Experimental Viral Hepatitis the Dog: Production of ... · The Journal of Clinical Investigation Vol. 46, No. 9, 1967 Experimental Viral Hepatitis in the Dog: Production of Persistent

The Journal of Clinical InvestigationVol. 46, No. 9, 1967

Experimental Viral Hepatitis in the Dog: Production ofPersistent Disease in Partially Immune Animals *

D. J. GOCKE, R. PREISIG, T. Q. MORRIS, D. G. MCKAY,ANDS. E. BRADLEY

(From the Departments of Medicine and Pathology, Columbia University College of Physiciansand Surgeons, New York)

Abstract. Experimental infection with canine hepatitis virus has been stud-ied in a series of 49 dogs. The pattern of response to infection was distinctlymodified by the immune status of the animal. All of 19 fully susceptible dogshad an acute, fulminating fatal hepatitis when infected with a standard dose ofvirus, and all of 19 dogs with high levels of immunity to the virus survivedwithout apparent illness. However, 11 dogs were spontaneously encounteredwith partial immunity to the infectious agent, and these animals developeddifferent, prolonged forms of hepatitis following infection. In four animalsdeath occurred in 8-21 days following what may be called a subacute course.The remaining seven dogs survived up to 8 months with evidence of chronichepatic damage. The subacute and chronic forms of hepatitis were repro-duced experimentally in seven of eight fully susceptible dogs which were pas-sively immunized against the canine hepatitis virus by administration of hy-perimmune serum. Although the virus could be found in sites of hepaticdamage in the early stages of the subacute and chronic diseases, it could notbe demonstrated in the later stages which were characterized by persistenthepatic damage and a marked chronic inflammatory reaction. Dogs withchronic hepatitis eventually developed extensive hepatic fibrosis. The patho-logic, physiologic, virologic, and immunologic features of these experimentalforms of viral hepatitis are described.

Introduction

The study of viral hepatitis in man is seriouslylimited by the long-standing difficulties in isolatingthe etiologic agent in vitro and in reproducing thedisease in animals. Canine hepatitis, however, isa viral disease of the liver which produces strikinghepatic damage in dogs and which has the experi-mental advantage of being caused by a cultivatableadenovirus. Viral hepatitis is widespread in thecanine population and is a common cause ofdeath. The disease was originally described in1947 by Rubarth (1), who established the viral

* Submitted for publication 9 January 1967; accepted7 June 1967.

This study was supported by a grant from the John A.Hartford Foundation, Inc.

Address requests for reprints to Dr. David J. Gocke,Department of Medicine, College of Physicians and Sur-geons, 630 West 168th Street, New York, N. Y. 10032.

etiology and demonstrated the pathologic findingsin dogs dying of the disease. In 1954 Cabasso andhis coworkers (2) reported the successful propa-gation of the canine hepatitis virus in vitro. Thisled to attenuation of the virus in tissue cultureand made possible the development of an effectivelive virus vaccine (3-5). Subsequent investiga-tion has shown the canine hepatitis virus to be anadenovirus which has partial antigenic cross-reac-tivity with human adenoviruses, but which is notknown to produce disease in man (6, 7). Cur-rent knowledge concerning this virus has been re-viewed by Cabasso (8).

The ability to isolate and grow the causativeagent of canine hepatitis in vitro makes it possibleto carry out the type of controlled experiment inthe dog which cannot be done at the present timein man. Thus, the canine disease has been em-ployed in this laboratory as a convenient model

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EXPERIMENTALVIRAL HEPATITIS IN THE DOG

for the experimental study of viral infection of theliver, and the correlation of pathologic and viro-logic events with alterations in hepatic function.In the course of this study, it became apparentthat the pattern of response to experimental in-fection was distinctly modified by the immunestatus of the animal. More specifically, a syn-drome of chronic progressive hepatic damage hasbeen observed in dogs which were only partiallyimmune to the virus. In addition, it has been pos-sible to reproduce such chronic liver disease insusceptible dogs by passive immunization to thecanine hepatitis virus. These observations arethe subject of this report.

Methods

The animals used in these studies were young pure-bred beagles, 12-20 wk old, obtained from the Cyn-PieraKennels.' The dogs were born and raised in an isolatedenvironment in order to avoid natural infection withcanine hepatitis virus. Approximately 1 month beforestudy, they were immunized against distemper with anattenuated live virus vaccine (Tissuevax, Pitman-MooreCo., Indianapolis, Ind.), and against leptospirosis with astandard heat-killed Leptospiral antigen (Leptospira Bac-terin, Pitman-Moore). The animals were transportedby air to this laboratory, subjected to control studies, andthen infected within 12 hr after removal from isolation.The following studies were carried out before infection,daily during the early acute phase, and then less fre-quently during the latter phases. The weight, rectaltemperature, and general condition of the animal werenoted. Biopsies of the liver were obtained with a Meng-hini needle for histologic and fluorescent antibody studies.The serum was examined for the presence of virus andantiviral antibody in a tissue culture system (see below).The white blood cell count, hematocrit, serum alkalinephosphatase, serum glutamic oxaloacetic transaminase(SGOT), and the serum glutamic pyruvic transaminase(SGPT) were determined by standard methods. Studiesof bromsulphalein transport maximum (BSP Tm) andstorage (S) were measured by the dual infusion tech-nique (9). The dogs usually were infected by inocula-tion of 300 TCID5o of canine hepatitis virus into the an-terior chamber of the eye. In a few animals the viruswas administered by the subcutaneous route (see Results).

Virologic techniques. Canine hepatitis virus was origi-nally supplied by Dr. Victor J. Cabasso (Lederle Labora-tories, Pearl River, N. Y.) as a 20% suspension of crudeliver homogenate from an infected dog. Subsequently,other infective liver homogenates were prepared fromdogs dying with fulminating hepatitis. The infected livertissue was finely minced and subjected to repeated freez-ing and thawing. Cellular debris and fibrous tissue were

1 Cyn-Piera, Inc., Freeville, N. Y.

centrifuged and filtered out, and the crude homogenateswere stored in the lyophilized state at minus 70'C.

Primary cultures of canine kidney were prepared bythe method of Bodian (10), or obtained from commer-cial sources.2 The growth medium was the minimumessential medium of Eagle (Microbiological Associates,Inc.) supplemented with 10% fetal calf serum (GrandIsland Biological Co., Grand Island, N. Y.) heated at560C for 30 min, penicillin 100 ,ug/ml, and streptomycin100 gg/ml. For maintenance, the serum concentrationwas reduced to 1%. Virus titrations were carried outby the tube technique under the conditions recommendedfor this system by Carmichael et al. (11). Titers wereexpressed in TCIDso as determined by the Karbermethod (12). Neutralizing antibody to the canine hepa-titis virus in serum was determined in a standard tissueculture neutralization assay, with 100 TCID5o of the virusas the test dose. Results were expressed in terms of thedilution of serum giving 50%o neutralization of the cyto-pathic effect.

Fluorescent antibody techniques. Sera containing hightiters of neutralizing antibody were conjugated withfluorescein diisothiocyanate by the method of Rinder-knecht (13). To minimize nonspecific staining, wepassed the conjugated antisera through a 10 X 2 cm col-umn of G25 Sephadex, and absorbed them 4 times with10 mg of acetone-dried liver powder per ml of serum.Needle biopsy specimens of the liver were rapidly frozenon dry ice. Sections were cut at 3 A in an Internationalmicrotome cryostat and fixed in acetone for 15 min, andfluorescein-conjugated antiserum was applied for 60 minat 37°C in a humid chamber. The specimens were washed3 times with phosphate-buffered saline (pH 7.2), and ex-amined microscopically for specific fluorescence using ul-traviolet illumination and the darkfield technique. Speci-ficity of the fluorescence for canine hepatitis virus wasestablished by the following control procedures: (a)pretreatment of frozen sections of infected liver withunconjugated antiserum (followed by fluorescein-conju-gated antiserum) resulted in blocking of the specificfluorescence; (b) infected liver exposed to fluorescein-conjugated normal dog serum (i.e., without antibody tothe virus) failed to show specific fluorescence; (c) normalliver treated with the labeled antiserum showed no fluo-rescence; (d) tissue culture cells infected with caninehepatitis virus showed characteristic fluorescence whenstained with labeled antiserum.

Results

I) EXPERIMENTALPRODUCTIONOF DISEASE

Observations in 49 dogs experimentally infectedwith canine hepatitis virus are summarized in Ta-ble I. Three patterns of response were seen:early death, survival with evidence of hepatic dis-

2 Microbiological Associates, Inc., Bethesda, Md. andBaltimore Biological Laboratories, Baltimore, Md.

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GOCKE, PREISIG, MORRIS, McKAY, AND BRADLEY

TABLE I

Summary of disease patterns after experimental infection with canine hepatitis virus

Staining withNo. fluorescent

of Titer of antiviral antiviralGroup dogs Course antibody* Hepatic pathology antibody

A 19 Death, day 4-9 <1/4 Massive hepatic necrosis Positivewith INI$

B Well, day 4-8 _ 1/16 . 1/500 Focal necrosis with IN II Positive1) 4 Death, day 8-21 Extensive hepatic Negative

necrosis with lymphocytes+ plasma cells, no INI+

2) 7 Survival, to 8 mos. Chronic inflammation Negativeand fibrosis

C 19 Well (all dogs) > 1/500 Normal Negative

*Values given are titers on the day of infection, except for certain Group B animals in which antibody first appeared4-5 days after infection (see text and Table II).

I INI, Intranculear inclusion body of the canine hepatitis virus.

ease, or survival without apparent illness. In gen-eral, the response correlated with the animal'slevel of immunity to the virus.

4) Acute disease in the fully susceptible dog

In 19 dogs (Group A, Tables I and II) thedisease was an acute, fulminating illness leadingto death in 4-9 days after infection. An exampleof the course of the disease in such a dog can beseen in Lydia. The dog appeared well for thefirst 3-4 days, except for slight temperature ele-vation (103.5° F) and development of cornealopacification in the infected eye. On the 5th day,viremia appeared, the temperature rose further

to 105 'F, and hepatic dysfunction was first evi-dent in depression of BSP Tm (70%o of control).By the 6th day, the animal was noticeably lethargicand anorectic, the SGOTwas now definitely ele-vated (95 Karmen units), and both BSP S andTm were reduced (80 and 65% of control, re-spectively). Thereafter, the disease process seemedto explode, and within 12 hr the dog expired be-fore further studies could be carried out. Neu-tralizing antiviral antibody was not found in de-tectable amounts (< a) before or during the ill-ness.

The earliest abnormality recognized in the liverof this dog was found on day 5 at the time when

FIG. 1. HEPATIC BIOPSY FROMLYDIA ON THE 5TH DAY AFTER INFECTION WHENHISTOLOGIC ABNORMALITIES WERE

FIRST RECOGNIZED. (a) Hematoxylin- and eosin-stained section showing an intranuclear inclusion body typical ofthe canine hepatitis virus in a Kupffer cell. X 1000. (b) Frozen section stained with fluorescein-labeled antise-rum to the canine hepatitis virus showing one of the brightly fluorescent bodies found along the hepatic sinusoid<.X 400.

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FIG. 2. HEPATIC BIOPSY FROMLYDIA ON DAY 6 AFTER INFECTION. (a) A small focus of hepatic necrosis withseveral cells containing typical intranuclear inclusions (arrows). Hematoxylin and eosin, X 400. (b) Frozensection stained with fluorescent antiviral antiserum showving a cluster of fluorescent viral material in a necroticfocus. x 400.

viremia was first detected. A diffuse increase ininflammatory cells in the hepatic sinusoids andthe appearance of characteristic intranuclear in-clusion bodies of the canine hepatitis virus, pre-dominantly in Kupffer cells, were observed (Fig.1 a). The presence of viral antigen in these cellswas demonstrated in frozen sections of the liverstained with fluorescein-labeled antiviral antibody(Fig. 1 b). By day 6, the liver was studdedwith small foci of parenchymal necrosis containingtypical intranuclear inclusion bodies (Fig. 2 a).Small clusters of viral material were seen in thesefoci on staining with fluorescent antibody (Fig.2 b). Finally, when examined at autopsy 12 hrlater, severe hepatic necrosis (Fig. 3 a and c) andwidespread fluorescent staining (Fig. 3 b) werefound, with intranuclear inclusions in almost everycell.

The disease pattern observed in all 19 of theacute animals was similar to that described abovefor Lydia. Table II summarizes clinical, bio-chemical, and serologic observations in the entiregroup of animals. The acute disease was charac-terized by development of fever and dense cornealopacification in the infected eye, followed by theonset of lethargy, anorexia, and abnormalities inserum alkaline phosphatase and SGOT, and thenrapid deterioration to coma and death in a matterof hours. All of these dogs had severe hepaticnecrosis on autopsy. The disease progressed soquickly that the severity of hepatic damage atdeath was not always reflected in biochemicalvalues obtained only 24 hr before. Dogs such as

Vem and Willy (Table II) in which blood wasobtained only 2-3 hr before death had markedelevations of SGOT and alkaline phosphatase.Measurement of other clinical and chemical param-eters commonly employed in the study of humanliver disease were not found to be useful becausethe fulminant course did not allow significantchanges to be observed. Significant elevation ofserum bilirubin was rarely observed because of thehigh renal clearance of bile pigments in the dog.

It was possible to carry out detailed studies ofBSP kinetics during the course of acute hepa-titis in 11 puppies. Each animal served as its owncontrol. Control values for S ranged from 10.8to 14 mg/mg% (averaged 12.8, SD 1.18), andfor Tm from 2.4 to 3.2 mg/min (average 2.9, SD0.26). After infection, a depression in one or bothvalues was observed as early as day 2 in two ani-mals. By the 4th day after infection, both S andTmwere usually decreased (average 65% of con-trol) in the nine animals studied at that time.Elevations in the SGOTand serum alkaline phos-phatase were always associated with changes inBSP kinetics, and in two animals a depression ofTm and S preceded enzymatic abnormalities.

It is emphasized that none of the dogs withacute disease had detectable antibody to the virusat the time of infection nor did they develop anti-body during the brief course of their illness. Theseanimals appeared to have been fully susceptibleand were overwhelmed by the virus before an im-mune response could occur. Viremia was ob-served as early as the 2nd day after infection in

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FIG. 3. FINDINGS AT AUTOPSY ON LYDIA WHODIED BETWEENDAY 6 AND 7 AFTER INFECTION. (a) Hematoxylinand eosin section of liver showing extensive hepatic damage. X 160. (b) Frozen section stained with fluorescentantiviral antiserum showing widespread fluorescent staining. X 100. (c) Higher magnification showing disruptionof hepatic cords, cells in varying stages of necrosis, and multiple intranuclear inclusions (arrows). Hematoxylin andeosin, x 400.

some cases, while in others it was not detecteduntil the 4th or 5th day. The onset of viremia was

accompanied by the finding of virus in the reticu-loendothelial system of the liver. Once this point

was reached the animal usually died within 48 hrwith extensive hepatic necrosis and abundant,easily demonstrable virus in the liver. It is to bereemphasized that none of the 19 dogs that died of

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TABLE II

Summary of clinical, biochemical, and serologic observations in canine hepatitis

Alk. phos- BSPphatase* SGOT*

Dog Course (KAU)j (KU)j Tmll SiO Titer antiviral antibody¶

mg/mix m/msg %Acute disease, Group A

Aaron Death day 5Boris Death day 5Eli Death day 6Favia Death day 4Gideon Death day 4Herod Death day 4Isis Death day 6Job Death day 4Lydia Death day 7Moses** Death day 6Noah** Death day 9Nick Death day 4Selma Death day 6Vem Death day 5Willy Death day 5Eros Death day 6Fairy Death day 6Illya Death day 6Moreen Death day 5

8516 8522' 36014 8516 10013 78

8 1201335 9546 38043 13035 80014 6350 100034 1500

17 650

1.3 6.92.6 12.42.2. 9.81.6 7.23.0 8.11.1 8.02.3 7.21.5 5.21.8 8.51.1 5.61.2 5.1

Subacute disease, Group BiKeto Death day 14Caleb Death day 21Rex Death day 8Quelle Death day 16

Chronic disease, Group B2Tally Death 7 mos.Uta Death 5 mos.Sol** Survived indef.Thor** Survived indef.Xerox Survived 7 mos.Chris Survived 8 mos.Fatal Survived 8 mos.

40 125 0.6 3.0 1/4038 110 3.5 7.2 <1/4 (1/20 day 4)28 150 1.0 4.6 <1/4 (1/20 day 5)28 105 1/100

181713172010

8

5870

106140170

7883

- 1/100< 1/4 (1/320 day 5)<1/4 (1/500 day 4)

- <1/4 (1/100day4)- <1/4 (1/36 day 5)

- 1/100- 1/100

* Values listed are the maximum deviations from normal observed during illness.$ KAU, King-Armstrong units (normal 4-12).§ KU, Karman units (normal 20-50).

l BSP control values: Tm = 2.9 i 0.26 mg/min; S = 12.8 41 1.18 mg/mg %.¶[ Values given are titers on the day of infection. Where antibody did not appear until after infection, the first

positive titer and the day of detection are given in parentheses. Group A animals never had detectable antibody beforedeath.

** Infected subcutaneously.

the acute disease had detectable levels of neutraliz-ing antibody to the virus prior to or during thecourse of infection (Table II).

B) Prolonged disease in the partially susceptibledog

11 dogs had a distinctly different and more pro-

longed illness after experimental infection (GroupB, Tables I and II). In four of these, death oc-

curred in 8-21 days after infection, following whatmay be called a subacute course (Group B11).The remaining dogs survived up to 8 months af-

ter infection with evidence of chronic hepaticdisease (Group B2). In all 11 dogs with pro-

longed disease, low levels of antiviral antibodywere detected in the serum at the time of infec-tion, or appeared very early after infection.

1) Subacute disease. Keto is representative ofthe dogs with subacute disease. This dog ap-

peared perfectly well between the 4th and 8th dayafter infection, at a time when fully susceptibleanimals died. There were no alterations inSGOT, serum alkaline phosphatase or BSP Tmor S. Then, toward the end of the 2nd wk after

<1/4<1/4<1/4<1/4<1/4<1/4<1/4<1/4<1/4<1/4<1/4<1/4<1/4<1/4<1/4<1/4<1/4<1/4<1/4

-11 IV

151


FIG. 4. HEPATIC BIOPSY FROMKETO ON THE DAY OF DEATH (DAY 14). (a) Widespread hepatic necrosis with in-tense inflammatory response especially about a dilated central vein. Hematoxylin and eosin, X 100. (b) Higher-power view showing the predominance of mononuclear cells and the absence of intranuclear inclusions. Hematoxylinand eosin, X 400.

infection, the dog became clinically ill. On days13 and 14 elevation of SGOTand marked de-pression of BSP S and Tmwere found. The dogdied on the 14th day. The preinfection serumfrom this animal contained antiviral antibody witha titer of 1/40. Viremia was never detected.

Hepatic biopsies on the 5th day after infectionrevealed small foci of necrosis similar to thatshown in Fig. 2. Virus was identified in theselesions by the presence of intranuclear inclusionbodies, and by specific staining with fluorescein-labeled antiviral antibody. In addition to the usualpolymorphonuclear leukocyte response, the ne-crotic foci contained significant numbers of lym-phocytes and plasma cells. The sections in Fig. 4 aand b were obtained on the 14th day after infection,just before the death of the animal. Widespread,severe hepatic necrosis was found. In addition,dense collections of lymphocytes and related formswere found about central veins and in portalareas. In contrast to the situation in dogs dyingwith the acute disease, the presence of the viruswas not demonstrated at death in Keto either bythe finding of inclusion bodies or by the fluores-cent antibody technique.

A similar subacute course was observed in threeother animals (Caleb, Rex, Quelle) in whichdeath occurred between 8 and 21 days after in-fection (Caleb appeared extremely ill and wassacrificed on the 21st day). In each, severe he-patic necrosis with lymphocytes and plasma cellswas found at postmortem examination, yet thevirus could not be demonstrated by the above

methods. In addition, each dog had low levelsof neutralizing antibody before infection, or ap-pearing early after infection. Clinical and bio-chemical data obtained in the animals with sub-acute disease- are presented in Table II (GroupB 1). In general, the functional abnormalitiesfound in the subacute illness resembled those de-scribed for the dogs with acute disease, exceptthat they were delayed in appearance.

?2) Chronic disease. Chronic hepatic damagewas observed in seven dogs with low antibodytiters who survived as long as 8 months after in-fection. Initially, there was minimal clinical orbiochemical evidence of illness in these animals.However, biopsies during the 1st wk after infec-tion revealed small foci of hepatic necrosis withinclusion bodies and positive fluorescent stainingindistinguishable from that seen in acute animals(Fig. 2). The animals survived this early phaseand over the following months the evolution ofchronic hepatic damage was observed. Fig. 5 a isfrom the 27th day after infection in the dog Tallyand shows one of the dense accumulations of lym-phoid cells and plasma cells scattered throughoutthe liver. Fig. 5 b was obtained 2 months afterinfection in this dog and shows an enlarged portalarea infiltrated with round cells and plasma cells.5 months after infection chronic inflammatory in-filtrates persisted and portal fibrosis was noted.Tally expired 7 months after infection, and atautopsy marked chronic inflammation and sig-nificant fibrosis were found (Fig. 6). Beyond the10th day after infection, virus was no longer iden-

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FIG. 5. (a) LIVER BIOPSY FROMTALLY ON 27TH DAY AFTER INFECTION SHOWINGONE OF THE COLLECTIONS OF LYM-PHOID CELLS SCATTERED THROUGHOUTTHE LIVER. Hematoxylin and eosin, X 400. (b) Biopsy from Tally 2 monthsafter infection showing an enlarged portal area infiltrated with mononuclear cells and plasma cells (arrow). Hema-toxylin and eosin, X 400.

FIG. 6. POSTMORTEMFINDINGS IN TALLY, WHODIED 7 MONTHSAFTER INFECTION, SHOWINGMARKEDCHRONIC IN-

FLAMMATORYREACTION ANDEXTENSIVE FIBROSIS. Hematoxylin and eosin, X 100.

tified in the hepatic lesions by the finding of inclu-sion bodies or by the fluorescent antibody tech-nique.

The remaining six dogs in the chronic groupdemonstrated progressive development of similarhepatic changes on serial biopsy. One of them(Uta) died 5 months after infection with markedhepatic fibrosis. The others have survived a min-imum of 7 months and continue to be studied. Itis to be reemphasized that antiviral antibody ti-ters between 1¼4 and %/o were present in thesedogs either at the time of infection or appearedquickly in 4-5 days after infection (Table II).As with the subacute animals, the presence ofvirus could not be demonstrated in the. liver be-yond the first 7-10 days after infection. Collectionof functional data in this group was limited be-

cause of belated recognition that the animals wereill.

C) Observations in the nonsusceptible animal

19 dogs (Group C, Table I) were infected andfailed to develop any sign of disease. They ap-peared well clinically after infection; there was noalteration in SGOT, serum alkaline phosphatase,or BSP Tm or S. The presence of the viruscould not be demonstrated by the fluorescent anti-body technique, and serial biopsies of the liverremained normal. These dogs all had neutralizingantibody to the canine hepatitis virus in the serumat the time of infection, usually with a titer greaterthan %/oo Thus, they appeared to have sufficientlevels of antiviral antibody to be protected frominfection.

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II) EXPERIMENTAL MODIFICATION OF THE DIS-

EASE

The observations described above on the oc-currence of the subacute and chronic forms ofhepatic disease after experimental infection wereinitially the results of chance encounters withpartially immune dogs. When the apparent re-lation between low titers of antibody to the virusand prolonged forms of hepatitis was recognized,experiments were undertaken to reproduce andconfirm these findings in a prospective fashion.This was done by passive administration of hyper-immune antiserum against the canine hepatitisvirus to fully susceptible dogs. On the basis ofthe estimated blood volume of the dog and thetiter of the antiserum, the amount of serum re-quired to produce approximately a 1/0 titer ofantibody in the animal was calculated. The anti-serum was administered intravenously, and 10-20min later the animal was infected with the stand-ard dose of virus. In the first eight attempts, thesubacute disease pattern has been observed inthree dogs and the chronic pattern has occurred infour, one of which died at 4 months. The re-maining dog died on day 5 with typical acute hepa-titis. In addition, a control group of five fullysusceptible dogs not given antiserum was inocu-lated in the same manner with the same dose andpreparation of virus, and all went on to die withtypical actue hepatitis. The hepatic changes seenin the passively immunized animals duplicatedthe histologic findings already described. Thus,the subacute and chronic disease patterns ob-served in dogs passively immunized to the hepa-titis virus before infection were indistinguishablefrom the patterns encountered in animals withpartial natural immunity.

Discussion

The acute form of canine hepatitis observed inthese studies was a fulminant, rapidly fatal in-fection in animals lacking an effective immune de-fense. The virus was first found in the Kupffercells of the liver, suggesting that the infectiousagent reached the liver by the blood stream, andwas filtered out by cells of the reticuloendothelialsystem. Once in the Kupffer cells, the virus un-derwent rapid multiplication, with an explosiveprogression of hepatic damage. In contrast to the

situation in the subacute and chronic diseases tobe discussed below, there was an easily recog-nized correlation between the demonstrable pres-ence of virus and the extent of hepatic necrosis.The degree of functional abnormalities correlatedwell in general with the severity of hepatic necro-sis. The pathophysiologic factors responsible fordeath in these animals have not been well studiedpreviously and are a subject of continuing in-vestigation. However, it has been extensivelyshown by Rubarth (1) and others, and confirmedin our experience, that although the virus can befound throughout the body, the liver is the onlysite of major injury.

None of the 19 animals that succumbed to thefulminating acute form of hepatitis (Group A)had detectable antibody to the canine hepatitisvirus in the serum either at the time of infectionor during the brief illness. The absence of neu-tralizing antibody was quite consistent with rapidprogression of the infection and death of the ani-mal before an effective immune defense could beraised. At the other extreme, the dogs that failedto develop any sign of illness (Group C) were al-ways found to have relatively high levels of neu-tralizing antibody to the virus in the serum at thetime of infection. No exception to these obser-vations has been encountered and, indeed, thiswould be the expected effect of specific immunityto an infectious agent on the outcome of theinfection.

The animals in Group B, however, demonstratean unexpected and untoward effect of host im-munity on the course of the disease. These dogshad low titers of antiviral antibody at the time ofinfection, or were able to produce antibody rapidlyin the first 4-6 days of illness. They survived theinitial phase but developed definite hepatic dam-age, sometimes leading to death, despite the pres-ence of antibody to the infectious agent. Thus,partial immunity to the virus seemed to have aclear, though unexplained, correlation with the de-velopment of persistent hepatic injury. This ob-servation was strongly supported by the success-ful reproduction of the subacute and chronic formsof hepatitis in seven of the eight dogs passivelyimmunized to the virus.

Certain notable features characterize the sub-acute and chronic disease patterns. Although theICH virus could be identified in small necrotic

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foci in the liver in the first few days after infec-tion, the rapid progression of hepatic necrosis seenin the acute disease did not occur. Instead, thevirus seemed to disappear, at least in so far as itcould be detected by the techniques available. Incontrast to Group A animals, the virus could notbe found in the animals even when severe hepaticnecrosis and death occurred. Whether the disap-pearance of the virus is real or only apparent isnot yet clear. The absence of intranuclear in-clusion bodies and the negative fluorescent anti-body test do not completely rule out the persistenceof virus in an incomplete or atypical form. At-tempts have been made to recover virus from theliver in tissue culture on 41 different occasions innine dogs with positive results only in the 1st wkafter infection. Additional recovery attempts arecurrently underway to extend these findings.

Despite the apparent absence of the virus, he-patic damage continued in both the subacute andchronic animals. The predominance of lymphoidelements was an outstanding feature. In thesubacute disease, severe and extensive hepaticnecrosis ensued, leading to death of the animal.The hepatic damage involved the entire liver lob-ule, and lymphocytes, monocytes, and plasma cellswere scattered profusely in the sinusoids and indense accumulations about central veins and in por-tal tracts. In the chronic disease, the animals sur-vived at least for several months with evidence ofpersistent hepatic injury. In the early weeksfocal collections of lymphoid cells were foundthroughout the liver, and in later stages thesechronic inflammatory cells were localized in por-tal areas in association with fibrosis. The findingof portal fibrosis in the later stages of the chronicdisease is of note. In some instances, fibrosis wasquite definite and had interlobular extensions.However, severe distortion of hepatic architectureand lobular regeneration were not prominent find-ings, nor was evidence of portal hypertensionavailable. What is noteworthy is the finding ofa chronic, prolonged form of hepatic damage withportal fibrosis as one of the outstanding features.

The source of the antiviral antibody in the ani-mals with partial immunity is of some interest.Carmichael et al. (14) found the half-life of ma-ternal neutralizing antibody to the ICH virus inyoung beagle puppies was 8.6 days. Thus, the ageof the animals used in this study ( 12-20 wk)

should have allowed for the disappearance of thebulk of maternal antibody. However, it is con-ceivable that occasional puppies were encounteredfrom sufficiently high titered mothers to providefor the persistence of low titers of antiviral anti-body in the offspring even at 5 months of age.Unfortunately, maternal serum was not availablefor titration. Also, it is possible that some of theanimals had previous unrecognized natural infec-tions. Perhaps the dogs that lacked antibodyinitially, but developed titers rapidly, were show-ing an anamnestic response. Another possiblesource of low levels of antibody may lie in theknown antigenic relationship of the ICH adeno-virus to some of the human adenoviruses. Car-michael and Baker (15) have made the interestingobservation that dogs inoculated intranasally withadenovirus type 4 developed neutralizing antibod-ies following an asymptomatic infection. Whensuch dogs were later inoculated with ICH virus, asecondary or anamnestic type of neutralizing anti-body response occurred to both viral antigens, andantibodies persisted for at least 340 days. Thus, itis conceivable that our animals had been previ-ously exposed to a human adenovirus while inisolation and thus sensitized to subsequent inocu-lation with ICH virus. The presence of smallamounts of antibody to the virus may simply per-mit the animal to survive the acute stages of in-fection, and then some other pathologic processsupervenes to produce further hepatic injury.However, it is not at all clear why a small amountof antibody should not simply suppress or attenu-ate the infection with eventual recovery, ratherthan predispose to continuing liver damage.

Although there is ample precedent for the per-sistence of virus in a host's tissues with produc-tion of latent or inapparent infection, in such in-stances the virus is demonstrable at sites of celldamage (16-18). Also, virus can be identifiedeasily in the liver in other types of viral hepa-titis (18-20). It would seem unlikely, therefore,that the canine hepatitis virus could produce theliver damage seen in the Group B animals withoutbeing easily detected. Conceivably, some otherlatent agent, such as herpes virus, could be ac-tivated by the initial infection with ICH virus andthen take over the cytopathic process in laterstages of the disease. Carmichael et al. havedescribed a fatal viral disease of newborn pups

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in which the etiologic agent was identified as aherpes virus (21, 22). Although this virus wasfound to cause severe illness and death only invery young pups (1-3 wk old), the possibility oflatent infection and subsequent reactivation, asrecognized with other herpetic infections, remainsto be evaluated.

The possible role of an immunologic process inthe pathogenesis of chronic hepatic damage hasreceived prominent consideration in human hepa-titis, and is suggested in the studies reported hereby the striking collections of round cells andplasma cells seen in biopsies from dogs with sub-acute and chronic disease. Antibodies againsthuman or animal liver antigens in acute hepatitisand other liver diseases have been demonstratedby a number of investigators (23). However,these antibodies have not been clearly shown tobe specific for liver antigen only, and it is diffi-cult to establish what role, if any, such "autoan-tibodies" play in the actual pathogenesis of hepaticdamage. Perhaps more pertinent to the presentstudies is the possibility that circulating antigen-antibody complexes may accentuate necrosis inthe liver initiated by the virus. Paronetto andPopper (24) have demonstrated the uptake ofantigen-antibody complexes by mildly damagedliver cells of mice when the Kupffer cells whichnormally take up such complexes failed to do so.This resulted in marked increase in the extent ofhepatocellular damage, and suggested that hepaticdeposition of cytotoxic immune complexes maymagnify preexisting minimal hepatic alterations.In light of this work it is interesting to note.thatabout 1-3 wk after recovery from infectiouscanine hepatitis, some dogs develop an intense an-terior uveitis with corneal opacification (evenwhen infected by other than the intraocular route).Carmichael (25) showed-that this reaction has thepathologic characteristics of Arthus-type hyper-sensitivity and occurred only when viral antigenand specific antibody were present concurrentlyin the eye tissues. The entire phenomenon wasreproduced by intraocular inoculations of ICHvirus-antibody complexes. Thus, it is conceivable,especially in dogs with subacute hepatitis, thatvirus-antibody complexes could be responsible forthe accentuation and extension of liver damage ini-tiated by the virus.

The experiments described in this report providenew information on the pathogenesis of virus-in-duced hepatic injury in the dog. They also dem-onstrate the potentially deleterious effect of par-tial immunity to the infectious agent on the courseof the disease. The possible relevance of thesefindings to viral hepatitis in man raises inter-esting questions which cannot be resolved at thepresent time. Despite certain similarities, hepati-tis in man and in the dog are different diseasescaused by different agents. No attempt is in-tended here to equate findings in the dog with thesituation in man. However, observations on thepathogenesis of virus-induced hepatic injury inthe dog may provide a different point of view onthe problem of human hepatitis, and perhaps sug-gest fresh experimental approaches.

References

1. Rubarth, S. 1947. An acute virus disease withliver lesion in dogs (hepatitis contagiosa canis).A pathologico-anatomical and etiological investi-gation. Acta Path. Microbiol. Scand., Suppl. 69:9.

2. Cabasso, V. J., M. R. Stebbins, T. W. Norton, andH. R. Cox. 1954. Propagation of infectiouscanine hepatitis virus in tissue culture. Proc. Soc.Exptl. Biol. Med. 85: 239.

3. Fieldsteel, A. H., and J. B. Emery. 1954. Cultiva-tion and modification of infectious canine hepa-titis virus in roller tube cultures of dog kidney.Proc. Soc. Exptl. Biol. Med. 86: 819.

4. Cabasso, V. J., M. R. Stebbins, and J. M. Avampato.1958. A bivalent live virus vaccine against caninedistemper (CD) and infectious hepatitis (ICH).Proc. Soc. Exptl. Biol. Med. 99: 46.

5. Burgher, J. A., J. A. Baker, S. Sarkar, V. Marshall,and J. H. Gillespie. 1958. Evaluation of acombined vaccine consisting of modified caninedistemper virus and modified infectious caninehepatitis virus for simultaneous immunization ofdogs. Cornell Vet. 48: 214.

6. Kapsenberg, J. G. 1959. Relationship of infectiouscanine hepatitis virus to human adenovirus. Proc.Soc. Exptl. Biol. Med. 101: 611.

7. Heller, L. A., and C. R. Salenstedt. 1960. One-sided immunologic relation between adenovirusand infectious canine hepatitis antigens. Virology.11: 640.

8. Cabasso, V. J. 1962. Infectious canine hepatitisvirus. Ann. N. Y. Acad. Sci. 101: 498.

9. Preisig, R., R. Williams, J. Sweeting, and S. E. Brad-ley. 1966. Changes in sulfobromophthalein trans-port and storage by the liver during viral hepa-titis in man. Am. J. Med. 40: 170

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10. Bodian, D. 1956. Simplified method of dispersionof monkey kidney cells with Trypsin. Virology.2: 575.

11. Carmichael, L. E., G. E. Atkinson, and F. D. Barnes.1963. Conditions influencing virus-neutralizationtests for infectious canine hepatitis antibody. Cor-nell Vet. 53: 369.

12. Finney D. J. 1952. Statistical method in biologicalassay. Hafner Publishing Company, New York.Exptl. Biol. Med. 109: 677.

13. Rinderknecht, H. 1962. Ultra-rapid fluorescentlabelling of proteins. Nature. 193: 167.

14. Carmichael, L. E., D. S. Robson, and F. D. Barnes.1962. Transfer and decline of maternal infectiouscanine hepatitis antibody in puppies. Proc. Soc.Exptl. Biol. Med. 109: 677.

15. Carmichael, L. E., and J. A. Baker. 1962. Sec-ondary serological response to infectious caninehepatitis virus produced by adenovirus type 4.Proc. Soc. Exptl. Biol. Med. 109: 75.

16. Hotchin, J. 1962. The biology of lymphocytic chorio-meningitis infection: virus-induced immune dis-ease. Cold Spring Harbor Symp. Quant. Biol.27: 479.

17. McNair Scott, T. F., and T. Tokumaru. 1965. Theherpesvirus group. In Viral and Rickettsial In-fections of Man. F. L. Horsfall and I. Tamm,editors. J. B. Lippincott Co., Philadelphia. 4thedition. 892.

18. Mims, C. A. 1959. The response of mice to largeintravenous injections of ectromelia virus. II. The

growth of virus in the liver. Brit. J. Exptl.Pathol. 40: 543.

19. Tigertt, W. D., T. 0. Berge, W. S. Gochenour, C. A.Gleiser, W. C. Eveland, C. V. Bruegge, and H.F. Smetana. 1960. Experimental yellow fever.Trans. N. Y. Acad. Sci. 22: 323.

20. Ruebner, B., and K. Miyai. 1962. The Kupffer cellreaction in murine and human viral hepatitis, withparticular reference to the origin of acidophilicbodies. Am. J. Pathol. 40: 425.

21. Carmichael, L. E., R. A. Squire, and L. Krook.1965. Clinical and pathologic features of a fatalviral disease of newborn pups. Am. J. Vet. Res.26: 803.

22. Carmichael, L. E., J. D. Strandberg, and F. D.Barnes. 1965. Identification of a cytopathogenicagent infectious for puppies as a canine herpes-virus. Proc. Soc. Exptl. Biol. Med. 120: 644.

23. Deinhardt, F., and A. W. Holmes. 1965. Epidemi-ology and etiology of viral hepatitis. In Progressin Liver Diseases. H. Popper and F. Schaffner,editors. Grune & Stratton, Inc., New York. 2:373.

24. Paronetto, F., and H. Popper. 1965. Aggravationof hepatic lesions in mice by in vivo localization ofimmune complexes (Auer Hepatitis). Am. J.Pathol. 47: 549.

25. Carmichael, L. E. 1965. The pathogenesis of ocularlesions of infectious canine hepatitis. Pathol. Vet.(Basel). 2: 344.

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Experimental Viral Hepatitis the Dog: Production of ... · The Journal of Clinical Investigation Vol. 46, No. 9, 1967 Experimental Viral Hepatitis in the Dog: Production of Persistent