable fran uninfected Vero cells. Progeny 20
virus fran pl-7 had CPE similar to stock CPI (-) virus; However, after
p8 the CPE of progeny virus was similar to stock CPI(+). At pi5,
virus titrations showed that the majority of infectious virus (>90%
was cell-free (data not shown).
Analysis of rate of cellular proliferation in CPI(-) and CPI(+)-
infected cells vs. uninfected control Vero cells shewed a mild
reduction with a mean yield of 9.3 x 10^ CPI(u-) cells/flask (n=43)
coipared to 11.03 x 10^ CPI(+) infected cells/flask (n-41) and 11.54
X 10^ uninfected cells/flask (n=20). Statistical analysis (Student's
t-test for unpaired variances) showed that cell replication in
persistent CPI(-) cell line was significantly reduced coipared to
either the CPI(+) cell line (P<0.05) or the control Vero cells
(P<0.025).
Viroloqical characterization of lytically and persistently
infected cells. Vero cells lytically infected with CPI(+) and CPI (-) parent viruses were 100% IF-positive 24 hr after infection. Essen tially, 100% of the cells in the persistent CPI(+) and CPI(-)
cultures contained viral antigen as determined by IF. The cyto plasmic fluorescence of CPI (+) and CPI (-) cells was of a fine granular pattern, with a few large granules in CPI(-) infected cells.
One hundred percent of cells lytically infected with CPI (+) were
Had positive, vAiereas only 10% of the CPI(-) lytically infected cells were Had positive. In contrast, from 1-10% of the persistently
infected CPI (+) cells were Had positive, vAïereas CPI(-) persistently infected cells during early (pl-7) persistence were Had negative. In the latter, several peaks of Had-positive cells were detected betveen 21
p8 to 12 (15%“80%). These increases in Had correlated with the onset
of syncytial giant cell-associated CPE. The number of Had-positive
cells once stabilized beyond p30 remained low (0-5%) throughout the
remainder of the study.
Interference by persistently infected cells with the replication of hcntologous and heterologous viruses. Persistently infected cells were tested for hanologous and heterologous interference by stperin-
fection (Table 2.2). Replication of lytic CPI (+) was severely depressed in persistent CPI(+) and CPI(-) cell lines, with a reduc tion of virus yield of 4 log 10. However, persistent CPI(-) and
CPI(+) cells were not protected against srçierinfection with lytic
R252-CDV virus.
Interference between lytic CPI (+) and lytic CPI(-) virus.
Interference was observed between lytic CPI(+) and CPI(-) virus.
Infection of a subconfluent monolayer with an inoculum containing 10^
TCID^q of both viruses resulted in the development of a strong- forming CPE, identical with the CPE of lytic CPI(-) infection alone.
Syncytial giant cell formation typical of CPI(+) virus was never observed. Titrations performed on progeny obtained from the mixed infection showed a CPI(-) typical CPE; no syncytial giant cell formation was present. Thus the yield of CPI (+) was reduced by co-infection with CPI (-) by 1 log 10.
Presence of interfering viral particles in persistently infected cell cultures. The data in Table 2.3 show that lytic parent CPI(+) virus did not contain detectable interfering material. At p21 the persistent CPI(+) cell line also shewed no interfering activity, yet 22 by p38 and 80 a significant reduction of parent virus yield (1.5 log
10) was observed. Materials harvested from persistent CPI(-) at p31,
53, 56, but not pl5 significantly reduced (1.5 log 10) lytic CPI(+) virus yield (Table 2.3).
Sensitivity to ultraviolet (ÜV)-irradiation was used to deter mine vAiether the interference was mediated ty virus particles. ÜV- irradiation of progeny virus fran persistent CPI(+) and CPI(-) cell lines corpletely abolished their interfering activity (Table 2.4).
Detection of low density viral particles. Gradient purified virions were isolated fran infected cultures and used to determine if viral persistence and the presence of interfering particles detected above were accatpanied ty ctianges in the density viral particles.
Lytic CPI(+) parental virus had a peak tuoyant density of 1.184 g/ml (Fig. 2.1). Virus recovered fran fractions t)etween 1.12 and
1.184 g/ml exhibited CPE typical of lytic stock virus (eg, syncytia and cytolysis). Fractions laelow 1.12 g/ml contained infectious virus vdiich did not ejdiibit typical CPI (+). Infectivity of the latter was detected only ty the forma-tion of viral cytoplasmic inclusions in fixed monolayers (eg, persistence). Profiles of labeled virions collected fran persistently infected CPI(+) cultures shaved a stiarp peak of radioactivity at a tmoyant density of 1.136 g/ml (p64) (Fig.
2.2).
Lytic CPI(-) parental virus was more heterogeneous than was 35 lytic CPI(+) virus. A broad plateau of incorporated S-mettiionine 23 was observed (1.08-1.184 g/ml) (Fig. 2.4). Progeny virus frcm per sistent CPI(-) cultures at p42 had a broad plateau of radioactivity
(1.08 to 1.19 g/ml) and 2 minor peaks at 1.19 and 1.125 g/ml. 24
DISCUSSION
Hie general focus of these experiments was to further character ize CPI (+) and CPI (-) viruses and to determine how each viral isolate evolved into the ^ vitro persistent infection state. ^ vitro persistent infection in Vero monolayers was induoed with both viral isolates using the technique of serial passage of surviving virus- infected cells (BaumgSrtner et , 1981). Evolution of this persis tent state was different for the 2 isolates studied. Lytic CPI (+) virus progressed through several distinct host-virus relationships during dn vitro passage in a fashion similar to that described for measles virus (Rapp and Robbins, 1981) and also CDV (Metzler et al.,
1984).
In contrast, the CPI(-) isolate immediately (pi) entered an in vitro persistent state. At p8, CPE more typical of parental CPI(+) virus was observed in these CPI(-) monolayers. At passage 31 a new stable host-cell virus relationship emerged with the in vitro charac teristics essentially identical to those obtained with CPI(+) virus at a similar (p29) passage level.
Lytic CPI(+) virions had a sharp peak of infectivity and radio activity at an average buoyant density of 1.185 g/ml. Higher buoyant density (1.25 g/ml) has been reported for lytic SV^ virions (18).
Lytic CPI(+) parent virus contained virus particles in fractions of buoyant densities below 1.12 g/ml. Fractions were infectious but did not induce overt CPE. These virions in stock lytic CPI(+) were not detected in the interference assay possibly because the assay is not 25
sensitive enough or because t h ^ do not possess interfering activity.
More iitportantly, they may be the original source of CPI(-) virus.
In general, in vitro persistence with either CPI(+) or CPI(-) viruses was characterized by viral particles of lower buoyant density. Thus, stabilization or establishment of the persistent
state was correlated with the production of low density particle virus. Similar observations are reported for measles and Sendai virus (Fisher, 1983; Fortner et al., 1975).
The role of these lower density viral particles in modulating viral CPE was investigated using a virus yield interference assay rather than the plaque reduction assay because CPI(-) virus could not be plaque titrated (Kawai and Matsumoto, 1982). Using this assay, interfering material was not detected in CPI(+) stock lytic virus nor in SL^)ematants from CPI(+) and CPI(-) persistently infected cell lines until after stability was achieved. In contrast, parental
CPI(-) virus readily interfered with the replication of lytic CPI(+) virus. As with the persistent CPI(+) cell line, interference was readily detected in supernatants frcm CPI (-) infected cells only after emergence of the stabilized persistently infected monolayers.
To further investigate interference, UV-irradiation experiments were performed to determine if the interference was due to viral particles or to a virus-induced soluble protein (ter Meulen et al.,
1976) . Cellular interferon as the modulator of viral persistence was not considered a factor in our studies since permanent Vero cell lines are known not to produce the substance (Desmyter et , 1968).
In a pilot study we determined the inactivation time for both lytic 26
CPI and CDV viruses. No infectious virus was present after 5 min UV e^qxosure. Since Dl-particles likely contained less PNA than the lytic parent virus and are thus more UV-resistant than the lytic virus (Bay et , 1979), the progeny virus was UV-irradiated for 45 min. After this manipulation, interfering activity was not demon strated in CPI(+) and CPI(-) sr:çeiniatants of persistently infected cells. These results suggested the presence of interfering particles and ruled out a soluble virus-induced protein (ter Meulen et al.,
1976) or interferon as the interfering mechanism. Finally, reduced buoyant density of interfering particles, as detected in our studies, is generally attributed to segmental loss of viral nucleic acid in these virions rather than the deletion of viral structural proteins
(Friedman and Ramseur, 1973; Lazzarini et ^ . , 1981). Thus, it is likely that presence of interfering particles in these persistently infected cell lines is a result of the emergence of gene-deficient mutant virus population(s) in these cells (Holland ^ al., 1982;
Perrault, 1981). Assanbly and release of these interfering particles undoubtedly contribute to the maintenance and observed stability of persistent iri vitro infection.
The biological significance of these observations regarding disease-causing potential of these altered CPI viruses for either dogs and ferrets remains to be determined. In general, paramyxovirus infections iri vivo are either acute and self-limiting or persistent and progressive in nature. A central issue in the study of paramyxo virus biology is how persistent infections evolve ^ vivo frcm acute lytic disease. For other members of the virus group, acute lytic 27 infections appear to be positively correlated with the ability of viral inocula to produce and incorporate significant quantities of envelope glycoproteins (eg, fusion and hemagglutinin glycoproteins) into the lipid envelope. The in vitro equivalent of this phenomenon is prominent syncytial giant cell formation and incorporation of these glycoproteins into cellular membranes (eg. Had positive). From this, it follows that viral inocula lacking these properties should be of reduced in vivo virulence (Rott, 1979). With this hypothesis in mind, experiments currently in progress in this laboratory have been designed to determine the virulence potential of both parental lytic (CPI(+) and CPI(-) viruses as well as viruses recovered frcm persistently infected cell lines developed frcm each. 28
SUMMARY
Two strains of canine parainfluenza (CPI) virus differing in their plaque-forming and cytopathic effects (CPE) were investigated.
Lytic stock virus (CPI+), originally recovered from cerebrospinal fluid (CSF) of a dog with neurologic disease exhibited prominent CPE
(eg, syncytia formation and cytolysis) in Vero cell monolayers. The non-syncytial giant cell-forming strain (CPI-) was isolated frcm brain tissues of a dog after experimental infection with syncytial giant cell-forming (CPI+) virus. Lytic CPI(+) virus had a buoyant density of 1.185 g/ml; in contrast CPI(-) virus exhibited a buoyant density profile of 1.080-1.184 g/ml. In mixed infection ezgeriments,
CPI(-) virus interfered with the replication of CPI(+) virus in that simultaneously infected cells showed strand-forming CPE typical of
CPI(-) and reduction of CPI (+) yield.
During evolution of in vitro persistence, cells in both infected monolayers were 90-100% positive for cytoplasmic viral antigen and exhibited 0-15% hemadsorption. The CPI(-) virus established immed iate in vitro persistence, followed by a severe crisis at passage (p)
8 and several similar crises until p31; no further crises occurred throu^out the remaining observation period (p80). The CPI (+) did not establish immediate in vitro viral persistence and experienced several lytic crises until p29. No further crises were observed throughout the observation period (plOO). Both persistently infected cell lines produced interfering viral particles. Buoyant density 29
studies demonstrated that interference in both lytic and persistently infected cells was due to viral particles with low buoyant density. 30 a Table 2.1 Virus yield in lytic and persistently infected cell lines.
Passage level Cell Lines of persistently infected cells CPI(+) CPI(-)
1o 6.75^'‘> 106.75^ Lytic infection
Persistent infection at passage level
12 lO^'S-lO^'O _d lo5.25 13 10^^5_iQ5.25C 15 -
21 --
28 - lO^'S lo2.0 35
38 0-10l'75= - lo2.5 41 -
51 lO^'O - lo2.5 57 - I0I .75 80 -
a _ TCID^q /0.1 ml. b = cell-associated and cell-released virus production. c = represents minimum and maximum virus yield of subsequent daily titrations of six days of cell-associated and cell-released virus at this passage level. d = -, not determined. 31
Table 2.2 Superinfection of persistently infected cells with lytic
CPI{+)^ and R252-CDV virus
Challenge Cell Line Passage Virus Titer^ Reduction^
e 106.75 Vero 180 CPI(+) lo2.0 C P K + ) 39 CPI(+) 4.75 lo2.00 CPI(+) 16 CPI(+) 4.75 lo4.25 Vero 185 R252-CDV -
CPI(-) 28 R252-CDV lO^'O 0.25 lo3.75 CPK+) 51 R252-CDV 0.5
a = Subconfluegt cellular monolayers in 25cm^ flasks were infected with 10 ' TCIDj.^ and virus yield was determined 48 hours post infection. b = Suteonfluent cell monolayers in 25cm^ flasks were infected with 10 * TCIDgQ and virus yield was determined 72 hours post infection. c = TCIDgg/0.1 ml. d = Differences in yield of lytic CPI(+) on CDV frcm mock and persistently infected cells. e = -, not determined. 32
Table 2.3 Hcmologous interference with replication of lytic CPI(+)
virus by CPI(+) or CPI(-) progeny virus of persistently
infected cells.
Passage level Interfering of progeny Challenge^ Material virus virus Titer^ Reduction^
d 106.25 MEME - CPI {+) lo6.25 Lytic CPK+) 11 C P K + ) 0
CPK+) 25 CPI(+) IOG'5 0
CPK+) 65 CPI(+) 104'75 1.5 lo5.0 C P K + ) 66 CPI (+) 1.25
CPI(-) 10 C P K + ) 104'75 1.5 lo4.75 CPI(-) 28 C P K + ) 1.5
1o 4.75 CPI(-) 40 CPI (+) 1.5
CPI(-) 70 CPK+) icf'O 1.25 a = 200 TCIDgQ in 200 yl MHyE. b = TCID^q/0.1 ml after 72 hours. c = Expressed in log.g as difference between yield of lytic virus mock and persistently infected cells. d = -/ not determined. 33
Table 2.4 Effect of ■ultraviolet (UV)-irradiation on inhibitory
material frcm persistently infected cells.
Lytic Interfering Challenge Material Passage Virus Titer^ Reduction
Medium — CPI(+) icf'5 -
CPI(+) Lytic - CPI(+) lof'S -
CPI(-) 56 CPI(+) lof'O 1.5
CPI(-)b 56 CPI(+) icf'5 0
CPK+-) 56 Medium 0 -
Medium - CPI(+) icf'O -
Medium - CPI(+)^ 0 - 1.5 CPI(+) 53 CPI(+) 10^*^ 106.0 CPI(+)b 53 CPI(+) 0
CPI(+) 53 Medium 10^'° -
CPI(+)^ 53 Medium 0 - a = TdDgg/O.l ml. b = UV-irradia-ted for 45 minu-tes under a germicidal lamp; distance 20 cm. Fig. 2.1 Equilibrium density gradient separation of CPI(+) in a
potassium tartrate gradient. Ten-fold virus dilutions of
650 yl firactions shews peak activity with a buoyant density
of 1.184 g/ml. CPE was characterized by syncytial giant
cell formation. Fractions with a buoyant density below
1.12 g/ml shewed no syncytial giant cell formation but
numerous cytoplasmic inclusion bodies (nunbers represent
buoyant densities in g/ml).
34 35
1 .184# 1.136
o lO
o
_J
10 15 20 23
FRACTION NUMBER
Fig. 2.1 Fig. 2.2 Equilibrium density centrifugation of lytic CPI(+) virus
(a) and CPI (+) progeny virus and frcm persistently infected
cells, passage level 64 (b). Infected confluent monolayers 35 were labeled with 20 yCi S-methionine per ml. Radio
activity of each fraction was determined. Lytic virus (a)
has a buoyant density of 1.185 g/ml; in contrast, at
passage level 64 the virus population has a lower buoyant
density with 1.136 g/ml (b). (Numbers represent buoyant
densities in g/ml).
36 COUNTS / min xio"^ COUNTS / min x 1 0 “ 2
I O ( O ^ U 1 0 > N ob(DO to 0) » tn «'J o o w o IÎm w t— *---- '------'----- :------■--- 1------1------1------1______I______■______i ■ u ~ t o -
#—' 00 01 Ô) I m -t k)
w 'si Fig. 2.3 Equilibrium density centrifugation of lytic CPI(-) virus
(a) and CPI(-) progeny virus frcm persistently infected
cells, passage level 42 (b). Virions were labeled with 35 S-msthionine as described under materials and methods.
Lytic CPI(-) virus (a) and progeny virus frcm passage 42
(b) show a similar distribution of the virus population,
vhich is very distinct from CPI (+). (Numbers represent
buoyant densities in g/ml).
38 39
7n
1.16 1.11 • 1.08 1.134 2
10 12 14 16 18 20
I o X 5 1.125 a 1.15 4 - •1.106 E 1.19 t-CO z 3 o o
8 10 12 14 16 18 20 FRACTION NUMBER
Fig. 2.3 40
REt'KKEÎKIES
Anren, R.C., Evermann, J.F., Truant, A.L., Lau^lin, C.A., Hallum,
J.V. (1977) Teaiperature sensitive mutants of measles virus
produced from persistently infected HeLa cells. Arch. Virol. 53,
121-132.
BaumgSrtner, W . , Krakoidca, S. (1986) A siirple method for the histo-
chemical demonstration of viral inclusion bodies in cell mono
layers in microtitration plates. Stain Tech. (in press).
BaumgSrtner, W.K., Metzler, A.E., Krakcudca, S., Koestner, A. (1981)
In vitro identification and characterization of a virus isolated
frcm a dog with neurological dysfunction. Infect. Immun. 31,
1177-1183.
BaumgSrtner, W.K., Krakcv^a, S., Koestner, A., Evermann, J.F. (1982)
Acute encephalitis and hydrocephalus in dogs caused by canine
parainfluenza virus. Vet. Pathol. 19, 79-92.
Bay, P.H.S., Reichmann, ME. (1979) UV-inactivation of the biological
activity of defective interfering particles generated by vesicular
stomatitis virus. J. Virol. 32, 876-884.
Choppin, P.W. (1981) Isolation of pararyxovirus frcm patient with
chronic neurologic disease. J. Infect. Dis. 143, 501-503.
Choppin, P.W. (1964) Multiplication of a myxovirus (SV^) with
minimal cytopathic effects and without interference. Virology 23,
224-233.
Caipans, R.W., Holmes, K.V., Dales, S., Choppin, P.W. (1964) An
electron microscopic study of moderate and virulent virus cell
interactions of the parainfluenza virus SV^. Virology 411-426. 41
Confer, A.W., Kahn, D.E., Koestner, A., Krakcvto, S. (1975) Biologi
cal properties of a canine distenper virus isolate associated with
daiyelinating encephalanyelitis. Infect. Immun. 11^, 835-844.
Desmyter, J., Melnick, J.L., Rawls, W.E. (1968) Defectiveness of
interferon production and of Rubella virus interference in a line
of African green monkey kidney cells (Vero). J. Virol. 2_, 955-961.
Evermann, J.F., Krakoto, S., McKeiman, A.J., BaumgSrtner, W. (1981)
Properties of an encephalitogenic canine parainfluenza virus.
Arch. Virol. 6S, 165-172.
Finney, D.I. (1964) Statistical methods in biology assay. Hafner
Publishing Co., New York.
Fisher, L.A. (1983) Characterization of four cell lines persistently
infected with measles virus. Arch. Virol. 77, 51-60.
Friedman, R.M., Ramseur, J.M. (1979) Mechanisms of persistent infec
tions by cytopathic viruses in tissue culture. Arch. Virol. 60,
83-103.
Goswami, K.K.A., Cameron, K.R., Russel, W.C., Lange, L.S., Mitchell,
D.N. (1984) Evidence of the persistence of paramyxoviruses in
human bone marrows. J. gen. Virol. 65, 1881-1888.
Holland, J., Spindler, K., Horodyski, F., Grabau, E., Nichol, S., Van
de Pol, S. (1982) Rapid evolution of RNA genomes. Science 215,
1577-1585.
Huang, S.A. (1973) Defective interfering viruses. Ann. Rev. Micro
biol. 27, 101-117. 42
Hsiung, G.S. (1972) Parainfluenza-5 virus. Progr. loed. Virol. 14,
241-274.
Huddlestone, J.R., Sipe, J., Braheny, S., Jensen, F.C., McMillan, R.,
Lanpert, P., Olds tone, M.B.A. (1979) Failure to isolate a trans
missible agent frcm the bone marrow of patients with multiple
sclerosis. Lancet, 415.
Johnson, R.T., McFarland, H.F., Levy, S.E. (1972) Age-dependent
resistance to viral encephalitis: Studies of infection due to
Sindbis virus in mice. J. Infect. Dis. 125, 257-262.
Kaaden, O.R., van Dawen, S. (1983) Persistierende Virusinfektionen
bei Tieren. Zbl. Vet. Med. B. 30, 1-21.
Kawai, A., Matsumoto, S. (1982) A sensitive bioassay system for
detecting defective interfering particles of rabies virus.
Virology 122, 98-108.
Lazzarini, R.A., Keeny, J.D., Schubert, M. (1981) The origin of
defective interfering particles of the negative-strand RNA
viruses. Cell 26, 145-154.
Metzler, A.E., Krakovdca, S., Axthelm, M.A., Goriiam, J.R. (1984) In
vitro propagation of canine distenper virus : Establishment of
persistent infection in Vero cells. An. J. Vet. Res. jS, 2211-
2215.
Mitdiell, D.N., Porterfield, J.S., Michele, H.R., Lange, L.S., Gos
wami, K.K.A., Taylor, P., Jacobs, J.P., Hockley, D.J., Salsbuiy,
A.J. (1978) Isolation of an infectious agent frcm bone marrows of
patients with multiple sclerosis. Lancet, 387-391. 43
Perrault, J. (1981) Origin and replication of defective interfering
particles. In Current Topics in Microbiology and Immunology 93,
151-207.
Portner, A., Scroggs, R.A., Marx, P.A., Kingsbury, D.W. (1975) A
temperature sensitive mutant of Sendai virua with an altered
hemagglutinin-neuraminidase polypeptide : Consequences for virus
assembly and cytqpathology. Virology 179-187.
Rapp, F . , Robins, S.J. (1981) Evolution of a human cell line persis
tently infected with measles virus. Intervirology 16, 149-159.
Robbins, S.J., Wrzos, H., Kline, A.L., Tenser, R.B., Rapp, F. (1981)
Rescue of a cytopathic paramyxovirus frcm peripheral blood leuko
cytes in subacute sclerosing panencephalitis. J. Infect. Dis.
143, 396-403.
Robbins, S.J., Rapp, F. (1982) Restriction of virus-specific protein
synthesis in a persistent paramyxovirus infection. Arch. Virol.
71, 85-91.
Rosenberg, F.J., Lief, F.S., Todd, J.D., Reif, J.S. (1971) Studies
on canine respiratory viruses: I. Experimental infection of dogs
with an SV^-like canine parainfluenza agent. Am. J. Epidem. 94,
147-165.
Rott, R. (1979) Molecular basis of infectivity and pathogenicity of
myxovirus. Arch. Virol. 59, 285-298. ter Meulen, V., Martin, S.J. (1976) Genesis and maintenance of a
persistent infection by canine distemper virus. J. gen. Virol.
32, 431-440. 44
Vainionpââ, R . , Salmi, A., Arstila, P. (1982) Characterization of a
hamster brain cell line persistently infected with measles virus.
Arch. Virol. 71, 311-322.
Wild, T.F., Dudgre, R. (1978) Establishment and characterization of
a subacute sclerosing panencephalitis (Measles) virus persistent
infection in BGM-cells. J. gen. Virol. 39, 113-124.
Youngner, J.S., Preble, O.T. : Viral persistence: Evaluation of viral
populations. Comprehensive Virology, (1980) Vol. 16, Ch. 2,
73-135. Plenum Press, New York, London.
Zakstelskaya, L.Ya., Zhdanov, V.M., Yakhno, M.A., Gushchin, B.V.,
Klimenko, S.M., Demidova, S.A., Konovalova, N.G., Gushchina, E.A.
(1976) Persistent SVg virus infection in continuous cell
cultures. Acta Virol. 20, 506-511.
Zhdanov, V.M. (1975) Integration of viral gencmesJ Nature 256,
471-473. CHAPTER III
VIRAL PERSISTENCE IN VERO CRT.TA DUALLY INFECTED WITH TWO
PARAMXXOVIRDSES: A MORPHOLOGICAL AND IMICNOELBCTRCN
MICROSCOPIC INVESTIGATION
I N T R ΠU C T i m
Subcellular anatomical localization of substances of biological importance can be achieved ty different approaches. The material can be labeled using polyclonal or monoclonal-specific antibodies or antibody fragments. Binding and hence localization is visualized by coupling the antibody to electron-dense substances such as ferritin or colloidal gold or via accumulation of enzymatic products (eg, diamino benzidine) secondary to reaction with a covalently linked enzyme such as horseradish peroxidase (Priestley et , 1984;
Sternberg, 1974). Essential to the latter is development of fixation methods for subsequent electron microscopy vdiich preserve ultra- structural details yet do not result in dénaturation of the antigen(s) involved.
Imrnunoelectron microscopic preembedding labeling of viral antigens in formalin or aldehyde-fixed cells has been acccnplished for other paramyxoviruses, including measles virus (Dubois-Dalcg et al., 1973), and canine distemper virus (Higgins et , 1982).
45 46
Post-embedding labeling techniques, with enzymes, ferritin or colloidal gold, result in good cellular preservation even though nonspecific staining and lack of antibody penetration limit the usefulness of these methods (Bendayan, 1984; Schwendemann et al.,
1982). Viral antigens have been demonstrated in post-embedded cells infected with Herpes Simplex virus type 1, simian virus 5 and Sendai virus (Garzon et al., 1982; Puvion-Dutilleul et al., 1985; Schwende mann et , 1982). The main obstacles of preembedding iirmuno- electron microscopy are limited permeability of cell membranes for antibodies and markers. Use of detergents or freeze-thawing improves permeability, yet loss of cellular integrity is unavoidable (Van den
Pol, 1984). Further, penetration of antibodies is restricted to 1-3 ym. The most ccmmonly used methods for improving cellular permea bility include saponin, Triton-X, digitonin or freeze-thawing
(Sternberg, 1974). Increased cytoplasmic penetration of cells and reduced background stain is achieved by using Fab fragments instead of v^ole immunoglobulins (Brandon, 1985; Kohama et al., 1981;
Kraehenbuhl and Jamieson, 1974).
Recently, Willingham (1983) introduced a gluteraldehyde- borohydride-saponin (GBS) procedure vdiich permitted penetration of cells with antibo^ yet ultrastructural features of the cell were preserved. Hall et (1978) obtained similar results with lympho cytes treated with saponin. Saponin, a plant glycoside, interacts with cholesterol-rich portions of cellular membrane and produces pores for antibody penetration (Hedmann, 1980; Vassy et al., 1984).
The major effects of borohydride are the neutralization of excess 47
aldehyde groups v M c h would cause nonspecific binding of the primary
antibody and the reduction of Schiff bases created by glutaralde-
hyde-protein cross-linking (Willingham, 1983).
In vitro ultrastructural labeling of canine distenper virus
(CDV) proteins during the viral replication cycle has previously been
reported (Higgins et , 1982). This procedure, \dien applied to the
study of canine parainfluenza (CPI) virus, was unsatisfactory.
Alternate fixation and labeling techniques were investigated and
applied to various CDV and CPI persistently infected cell lines. One
of the cell lines contained both CDV and CPI viruses. Conventional
ultrastructural examination of the latter revealed 2 distinct forms
of viral nucleocapsids (NC). Labeling these NC by virus-specific
antibody was necessary to conclusively identify the viral origin of
each and would also allow us to determine if jdienotypic mixing (eg, viral hybrid envelopes and/or heterologous envelope-NC virions)
occurred during the viral replication cycles in these monolayers.
Thus, the objectives of this study were to determine: the effects of different pre-embedding fixation methods upon preservation of cellular viral structures; and the effects of fixation ipon antibody penetration and subsequent imnunocytochemical labeling of viral antigens in the various cell lines using an avidin-biotin cotplex (ABC) marker and, frcm these data, to investigate further the viral replication cycle and opportunities for phenotypic mixing in cells dually infected with CDV and CPI viruses. 48
MAIERIAIg AND METHODS
Cells and viruses; African green monkey (Vero) cells persis tently infected with canine parainfluenza (CPI) virus were siper- 4 25 infected with 10 * TCID^g/ml R252-canine disteitper virus (CDV) at passage level 14 (BaumgSrtner et , 1981; Confer et 1975).
The doubly infected cells were passed weekly thereafter methods previously described (BaumgSrtner et , 1981). Viral infection status was monitored by indirect immunofluorescence staining of acetone-fixed monolayers using rabbit origin primary antibody (see below) and commercial goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (Cappel Laboratories).
For electron microscopic studies, infected and uninfected Vero cells were seeded in 6-well tissue culture plates at 7 x 10^ cells/ well with growth medium and maintained until confluency. In later experiments, cells were seeded at 4 x 10^ cells/well onto 4-well glass microscope slides (Lab-Tek Products, Westmont, IL) and main tained until subconfluency. Vero cells lytically or persistently infected with CDV or CPI viruses alone were also used in the evaluation of the various fixation and embedding techniques.
Virus purification and polyclonal antibody preparation; Rabbit anti-CPI and anti-CDV polyclonal antibodies were immunization with purified virus preparations. Briefly, 75 cm^ flasks with subcon fluent monolayers were infected at a multiplicity of 0.01 TCID^q .
After 1 hr absorption, monolayers were washed with PBS and -the cells were supplemented with growth medium. When virus-specific CPE was 49 optimum, the cultures were frozen and thawed and clarified by low speed centrifugation at 1,000 g for 20 minutes. The virus was pelleted by ultracentrifugation of the supernatant at 58,000 X g for
40 minutes (Beckman, SW41). The pellet was resuspended in 5 ml 2.5 iiM Tris-buffer in saline, clarified by centrifugation at 1,800 g for
30 minutes, aliqucted and stored at -70°C. New Zealand vdiite rabbits were immunized according to the following procedure; 2X intraperitcn- 6 72 eal and 2X subcutaneous inoculation using 10 * TCID^q per inocula tion site for CPI and 10^'^^ TCXD^q for CDV, respectively. The animals were boosted 10 days later with virus protein suspended in ccnplete Freund’s adjuvant, followed a second boost with incom plete Freund's adjuvant 10 days later. Ihe animals were exsanguin ated vben antibody titers were highest. To remove anti-cellular antibodies, 10 ml (1:10 diluted in PBS) rabbit serum was incubated with Vero cell lysate (20 x 10^ cells at 37 ®C) 1-2 hrs. Bnmune cdtplexes were removed by high speed centrifugation at 15,000 X g for
30 minutes. Absorbed serum was aliquoted and stored at -70®C.
Preanbeddinq Fixation Techniques
Saponin pretreatment: Confluent Vero cell monolayers were washed twice in PBS and then fixed situ with 4 ml 0.1% glutaraldehyde in
O.IM cacodylate-buffer pH 7.4 for 4 minutes, 22°C. After rinsing with Dulbecoo's PBS, cells were incubated with 1.0% saponin (v/v) in double distilled (dd-) H 2O for 100 minutes at 55 °C in a water bath
(Hall et , 1979).
GBS technique: Confluent cellular monolayers were washed in PBS and fixed in situ in 0.4% glutaraldehyde in O.IM oacodylate buffer pH 50
7.4 for 10-15 minutes, 22°C. Monolayers were washed for 20 minutes in PBS, treated with sodium borot^dride (0.5 mg/ml) for 5 minutes, washed and incubated with 0.1% saponin buffer (0.1% saponin w/v, 0.1 mM ethylene glycol tetraacetic acid in PBS) for 30 minutes, 22°C
(Willingham, 1983).
Modified GBS fixation; Subconfluent cellular monolayers were washed twice in PBS and then fixed in 0.3% glutaraldehyde in O.lM cacodylate-buffer pH 7.4 for 15 minutes, 22°C. After a 20 minute wash with PBS, cells were treated with sodium borohydride (0.5 mg/ml) for 5 minutes, washed and treated with 0.1% saponin buffer (0.1% saponin 1 mM EGTA in PBS) for 30 minutes, 22°C and washed in PBS for
20 minutes.
Immunocytochemistry; Primary antibodies, biotinylated antibocty and avidin-biotin coiplex (ABC) were diluted in NTS (0.9% NaCl w/v,
0.5M Tris base, 0.2% saponin w/v dd H2O fdl 7.2 buffer (23). Cells were incubated with diluted (1:50 to 1:2000) rabbit anti-CDV or rabbit anti-CPI antibody, 4°C for 12-24 hours with the modified GBS method, or 37°C for 1 hour, washed 3 x 20 minutes in PBS, and incuba ted with biotinylated antibody and ABC corplex, the latter according to the manufacturer' s instructions (Babbit IgG, Vectastain-Kit,
Vector Laboratories). Positive reactions were demonstrated by diamino benzidine (DAB) precipitation after 10 minutes (Graham et al., 1965). 51
Elec±ron Microscopy
For electron microscopy, cells were post-fixed with 3.0% glutaraldehyde, 4°C for 30 minutes, washed and post-fixed with 1.33% osmium tetraoxide in S-collidine buffer pH 7.4 for 30 minutes at 4°C in the dark. After dehydration in alcohol the plates or glass slides were embedded in a mixture of Medcast (Ted Pella, Inc., Tustin, CA)
100% ethanol (1:1) for 15 minutes, followed by a second overnight embedding with the same mixtures at 4°C. Embedding medium was discharged and replaced by 100% Medcast at 4°C for 6 hours, followed by incubation of fresh 100% Medcast for 2 hours at 60°C and 72 hours at 40 °C. Plastic embedded cells were separated from the plates or glass slides by snap freezing in liquid nitrogen. Selected areas were mounted on copper grids and examined unstained or after staining with uranium acetate alone or with uranium acetate and lead citrate with a Philips 200 electron microscope. 52
RESULTS
Antibody Specificity
Anti-CPI and anti-CDV antibodies were tested for specificity iy
tbe indirect imnunofliaorescence (IF) technique on homologous and
heterologous virus-infected and uninfected Vero cells (BaumgSrtner et
al., 1981). Specificity was confirmed by blocking assays performed
with canine-anizL-CPI and canine-anti-CDV convalescent serum. Both
an-tibodies stained only homologous virus-infected cells; viral cross
reactivity was not observed. Blocking assays with unlabeled homo
logous serum severely reduced virus-specific staining. Uninfected
Vero cells were IF-negative.
Ul-fcrastructxural Preservatu.on of Cells
Ultrastzuctural integrity was most severely affected iy saponin treatment. Most cells showed a marked loss of cellular organelles,
fragments of vacuolated rough endoplasmic reticulum (rER) and dis rupted mitochondria. Cells possessed an intact plasma membrane with
few microvilli and an incaiplete layer of sukmenbranous microfila ments and an intact nucleus (Fig. 3.1).
In contrast, GBS-treated cells had good ultrastructural preser vation and were catparable to non-treated hi situ glutaraldehyde-
fixed cells. The inter- and intracellular details were well preserved and the cytoplasm contained abundant rER, mitochondria, microtubules, intermediate filaments and microfilaments (Fig. 3.2).
Cells treated with the mGBS technique shewed mild to moderate 53
cytoplasmic vacuolization with infrequent loss of organelles (Fig.
3.3, 3.10).
Light Microscopic and Snmunoelectron Microscopic Demonstration of
Viral Antigen
Light microscopy; Saponin treated CPI-infected cells shewed a diffuse cytoplasmic yellowish-brown DAB precipitation product. After
GBS treatment, a distinct yellowish granular cytoplasmic pattern was observed. In contrast, CDV-infected saponin-treated cells had granular dark brown-to-black cytoplasmic inclusions. No CDV inclusion bodies were stained after GBS fixation. Both CPI and CDV
inclusion bodies and membrane-bound viral antigen were readily detected by the dark brown DAB-precipitate in cells fixed in mGBS.
Electron microscopy; In saponin treated monolayers, heavily labeled electron-dense CDV inclusion bodies, varying in size, were found throughout the cytoplasm, with predilection for the perinuclear region (Fig. 3.1). No unlabeled viral NC were seen. Cells cut transversely possessed positively labeled inclusions throughout the cytoplasam. Segmental labeling of the cell membrane with and without associated virus budding was present. In contrast, saponin-treated
CPI-infected cells showed only a few positively stained cytoplasmic
NC, membrane segments and budding virions. The majority of the unlabeled NC had a diffuse cytoplasmic distribution.
After fixation in GBS; Positively labeled CPI viral structures included cytoplasmic NC, budding virions and segments of the plasma membrane (Fig. 3.2) . In large inclusion bodies, centrally placed NC were weakly stained, vAereas peripheral NC were strongly stained 54
(Fig. 3.11). Solitary NC in the cytoplasm were not labeled. For
CDV, membrane antigens were stained, but cytoplasmic NC remained unlabeled.
Viral antigens fran both viruses were positively labeled after fixation with the mGBS method. The CPI antigens, membranous and cytoplasmic, were marked by DAB-precipitation product and could be clearly distinguished from the surrounding cytoplasm. The CDV antigen was caipletely labeled on cell membranes, and cytoplasmic NC were also labeled (Fig. 3.10). The results of fixation and subse quent labeling are summarized in Table 3.1.
Ultrastructural Observations of the CPI-CDV-infected Cell Line
The dually (CPI-CDV)-infected cell line was ultrastructurally observed at in vitro passage levels 24, 40, 52, 63, 68 and 75. Cells contained one or both of two types of viral NC (Fig. 3.4-3.7).
Smooth NC, had a filamentous structure with 15 nm in diameter and an inner electrolucent core of 5 nm. This NC had a multifocal and/or diffuse cytoplasmic distribution. Hie structures were similar to NC observed in CPI-infected cells. The second type of NC was rough and of 25 tx) 30 nm in diametier. These rough N C were found predominantJ.y in perinuclear regions and were similar to NC s-tructures identified in CDV-infected cells. Confirmation of the identity of these NC was achieved ly labeling the dually infected cell monolayers with anti-
CPI and anti-CDV antibodies, respectively. CPI antibody stained only smooth NC; CDV antibody stained rough NC exclusively (Fig. 3.8-3.13).
Both CDV and CPI viral NC occurred together in the cytoplasm of individual virus-infected cells. In the early passages, large areas 55 of the cytoplasm were occiçied inclusion bodies (Fig. 3.4-3.6).
These inclusions decreased in size at hi^er passage levels. The CPI and CDV NC were present in different cell caipartments and were well demarcated from each other (Fig. 3.7). The frequency of budding viral particles varied with each passage level but was, in general, very low. Viral pairticles of both viruses were released ffcm single or syncytial giant cells. Generally, CDV or CPI positively labeled virions were underlined hy hcmologous NC, hovrever, heterologous NC were occasionally present in the vicinity of viral budding (Fig.
3.10).
Finally, several types of nuclear bodies (NB) were observed in infected and uninfected cells. Fibrillary nuclear bodies were present frequently in nuclei of infected and uninfected cells (Fig.
3.5). Praninent fibrogranular nuclear bodies were infrequently present in the doubly infected cell line but not in uninfected or singly infected cell lines (Fig. 3.7). Neither CDV nor CPI antisera stained the NB structures. 56
DISCÜSSICN
The first objective of the present study was to evaluate 3 different fixaticn methods for applicability to pre-embedding irtmuno- cytochemistry. Hall et a l . (1978) found good ul-fcrastructxiral preser vation of sheep lyirphocytes after saponin treatment. Vfe found that
Vero cells treated in the same manner showed insufficient ultrastruc tural preservation, with loss of cellular organelles, vacuolization and partial disruption of rER and mitochondria. Nuclear and cellular membranes were less affected. Even though ultrastructural details were poor, CDV-associated cytoplasmic and membrane-bound antigens were readily detected in saponin-treated cells, and sufficient penetration was achieved in that transverse sections revealed ccmpletely labeled viral NC. The paucity of positively labeled CPI antigen after saponin treatment was surprising. Several explanations are possible. Saponin may damage CPI NC, thereby destroying the antigenicity. In formalin-fixed tissues we have noted that CPI an-tigen is largely destroyed b y enzymes (e.g. trypsin and protease), vhereas CDV treated by the same protocol showed an improved visi bility (W. BaumgSrtner, personal observation, 1985). It is also possible that the bulk of NC -together with cell organelles are lost during the washing procedure through saponin-created membrane pores.
The GBS-fixed cells were well preserved as reported by Willingham
(1983). The CPI-infected Vero cells -trea-ted by the GBS method showed mul-tifocal cytxplasmic electron-dense inclusions, segmen-tal 57 membranous labeling and virus budding. However, intracellular CDV NC were not labeled.
Improved imnunocytochemical staining for both CDV and CPI viruses was achieved by changing three steps of the original GBS protocol. To increase antibo All three methods were then applied to an iji vitro evaluation of dually infected Vero cells. Both viruses were shewn to replicate within the same cell. Smooth and fuzzy NC inclusion bodies were observed; the smooth NC represented the CPI virus and the fuzzy NC the CDV virus. These NC occupied separate cellular coipartments. The size of the cytoplasmic inclusions of both CDV and CPI viruses decreased during the course of viral persistence. Immunoelectron microscopy shewed that CDV virions budded from CDV NC cell sites and CPI virions budded from CPI NC cells sites. These observations 58 indicate that phenotypic mixing is a rare event in doubly infected cells. Ultrastructural studies with other paran^oviruses have demon strated that a single form of NC is found in the cytoplasm (Robbins et al., 1980). In contrast, Dubois-Dalcg et al. (1974) have clearly demonstrated that cells infected with measles/SSPE virus possess both fuzzy and smooth NC. Our CPI-CDV cell line possesses certain ultra structure features of SSPE cell line including tsro types of NC and NB. The latter have been observed in a wide variety of tissues and are considered normal cons-tituents of the nucleus which can increase in size and number under certain stimuli (Ghadially, 1975). Other cell lines doubly infected wi-fch unrelated viruses have been ultrastructurally and serologically studied (Choppin and Caipans, 1970; Zavada, 1976)). Evidence for phenotypic mixing is detected ty reciprocal virus neu-fcralization assays. Ultrastructural observatzions of doubly infected cells revealed that different viruses have different cellular budding sites (Rindler et , 1984). In our studies, the separate and distinct subcellular distribution of viral NC from each virus within the cell suggests that replication is independently regulated, even though both agents presumably utilize caimon host cell protein synthesis pathways. Establishment of this dually infected cell line will permit us to investigate regulation of viral protein synthesis at the molecular level. 59 SUMMARY Vero cells persistently infected with canine parainfluenza (CPI) virus and canine distesn:per virus (CDV) contained two types of viral nucleocapsids (NC). Smooth NC were multifocally or diffusely distributed in cytoplasm and were 15 nm in diameter. Fuzzy NC were located largely in perinuclear cytoplasm and were 25 to 30 nm in diameter. Conclusive identity of the viral NC in dually infected cells was determined by application of imnunocytochatiical labeling techniques. Smooth NCs were identified as CPI-virus subunits and fuzzy NCs were CDV subunits. For this, three different fixation methods were evaluated: (i) 1% (v/v) saponin 55°C (saponin), (ii) glutaraldehyde-borohydride-saponin (GBS), and (iii) a modified GBS (mGBS) method. Ultrastructurally, saponin-treated monolayers shewed, modera-te to severe cytoplasmic disruption. With saponin, CDV-associa-ted cytoplasmic and membrane viral antigens were readily labeled, vhereas CPI-associated viral antigens were largely unlabeled. Even though GBS-treatod cells shewed an excellent ultrastructural preservation, labeling was unsatisfactory; the CPI-NC were incenpletely labeled, and CDV-NC were unlabeled even though membrane viral protoins of both viruses survived GBS fixation. After fixation witdi mGBS, preserva tion of cellular architecture and ul-trastzuctural labeling of NC and membrane-associated viral proteins of both viruses was achieved. 60 Table 3.1 Suintiarized effects of 3 different preanbedding treatments on glutaraldehyde-fixed cells on cellular integrity and labeled viral antigen presentation. Modified 1% Saponin^ GBS Methocÿ* GBS Method CDV CPI CDV CPI CDV CPI Li^t microscopy visibility of viral antigen + 4 + + — ++ 4 4 4 - 4 4 4 - Transmission electron microscopy cellular preservation +/- +++ 4-4- Labeled membrane-bound viral antigen +++ + ++ 444- 444- 444- Labeled cytoplasmic viral antigen +++ +/— 44- 44- 444- Percentage of labeled viral antigen 100% <1% <1% 60-80% 60-80% 100% ^As described Hall et al. (1984) ^As described by Willingham et (1983) ^As described in this paper +++, ++, +, Excellent, good, moderate, poor visibility or ultra- structural preservation. Fig. 3.1 Vero cells persistently infected with CDV virus after saponin treatment. Membrane ^ budding viral particles (arrowhead), and cytoplasmic viral antigen (arrow) are labeled (anti-CDV 1:200). Note loss of cytoplasmic organelles and disruption of mitochondria, N = nucleus, M = mitochondria (osmic acid only). Original magnification X 7800). 61 62 n m Fig. 3.1 Fig. 3.2 Vero cell lytically infected with CPI virus, after GBS treatment. Sutmotibraneous viral antigen and budding viral particles are labeled (arrows) N - nucleus (osmic acid only). Original magnification X 4800. 63 64 Fig. 3,2 Fig. 3.3 Vero cells persistently infected with CPI virus, after >t-GBS treatment. Cytoplasmic viral antigens are labeled (arrows) N = nucleus (anti-CPI 1:100) (osmic acid only). Original magnification X 17,700. 65 66 Fig. 3.3 Fig. 3.4 Vero cell persistently infected with CPI-CDV virus, passage 27. Both smooth (sNC) CPI and fuzzy (fNC) NC are present in the cytoplasm (osmic acid, utanyl acetate and lead citrate counterstain). Original magnification, X 17,000. 67 68 m Fig. 3.4 Fig. 3.5 Vero cells persistently infected with CPI-CDV virus, passage 27. Cytoplasmic accumulation of both smooth (^C) CPI virus and fuzzy (fNC) CDC nucleocapsids. Several fibrillary nuclear bodies are present in the nucleus (osmic acid, uranium acetate and lead citrate). Original magnifi cation X 10,800. 69 70 « (g j # # i M & Fig. 3,5 Fig. 3.6 Vero cells persistently infected with CPI virus^ passage 27. Borderline between smooth (sNC) CPI virus and fuzzy (fNC) CDV nucleocapsids (osmic acid, uranium acetate and lead citrate). Original magnification X 123,000. 71 72 m m % ft sJw. K# / - cr- #_ Fig. 3.6 Fig. 3.7 Vero cells persistently infected with CPI vims, passage 27. Multilobulated nuclei in syncytium giant cells with prominent fibrogranular nuclear bodies (vAiite arrovAead), smooth (arxodiead) and fuzzy (arrows) nucleocapsids (osmic acid, uranium acetate and lead citrate). Original magnification X 10,800. 73 74 ^ 9 e Fig. 3.7 Fig. 3.8 Vero cell persistently infected with CPI-CDV. Saponin treatment, p40, labeled cytoplasmic CDV antigen (arrow) and unlabeled CPI NC (arrovAead). (Stained with osmic acid and heavy metals.) Original magnification X 13,400. 75 76 " ; 'c% ; ^ p - ## ill mm Fig. 3.8 Fig. 3.9 Vero cell persistently infected with CPI-CDV^ passage 40, saponin treatment. Perinuclear positively labeled CDV NCs (arrow) (anti-CDV 1:200) with adjacent diffusely distrib uted unlabeled smooth CPI NCs (arrovdiead), N = nucleus. Original magnification X 69,000. 77 78 m m m ^ 'ft' & •! % ■ f é f VO 0 ^ Fig. 3.9 Fig. 3.10 Vero cell persistently infected with CPI-CDV, p75. Labeled cytoplasmic CDV NC and budding viral particles (arrows) (anti-CDV 1:200} adjacent to unlabeled CPI NCs (arrovdiead) M-GBS method (osmic acid and uranyl acetate only). Original magnification X 9300. 79 so & 4l,‘ Fig. 3.10 Fig. 3.11 Vero cell persistently infected with CPI-CDV (CBS method) (p63). Labeled cytoplasmic CPI antig^ (anti-CPI 1:100) (large arrows) and imlabeled CDV antigen (large arrovAeads) in adjoining cells (stained with osmic acid and heavy metal counterstain) N = nucleus, small arrovAeads = plasma membrane. Original magnification X 13,500. 81 82 mmi .e-'" A4*, aw Fig. 3.11 Fig. 3.12 Segment of a Vero cell persistently infected with CPI-CDV, p75 (M-GBS treatment). CPI antigen is labeled with anti- CPI (1:200) (arrovdieads) and the adjacent, fuzzy CDV NC ranains unlabeled (arrows) (stained with osmic acid and heavy metal counterstain). Original magnification X 90,000. 83 84 % : % 0 Fig. 3.12 Fig. 3.13 Vero cell persistently infected with CPI-CDV, p68. Sufcmesiibranous positively labeled CPI antigens (anti-CPI, 1:75) (arrows) and unlabeled perinuclear fuzzy CDV NCs (arrovdiead). N = nucleus, GBS method (stained with osmic acid and heavy metal counterstain). Original magnification X 11,960. 85 86 m f Fig. 3.13 87 REFERENCES Baumgârtner WK, Metzler AE, Krakov^ S, Koestner A. (1981) In vitro identification and characterization of a virus isolated fron a dog with neurological dysfunction. Infect. Iirinun. 31(3) : 1177-1183. Bendayan M. (1984) Enzyme-gold electron microscopic cytochemistry: A new affinity approach for the ultrastructural localization of macrcmolecules. J. Electron. Microsc. Tech. 1:349-372. Brandon C. (1985) Improved imnunocytochemical staining through the use of Fab fragments of primary antibody. Fab specific second antibo(fy, and Fab-horseradish peroxidase. J. Histochem. Cytochem. 33:715-719. Choppin PW, Coitpans FW. (1970) Phenotypic mixing of envelope proteins of -the parainfluenza virus SV^ and vesicular stcmatitis virus. J. Virol. 5:609-616. Confer AW, Kahn DE, Koestner A, Krako^ka S. (1975) Biological properties of a canine disteaftper virus isolate associated with denoyelinating encephalotyelitis. Infect. ]jnmun. 11 (4) : 835-844. Dubois-Dalcq M, Barbosa LH, Hamilton R, Sever JL (1973) Corparison between productive and latent subacutze sclerosing panencephalitds viral infection in vitro. Lab. Invest. 30:241-250. Garzon S, Bendayan M,Kurs-tak E. (1982) Ul-trastructural localization of viral antigens using protein A-gold -technique. J. Virol. Methods 5:67-75. Ghadially FN. (1975) Ultras-tructure Pathology of the Cell (1975) (Butterworth, London and Boston). 88 Graham HC, Kamovsky MJ. (1965) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry ty a new technique. J. Histochem. Cÿtochem. 14(4) :291-302. Hall JG, Birbeck MSC, Robertson D, Peppard J, OrIans E. (1978) The use of detergents and irrmunoperoxidase reagents for the ultra- structural demonstration of internal inmunoglobulin in lyrtph cells. J. Irtitiunol. Methods 19:351-359. Hedmann K. (1980) Intracellular localization of firbonection using inmunoperoxidase cytochemistry in liÿit and electron microscopy. J. Histochem. Cytochem. 28(11):1233-1241. Higgins RJ, Krakovdca S, Metzler AE, Koestner A. (1982) Immunoperox- idase labeling of canine distemper virus replication cycle in Vero cells. Am. J. Vet. Res. 43(10:1820-1824. Kohama MT, Cardenas JM, Seto JT. (1981) htinunoelectron microscopic study of the detection of the glycoproteins of influenza and sendai virus in infected cells the inmunoperoxidase method. J. Virol. Methods 3:293-301. Kraehenbuhl JP, Jamieson JD. (1974) Localization of intracellular antigens by immunoelectron microscopy. Int. Rev. Experiment Pathol. 13:1-52. Priestley J, Polak JM, Vardell JM. (1984) Immunolabelling for Electron Microscopy, Chapter 4, 37-52. In: Elsevier Sciences Publishers, Amsterdam, New York, Oxford. 89 Puvion-Dutilleul F, Laithier M, Scheldrick P. (1985) Ultrastructural localization of the Herpes Sirtplex virus major ENA-binding protein in the nucleus of infected cells. J. gen. Virol. 66:15-30. Rindler MJ, Ivanov IE, Plesken H, Rodriguez-Boulan E, Sabatini DD. (1984) Viral glycoproteins destined for apical or basolateral membrane domains traverse the same Golgi apparatus during their intracellular transport in doubly infected Madin-Darty canine kidney cells. J. Cell. Biol. 98:1304-1319. Bobbins SJ, Bussel BH, Bapp F. (1980) Isolation and partial charac terization of two forms of cytoplasmic nucleocapsids frcm measles virus infected cells. J. gen. Virol. 47:301-310. Schwendemann G, Wblinsky JS, Hatzidimitriou G, Merz DC, Waxham MN, (1982) Postembedding iirmunocytochemical localization of paranyxo- virus antigens by light and electron microscopy. J. Histochem. Cytochem. 30(12):1313-1319. Sternberg LA. (1974) Immunocytochemistry. Sec. Edition, John Wiley & Sons, New York, Chichester, Brisbane, Toronto, 67-103. Van den Pol AN. (1984) Colloidal gold and biotin-avidin conjugates as ultrastructural markers for neural antigens. J. Ibp. Physiol. 60:1-33. Vassy J, Bissel M, Kraemer M, Foucrier J, Guillouzo A. (1984) Ultra- structural indirect iramunolocalization of transferrin in cultured hepatocytes permeabilized with Saponin. J. Histochem. Cytochem. 32(5):538-540. 90 Willingham MC. (1983) An alternative fixation - procession method for preembedding ultrastructural immunocytochemistry of cyto plasmic antigens. J. Histochem. Cytochem. 31(6) ;791-798. Zavada J. (1976) Viral pseudotypes and phenotypic mixing. Arch. Virol. 50:1-15. CHAPTER IV PERSISTENT INFECTION OF VERO CKT.Tfi BY PARAMXXOVERDSES INTRCDUCTICN Both canine parainfluenza virus (CPI) and canine distemper virus (CDV) are maribers of the family Paramyxoviridae. The CPI is closely related to human parainfluenza-2 and simian virus 5 (SV^) (Baumgart ner et , 1981; Goswami et al ., 1984); CDV is a morbillivirus closely related to measles virus (MV) (Metzger et , 1980). In vivo concurrent infection of dogs with both canine viruses have been reported in the respiratory tract, but not in extra pulmonary locations (Binn, 1970; Cornwell et al ., 1976). Ifciwever, CPI virus has been occasionally isolated frcm non-respiratory tissue including cerebrospinal fluid, intestine, kidney and liver (BaumgSrtner ^ , 1981; Binn, 1970; Macartney et al., 1985). Simultaneous infection of the human counterparts, SV^-like viruses and MV, have been demon strated in patients with Paget's disease and subacute sclerosing panencephalitis (SSPE) (Basle et al., 1985; Robbins et al., 1981). Canine distemper virus causes acute and chronic central nervous ^stem (CNS) infection in its natural host. The mechanisms lending to persistent infection are unkncwn, however, to vitro studies 91 92 shewed that CDV persistence is mediated by Dl-particle production and/or a virus-coded nonstructural protein (ter Meulen and Martin, 1976; Tdbler and Imagawe, 1984). Chronic vivo infection has not been associated with either of these mechanisms. Recently, we reported that interfering, low density viral particles occur in persistent CPI cell lines (Baumgârtner et , sufcmitted, 1986). Using the same cell and assay system, we investigated possible mechanisms of CDV persistence and the effect of a second virus vpon the development of vitro persistence. Ihe results of that study form the basis for this report. 93 MATERIALS AND METHODS Viruses. A syncytial giant cell and plaque-forming canine parainfluenza virus designated CPI (+), originally isolated from the CSF of a dog with neurological dysfunction, and the canine distotper virus R252-CDV have been described previously (Baumgârtner et al ., 1981; Confer et al., 1975). Cell cultures and virus titrations. African green monkey kidney (Vero) cells were maintained as described (Baumgârtner et , 1981). Virus titrations were performed in a microtiter system by adding 4 serial 10-fold dilutions of viral suspension to Vero cells (1.5 x 10 cells/vrell) with incubation for 7 days. Plates were fixed and stained by the Bouin ' s-formalin-Giemsa method as described (Baumgârtner et , in press). Virus titrations eigressed as TCID^g were calculated by the formula of Spearman-Kârber (Finney, 1964). Establishment of persistently infected cells. Vero cells were infected with virus stocks, CPI(+) or CDV at a multiplicity of infection (M.O.I.) of 1.0 or 0.1 and maintained in the same flask until confluency. Medium was changed every three days. Surviving virus-infected cells were maintained until confluence and then passed weekly thereafter seeding 1.5 x 10^ cells onto 25 cm^ flasks. Cells infected with CPI (+) virus were srperinfected with CDV at a M.O.I. of 0.1 at pl3. Light microscopy and imnunofluorescence (IF). To evaluate viral 4 CEE, 4.0 X 10 persistently infected cells per well were propagated on glass microscope slides (Lab-Tek Products, Westmont, IL). After 3 94 days, cell monolayers were fixed in Bouin's fixative for 10 min, dehydrated and stained with hematoxylin and eosin (H&E). Indirect IF on acetone-fixed monolayer replicates was performed as described (Baumgartner et , 1981). Hemadsorption (Had). Had for CPI-encoded cell membrane glyco proteins was performed as described (Baumgartner et , 1981). The number of Had-positive cells were determined by counting the number of Had-positive cells/300 cells. Interference assay. Supernatants frcm persistently infected cultures were tested for interfering activity with 200 TCID^q of lytic CPI(+) virus in 200 ul MEME. Equal volumes (0.2 ml) of lytic virus plus interfering material were mixed and inoculated onto a 25an2 flask of subconfluent Vero cells. After incubation at 37°C for 72 hours, the cells were scraped into the medium, virus was harvested after 3 freeze-thaw cycles and assayed for titratable virus iy 10- fold dilution. Controls included mixtures of lytic virus with lytic virus or growth medium. Virus purification and equilibrium density gradient centri fugation. Persistently or lytically infected Vero cells were labeled for 24 hours with 80 yCi L-methionine in 25cm^ flasks (NEN Research Products, Dupont, specific activity 1022 %i/nmol). Persis tently infected cells were labeled 2-3 days after passage; lytically infected cells were labeled vhen CPE cotmenced. Growth medium was replaced by methionine-deficient medium supplemented with dialyzed 35 PCS; 80 yCi S-L-methionine was added 2 hours later. Virus was harvested ly 3 cycles of freezing and thawing. Cellular debris was 95 removed by centrifugation for 10 min at 2,200 x £ and the resultant virus suspensions were mixed with polyethylene glycol 6000 as described (Fisher, 1983). The virus eluates were further purified by a zonal centrifugation in 40% potassium tartrate in OTE and 15% sucrose (w/w) in NTE for 2 hours at 21,000 rpm (SW41 Rotor, Beckman, Inc.). The virus-containing band at the surface was collected with a syringe and sedimented to equilibrium on a 0-50% potassium tartrate gradient in NTE buffer (w/w) for 16 hours at 21,000 rpm. Tubes were punctured with a needle at the bottcm and 14 drop fractions (650 yl) were collected. One hundred yl of each fraction were wei^ied to determine buoyant density. Radioactivity of each fraction was determined by adding 10 yl of each fraction to 5 ml Aguasol-2 (New England Nuclear) and 500 yl distilled water, and counted by liquid scintillation. 96 RESULTS CDV virus. Subconfluent Vero cell monolayers in 25cm^ flasks 5 25 were infected with 10 ’ TCID^q . Three days after infection a progressing CPE characterized by single cell necrosis, cellular detachment and a few small syncytial giant cells developed. After two weeks, surviving cells formed a sparse monolayer. Throughout the observation period numerous lytic crises occurred in which severe cellular destruction and syncytial giant cell formation alternated with short periods of steady cell growth. Yield of progeny virus (eg, titer) dropped continuously during the chronic infection (Table 4.1). By p25, the majority of progeny virus (>90%) was cell released (data not shewn). The CPE of the persistent CDV was similar to the standard CDV. Aged, 3 week old, chronically infected monolayers shewed numerous multifocal small multilayered foci (Fig. 4.1, 4.2). CPI-CDV virus. Vero cells infected with CPI(+) virus were 5 25 superinfected with 10 * TCJD^q CDV. Three days post-inoculation (PI) a CPE typical of CDV developed. As with the CDV cultures above, numerous lytic episodes changes with episodes of normal cell growth. The CPE was characterized by syncytial giant cell formation and single cell necrosis. Throughout the observation period (p75) a steacfy reduction of the yield of progeny virus was observed (Table 4.1). By p24, the majority (>90%) of progeny virus was cell released (data not shown). Aged, 3 week old, persistently infected monolayers showed numerous large multilayered foci (Fig. 4.1). 97 Uninfected Vero cells had a mean cellular yield of 11.54 x 10^ viable cells/flask (n=20) ccnpared with 7.3 x 10^ viable CDV-infected cells/flask (n=44) and 6.9 x 10^ viable CPI(+)-CDV-infected cells/ flask (n=44). The mean cellular yield of the CDV and CPI (+) -CDV cell line was statistically significantly reduced (p<0.05) compared with uninfected Vero cells (Student's t-test for unpaired variances). Viroloqical characterization of persistently infected cells. Essentially, 100% of the cells infected with CPI (+) -CDV showed cytoplasmic viral antigen as determined by IF. The percent CDV antigen-positive cells varied weekly fron 1 to 100% in both CDV and CPI (+)-CDV cell lines. The percentage of Had-positive CPI(+)-CDV cells varied from 10 to 80%. Interference by persistently infected cells with the replication of homologous and heterologous viruses. Replication of lytic CPI(+) virus was severely depressed in CPI (+) -CDV cell lines (but not in CDV-infected cells). In contrast, both cell lines, persistently infected with CPI(+)-CDV or CDV alone were protected against superin fection with lytic CDV. At p30, CPI (+) -CDV cells were not protected against lytic CDV infection; however, this result could not be substantiated in several other challenge experiments (Table 4.2). Interfering viral particles in persistently infected cells. Progeny virus frcm CDV and CPI (+) -CDV cells showed no interfering activity with lytic CDV (Table 4.3). No taiperature-sensitive mutants were detected vhen titrations of progeny virus were performed at 32®C and 37®C (data not shown). Progeny virus frcm CPI (+) -CDV interfered 98 slightly with the replication of lytic CPI(+) virus at pll and p21 but not at p59 and p60 (Table 4.4). Detection of lew density viral particles. Lytic CDV virus had a peak buoyant density of 1.175 g/ml. Profiles of labeled virions collected frcm persistently infected CDV cultures shewed a sharp peak of radioactivity at a buoyant density of 1.164 g/ml (p51) and 1.161 g/ml (p60). Progeny virus fron CPI (+) -CDV showed peaks of radio activity at a buoyant density of 1.181 g/ml (p44) and 1.179 g/ml (p63) (Fig. 4.4). 99 DISCUSSION Persistent CDV infection of Vero cells was achieved propa gating surviving cells fron a lytic infection. Ihe observed unstable virus-host cell relationship, including fluctuation of CPE and percentage of cytoplasmic CDV-positive cells, similar to other persistently infected cell lines (Metzger et , 1984). The resis tance of both persistently infected cell lines to si:perinfection with hotiologous but not heterologous viruses and the drop in the release of infectious virus are also characteristic of persistent viral infection (Youngner and Preble, 1980). In general, ^ vitro viral persistence is associated with production of defective-interfering viral particles, tenperature-sensitive mutants and interferon (Friedman and Rumseur, 1979). However, mechanisms of CDV-induced in vitro and vivo persistence remains unclear, ter Meulen et al. (1976) found a labile component with interfering activity in the medium of Vero cells persistently infected with 03V. Interferon has been proposed to be an vivo marker for in vivo persistence but these results could not be substantiated in follow-up experiments (Tsai et ^ . , 1979; Friedlander et ^ . , 1985). Since our studies were carried out in Vero cells, vdiich do not produce interferon, this does not seem to be a factor in the system. Tdbler et (1984) attributed CDV in vitro persistence as to Dl-particles in one cell line and to cell-associated viral-particles in a second line. Gorman (1983) showed that antibody and corplement modulate viral persistence under in vitro conditions. 100 Hie present study failed to demonstrate Dl-particles or t^ mutants (32°C and 39°C, data not shown) as a mechanism for CDV persistence. Similar results are reported for sane MV cell lines (Rafp and Robbins, 1981; Sorodoc et , 1980). Hie possible produc tion of CDV Dl-particles cannot be ruled out from our data. Varia tions in the sensitivity of various interference-bioassay systems are described (Kawai and Mutsowoto, 1982). Further studies, including investigations of the genomic RNA and oligonucleotide fingerprinting are necessary to detect or rule out defective viral particles. TO our knowledge, low density viral particles in CDV persistence have not been described. Changes in the buoyant density presumably reflect a change in the viral ENA content since all virion polypep tides are produced in these cells (Perrault, 1981) . Hiough the heterogenous nature of paran^oviruses makes separation of virus subpqpulations by buoyant density studies difficult, the repeated demonstration of low density viral particles in the CDV cell line was considered a significant finding. Low density viral particles are described for other paramyxoviruses (Fisher, 1983; Portner et al., 1975; Perrault, 1981). Hie present study was performed parallel with the investigation of CPI in vitro persistence (BaumgSrtner, to be published). Dl- particles were readily demonstrated in the latter systan. Similarly, persistently infected CPI-CDV cells showed slight and infrequent interference with lytic CPI(+) virus. Hie appearance of Dl-particles in the CPI cell line correlated with loss of virus-induced CPE, therefore the reduced production of interfering particles in the 101 CPI-CDV cell line can be ea^lained by the unstable virus-host cell relationship. Similar instabilities are reported by Barski et al. (1969) with a Herpes-SV^ cell line. The results of this study suggest that CDV persistence in both cell lines is due to cell- associated particles and not due to cell-free Dl-particles. The importance of a dynamic virus-cell relationship for in vitro viral persistence has been stressed by others (Rapp and Robins, 1981; Sorodoc et al., 1980; Wild and Dugre, 1978). Both persistent cell lines, CDV and CPI-CDV, shewed a marked effect on cell monolayer mor0iology and cell replication. In contrast, CPI cell lines showed no effect on cell morphogenesis (BaumgSrtner, to be published). Reductions of cell growth has been reported with BC3Î cells persis tently infected with SSPE virus (Wild and Dugre, 1978). Sorodoc et al. (1980) described a calf kidney cell line persistently infected with measles virus with similar multilayered foci. The authors suspected a host cell factor and/or a possible in vitro transforma tion as the underlying cause for viral persistence. Minagawa et al. (1974) reported a non-virion-associated ccnponent with host-cell DMA synthesis suppressing activity. The doubly infected cell lines showed characteristics of the single infected cell line, including lack of CDV interfering activity and unstable virus-host-cell relationship. In contrast, the CPI characteristic (Dl-particle, stable virus-host-cell relationship) were basically absent in the doubly infected cell line. Furthermore, increased Had in the doubly infected cells cotpared with the single CPI-infected cells, indicate increased CPI-H protein incorporation in 102 the cell membrane. Buoyant density studies shewed that progeny viruses from the doubly infected cells have buoyant densities in the range of the lytic viruses ccnpared with single infected CDV and CPI (BaumgSrtner, to be published) cell lines with low buoyant density viral particles. Ihe significance of this observation is unclear. Further studies are warranted to understand this phenomenon. 103 SUMMARY Two Vero cell lines persistently infected with canine disteitper virus (CDV) or with both CDV and canine parainfluenza (CPI) viruses vere investigated. Cells in the CPI-CDV cell line \rere 90-100% positive for cytoplasmic CPI antigen and exhibited 10-80% CPI-hemad- sorption. Cytoplasmic CDV antigen expressed in both singly and dually infected cultures varied v^ekly frcxn 1-100%. Numerous cyto lytic crises vrere observed in both cell lines throughout the obser vation period (80 passages). Cell replication was severely depressed in both cell lines coipared with uninfected Vero cells. Interfering CDV viral particles were not detected in the CDV or CPI-CDV cell line, however, mild interfering activity against lytic CPI virus was present in the CPI-CDV cell line. Buoyant density studies showed that the viral population in the persistent CDV cell line had a slightly lower mean buoyant density coipared to the lytic CDV stock virus. Low density particles were not detected in the doubly infected cell line. 104 Table 4.1 Virus yield in lytic and persistently infected cell lines. Cell Lines Passage No. CDVCPI-CDV lo5.25 Lytic infection - Persistent infection at passage level 6 10^*^ 10^"^ lo3.75 lo3.0 11 lo2.0 21 _ 24 0-10^'^^ 0_loO'75 25 — 30 - 10^'^ 35 10°*^ - 1q 3.0 37 - I0O .75 38 - 57 - <10^*° 60 <10^ 101.75 65 a = ej^ressed in TCID^q /O.I ml. b = cell-associated and cell-released virus production. c = represents minimum and maximum virus yield of subsequent daily titrations of six days of cell-associated and cell-released virus at this passage level. 105 Table 4.2 Superinfection of persistently infected cells with the lytic C P I (+)^ and CDV virus.^ Challenge Cell Line Passage Virus Titer^ Reduction^ Vero 180 C P K + ) IOG'75^ - CPI (+) -CDV 25 C P K + ) 0 6.75 103.25 CPI(+)-CDV 34 CPI(+) 3.5 CDV 25 C P K + ) 10^-° 0.75 lo4.25 Vero 185 CDV - 1o 3.75 CPI (+) -CDV 30 CDV 0.5 CPI(+)-CDV 34 CDV 10^'^ 1.75 CDV 27 CDV 10^'^ ;.75 Subconfluent cellular monolayers in 25cm^ flasks were infected with 10 6.75 and virus yield was determined after 48 hours. b = Subconfluent cell monolayers in 25cm^ flasks were infected with 4 55 * TCID__ and virus yield was determined 72 hours post infection. c = TCID^q /0.1 ml. d = Differences in the yield of lytic CPI(+) or CDV after propa gation on mock-infected or persistently infected Vero cells, expressed in log^Q. 106 Table 4.3 Interference with replication of lytic CDV virus by CDV and CPI-CDV progeny virus of persistently infected cells. Passage level Interfering of progeny Challenge^ Material virus virus Titer^ Reduction^ lo3.25 Medium — CDV - lo3.25 CDV 11 CDV 0 lo3.25 CDV 25 CDV 0 102.75 CDV 65 CDV 0.5 CDV 66 CDV lO^'OO 0.25 lo3.25 0 CDV 70 CDV lo3.25 CPI-CDV 10 CDV 0 lo3.25 CPI-CDV 28 CDV 0 CPI-CDV 40 CDV lO^'OO 0.25 a = 200 TCID^q in 200 yl MEME. b = TdDgg/O.l ml after 72 hours. c = Reduction in yield of lytic CDV after mixed infection with persistent virus expressed in log^^Q. 107 Table 4.4 Interference with replication of lytic CPI(+) virus by CPI-CDV progeny virus of persistently infected cells. Passage level Interfering of progeny Challenge^ Material virus virus Titer^ Reduction^ lo5.75 Medium - C P K + ) - 1o 4.75 CPI (+) -CDV 11 CPK+) 1.0 10"'75 CPI (+) -CDV 21 CPI(+) 1.0 CPI (+) -CDV 59 C P K + ) 10^*^ 0.25 1o 6.25 CPI (+) -CDV 60 CPI(+) 0 a = 200 TCIDgQ in 200 yl MEME, b = TCID^q /0.1 ml after 72 hours. c = Reduction in yield of lytic CPI (+) virus after mixed infection with persistent virus expressed in log^Q- AJB Fig. 4.1 Catparison of monolayer morphology of aged, 3 week old, monolayers fron (A) uninfected Vero cells, (B) persistent CPI-infected, (C) CPI-CDV infected cells (hiÿi power magnification, phase contrast), and (D) CDV-infected cells. 108 109 Fig. 4.1 Fig. 4.2 Ccnparison of monolayer morphology of aged, 3 week old monolayers from (A) uninfected Vero cells, and (B) persistent CDV infection. Vero cells persistently infected with CDV show numerous small multilayer foci (high dry magnification). 110 Fig. 4.3 Equilibrium density centrifugation of lytic CDV (A) and CDV progeny virus from persistently infected cell lines from passage level p51, (B) infected confluent monolayers were labeled with 20 yCi ^^S-methionine/ml. Lytic virus has a buoyant density of 1.175 g/ml; in contrast, at passage level 51 the virus population has a lo^ær buoyant density with 1.165 g/ml (numbers represent buoyant densities in g/ml). 112 COUNTS / min x10"2 COUNTS / min x10“ 2 O)* (Û o> A O' O' O'* Fig. 4,4 Equilibrium density centrifuga-tion of progeny viruses fron Vero cells doubly infected with CPI-CDV from passage level 63 (numbers represent buoyant densities in g/ml). 114 COUNTS / min xIO"^ o z z c S s m m if X 0)' 0 0 " V i 116 REFERENCES Barski, G., LePrancois, , Youn, J.K. (1969) Infection latente double avec SV^ et virus de herpes en culture de cellules de hamster. Ann. Inst. Pasteur 117, 420-433. Basle, M.F., Russel, W.C., Goswami, K.K.A., Rebel, A., Giraudon, P., Wild, F., Filmon, Q. (1985) Paramyxovirus antigens in osteoclasts from Paget's bone tissue detected hy monoclonal antibodies. J. gen. Virol. 66, 2103-2110. BaumgSrtner, W., Krakcxidca, S. A simple method for the histochanical demonstration of viral inclusions in cell monolayers in micro- titration plates. Stain Tech. (in press). BaumgSrtner, W.K., Metzler, A.E., Krakovdca, S., Kbestner, A. (1981) In vitro identification and characterization of a virus isolated from a dog with neurological dysfunction. Infect. Iramun. 31, 1177-1183. Binn, L.N. (1970) A review of viruses recovered from dogs. J.A.V.M.A. 156, 1672-1675. Confer, A.W., Kahn, D.E., Kbestner, A., Krakowka, S. (1975) Biolog ical properties of a canine distemper virus isolate associated with demyelinating encephalomyelitis. Infect. Iramun. 11, 835-844. Cornwell, H.J., McCandlish, I.A.P., Thompson, H., Laird, H.M., Wright, N.G. (1976) Isolation of parainfluenza virus SV^ from dogs with respiratory disease. Vet. Rec. 98, 301-303. Desmyter, J., Melnick, G.L., Rawls, W.E. (1968) Defectiveness of interferon production and of Rubella virus interference in a line 117 of African green monkey kidney cells (Vero). J. Virol. 2, 955-961. Finney, D.I. (1964) Statistical methods in biological assay. Hafiier Publishing Co., New York. Fisher, L.A. (1983) Characterization of four cell lines persistently infected with measles virus. Arch. Virol. 77, 51-60. Friedlander, J.M., Summers, B.A, i^pel, M.J.G. (1985) Persistence of virulent canine distenper virus in lyitphoblastoid cell lines. Arch. Virol. 86, 47-62. Friedman, R.M., Ramseur, J.M. (1979) Mechanisms of persistent infec tion by cytopathic viruses in tissue culture. Arch. Virol. 60, 80-103. Gorman, N.T. (1983) The interaction of cells persistently infected with canine distemper virus with antiviral antibody and caiple- ment. Cell. Immunol. 77, 242-248. Goswami, K.K.A., Lange, L.S., Mitchell, D.N., Cameron, K.R., Russel, W.C. (1984) Does simian virus 5 infect humans. J. gen. Virol. 65, 1295-1303. Higgins, R.J., Krakc^^to, S., Metzler, A.E., Koestner, A. (1982) Immunoperoxidase labeling of canine distemper virus replication cycle in Vero cells. Am. J. Vet. Res. 43, 1820-1824. Kawai, A., Matsumoto, S. (1982) A sensitive bioassay system for detecting defective interfering particles of rabies virus. Virology 122, 98-108. Kosch, K. (1980) Persistent paramyxovirus infections and bahaviour of B-adrenergic receptors in C-6 rat glicma cells. J. gen. Virol. 47, 513-517. 118 Macartney, L., Cornwell, McCandlish, T.A.P., Thcnpson, H. (1985) Isolation of a novel paramyxovirus from a dog with enteric disease. Vet. Rec. 117, 205-207. Metzger, A.E., Higgins, R.J., Krakovto, S., Ifoestner, A. (1980) Persistent in vitro interaction of virulent and attenuated canine diststper virus with bovine cells. Arch. Virol. 66, 329-339. Metzger, A.E., Krakovdca, S., Axthelm, M.A., Gorham, J.R. (1984) In vitro propagation of canine distemper virus : Establishment of persistent infection in Vero cells. Am. J. Vet. Res. 45, 2211- 2215. Minagawa, T., Nakaya, C., lida, H. (1974) Host DNA synthesis- suppressing factor in culture fluid of tissue cultures infected with measles virus. J. Virol. 13, 1118-1125. Perrault, J. (1981) Origin and replication of defective interfering particles. In Current Topics in Microbiology and Immunology 93, 151-207. Portner, A., Scorggs, R.A., Marx, P.A., Kingsbury, D.W. (1975) A taperature-sensitive mutant of Sendai virus with an altered hemagglutinin-neuraminidase polypeptide: Consequences for virus assenibly and cytopathology. Virology 47, 179-187. Rapp, F., Robbins, S.J. (1981) Evolution of a human cell line persistently infected with measles virus. Intervirology 16, 149-159. Robbins, S.J., Wrzos, H., Kline, A.L., Tenser, R.B., Rapp, F. (1981) Rescue of a cytopathic paramyxovirus fron peripheral blood 119 leukocytes in subacute sclerosing panencephalitis. J. Infect. Dis. 143, 396-403. Sorodoc, I., Merca, I., Cajal, N., Dumitrescu, S.M., Constantinescu, 0., Cemescu, C. (1980) Peculiarities of the virus-host cell relationship in a calf kidney cell line persistently infected with measles virus. Acta Virol. 24, 253-264. ter Meulen, V., Martin, S.J. (1976) Genesis and maintenance of a persistent infection by canine distenper virus. J. gen. Virol. 32, 431-440. Tobler, L.H., Imagawa, D.T. (1984) Mechanism of persistence with canine disteaiper virus; Difference between a laboratory strain and isolate from a dog with chronic neurological disease. Inter virology 21, 77-86. Tsai, S.C., i^pel, M.J.G. (1979) Interferon in duction in dogs. Am. J. Vet. Res. 40, 356-361. Wild, T.F., Dugre, R. (1978) Establishment and characterization of a subacute sclerosing panencephalitis (measles) virus persistent infection in BGM cells. J. gen. Virol. 39, 113-124. Younger, J.S., Preble, O.T. Viral persistence: Evolution of viral populations. In Ccnprehensive Virology Vol. 16, Chap. 2, 73-135. Plenum Press, New York, London. CHAPTER V NEUROPATHOLOGY OF CANINE PARAINFLUENZA VIRUS AFTER INTRACEREBRAL INFECTICN IN FERRETS INTRœUCTICN Canine parainfluenza (CPI) virus is closely related to Simian VirusS (SVg), human mumps virus and human parainfluenza virus II (BaumgSrtner et , 1981; Goswami and Russel, 1982; Rosenberg et al., 1971). Natural viral infection in dogs produces an acute self- limiting tracheo-bronchitis (Wagener et , 1983) . However, CPI has been isolated fron extrapulmonary tissues including intestine, spleen, kidney and brain (Binn, 1970; Evermann et , 1980; Macartney et , 1985), suggesting that viral infection, on occasion, may produce systemic disease. Human parainfluenza virus infection is also thought to be restricted to the respiratory tract; however, several recent reports documented the isolation of SV^-like virus from bone marrow cells of humans with multiple sclerosis (Goswami et al., 1984; Mitchel et al., 1978) and also from peripheral lynphocytes obtained from a patient with subacute sclerosing paren- cephalitis (Robbins et , 1981). Recently, a CPI virus was isolated from the cerebrospinal fluid of a dog with transient posterior paresis (Evermann et , 1980). 120 121 To determine the potential neurovirulence and hence the significance of this viral isolate as an etiologic agent in suspect cases of viral encephalitis in dogs, this CPI was intracerebrally inoculated into seronegative gnotobiotic dogs (Baumgârtner et al., 1982a,b). Infec tion resulted in an acute nonstçpurative meningoencephalitis and ventriculitis. In fatally infected animals, viral antigen was demonstrated in ependymal cells, paraventricular glia and occasional cerebrocortical neurons. Five of 6 convalescent dogs developed moderate to severe internal hydrocephalus. These jji vivo data suggest that CPI may be a significant cause of self-limiting encegAi- alitis and/or hydrocephalus in the canine species. Ferrets are extremely susceptible to canine viral pathogens such as canine distemper virus (i^jpel and Gillespie, 1972) and also many human paraityxovirus pathogens including measles virus (Brown et , 1985; Wisniewslqr et ^ . , 1983). During the course of ^ vitro study with CPI virus, a need to develop an animal infection model alterna tive to the gnotobiotic dog for neurovirulence evaluation of both large and small plaque variants of CPI became apparent. For this reason, ferrets were experimentally infected with stock (lytic) CPI virus. The objectives of this study were to describe the resultant CPI infection-induced lesions within the CNS, to correlate the lesions with the distribution of viral antigen (s) as determined by immunoperoxidase staining and to carpare the resultant pattern of CPI disease in ferrets to that observed to that obtained in the gnoto biotic dog. The results of that study are reported here. 122 MATERIALS AND METHODS Maintenance and Inoculation of Ferrets A total of 28 seronegative 7^week-old ferrets were used in this study. Ferrets were purchased fron a ccmmercial dealer (Marshall Ltd., NY). Animals were not vaccinated for canine distenper virus (CDV). The ferrets were anesthetized with ketamine-xylazine and inoculated into the left cerebral hemisjiiere. Five separately housed control animals received 150 yl cell lysate from uninfected control Vero cells. The remaining 23 animals received 150 yl of CPI (10^*^^ TCID^q /0.1 ml). The sacrifice schedule is outlined in Table 5.1. CPI Virus The viral variant (78-238) of CPI virus isolated from the cerebrospinal fluid of a dog with posterior paresis was used in this study. Methods for in vitro propagation of the agent has been described elsevdiere (BaumgSrtner et , 1981). Clinical and Inmunological Studies Ferrets were examined daily for neurological dysfunction. Sera were collected fron the ferrets at the day of infection and at weekly intervals thereafter. Virus neutralization tests were performed on sera as previously described (BaumgSrtner et , 1981). Virus Purification and Polyclonal Antibody Preparation Rabbit anti-CPI polyclonal serum was prepared by standard tech niques. Briefly, 75 cm^ flasks with subconfluent monolayers were infected at a multiplicity of 0.01 TCID^q . After 1 hour absorption, monolayers were washed with PBS and the cells were supplemented with 123 growth medium. When viral CPE was maximum, the cultures were frozen and thawed and clarified by Icw-speed centrifugation at 1,000 X £ for 20 minutes. The virus was pelleted by ultracentifugation of the si:pematant at 58,000 X £ for 40 minutes (Beckman SW41). The pellet was resuspended in 5 ml 2.5 mM Tris-buffer in saline, clarified by centrifugation at 1,800 X £ for 30 minutes, aliquoted and stored at -70 °C. New Zealand White rabbits were immunized according to the following schedule: 2X intraperitoneal and 2X subcutaneous inocula tion using 10^’^^ TCID^q per inoculation site. The animals were boosted 10 days later with virus protein suspended in caiplete Freund's adjuvant, followed by a second boost with incarplete Freund's adjuvant 10 days later. To remove non-viral antibocfy, 10 ml of diluted rabbit serum (1:10) was absorbed wi t h Vero cell lysate (20 X 10^ cells) at 37°C for 1 hr. Immune complexes were removed by high-speed centrifugation at 15,000 X £ for 30 minutes. Absorbed serum was aliquoted and stored at -70®C. Histopatholoqy Samples from various tissues, including brain and spinal cord, were collected and fixed in 10% buffered formalin. In addition, 10 ferrets were perfused with 4% (w/v) paraformaldehyde followed by 5% (v/v) glutaraldehyde for subsequent light microscopic evaluation (Williams et al., 1978). Sites routinely examined for the presence of virus were the olfactory bulb, frental and temporal cortex, hippocampus, thalamus, midbrain, cerebellar cortex, choroid plexus, cerebellar peduncles and medulla oblongata. Removed organs were stored in 2% glutaraldehyde at 4°C. For histological examination. 124 tissue was dehydrated, embedded in paraffin, cut at 6 ym and stained with hemato:Qrlin and eosin. Inmunocytochemistiv Six ym thick, formalin-fixed and paraffin-embedded, unstained sections were deparaffinized in xylene and réhydrated in alcohol and stored in Tris buffer (0.05M Tris-base, 0.8% NaCl) pH 7.6 for 3 hours at room temperature. Endogenous peroxidase was blocked by 0.5% '^■P2 in methanol for 20 minutes at roan tenperature, and washed in PBS, pH 7.2 for 20 minutes. Sections were then incubated for 20 minutes at 37 ®C with diluted normal goat serum followed by incubation with rabbit anti-CPI polyclonal antibody diluted 1:75 in NTS buffer (0.9% NaCl, 0.5M Tris-HCl, 0.2% saponin in double distilled water, pH 7.6) for 24 hours, 4°C. Sections were then washed in PBS and incubated sequentially with biotinylated goat anti-rabbit IgG antibody and ABC-Caiplex according to the manufacturer's instructions (Rabbit IgG, Vectastain Kit, Vector Laboratories). Positive reactions were demon strated by DAB precipitation after 10 minutes. Sections were washed in tap water for 5 minutes, stained with 1% Giemsa (v/v) for 10 min, dehydrated, coverslipped and examined by conventional liÿit micros copy. Controls included incubation with preinoculation sera, deletion of the primary or secondary antibody staining steps in the staining protocol and hy staining uninfected control sections. 125 RESULTS Clinical Findings Neurological signs were not obseirved in control ferrets or in 21 ferrets inoculated with CPI virus. In the remaining 2 CPI-infected ferrets, clinical signs of depression and intermittent rolling, mild truncal ataxia and mild unintentional fine head tremors were observed between PID 5 and 11. The clinical signs did not progress in severity except in one ferret (No. 21) vàiich showed anorexia, depres sion, progressive incoordination, circling to the right and rolling. This ferret was moribund on PID 8. Light Microscopy of Brain and Spinal Cord Ferrets sacrificed on PID 1, 2 and 4 did not show histological alterations. By PID 8, 1 (No. 21) of 3 ferrets examined showed inflammatory and degenerative changes predcminently in the fourth ventricle and the cervical spinal cord. The changes consisted of widespread ependymal cell degeneration and infiltration of mono nuclear cells into paraventricular perivascular spaces. Nests of detached ependymal cells and macrophage-like cells were found in the venxtricular space in the vicinity of the ventricular wall (Fig. 5.1, 5.2). The ependymal cells shewed patchy loss of cilia. These changes were most prominent in the cervical spinal cord and in the fourth ventricle including the lateral and medial recesses. The paraventricular vessels showed a mild-to-moderate histiocytic- lynphocytic infiltrates in Virchcw-Robin spaces, including the vessels of the raphe of the reticulum formation. Similar mild 126 cellxilar infiltrates were present in the overlying meninges. Lesions in the mesencephalon included multifocal Wallerian degeneration with swollen myelin sheaths, loss of axons and infrequent intra-axonal gitter cell infiltration (Fig. 5.3). By PID 11 and 16, a more widespread involvement of the ventricu lar and paraventricular ^sterns was present. Hie choroid plexus of the lateral ventricles eidiibited diffuse lynphocytic cellular infil trates. All three ferrets (PID 16) shewed ependymal changes in the ventricles and cervical spinal cord similar to that described above (Fig. 5.4). Infrequent fociof neuronophagia were present in the reticulum formation. By PID 22, infrequent foci of minimal ependymal cell degener ation and paraventricular perivascular lymphocytic-monocytic infil trates were present. One ferret sheared moderate focal malacia with axonal degeneration and diffuse astrogliosis in the vestibular nucleus. After PID 22, degenerative and inflammatory changes were no longer detected. The only change observed was slight dilation of the fourth ventricle in 1 animal examined on PID 60. Inmmocytochemistry Sections from control ferrets were negative for CPI antigen and of CPI antigen-positive brain sections with normal rabbit serum were unstained. Virus-specific antigen was first detected 4 days after infection, vhen foci of viral antigen were seen in the cytoplasm of ependymal cells and less frequently in tanycytes of the fourth ventricle and the cervical spinal cord canal (Figs. 5.4, 5.5, 5.6). The DAB-precipitation product appeared as spherical cytoplasmic 127 inclusion bodies or shewed a diffuse cytoplasmic involvement. Viral antigen was rarely detected in the ependymal cells of the lateral ventricles. Sylvian duct, and never in the choroid plexus, blood vessels, infiltrating inflamnatory cells, neuropil and meninges (Fig. 5.7). Virus replication was restricted to ependymal and subependymal cells. However, occasionally viral antigen was present in subepen dymal processes in the cervical spinal cord (Fig. 5.5). These processes could be of ependymal cell or astrocytic origin. By PID 8, only a few faintly positive ependymal cells were seen. The CNS tissues examined after PID 11 were devoid of viral antigen. Development of Antibodies Preinoculation sera were negative for virus neutralizating antibody. Control animals inoculated with Vero cell lysates did not develop CPI antibodies. In inoculated animals, CPI-neutralizing antibodies were first detected in 2 of 3 animals on PID 8. Antibody levels in all remaining animals rose thereafter to final titers of 1:16. 128 DISCUSSION Several paramyxoviruses (eg, CDV, measles, munps, Sendai virus, Nariva virus and PI-3) are known for their neurqpathogenicity in their natural host and also in laboratory animals (Johnson and Johnson, 1972; Krakowka and Itoestner, 1976; Kristensson et al., 1984; Love ^ , 1985; Schwendemann and Kohler, 1979; Shibuta et al., 1985; Poos and Wollmann, 1979) ). Paramyxoviruses attract special attention because of their known involvement in sane CNS diseases (eg, subacute sclerosing panencephalitis and chronic disteiper encejtolaryelitis) and their suspected participation in others (eg, multiple sclerosis, Landry-Guillain-Barre syndrome) (Mitchel et al., 1978; Robbins et ^ . , 1981). Ihe recent isolation of SV^-like viruses from individuals with CNS disorders pronpted us to further investigate a canine paraityxovirus isolate (Mitchell et , 1978; Robbins et al., 1981; Goswami et al., 1984). The present paper described the events following the intra cerebral infection of 7 week-old ferrets with canine parainfluenza virus. Virus replication was most prominent in the ependymal cells of the fourth ventricle and the cervical spinal canal. Viral antigen was first detected on PID 4 and declined rapidly thereafter. Similar results were reported for dogs after experimental CPI infection (BaurogSrtner et al., 1982a,b). The more caudal distribution of the virus replication in the ferret system might represent an injection artifact. The injection sites reached to the diencephalon and mesencephalon in several of the animals. 129 Histological changes of acute encejdialitis occurred between PIDs 8 throu^ 22. The lesions were characterized by degeneration of ependymal cells, non-suppurative paraventricular perivascular inflam matory response and axonal degeneration in two ferrets. Similar lesions were present in dogs eaqjerimentally infected with CPI (BaumgSrtner et , 1982a,b). However, gnotobiotic piç>pies showed a great variation in the severity of lesions, including laminar cortical necrosis. In the latter, viral materials were demonstrated ultrastructurally in neurons within these sites. The difference in age and species may have contributed to the observed differences in CPI pathogenicity in ferrets vs. dogs. Resistance to Sindbis virus encephalitis develops abruptly during the second week of life in mice (Johnson et , 1971). Similar age-dependent differences in the outcome and susceptibilii^ has been described for canine distorper virus (Krakowka ^ , 1976). In ferrets, CPI virus-induced lesions and viral antigen distri bution were restricted to the ependymal lining of the ventricles and the immediate paraventricular areas. Schwendemann and Lfihler (1979) describe coirparable findings after intracerebral infection of mice with Sendai virus. Experimentally, human wild-t^pe mumps virus, a virus similar to CPI, also is largely restricted to ependymal cells. Neurovirulent variants of this virus have been described vhich also replicate in neurons (Love et al., 1985). Using cellular distribu tion as a criterion of virulence, it appears that the CPI isolate used in this study is of limited neurovirulence for ferrets. 130 Another difference between the ferret and the dog is lack of residual lesions (eg, internal hydrocephalus) in the former. The degree of virus-associated hydrocephalus in convalescence is largely a reflection of the extent of virus-induced ependymal damage (and the age of the animal) during the acute phase of infection. Neonatal gnotobiotic dogs developed a severe internal hydrocephalus with aqueductural stenosis after CPI virus inoculation, vàiereas 6 month old puppies developed a mild internal hydrocephalus without aque- ductal stenosis (BaumgSrtner et , 1982). Similar age-related phenomena have been reported for Mycoplasma sp. and Kaolin-induced hydrocephalus (Hochwald et al., 1972; Kohn et al., 1977). Therefore, the lack of prominent virus-associated changes of the ventricular system in convalescent ferrets is most likely due to the age of the ferrets at the time of incoulation. In sunmary, the ferret was shown to be susceptible to intra cerebral infection with CPI virus. Lesions observed were moderate and self-limiting. The spectrum of lesions observed in ferrets were similar to that seen in the dog though less severe. Internal hydro cephalus was not a residual effect of viral ependymitis. 131 SUMMARY Young seronegative ferrets were intracerebrally inoculated with a neurotropic strain of canine parainfluenza (CPI) virus and serially sacrificed at intervals after infection for subsequent viral innuno- peroxidase and light microscopic evaluation. CPI virus infection resulted in a self-limiting nonsx:çpurative lynphocytic ependynitis and choroiditis with associated ependymal cell degeneration and necrosis. These changes were accorpanied by paraventricular perivascular cellular infiltrates of lyitphocytes and monocytes predcminantly in the fourth ventricle and the cervical spinal cord and less frequently in the lateral ventricles and Sylvian duct. The inflammatory lesions were first detected on postinoculation day (PID) 8 and had largely resolved after PID 22. Two animals showed multi focal, axonal degeneration in the mesencephalon and reticulum formation, respectively. Mild dilation of the fourth ventricle was observed as a residual lesion in one ferret (PID 60). Immunohisto- cytochemistry showed that virus replication was restricted to ependymal and subependymal cells and was most prominent on PID 4. Virus neutralizing CPI antibodies developed by PID 9 and increased slowly thereafter. Table 5.1 The Time-Dependent Occurrence of Microscopic Lesions and Viral Antigen in Ferrets Intracerebrally Inoculated with Canine Parainfluenza Virus^ Days Post-Inoculation Inoculum 1 2 4 8 11 16 22 60 120 150 CPI Virus Microscopic lesions 0/2^ 0/2 0/2 1/3 3/3 3/3 1/2 0/2^ 0/2 0/2 Viral antigen 0/2 0/2 2/2 0/3 0/3 0/3 0/2 0/2 0/2 0/2 Vero Cell Lysate Microscopic lesions 0/1 0/1 0/1 0/1 0/1 -- - - Viral antigen 0/1 0/1 0/1 0/1 0/1 — — - - ^ Ferrets were inoculated with 150 vil of 10^'^^ TCID50/O.l ml CPI virus or cell lysate from uninfected Vero cells. ^ Expressed as number of ferrets with microscopic lesions (numerator)/number of ferrets examined (denominator). ^ One animal had a mild dilation of the fourth ventricle. - = not done. w ro Figure 5.1 Ventricular lining cells of the fourth ventricle PID 8. Focal loss of ependymal cells and moderate subependymal; supraependymal and mild perivascular infiltration, H&E (X 250). 133 134 Figure 5.2 Fourth ventricle fron same ferret as in Fig. 1 with focal loss of ependymal cells and mononuclear infiltration, 1 = fourth ventricle, H&E (X 250). 135 136 Figure 5.3 Mesencephalon PID 8. Focal axonal degeneration with dilation of myelin sheaths and gitter cell infiltration, H&E (X 250). 137 Figure 5.4 Cervical spinal cord canal, PID 11. Mild to moderate mononuclear infiltration of the leptcmeninges and Virchov Robin spaces. Moderate diffuse paraventricular gliosis and intraventricular accumulation of mononuclear cells, 1 = cervical spinal cord canal, H&E (X 100). 139 Figure 5.5 linnnunqperoxidase labeling of CPI viral antigen with poly- glonal anti-CPI serum (1:75). Ependymal cells of the fourth ventricle PID 4. Reaction product is present in cytoplasm of ependymal cells, 1 = fourth ventricle (X 250). 141 142 % ISA Fig, 5.5 Figure 5.6 ütinunqperoxidase labeling of CPI viral antigen with poly- glonal anti-CPI serum (1:75). Cervical spinal cord canal (A) of ferret on PID 4. Reaction product is present in cytoplasm of ependymal cells and cellular processes of subependymal cells (X 400). 143 Figure 5.7 Imrnunoperoxidase labeling of CPI viral antigen with poly- glonal anti-CPI serum (1:75). Section of fourth ventricle and choroid plexus of same ferret as Fig. 5. Reaction produced is exclusively in cytoplasm of ependynal cells (arrows) but not in the choroid plexus cells, 1 = choroid plexus, 2 = fourth ventricle (X 100). 145 146 icaoft Fig. 5.7 147 REFERENCES ^pe l M.J.G., Gillespie J.H. (1972) Canine distemper virus, in Virol Monogr II, Springer-Verlag, New York, Wien. BaumgSrtner, W . , Metz 1er, A.E., Krakcxdca, S., Kbestner, A. (1981) In vitro identification and characterization of a virus isolated frcm a dog with neurological dysfunction. Infect. Immun. 31:1177-1183. BaumgSrtner, W.K., Krakowka, S., Koestner, A., Evermann, J. (1982) Ultrastructural evaluation of acute encephalitis and hydrocephalus in dogs caused by canine parainfluenza virus. Vet. Pathol. 19: 305-314. BaumgSrtner, W . K . , Krakovdca, S., Koestner, A., Evermann, J. (1982) Acute encephalitis and hydrocephalus in dogs caused by canine parainfluenza virus. Vet. Pathol. 19:79-92. Binn, L.N. (1970) A review of viruses recovered fron dogs. J. Am. Vet. tfed. Assoc. 156:1672-1677. Brown, H.R., Pessolano, T.L., Nostro, A.F., Thormar, H. (1985) Demonstration of immunoglobulin in brains of ferrets inoculated with an SSPE strain of measles virus: Use of protein A conjugated to horseradish peroxidase Acta Neuropath. 65:195-201. Evermann, J.F., Lincoln, J.D., MdKieman, A.J. (1980) Isolation of a paraiTQ^xovirus frcm the cerebrospinal fluid of a dog with posterior paresis. J. Am. Vet. Med. Assoc. 177:1132-1134. Goswami, K.K., Russel, W.C. (1982) A ccmparison of paramyxoviruses by immunoprécipitation. J. Gen. Virol. 60:177-183. 148 Goswami, K.K.A., Cameron, K.R., Russel, W.C., Lange, L.S., Mitchell, D.N. (1984) Evidence of the persistence of paramyxoviruses in human bone marrow cells. J. Gen. Virol. 65:1881-1888. Hochwald, G.M., Eîpstein, P., îfethan, C . , Ransohoff, J. (1972) The role of the skull and dura in experimental feline hydrocephalus. Dev. Med. Child Neurol. 14(Suppl)27:65-69. Johnson, R.T., McFarland, H.F., Levy, S.E. (1972) Age dependent resistance of viral encejdialitis : Studies of infections due to Sindbis virus in mice. J. Infect. Dis. 125:257-262. Johnson, K.P., Johnson, R.T. (1972) Granular epen^miitis. Am. J. Pathol. 65:511-521. RDhn, D.F., Kirk, B.E., Chou, S.E. (1977) jXÿcoplasma-induced hydro cephalus in rats and hamsters. Infect. Immun. 16:680-689. Krakc^d^, S., KOestner, A. (1978) Age-related susceptibility to canine distemper virus infection in gnotobiotic dogs. J. Infect. Dis. 134:629-632. Kristensson, K., Leestma, J., Lundh, B., Nbrrby, E. (1984) Sendai virus infection in the mouse brain: Virus spread and long-term effects. Acta Neuropath. 63:89-95. Lfive, A., Rydbeck, R., Kristensson, K., Orvell, C., Norrby, E. (1985) Hemagglutinin-neuraminidase glycoprotein as a determinant of pathogenicity in muirps virus hamster encephalitis: Analysis of mutants selected with monoclonal antibodies. J. Virol. 53:67-64. Macartney, L., Cornwell, H.J.C., McCandlish, I.A.P., Thaipson, H. (1985) Isolation of a novel paramyxovirus frcm a dog with entzeric disease. Vet. Rec. 117:205-207. 149 Mitchel, D.N., Porterfield, J.S., Micheletti, R., Lange, L.S., Gos wami, K.K.A., Taylor, P., Jacobs, J.P., Hockley, D.J., Salsbiary, A.J. 1978) Isolation of an infectious agent frcm bone marrows of patients with multiple sclerosis. Lancet ii:387-391. Robbins, S.J., Wrzos, H., Kline, A.L., Tenser, R.B., Rapp, F. (1981) Rescue of a cytqpathic parairyxovirus frcm peripheral blood leukocytes in subacute sclerosing panencephalitis. J. Infect. Dis. 143:396-403. Reman, G., Phillips, A., Poser, M. (1978) Parainfluenza virus type 3. Isolation frcm CSF of a patient with Guillain Barre syndrome. J. Am. Med. Assoc. 16:1613-1615. Roos, R.P., Wollmann, R. (1979) Non-productive paranQpcoinfection: Nariva virus infection in hamsters. Arch. Virol. 62:229-240. Rosenberg, F.J., Lief, F.S., Todd, J.D., Reif, J.S. (1971) Studies of canine respiratory viruses. I. E>q)erimental infection of dogs with an SV^-like canine parainfluenza agent. Am. J. Epidemiol. 94:147-165. Schwendemann, G., Lfihler, J. (1979) Pathological alterations of ependyma and choroid plexus after experimental cerebral infection of mice with Sendai virus. Acta Neuropathol. 46:85-94. Shibuta, H., Kanda, T., Nozawa, A., Sato, S., Kumanhishi, T. (1985) Experimental parainfluenza virus infection in mice: Growth and spread of a high pathogenic variant of parainfluenza 3 virus in the mouse brain. Arch. Virol. 83:43-52. 150 Wagener, J.S., Mimich, L., Sobonya, R., Taussig, L.M., Ray, C.G., Fulginiti, V. (1983) Parainfluenza type II infection in dogs. Am. Rev. Resp. Dis. 127:771-775. Williams, R.M., Krakowka, S., Koestner, A. (1978) A procedure for processing central nervous system tissue for immunofluorescence, light and electron microscopic evaluation. Am. J. Vet. Res. 39: 1946-1949. Wisniewsky, H.M., Brcwn, H.R., Thoumar, H. (1983) Pathogenesis of viral encephalitis: Demonstration of viral antigens in the brain endothelium. Acta Neuropathol. 60:107-112. CHAPTER VI CANINE PARAINFEUÏNZA VIRUS INTRCOUCTICN Canine parainfluenza (CPI) virus, an enveloped RNA virus of the paramyxovirus group, is an upper respiratory viral pathogen of dogs. It is closely related to simian virus (SV-5), which was first isolated from rhesus and cynanolgous kidney cell cultures in 1956 (Hull et , 1956). Between 1967 and 1970, numerous instances of CPI virus isolations (synonyms: SV-5-like virus, parainfluenza-2 virus) fron dogs with respiratory disease were reported (2^>pel and Percy, 1970; Binn et al., 1967; Crandell et al., 1968). Clinically, affected dogs eshibited signs of sudden onset of fever, malaise, coughing, and occurrence of copious amounts of nasal discharge. This virus has been shown to be one of several etiologic agents of kennel cough, an inportant clinicopathologic entity of dogs. Recently, Evermann et (1980) isolated a CPI virus fron the cerebrospinal fluid (CSF) of a dog with neurological dysfunction. This viral isolate was subsequently found capable of inducing acute and chronic central nervous system (CNS) lesions under experimental conditions (Baumgartner et , 1982a,b) . 151 152 Characterization of Canine Parainfluenza (CPI) Virus Canine parainfluenza vinos is a typical member of the genus Paramyxovirus. Ihe paranyxovirus virion consists of a membrane envelope, covered with surface projections that enclose a helical ribonucleoprotein nucleocapsid. Most of the spherical virions are 150 to 200 nm in diameter; however, large particles of 500 to 700 nm also occur (Choppin and Stockenius, 1984; Baumgârtner et , 1981) . The virion is covered with spikes 8 toi3 nm in length. The nucleo- capsids are flexible helical structures 17 to 18 nm in diameter with a central hole about 5 nm in diameter. The nucleocapsid is single- stranded with a length of approximately 1 um, and contains 4 to 5% RNA. Paramyxoviruses have six to seven proteins with a molecular weight between 74,000 and 38,000 daltons. The major proteins are the envelope associated matrix protein (M-protein), two glycoproteins, a larger and a smaller one, a nucleocapsid protein (NC-protein), and the P-protein. Hemagglutinin and neuraminidase activity are associated with the larger glycoprotein (HN-protein). The smaller glycoprotein (F-protein) is responsible for hemolysis, cell fusion and virus penetration (Chopin et al., 1975; Roh, 1978). The virus replicates readily in primary cultures of canine kidney cells and in many different continuous cell lines (Vero cells, Madin Darby Canine Kidney, BHK-21, etc.). Forty-eight hours after infection, multinucleated syncytial giant cell formation begins. With time, the syncytial cells enlarge in size and number. Assoc iated with this progressing cytopathic effect (cpe), individual cells become vacuolated and rounded before detachment from the culture 153 vessel. By 6 days postinfection, approximately 95% of the cells detach fron the monolayer. In addition, small syncytia and/or no cpe (persistent) types of SV-5 and CPI virus infection in tissue culture had been reported (Rhim and Schell, 1967; Cotpans et ^ . , 1964). Virus-infected cells show hemadsorption (Had) at 4°C with guinea pig, chicken, dog, and human (type 0) erythrocytes. Furthermore, eosinophilic intracytoplasmic inclusion bodies were observed (Fig. 6.1); occasionally intranuclear inclusion bodies are found in the late stage of infection with the CNS-origin CPI virus (BaumgSrtner et al., 1981). Vero cells, persistently infected with CPI, are readily established fron cells surviving a lytic infection. BetJæen 85 and 100% of cells persistently infected with CPI contain viral antigen as determined by immunofluorescence (Fig. 6.2). Roughly 10 to 15% of these persistently infected cells are Had-positive. Inrnune Response Humoral antibodies induced by CPI infection are determined virus-neutralization, hemagglutination inhibition (HAI), or CPI- specific ELISA assays. To enhance hemagglutination (HA) titers of viral preparations, the virus is treated with Tween-80 and ether (John and Fulginiti, 1966). The HAI tests are performed at 4°C to reduce neuraminidase activity. Canine parainfluenza virus cross- reacts with SV-5 virus and, to a lesser extent, with human rnunps virus; the latter is a one-way reaction that does not occur vdien CPI virus is tested with antisera against mumps virus (Rosenberg et al., 1971). Therefore, reports (Binn, 1970) of muitps virus infection in dogs based upon serology only should be cautiously interpreted. 154 Several studies have evaluated the epizootiology of CPI infec tion in various canine populations. A survey in eastern Washington showed that 19% of the dogs had antibodies against CPI virus (Fulton et a l ., 1974). Others found 14% of the dogs were seropositive in various geographic areas of the U.S. (Bittle and Qnery, 1970). It has been shown that CPI virus is hi^ly catntunicable within a closely confined kennel population as demonstrated by increase in the nxmiber of seropositive dogs from 3 to 72% within 3 weeks after a CPI virus infection outbreak (Binn and Lazar, 1970). Thirty percent of 456 canine sera, including 320 sera from kennels with respiratory disease problems, had antibodies against CPI, as shown by a study conducted in West Germany (Bibrack and Benary, 1975). Under ejq^erimental conditions, seroconversion occurred between 7 and 11 days post infection (PID), reached peak values 4 to 7 weeks postinfection and then declined thereafter, though still detectable 5 months later (Baumgârtner et al., 1982; Rosenberg et al., 1971). Clinical and Pathological Findings in Dogs with CPI Virus Infection Two recognized clinicopathologic forms of disease (upper respiratory and CNS) associated with CPI virus infection are known to occur. The respiratory form occurs under natural conditions and within the canine population is by far the most ccmmon and iirportant manifestation of disease. Two to three days after aerosol exposure, 100% of infected dogs without preexisting antibodies develop a rise in temperature (0.5 to 1.0°C), vdiich lasts for 2 days. This is accatpanied by subsequent slight nasal discharge and a tracheo bronchitis characterized by spontaneous cough of 2 to 12 days 155 duration. Histopathologic lesions include catarrhal rhinitis, inter stitial pneumonia, bronchopneumonia, bronchitis, and bronchiolitis (l^pel and Percy, 1970; Rosenberg et al., 1971) (Fig. 6.3). With the fluorescent antibo<^ test, viral antigen was found in the epithelium of the nasal mucosa, trachea, lungs, tonsils, and pharynx. Under natural conditions CPI virus infections occur in conbination with other agents, e.g., Bordetella bronchiseptica, canine adenovirus-2, canine disteitper virus, and canine herpesvirus (Binn et ^ . , 1967; Bibrack and Benary, 1975). Furthermore, the clinical signs of CPI-induced respiratory infection are unspecific and not distinguish able fron other causes of acute respiratory infections in dogs. Once acquired vaccination or natural infection, circulating antibodies prevent reinfection and multiplication of virus (Binn et ^ . , 1967; Rosenberg et al., 1971; Comvell et al., 1976). In the experimental and clonical cases described above, it is inportant to note that an overtly viremic phase of infection was not apparent. However, reports of successful isolation of CPI frcm cerebrospinal fluid (Evermann et al., 1980), spleen, kidney, and liver (Binn et , 1967) iitply that the virus, even under natural conditions, will spread beyond the upper respiratory tract. Ihe second or neurological form of CPI-related disease is inade quately defined at this time. A CPI virus was isolated from the CSF of a dog with a history of incoordination and posterior paresis of 2 weeks duration. Subsequently, loccmotor activity returned to normal (Evermann et al., 1980). Intracerebral inoculation of dogs with this 156 neviropathogenic CPI virus resulted in the development of a nonsvçpur- ative meningoencephalitis with focal necrosis 7 to 10 PID in half of the infected animals (Fig. 6.4). The diagnosis of CPI virus infec tion was confirmed by seroconversion by PID 7, by virus reisolation from brain tissue, and by demonstration of virus iN CNS tissues by immunofluorescence and ultrastructural examination (BaumgSrtner et al., 1982a,b). Surviving animals developed internal hydrocephalus (Fig. 6.5) with and without aqueductal stenosis (BaumgSrtner et al., 1982b). Since the virus was shown to replicate in ependymal lining cells it is likely that hydrocephalus developed subsequently to this effect. Generally, the respiratory form has a mild to moderate course and thus does not necessitate treatment. More severe cases are complicated by secondary bacterial infections that will respond to proper antibiotic and symptanatic therapjeutic regimens. Occasional fatalities attributed solely to CPI respiratory infection have been reported (Ajiki et al., 1982). To prevent CPI virus infections and to avoid cotplicated secondary infections, a modified-live canine p>arainfluenza virus vaccine has been developed. Dogs vaccinated i.m. developed a significantly greater immune response than dogs given the same dose subcutaneously (Einery et , 1976). Attenuated CPI virus is frequently included in the multiple agent vaccine products currently used by veterinarians. Fig. 6.1 Multinucleated syncytial giant cell with intracytoplasmic viral inclusion bodies (arrow) in a Vero cell monolayer infected 36 hr previously with CPI virus. 157 158 Fig. 6.2 Vero cell monolayers with persistent CPI virus infection. Viral antigen is demonstrated by indirect immunofluores cence methods. 159 Fig. 6.3 Interstitial pneumonia and atelectasia (PID 10) induced by CPI virus infection in a gnotobiotic pc^)py inoculated intracerebrally at 7 days of age. 161 Fig. 6.4 Acute encephalanalacia (arrows) and meningoencephalitis induced in a dog inoculated with CPI virus (PID 10). 163 Fig. 6.5 Severe internal hydrocephalus that developed 6 ironths after intracerebral infection with CPI virus. 165 167 REFERENCES Ajüd., M . , Takaraura, K . , Kiramatsu, K . , Nakai, M . , and Susaki, N. (1982) Isolation and characterization of parainfluenza 5 virus fron a dog. Jpn. J. Vet. Soi. 44(4):607-618. i^jpel, M.J.G. and Percy, D.H. (1970) SV-5-like parainfluenza virus in dogs. J. Am. Vet. Med. Assoc., 156:1778-1781. BaumgSrtner W.K., Metzler, A., Krakovdca, S., and Koestner, A. (1981) In vitro identification and characterization of a virus isolated fron a dog with neurological dysfunction. Infect. Itnnunol. 31: 1177-1183. BaumgSrtner, W.K., K r a kc^a, S., Koestner, A., and E^rermann, J. (1982a) Acute encephalitis and hydrocejdialus in dogs caused by canine parainfluenza virus. Vet. Pathol. 19:79-92. BaumgSrtner, W.K., Krakovdca, S., Koestner, A., and Evermann, J. (1982b) Ultrastructural evaluation of acute encephalitis and hydrocephalus in dogs caused by canine parainfluenza virus. Vet. Pathol. 19:305-314. Bibrack, B. and Benary, F. (1975) Seroepizootologische Untersuch- unger tlber die Bedeutung von parainfluenza-2-infectionen beim Zwingerhusten in Deutschland. Zentralbl. Vet. Med. B. 22:610-614. Binn, L.N. (1970) A review of viruses recovered fron dogs. J. Am. Vet. Med. Assoc. 156:1672-1677. Binn, L.N. and Lazar, E.G. (1970) Ccnments on epizootiology of para influenza SV-5 in dogs. J. Am. Vet. Med. Assoc. 156:1774. 168 Binn, L.N., Eddy, A., Lazar, E.C., Helms, J., and Mumane, T. (1967) Viruses recovered frcm laboratory dogs with respiratory disease. Proc. Soc. Ejq>. Biol. Med. 126:140. Bittle, J.L. and Emery, J.P. (1970) The epizootiology of canine parainfluenza. J. Am. Vet. Med. Assoc. 156:1771-1777. Choppin, P.W. and Stoeckenius, W. (1964) The morphology of SV-5 virus. Virology 23:195-202. Choppin, P.W., Conpans, R.W., Wagner, R.R., and Bader, J.P. in Comprehensive Virology, Vol. 4, Fraenkel-Conrat, H., and Wanger, W.W., Eds., Plenum Publishing, New York, 1975, p. 95-178. COrpans, R.W., Holmes, K.V., Sales, S., and Choppin, P.W. (1964) An electron microscopic study of moderate and virulent cell inter action of the parainfluenza virus SV-5. Virology 30:411-426. Cornwell, H.I.C., McCandlish, I.A.P., Thotpson, H., Laird, H.M., and Wright, N.G. (1976) Isolation of parainfluenza virus SV-5 frcm dogs with respiratory disease. Vet. Rec. 98:301-303. Crandell, R.A., Brumlcw, W.B., and Davison, V.E. (1968) Isolation of parainfluenza virus from sentry dogs with upper respiratory disease. Am. J. Vet. Res. 29:2141-2147. Emery, J.B., House, J.A., Bittle, J.L., and Spotts, A.M. (1976) A canine parainfluenza viral vaccine: imnunogenicity and safety. Am. J. Vet. Res. 37:1323-1327. Evermann, J.F., Lincoln, J.D., and McKeiman, A.J. (1980) Isolation of a paramyxovirus frcm the cerebrospinal fluid of a dog with posterior paresis. J. Am. Vet. Med. Assoc. 177:1132-1134. 169 Fulton, R.W., Ott, R.L., Duenwald, J.L., and Gorham, J.R. (1974) Serum antibodies against canine respiratory viruses: Prevalence among dogs of eastern Washington. Am. J. Vet. Res. 35:853-855. Hull, R.N., Minner, J.R., and Smith, J.W. (1956) New viral agents recovered frcm tissue cultures of monkey kidney cells. Am. J. Hyg. 63:204. John, T.J. and Fulginiti, V.A. (1966) Parainfluenza 2 virus: Increase in hemagglutinin titer on treatment with (Rveen 80 and ether. Proc. Soc. Exp. Biol. Med. 121:109-111. Packard, M.E. and Grafton-Packard, B. (1979) The effect of canine parainfluenza vaccine on the spread of tracheobronchial coughs in a boarding kennel. Am. Anim. Hosp. Assoc. 15:241-244. Rhim, J.S. and Schell, K. (1967) Cytopathic effects of the para influenza virus SV-5 in Vero cells. Nature (London) 216:271. Rott, R. (1978) Molecular basis of infectivity and pathogenicity of ityxovirus. Arch. Virol. 59"285-298. Rosenberg, F.J., Lief, F.S., Todd, J.D., and Reif, J.S. (1971) Studies of canine respiratory viruses. I. Experimental infection of dogs with an SV-5-like canine parainfluenza agent. Am. J. Epidemiol. 94:147-165. BIBLIOGRAPHY Ajiki, M., Takamura, K., Kiramatsu, K., Nakai, M., and Susaki, N. (1982) Isolation and characterization of parainfluenza 5 virus fron a dog. Jpn. J. Vet. Soi. 44(4) :607-618. Appel, M.J.G. and Percy, D.H. (1970) SV-5-like parainfluenza virus in dogs. J. Am. Vet. Med. Assoc. 156:1778. Appel, M.J.G. and Gillespie, J.H. (1972) Canine distatper virus, in Virol. Monogr. II, Springer-Verlag, New York, Wien. ippel, M. and Robson, D.S. (1973) A Microneutralization Test for Canine Distemper Virus. Am. J. Vet. Res. 34:1459-1463. Armen, R.C., Evermann, J.F., Truant, A.L., Laughlin, C.A. and Halim, J.V. (1977) Temperature sensitive mutants of measles virus produced from persistently infected HeLa cells. Arch. Virol. 53:121-132. Barski, G., LeFrancois, and Youn, J.K. (1969) Infection latente double avec SV^ et virus de herpes en culture de cellules de hamster. Ann. Inst. Pasteur 117:420-433. Basle, M.F., Russel, W.C., Goswami, K.K.A., Rebel, A., Giraudon, P., Wild, F. and Fihnon, Q. (1985) Paramyxovirus antigens in osteo clasts frcm Paget's bone tissue detected by monoclonal antibodies. J. gen. Virol. 66:2103-2110. Baumgârtner, W.K., Krakcvka, S., Koestner, A., and Evermann, J. (1982) Ultrastructural evaluation of acute encephalitis and hydrocephalus in dogs caused by canine parainfluenza virus. Vet. Pathol. 19:205. Baumgârtner, W. and Krakotka, S. (1986) A simple method for the histochemiical demonstration of viral inclusion bodies in cell monolayers in microtitration plates. Stain Tech. (in press). Baumgârtner, W.K., Metzler, A.E., Krakovka, S. and Koestner, A. (1981) In vitro identification and characterization of a virus isolated frcm a dog with neurological dysfunction. Infect. Tnmun. 31:1177-1183. /' 170 171 Baumgârtner, W.K., Krakowka, S., Itoestner, A. and Evermann, J.F. (1982) Acute encephalitis and hydrocephalus in dogs caused by canine parainfluenza virus. Vet. Pathol. 19:79-92. Bay, P.H.S. and Reichmann, ME. (1979) UV-inactivation of the biolog ical activity of defective intzerfering particles generated by vesicular stomatitis virus. J. Virol. 32, 876-884. Bendayan, M. (1984) Enzyme-gold electron microscopic cytochemistry: A new affinity approach for the ultrastructural localization of macrcmolecules. J. Electron Microsc. Tech. 1:349. Bibrack, B. and Benary, F. (1975) Seroepizootologische Untersuchunger tlber die Bedeutung von parainfluenza-2-infectionen beim Zwinger husten in Deutschland. Zentralbl. Vet. Med. B. 22:610. Binn, L.N., Eddy, A., Lazar, E.C., Helms, J., and rkxmane, T. (1967) Viruses recovered fron laboratory dogs with respiratory disease. Proc. Soc. E j ç ). Biol. Med. 126:140. Binn, L.N. and Lazar, E.C. (1970) Comments on epizootiology of para influenza SV-5 in dogs. J. Am. Vet. Med. Assoc. 156:1774. Binn, L.N. (1970) A review of viruses recovered fron dogs. 156:1672- 1677. Bittle, J.L. and Emery, J.P. (1970) The epizootiology of canine parainfluenza. J. An. Vet. Med. Assoc. 156:1771. Brandon, C. (1985) Improved imraunocytochemical staining through the use of Fab fragments of primary antibody. Fab specific second antibody, and Fab-horseradish peroxidase. J Histochem Cytochem 33:715. Brown, H.R., Pessolano, T.L., Nostro, A.F. and Thormar, H. (1985) Demonstration of imiiunogldbulin in brains of ferrets inoculated with an SSPE of measles virus: Use of protein A conjugated to horseradish peroxidase. Acta Neuropath. 65:195-201. Carandell, R.A., Brumlcw, W.B., and Davison, V.E. (1968) Isolation of parainfluenza virus from sentry dogs with upper respiratory disease. Am. J. Vet. Res. 29:2141. Chogpin, P.W. (1981) Isolation of paramyxovirus fron patient with ohronic neurologic disease. J. Infect. Dis. 143:501-503. Choppin, P.W. (1964) Multiplication of a myxovirus (SVg) with minimal cytqpathio effects and without interference. Virology 23:224-233. Choppin, P.W. and Stoeckenius, W. (1964) The morphology of SV-5 virus. Virology 23:195. 172 Choppin, P.W., Corpans, R.W., Wagner, R.R., and Bader, J.P. (1975) in Cctnprehensive Virology, Vol. 4, Fraenkel-Conrat, H., and Wanger, W.W., Eds., Plénum Publishing, New York, p. 95. Choppin, P.W. and Compans, R.W. (1970) Phenot^ic mixing of envelope proteins of the parainfluenza virus SVj. and vesicular stanatitis virus. J. Virol. 5:609. Ccnpans, R.W., Holmes, K.V., Dales, S. and Chojpin, P.W. (1964) An electron microscopic study of moderate and viinilent virus cell interactions of the parainfluenza virus SV^. Virology 30:411-426. Confer, A.W., Kahn, D.E., Koestner, A. and Krakovdca, S. (1975) Biological properties of a canine distenper viirus isolate assoc iated with derayelinating ence^Aialcmyelitis. Infect. Iramun. 11:835-844. Cornwell, H.J., McCandlish, I.A.P., Thcnpson, H., Laird, H.M. and Wright, N.G. (1976) Isolation of parainfluenza virus SV^ frcm dogs with respiratory disease. Vet. Rec. 98:301-303. Desrayter, J., Melnick, J.L. and Rawls, W.E. 1968 Defectiveness of interferon production and of Rubella virus interference in a line of African green monkey kidney cells (Vero). J. Virol. 2:955-961. Dubois-Dalcq, M. and Barbosa, L.H. (1973) J. Virol. 12(4) :909. Dubois-Dalcq, M., Barbosa, L.H., Hamilton, R. and Sever, J.L. (1974) Caiparison between productive and latent subacute sclerosing panencephalitis viral infection in vitro. Lab. Invest. 30:241. Emery, J.B., House, J.A., Bittle, J.L., and Spotts, A.M. (1976) A canine parainfluenza viral vaccine: immunogenicity and safety. Am. J. Vet. Res. 37:1323. Evermann, J.F., Krakcudca, S., McKeiman, A.J. and Baumgârtner, W. (1981) Properties of an encephalitogenic canine parainfluenza virus. Arch. Virol. 68:165-172. Evermann, J.F., Lincoln, J.D., and McKeiman, A.J. (1980) Isolation of a paramyxovirus from the cerebrospinal fluid of a dog with posterior paresis. J. Am. Vet. Med. Assoc. 177:1132. Finney, D.I. (1964) Statistical methods in biological assay. Hafiier Publishing Co., New York. Fisher, L.A. (1983) Characterization of four cell lines persistently infected with measles virus. Arch. Virol. 77:51-60. Freshney, R.J. (1983) Culture of Animal Cells. A Manual of Basic Technique. Alan R. Liss, Inc. New York. 173 Friedman, R.M., Ramseur, J.M. (1979) Mechanisms of persistent infec tions by cytopathic viruses in tissue culture. Arch. Virol. 60:83-103. Friedlander, J.M., Sumners, B.A, i^pel, M.J.G. Persistence of virulent canine distemper virus in lyitphoblastoid cell lines. Arch. Virol. 86, 47-62, 1985. Fulton, R.W., Ott, R.L., Duenwald, J.L., and Goiiiam, J.R. Serum antibodies against canine respiratory viruses: Prevalence among dogs of eastern Washington. Am. J. Vet. Res. 35:853, 1974. Garzon, S., Bendayan, M. and Kurstak, E. (1982) Ultrastructural localization of viral antigens using protein A-gold technique. J. Virol. Methods 5:67. Ghadially, F.N. (1975) Ultrastructure Pathology of the Cell (Butter- worth, London and Boston). Gorman, N.T. (1983) The interaction of cells persistently infected with canine distemper virus with antiviral antibody and coiple- ment. Cell. Immunol. 77:242-248. Goswami, K.K. and Russel, W.C. (1982) A corparison of paramyxoviruses by immunoprécipitation. J. Gen. Virol. 60:177-183. Goswami, K.K.A., Lange, L.S., Mitchell, D.N., Cameron, K.R. and Russel, W.C. (1984) Does simian virus 5 infect humans. J. gen. Virol. 65:1295-1303. Goswami, K.K.A., Cameron, K.R., Russel, W.C., Lange, L.S. and Mitchell, D.N. (1984) Evidence of the persistence of paramyxo viruses in human bone marrows. J. gen. Virol. 65:1881-1888. Graham, R.C. and Kamovsly, M.J. (1965) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14(4):281. Hall, J.G., Birbeck, M.S.C., Robertson, D., Peppard, J. and OrIans, E. (1978) The use of detergents and immunoperoxidase reagents for the ultrastructural demonstration of internal immunoglobulin in lymph cells. J. Immunol. Methods 19:351. Hedmann, K. (1980) Intracellular localization of fibronectin using imnunoperoxidase cytochemistry in light and electron microscopy. J. Histochem. Cytochem. 28(11): 1233. Higgins, R.J., Krakovka, S., Metzler, A.E. and Koestner, A. (1982) Lmmunqperoxidase labeling of canine distemper virus replication cycle in Vero cells. Am. J. Vet. Res. 43:1820-1824. 174 Hochwald, G.M., Epstein, F., Nathan, C. and Pansohoff, J. (1972) The role of the skull and dura in experimental feline hydrocephalus. Dev. Ned. Child Neurol. 14 (Suppl) 27:65-69. Holland, J., Spindler, K., Horodyski, F., Grabau, E., Nichol, S. and Van de Pol, S. (1982) Rapid evolution of RNA genanes. Science 215:1577-1585. Huang, S.A. (1973) Defective interfering viruses. Ann. Rev. Nicro- biol. 27:101-117. Hull, R.N., Ninner, J.R., and Smith, J.W. (1956) New viral agents recover®! fron tissue cultures of monkey kidney cells. Am. J. Hyg. 63:204. Hsiung, G.S. (1972) Parainfluenza-5 virus. Progr. med. Virol. 14:241-274. Huddles tone, J.R., Sipe, J., Braheny, S., Jensen, F.C., NcNillan, R., Lairpert, P. and Olds tone, N.B. A. (1979) Failure to isolate a transmissible agent fron the bone marrow of patients with multiple sclerosis. Lancet, 415. John, T.J. and Fulginiti, V.A. (1966) Parainfluenza 2 virus: Increase in hemagglutinin titer on treatment with Tween 80 and ether. Proc. Soc. EîÇ). Biol. Ned. 121:109. Johnson, R.T., ffcFarland, H.F. and Levy, S.E. (1972) Age-dependent resistance to viral encephalitis: Studies of infection due to Sindbis virus in mice. J. Infect. Dis. 125:257-262. Johnson, K.P. and Johnson, R.T. (1972) Granular ependymitis. Am. J. Pathol. 65:511-521. Kaaden, O.R. and van Dawen, S. (1983) Persistierende Virusinfektionen bei Tieren. Zbl. Vet. Ned. B. 30:1-21. Kawai, A. and Natsumoto, S. (1982) A sensitive bioassay system for detecting defective interfering particles of rabies virus. Virology 122:98-108. Kohama, N.T., Cardenas, J.N. and Seto, J.T. (1981) Immunoelectron microscopic study of the detection of the glycoproteins of influenza and sendai virus in infected cells by the immunoperoxi- dase method. J. Virol. Nethods 3:293. Kchn, D.F., Kirk, B.E. and Chou, S.E. (1977) îtycoplasma-induced hydrocephalus in rats and hamsters. Infect. Imnun. 16:680-689. 175 Kbsch, K. (1980) Persistent paranyxovirus infections and behaviour of B-adrenergic receptors in C-6 rat gliona cells. J. gen. Virol. 47:513-517. Kraehenbuhl, J.P. and Jamieson, J.D. (1974) Localization of intra cellular antigens by immunoelectron microscopy. Int. Rev. Esqseri. Pathol. 13:1-52. Lazzarini, R.A., Keeny, J.D. and Schubert, M. (1981) The origin of defective interfering particles of the negative-strand RNA viruses. Cell 26:145-154. Krakovdca, S. and Kbestner, A. (1978) Age-related susceptibility to canine distenper virus infection in gnotcbiotic dogs. J. Infect. Dis. 134:629-632. Kristensson, K., Leestma, J., Lundh, B. and Nbrrby, E. (1984) Sendai virus infection in the mouse brain: Virus spread and long-term effects. Acta Neuropath. 63:89-95. Love, A., %dbeck, R., Kristensson, K., Orvell, C. and Norrby, E. (1985) Hemagglutinin-neuraminidase glycoprotein as a determinant of pathogenicity in munps virus hamster encephalitis : Analysis of mutants selected with monoclonal antibodies. J. Virol. 53:67-64. Macartney, L., Cornwell, H.J.C., McCandlish, I.A.P. and Thotpson, H. (1985) Isolation of a novel parairyxovirus fran a dog with enteric disease. Vet. Rec. 117:205-207. Metzger, A.E., Higgins, R.J., Krakovdca, S. and Koestner, A. (1980) Persistent in vitro interaction of virulent and attenuated canine distenper virus with bovine cells. Arch. Virol. 66:329-339. Metzger, A.E., Krakov^, S., Axthelm, M.A. and Gorham, J.R. (1984) In vitro propagation of canine distemper virus : Establishment of persistent infection in Vero cells. Am. J. Vet. Res. 45:2211- 2215. Macartney, L., Cornwell, H.J.C., McCandlish, T.A.P. and Thotpson, H. (1985) Isolation of a novel paramyxovirus fran a dog with enteric disease. Vet. Rec. 117:205-207. Mayr, A., Bachmann, P.A., Bibrack, B. and Wittmann, G. (1977) Virolo- gische Anbeitsmethoden, Band II, Gustav-Fischer-Verlag. Stutt gart, New York. Metzler, A.E., Krakc^dca, S., Axthelm, M.A. and Gorham, J.R. 1984 In vitro propagation of canine distenper virus : Establishment of persistent infection in Vero cells. Am. J. Vet. Res. 45:2211- 2215. 176 Minagawa, T., Nakaya, C. and lida, H. (1974) Host DNA synthesis- suppressing factor in culture fluid of tissue cultures infected with measles virus. J. Virol. 13:1118-1125. Mitchell, D.N., Porterfield, J.S., Michele, H.R., Lange, L.S., Goswami, K.K.A., Taylor, P., Jacobs, J.P., Hodkley, D.J. and Salsbury, A.J. (1978) Isolation of an infectious agent fran bone marrows of patients with multiple sclerosis. Lancet, 387-391. Packard, M.E. and Grafton-Packard, B. (1979) The effect of canine parainfluenza vaccine on the spread of tracheobronchial coughs in a boarding kennel. 5m. Anim. Hosp. Assoc. 15:241. Perrault, J. (1981) Origin and replication of defective interfering particles. In Current Topics in Microbiology and Immunology 93: 151-207. Portner, A., Scroggs, R.A., Marx, P.A., Kingsbury, D.W. (1975) A taiperature sensitive mutant of Sendai virua with an altered hemagglutinin-neuraminidase polypeptide : Consequences for virus assembly and cytqpathology. Virology 47:179-187. Priestley. J., Polak, J.M. and Vardell, J.M. (1984) Immunolabelling for Electron Microscopy, Chapter 4, 37-52. In Elsevier Sciences Publishers, 5msterdam, New York, Oxford. Puvion-Dutilleul, F., Laithier, M. and Scheldrick, P. (1985) Ultra- structural localization of the Herpes Sinplex virus major ENA- binding protein in the nucleus of infected cells. J. gen. Virol. 66:15. Rapp, F. and Robins, S.J. (1981) Evolution of a human cell line persistently infected with measles virus. Intervirology 16:149- 159. Rhim, J.S. and Schell, K. (1967) Cytopathic effects of the parainflu enza virus SV-5 in Vero cells. Nature (London) 216:271. Rindler, M.J., Ivanov, I.E., Plesken, H., Rodriguez-Boulan, E. and Sabatini, D.D. (1984) Viral glycoproteins destined for apical or basolateral membrane domains traverse the same Golgi apparatus during their intracellular transport in doubly infected Madin- Darl^ canine kidney cells. J. Cell. Biol. 98:1304. Robbins, S.J., Wrzos, H., Kline, A.L., Tenser, R.B., Rapp, F. (1981) Rescue of a cytopathic paramyxovirus from peripheral blood leukocytes in subacute sclerosing panencephalitis. J. Infect. Dis. 143:396-403. Robbins, S.J., Wrzos, H., Kline, A.L., Tenser, R.B., Rapp, F. (1981) Fescue of a cytopathic paranyxovirus from peripheral blood Ill leukocytes in subacute sclerosing panencephalitis. J. Infect. Dis. 143:396-403. Robbins, S.J., Bussel, R.H. and Rapp, F. (1980) Isolcition and partial characterization of two forms of cytoplasmic nucleocapsids from measles virus infected cells. J. gen. Virol. 47:301. Robbins, S.J. and Rapp, F. (1982) Restriction of virus-specific protein synthesis in a persistent paramyxovirus infection. Arch. Virol. 71:85-91. Reman, G., Philips, A. and Poser, M. (1978) Parainfluenza virus type 3. Isolation from CSF of a patient with Guillain Barre syndrome. J. An. Med. Assoc. 16:1613-1615. Roos, R.P. and Wollmann, R. (1979) Non-productive paranyxoinfection: Nariva virus infection in hamsters. Arch. Virol. 62:229-240. Rosenberg, F.J., Lief, F.S., Todd, J.D. and Reif, J.S. (1971) Studies of canine respiratory viruses. I. Experimental infection of dogs with an SVq-like canine parainfluenza agent. An. J. Epidemiol. 94:147-165Ï Rott, R., Reinacher, M., Orlich, M. and Klenk, H.D. (1980) Cleava- bility of hemagglutinin determines spread of avian influenza viruses in the Chorioallantoic membrane of chicken embryo. Arch. Virol. 65:123-133. Rott, R. (1979) Molecular basis of infactivity and pathogenicity of ityxovirus. Arch. Virol. 59:285-298. Sheehan, D.C. and Hrapchack, B.B. (1980) Theory and practice of histotechnology. Sec. edition. C.V. Mosby Ccnpany. St. Louis, Toronto, London. Schwendemann, G., Wolinsky, J.S., Hatzidimitriou, G., Merz, D.C. and Waxham, M.N., (1982) Postembedding iitmunocytodiemical localization of paramyxovirus antigens light and electron microscopy. J. Histochem. Oytochem. 30(12):1313. Schwendemann, G. and Lôhler, J. (1979) Pathological alterations of ependyma and choroid plexus after ejpjerimental cerebral infection of mice with Sendai virus. Acta Neuropathol. 46:85-94. Shibuta, H., Kanda, T., Nozawa, A., Sato, S. and Kumanhishi, T. (1985) Experimental parainfluenza virus infection in mice: Growth and spread of a high pathogenic variant of parainfluenza 3 virus in the mouse brain. Arch. Virol. 83:43-52. Sorodoc, I., Aderca, I., Cajal, N., Dumitrescu, S.M., Constantinescu and 0., Cemescu, C. (1980) Peculiarities of the virus-host cell 178 relationship in a calf kidney cell line persistently infected with measles virus. Acta Virol. 24, 253-264. Sternberg, L.A. (1974) Inmunocytodiemistry. Sec. Edition, John Wiley & Sons, New York, Chichester, Brisbane, Toronto, 67-103. ter Meulen, V. and Martin, S.J. (1976) Genesis and maintenance of a persistent infection by canine distenper virus. J. gen. Virol. 32:431-440. Tabler, L.H. and hnagawa, D.T. (1984) Mechanism of persistence with canine distenper virus: Difference between a laboratory strain and isolate from a dog with chronic neurological disease. Inter virology 21:77-86. Tsai, S.C. and ^pel, M.J.G. (1979) Interferon in duction in dogs. Am. J. Vet. Res. 40:356-361. VainionpSâ, R . , Salmi, A. and Arstila, P. (1982) Characterization of a hamster brain cell line persistently infected with measles virus. Arch. Virol. 71:311-322. Van den Pol, A.N. (1984) Colloidal gold and biotin-avidin conjugates as ultrastructural markers for neural antigens. J. Ejp. Physiol. 60:1. Vassy, J., Rissel, M., Kraemer, M., Foucrier, J. and Guillouzo, A. (1984) Ultrastructural indirect irmunolocalization of transferrin in cultured hepatocytes permeabilized with Saponin. J. Histochem. Cytochem. 32(5): 538. Wagener, J.S., Minnich, L., Sobonya, R., Taussig, L.M., Ray, C.G. and Fulginiti, V. (1983) Parainfluenza type II infection in dogs. Am. Rev. Resp. Dis. 127:771-775. Wild, T.F. and Dudgre, R. (1978) Establishment and characterization of a subacute sclerosing panencephalitis (Measles) virus persis tent infection in BQl-cells. J. gen. Virol. 39:113-124. Williams, R.M., Krakc%d Willingham, M.C. (1983) An alternative fixation - procession method for preembedding ultrastructural iratnunocytochemistry of cyto plasmic antigens. J. Histochem. Cytochem. 31(6):391. WisniewsJq^, H.M., Brown, H.R. and Thoumar, H. (1983) Pathogenesis of viral encephalitis: Demonstration of viral antigens in the brain endothelium. Acta Neuropathol. 60:107-112. 179 Wolinsl^, J.S., Wasdiam, M.N. and Server, A.C. (1985) Protective effects of glycoprotein-specific monoclonal antibodies on the course of ejqjerimental murrps virus meningoencephalitis. J. Virol. 53:727-734. Youngner, J.S. and Preble, O.T. (1980) Viral persistence: Evaluation of viral populations. Carprehensive Virology, Vol. 16, Ch. 2, 73-135. Plenum Press, New York, London. Zakstelskaya, L.Ya., Zhdanov, V.M., Yakhno, M.A., Gushchin, B.V., Klimenko, S.M., Demidova, S.A., Konovalova, N.G. and Gushchina, E.A. (1976) Persistent SVg virus infection in continuous cell cultures. Acta Virol. 20:506-511. Zavada, J. (1976) Viral pseudotypes and phenotypic mixing. Arch. Virol. 50:1. Zhdanov, V.M. (1975) Integration of viral genanes. Nature 256:471- 473.