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~MICROCOPY RESOLUTION TEST CHART

NAIIONAL BUREAU OF STA NOAR DS- 1963~~

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• OFFICE OF NAVAL RESEARCH

CONTRACT NO. N00014-76-C-0247

WORK UNIT # NR 204-014

3 TECHNICAL REPORT NO. 5

/ _..i CULTIVATION OF HEPATITIS VIRUS IN TISSUE CULTURE• I •/~~ L~-

_ BY

G.L. Gitnick , M.D.- University of California at Los Angeles

Department of Medicine_________ Los Angeles, Cali fornia 90024

Wi le ~eclll

~~ ~~~~ ~ March 1 , 1978

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Reproduction in whole or in part is permitted for0 any purpose of the United States Government

t This document has been apprcied for public release; itsdistribu tion is unlimited

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INTRODUCTIONThe forms of acute viral hepatitis include hepatitis A , hepatitis B, and the

most coninon form of hepatitis has been designated non A non B hepatitis. Hepatitis -A generally carries with it a short incubation period and is usually transmitted by

f fecal or oral contamination. Hepatitis B is the least coninon form of viralhepatitis, is usually transmitted by contaminated blood products or needles butmay be transmitted by the fecal-oral route and often has a longer incubation period .The most comon form of hepatitis, non A non B hepatitis may represent vira l diseaseof one or more etiologies . It has an incubation period varying from 6 to 24 weeks,may be transmitted either by blood products, needles or the fecal-oral route andis clinically indistinguishab le from the other forms of hepatitis. This conwnonform of hepatitis requires additional scientific evaluation but this has beenhampered by the absence of serologic assays for its detection.

The average period of disability for any of the forms of acute viral hepatitisis approximately 5 weeks. In addition , between 1 and 3% of patients will die withacute viral hepatitis due to the development of fulminant fatal hepatitis.Approximately 14% of patients will develop either chronic persistent or chronicactive hepatitis. Of those who develop chronic active hepatitis most will developpost necrotic cirrhosis. Because of the significant morbidity and eventualmortality associated with these illnesses it is important that efforts be directedat the cultivation of hepatitis viruses and eventually at the development ofeffective and safe vaccines.

Recently, Watana be, et al,6 developed a new technique for the cultivation ofhepatitis B virus. They utilized oval hepatocytes which flow freely in the mediaafter liver explants are planted. These hepatocytes maintain the function ofnormal liver cells including the production of albumi n and alpha—1—fetoprotein.They are intermediate in appearance between an epithelial cel l and a fibrobl astand have an oval shape. On initial passage, ininunofluorescent studies demonstratedthe presence of hepatitis B surface antigen in these hepatocytes. In our laboratorywe have previously been unable to demonstrate cytopathic change or increase inantigen titer using standard assay procedures. However, Watanabe uniquely appliedininunofluorescence to his assay system and was able to demonstrate persistence ofviral antigen. Furthermore, he was then able to subculture oval cells up to 14passages and demonstrate the continued presence of specific ininunofluorescence wi theach subpassage. We have recently undertaken similar studies.

During the past year we have developed methods for the detection of blood unitslikely to contain the agent of non A non B hepatitis. We have recently undertakenthe appl ication of these new methods to the needs of this hepatitis vaccinedevelopment program.

METHODS

1. Sources of Non A Non B Virus and Antigen

Patients at the Univers ity of California, Center for the Health Sciences ,Los Angeles, were entered into a prospective study of PTH. Criteria for inclusionwere the absence of a history of jaundice, hepatitis, clinical or biochemi calevidence of l iver disease, blood transfusion within the previous nine months,hepatotoxins , or a life expectancy of less than nine months. A prerequisite forenrollment was a normal SGPT and CEA level in the patients ’ blood samples prior totransfusion. After transfusion , blood specimens were obtained every two weeks for

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ten months. These samples were tested for HBsAg (Ausria II , Abbott Laboratories,North Chicago, Ill inois), SGPT (Beckman Automated , kinetic method), and CEA(CEA Roche, Hoffmann-La Roche, Inc., Nutley, New Jersey). The diagnosis of PTHwas made if the patient was found to have two SGPT levels greater than five timesthe upper limit of normal drawn five to 21 days apart. The episode was termednon A non B PTH if the patients were negative for HBsAg during the acute phaseand failed to develop either anti-HBs or anti-HA during convalescenc~.~.

The donors of blood uni ts with elevated CEA and SGPT levels from the paststudy, implicated in the transmission of non A non B PTH, were contacted Bloodsamples were drawn in EDTA for CEA testing and as serum for HBsAg, anti-HBc andSGPT testing. Those donors with persistently abnormal CEA and SGPT were asked toundergo a single unit plasmaphoresis. Transfusion recipients who developed non Anon B PTH during this study, who had persistently abnormal SGPT (greater thdfl45 I.U.) with or without CEA elevation were asked to undergo a one unit plasma-phoresis. All such plasma was coded and stored at -70 C.

All outdated plasma from the UCLA Blood Bank was provided to our group. Allunits were screened for SGPT. All HBsAg negative units with SGPT levels greaterthan 50 I.U. were assayed for CEA. Units with SGPT levels greater than 50 I.U.and CEA level s greater than 4.9 ng/ml were coded for use as a source of antigen.In addi tion, HBsAg negative units with SGPT levels greater than 100 I.U. werecoded and stored at -70 C as an alternative source of antigen regardless of CEAlevels.

Blood specimens (10 ml) were obtained in EDTA at the time of voluntary blooddonations for future CEA testing. An additional blood specimen was obtained forSGPT testing from all donors presenting to the UCLA Blood Donor Center d8ring a90 day period in each calendar year. Those specimens were stored at -70 C andretrospectively tested over a two week period following transfusion. During thatperiod all recipients of blood from the UCLA Blood Donor Center were evaluated.Those who lacked evidence of malignancy, who were receiving no hepatotoxic drugs,who had normal SGPT levels prior to transfusion, who denied any history of prior

-. liver, disease and who were willing to participate in a prospective study wereenl isted. All patients receiving one or more blood units with an elevated CEAlevel were matched with patients of the same race, age, sex and smoking history,who had a similar disease state and who had received all volunteer blood In whichno units had CEA elevation. Similarly, all patients receiving a blood unit with anSGPT level over 50 1.1.1. were matched in the same way with patients receiving bloodunits without SGPT elevation. Patients were then followed at two week intervalsfor 10 months. Blood specimens were tested for SGPT and for CEA.

Donors with both SGPT and CEA elevation or with SGPT elevation alone, implicatedIn the transmission of non A non B PTH in this prospective study were reassayed forSGPT and CEA. If persistently abnormal , they were asked to undergo a one unitplasmaphoresis.

Patients developing non A non B PTH during prospective study had 60 ml ofblood drawn weekly (10 ml in EDTA) for the duration of their acute illness. Ifsuch patients had persistent SGPT elevation for greater than six months, they wereasked to undergo a one unit plasmaphoresis.

All serum and plasma were stored at -70°C pending further processing. Thus,the potential sources of non A non B PTH virus/antigen available for examinationIncluded :

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1) Donors with an elevated CEA and SGPT impl icated in the transm ission ofnon A non B PTH.

2) Donors wi th an elevated SGPT but wi th normal CEA implicated in thetransmission of non A non B PTH associated with a normal CEA levelduring the acute phase of illness.

3) Recipients of such blood with acute and chronic non A non B PTH.

4) Outdated bank plasma with CEA elevation and/or SGPT elevation

2. Sources of Non A Non B PTH Antibody

Patients with non A non B PTH identified by the prospective studies werefollowed for resolution of their hepatitis (return of the SGPT l evel to nor”:l).After six weeks to six months convalescence from non A non B PTH, patients wereasked to undergo one-unit p~asmaphoresis at the UCLA Blood Donor Center. Plasmawas coded and stored at -20 C. These samples will provide the source of non Anon B antibody.

3. Controls

Age and sex matched donor controls and outdated plasma controls with normalSGPT and CEA levels were coded and stored at -20 C. Age , sex and disease statematched hospital controls who were either not transfused or transfused withblood without an elevated SGPT or CEA level , were part of the above describedprospective study. Their serum samples, stored at -70°C are available forcontrolled comparison.

4. Tissue Cul ture

Serum samples likely to contain non A non B PTH virus (see 1 above) andvirus-like material derived from affinity chromatography and ultracentrifugationwill be inoculated into tissue culture cell lines available and in use in thislaboratory.

a. Hepatocyte Cultures

Hepatocyte cultures will be established using explants of adult and fetalliver tissues. Explants will be cultures in growth medium consisting of RPMI1640 med ium fortified with 20% fetal calf serum, 600 mgm/L bactopeptone, 300mgm/L of L—glutamine and 136 Mu/L of crystalline insulin. Cultures will begrown in a CO incuba tor at 37°C and the liberated floating cells will be collectedon the 8th da~. They will then be centrifuged in Ficol-Corary (24:10) for 30minutes at 1500 rpm. Oval cells will be distributed under the lymphocyte layer.The oval cell fraction will be removed and will be used as a substrate forsubsequent studies.

0~aj cell suspension cul tures will be used to evaluate the report of Watanabe,et al , ‘ regarding the reproducibl e subpassage of hepatitis B virus in oval cells.The inocula described above will be used for these studies. When and if confirmed,add itional hepatocyte culture experiments will be undertaken. The oval cell richfraction will be planted as an explant culture in falcon plastic dishes. Once anadequate monolayer has been developed using an explant technique of a previouslyIsolated oval cell rich fraction, the growth medium described above will be alteredIn that the concentration of fetal calf serum will be reduced to 2% for maintenance.

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In~ addi tion to the employment of monolayer cultures,oval cells will be grownin co-cultivation with human dip loid lung cells, Hep-2 cells and HeLa cells. Forea~h of these studies , one volume of hepatocyte cells containing approximately10 via ble oval cells per ml wi l l be plan ted in a speciall y devised falconplastic dish separated by a small elevated line which passes through the middleof the d ish. On the other side of the dish , cel l s such as HeLa , Hep-2 or humandi ploid lung cells will be planted in the same concentrations. Each monolayerwill be grown in the same dish and will be bathed by the same growth andmaintenance media. The hepatocytes will be inocul ated with each of the inoculalisted below in the hope that viruses may repl icate wi thin the hepatocytes andmay then pass to the other tissues where they may more easily demonstratecytopathic change or positive imunofluorescence. Because of differences ingrowth rates, it may be necessary either to plant on different days or to alterthe frequency of fluid changes. Control cultures will include cultures of tissuesubstrates only and each of the standard tissue cul tur~~separately. Cul tureswill be examined daily for evidence of cytopathic change and a portion of eachgroup of cultures will be examined every four days for specific imunofluorescence.Any cul tures showing cytopathic change or positi ve immunofluoréscence wi ll beseriall y passaged to establish a viral pool of sufficient size for further stud ies.If control cultures reveal the presence of adventitious agents, such as Simianvirus (SV-40), that line of tissue will be discarded and fresh cultures will bereinoculated with the previous passage level . Tissue culture aliquots from eachpassage l evel will be stored in liquid nitrogen for future reference and ifnecessary for replanting. Irnmunofl uorescence, electron microscopy and imunoelectronmicroscopy will be used to search for evidence of viral growth. Indirect imuno-fluorescence wi ll be employed in selected cul tures using the sera of patients withhigh l evels of hepatitis B surface antibody as determined by the hepatitis Bsurface antibody radioimmunoassay. Human antibody to hepatitis B antigen and goatantibody to hepatitis B antigen are also available for use. Selected cultureswill be examined by electron-microscopy and by innunoelectron microscopy forevidence of viral replication. Cultures will be removed at eight day intervals forelectron microscopic studies. If any agent is detected , or if evidence of viralgrowth is demonstrated , attempts will be undertaken to neutral ize the agents using

- convalescent hepatitis sera. If feasible , the virus will be adapted to and seriallypassaged in Riff free chick embryo culture in order to produce a viral vaccineappropriate for federal licensure.

b. Viral Inocula

Watanabe has used plasma from patients with fulminant hepatitis B for hissuccessful studies of hepatitis B surface antigen cultivation. We have availableto us a unique source of sera from such patients. This large medical center in1975 and 1976 cared for more patients with fulminant hepatitis than any other medicalcenter in the United States and entered more patients in the National Fulm inantHepatic Failure Collaborative Program. We have available to us sera from suchpatients rich in hepatitis B surface antigen and rich in Dane particles and wepropose to use representative plasma for these viral inoculation studies. Inaddition , we have collected sera from patients found to have hepatitis A. Serahas been collected at seven day intervals from patients who initially lackedhepatitis A antibody but subsequently, after exposure, developed acute viral hepatitisand eventually developed high levels of anti—hepatitis A. Such serial plasmspecimens will be available for these viral cultivation studies. Finally, we alsohave available al iquots from blood units which produce non A non B hepatitis onprospective follow-up of recipients as previously described .

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C.’ Non A Non B Culture Inoculation

Serum inocula will be dilu ted 1:4 in growth medium and a O~3 ml of inoculum wi ll beused per tube or flask. Pelleted , virus-like material derived from affinitychromatography and ultracentrifugation will be resuspended in , and dialyzed in ,three changes of normal saline. 0.3 ml of this material will be inoculated intoeach of four tissue culture tubes and flasks. Control sera, similarly diluted andsimilarly processed by column chromatography and ultracentrifugation will be usedas control inocula. Viral replication will be assessed by daily microscopicobservation and , after one to two weeks, by electron microscopy of fixed monolayers.

RESULTS

In a pi lot program, we evaluated nieans of preventing non A non B hepatitis.Prior to transfusion, patients were identified who lacked evidence of malignancyor chronic l iver disease, who had no history of blood transfusions in the prior12 months , who had not been adm inistered hepatotoxic drugs , and who were likel yto survive for a ten month period of follow-up. Blood units were obtained fromboth commercial and volun tary donors. Al iquots from the transfused un~,ts wereobtained in ethylenediaminotetraacetic acid (EDTA), were stored at -70 C and wereretrospectively screened using the CEA radioirnniunoassay of Hansen , et al (CEA Roche,Hoffmann-La Roche, Inc., Nutley , New Jersey). We had previously observed thefrequent development of this fetal antigen during PTH. After identification ofCEA positive blood units , the recipients of these units were sought. Recipients ofCEA positive blood were selected on the basis of having received units containingCEA in concentrations greater than 2.5 ng/ml and subsequently were divided intothose with CEA levels between 2.5 and 4.9 ng/ml and those above 4.9 ng/ml . All ofthe other blood units transfused to these patients as wel l as all blood unitstransfused to patients in the control groups were then screened for the presenceof CEA .

For each recipient of CEA positive blood , two control subjects were randomlyselected. One control subject was a recipient of the same number of blood units ,all of which were negative for CEA. These controls were matched for sex, age,race and the total number of blood units , coninercial and otherwise received . Theother control subject was a patient without malignant disease of the same race, age ,and sex, who was hospitalized but who received no blood transfusions.

All blood units were free of HBsAg as determined by RIA (Ausria II , AbbottLaboratories, North Chicago, Ill inois). A pre-transfusion specimen was obtained inEDTA and 30 ml of plasma was removed from every blood unit administered to eachstudy patient and also placed in EDTA for future CEA testing. In calculating the CEAresults, the dilutional effect of the acid citrate dextrose anticoagulant of theblood was taken into account. All patients and controls were followed for tenmonths. Plasma specimens in EDTA and serum specimens were obtained prior totransfusion and at two week intervals for the ten months of follow-up. Serumspecimens were tested for HBsAg , anti-HBs (Ausab, Abbott Laboratories , North Chi cago ,Ill inois), bilirubin , alan ine amino transferase (SGPT) and antibody to hepatitis A(RIA , Abbott Laboratories, North Chica go, Ill inois).

Hepatitis was defined as an SGPT greater than five times the upper limit oflaboratory normal (upper l imit normal = 45 I.U.) on one occasion and at leasttwo times the upper l imit of laboratory normal on a separate occasion at least fivedays apart in the absence of any other likely cause of liver disease. Type B viral

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hepati1~is was defined as the development of HBsAg during the acute phase ofhepatitis as described above or the development of anti-I-lBs during convalescence .

The initial screening of 486 HBsAg negative units revealed 86 (18%) withCEA concentration over 2.5 ng/ml , 50 (10%) of the~~un its had CEA concentrationsover 4.9 ng/ml . Among the 86 recipients of this blood with abnormally elevatedCEA , 29 (34%) developed non A non B PTH. All 29 of these patients had transientelevation of their CEA l evels to above 2.5 ng/ml duri ng the acute phase of theirillness. Using the higher cut off for abnormal CEA of 4.9 ng/ml. we found that23 (46%) of these recipients developed non A non B PTH with transient CEAelevation. Of the 86 recipeints in the control group, al l of whom received bloodwith CEA levels below 2.5 ng/ml , 8 (9%) developed non A non B PTH. In none ofthese eight cases was there an elevation of the recipients ’ CEA level to above2.5 ng/ml during their hepatitis. Among the non-transfused hospital controls ,there were no cases of hepatitis.

Because blood donors whose blood units transmi t hepatitis may themselveshave asymptomatic l iver disease, the transfused blood was also screened for SGPT.Of 16 recipi ents of blood uni ts with SGPT levels over 50 I.U. and CEA levelsover 4.9 ng/ml , all developed hepatitis while ten recipients of blood with elevatedSGPT and normal CEA levels failed to develop hepatitis.

DISCUSSION

Viral hepatiti s remains a critical national health hazard . By recentestimates there are over 30,000 cases of post transfusion hepatitis (PTH) each yearresulting in approximately 3,000 deaths and additional unestimated morbidity fromchronic hepatitis. Historically two forms of viral hepatitis were distinguishedby their lack of cross-irnmun~t~ in human transmission experiments and otherepidemiological differences. Infectious hepatitis (type A viral hepatitis)was found to have a short incubation period of from two to six weeks, and wastransmitted by contaminated food, water and by the fecal-oral route. In contrast,homologous serum jaundice (type B viral hepatitis) was found to have a longerincubation period of three weeks to six months and was mainly transmi ttedparenteral ly by contaminated serum. Later, detailed epidemiologica l studiesof PTH (the “homologous serum jaundice” described by McCallum , et al , Paul , et al ,and Neefe, et al) by Allen and Sayman, revealed a skewed unimodal distri butionof incubation periods , not the bimodal curve one would anticipate if PTH were theresult of a combina tion of types A and B hepatitis. However, advancement in thisarea of research was not possible until the discovery of the Australia antigen byBlumberg , et al , and its eventual identification as the hepatitis B surfaceantigen (HBsAg). With the development of successively more sensitive tests forthe detection of HBsAg and other hepatitis B viral markers, particularly . artibodyto the hepatitis B core antigen (anti-HBc), it became evident that 50%-70% of PTHwas not a consequence of hepatitis B virus infection. Furthermore, epidemiologicalevidence ind icated that hepatitis A was also not responsible and objective serolpgyexcluded the Epstein-Barr viru s and cytomegalovirus as etiologic agents. Thediscovery by Feinstone, et al , of the hepatitis A antigen in the stool ofexperimentally infected humans by immune electron microscopy and the resultingobjective serological test for hepatitis A finally provided firm serologicalevidence that non B PTH was also not type A viral hepatitis. These data have beenamply confirmed at several centers using the newer assays for antibody to thehepatitis A antigen , imune adherence hemagglutination and radioimunoassay (RIA).

Several studies have assessed the reduction in PTH resulting from the use ofsensitive methods for HBsAg screening , the use of volun teer as opposed to commercial

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blood donors and the use of prophylactic gamaglobulin to prevent PTH. Testingfor UBsAg by RIA or a test of equivalent sensitivity has resulted in at least

• 50% and possibly a 90% reduction in type B PTH but has had no impact on non Anon B PTH. The substi tution of vol unteer blood donors resul ted in a markeddecrease in both types B and non A non B PTH. But even using both a volunteerblood supply and RIA testing for HBsAg , there was left a 5%-15% incidence of non Anon B PTH among transfused patients. While the prophylactic use of human immuneserum globu lin has reduced the icteric , overt, acute phase of PTH , the overallincidence has remained unchanged . The morbidity of this disease is increasedby the frequent progression of acute non A non B PTH to chronic hepatitis. The

F . reported incidence of chron ic hepatiti s follow i ng non A non B di sease var ies from23% in the study of Knodell , et al , in which some patients received prophylacticgammaglobulin to 70% in the study of Koretz, et al , in which 85% of bloodtransfused was commercial and in which gamaglobul i n was not used. The reportby Knodel l , et al , that prophylactic human immune serum globul in may reducethis progression to chronicity needs to be confirmed . But even with gammaglobulinprophylaxis , substantial progression to chronicity remains. Many of these patientshad biopsy features of chroni c hepatiti s and cirrhosis, diseases which in otherinstances have been shown to progress relentlessly.

The close epidemiological and clinical similarity between type B hepatitisand non A non B hepatitis makes it likely that the latter is also associated witha chronic viremia or chronic carrier state. Evi dence of such a chronic carrierstate was reported by Hoofnagle , et al , who found , in reanalyzing the humantransmiss ion experiments by Murray, et al , that three donors implicated in thetransmiss ion of non A non B hepatiti s transm itted the di sease on separate occasionsas much as 385 days apart. These data suggest that the reservoir for non A nonB PTH is found in the l iver and circulation of chronic carriers making carrierblood an appropriate target for investigations which might result in theidentification and isolation of the presumed viral agent or agents of this disease.However, there is no reason to believe that the viremia of non A non B PTI-1 willhave any of the un ique features of hepatiti s B which made the latter assayble byrelatively insensitive means. Thus , the mi ll igram quan titi es of excess HBsAgcircula ting in chronic hepatitis B are un ique in vi rology. In contrast, theviremia of other human diseases comprise quan titi es of whole virus not assayabl eby even the most sensi tive methods , such as RIA , in unconcentrated serum specimens.Instead the isolat ion of non A non B hepatiti s antigen from blood would be expectedto require biophysical and immunological concentration as was required to isolatethe core of the complete hepatiti s B virus from plasma .

Reproducible and reliable techniques were developed both for short and longterm maintenance of hepatocytes in monolayer tissue cul tures. These cultureswere free of fibroblastic cells and retained epithelial characteristics . They weremaintained for periods i n excess of ten weeks. The development of this substrateconstituted the in iti al phase of a program wi th the eventual goal being thedevelopment of a vi ral vaccine for hepatiti s B and for other forms of hepatiti s.The second phase of this program undertook to cultivate hepatitis virus inhepatocyte tissue cultures . Initial studies demonstrated electron-microscopicevidence of hepatitis B antigen within tissue culture cells during the first weekafter inoculation. Serological studies demonstrated evidence of hepatitis Bsurface antigen in pour off fluids for periods ranging from four to ten days afterinoculation. Recent studies aimed at the long term maintenance of infected ovalcell cultures have not yet revealed direct evidence of viral replication in ovalcell suspension cul tures. However, in related studies it has been found that blood

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units with elevated SGPT and CEA l evels usually transmit hepatitis. Aliquots fromun its known to have transmi tted hepatitis have been obtained and these will beused as inoculum for new tissue cul ture studi es.

Epidemiological evidence suggest that there may be more than one virus ofnon A non B PTH. Thus , Mosley , et al , reported three patients with four distinctbouts of hepatitis. In each case two of these four bouts could not be related toeither the hepatitis A or B viruses by sensitive serological assays. Most ofthese attacks occurred in the context of intravenous drug abuse. Independentev idence to support the existence of more than one non A non B hepatiti s viruswas developed by our group which showed two di sti nctive serolo gi cal patterns inthis d isease. In one form of non A non B PTFI , implicated donor blood was found tohave elevated carcinoembryonic antigen (CEA) levels above 4.9 ng/ml . Thetransfus ion of such bloo d resul ted i n a form of non A non B PTH whi ch wasassociated wi th abnormally elevated CEA level s in the recipient during the acutephase of the disease. When other patients , transfused with blood containing normallevels of CEA , developed non A non B PTFI it was invariably without CEA elevation .There was no “cross-over” betwe..~rm these two groups , that is blood with a normal CEAl evel never resulted in hepatiti s assoc iated with an acutely increased CEA level ,while bl ood with an elevated CEA level never resulted in PTH with normal CEAlevels during the acute illness.

Research in non A non B hepatitis is currently at a point similar to thatof hepatiti s B research before the d iscovery and understand ing of the Austral iaantigen (HBsAg) or hepatitis A research before the detection of the hepatitis Aantigen. The disease has been defined epiden riologically, clinically and by theexclusion of other etiologies but further progress in this field awaits theidentification and isolation of the responsible agents or their antigenic markers .The overall pro blem can be reduced to that of identifying sources of non A non Bhepatitis antigens and antibodies followed by isolation in sufficient quantitiesto allow for spec i f ic serologi c assays.

- CONCLUSIONS

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In summary, in our earlier work we were abl e to develop reproducibl e andreliable techniques for the long term maintenance of adult and fetal hepatocytesin monolayer cultures . These hepatocytes were shown to produce albumin andsubsequently to produce alpha-l-fetoprotein. In these studies , persistence ofhepatitis B surface antigen could be demonstrated for a period of about ten daysbut evidence of viral replication was not obtained . Recently, a new hepatocytecultivation technique employing the use of oval cells in suspension cultures ofliver explants suggested the persistence of hepatitis B antigen when studiei byininunofluorescence. Thus far in ou.r studies only non-specific irnmunofl uorescencehas develo ped and ev idence of actual v iral repl ication is lac king. However ,recent ev idence has shown that blood units whi ch contain eleva ted SGPT levelsand elevated CEA levels have produced hepatitis in each of 16 recipients of suchunits. Al iquots from these units have been frozen away for testing in tissueculture systems to determine if the systems will support hepatitis virus growth.

ACCOMPLISHMENTS TO DAT E

This laboratory has developed unique techniques for the cultivation of hepato-cytes in monolayer culture . We have methodically moved through a series ofdevelopmental phases wi th the eventual goal being the development of liier attenuatedhepatitis virus vaccines. Initially, techniques for the successful cultivation offetal hepatocytes and subsequently techniques for the cultivation of adult hepato-cytes were developed. We documented the first evidence of long-term cultivation of

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fetal hepatocytes for periods of several weeks . These long term hepatocyte cultureswere shown to produce al bumin and a l pha-1-fetog lobulin. Studies identifying the cor~1itionsneces sary for v i ral attac hment, infection and replication were undertaken. Optima lconditions for growth of hepatocytes including temperature , CO2 concentration andserum concentrations were determined.

Once the technology for the maintenance of long-term hepatocyte cultures wasestabl ished , attempts were made to use these cultures as a substrate for the arowthof hepatitis viruses. Hepatitis B infective material including sera rich in wholevirus particles were inoculated into tissue culture under a var~ety of conditionsand evidence of viral persistence for a period of about ten days was established .However , definitive evidence of viral replication was lacking. In addition ,filtrates of stools of patients with hepatitis A were used as inocula for hepatocytecultures but these failed to demonstrate cytopathic change. Liver biopsies obtainedfrom patients with hepatitis B surface anti gen positive chronic aggressive hepatitiswere grown in monolayer culture and in co-cultivation both with normal hepatocytesand with other standard tissue culture substrates. These, however, failed todemonstrate evidence of viral replication. The adaptation of “slow v i rus tec hniq ues ”including co-cultivation , feeder layer cultures , and long-term maintenance culturesalso failed to yield evidence of viral growth as did the use of “complexes ” ofvirus , antibody and complement.

We have undertaken to develop systems for the production of tissue culturesubstrate thought to be likely to support hepatitis virus growth. We have evaluatedthese new tissue culture systems in carefully controlled studies. The followingdescribes the results of this program over the past six months.

Hepatocyte cultures were established using explants of adult and fetal livertissue. Explants were grown in a C02 incubator at 37°C and the liberated floatingcells were collected on the eighth day. They were centrifuged for 30 minutes at1800 rpm. The oval cell fraction was removed and was used for a substrate forsubsequent studies. In these studies the oval cell fraction was planted as an 6explan t culture in Falcon plas ti c di shes us ing the techni que descr ibed by Wa tanabe.

Cultures were inoculated with plasma from patients with fulminant hepatitis B richin hepatitis B surface antigen. The cultures were then examined daily for the develop-ment of cytopathic change and a portion of each group of cultures was examined forspecific immunofluorescence. Cells were also embedded in Epon and examined forelectron microsco pi c ev idence of v i ral particles .

Because bl ood donors whose b lood units transmit hepatiti s may themselves haveasymptomatic liver disease , it thus , woul d be a valuable resource for cultivationof non A non B hepatiti s v i rus. Trans fused blood was screen ed for SGPT and forCEA the latter hav ing been shown to develop dur ing the course o f acute hepatiti s.The units of blood conta ining CEA and SGPT eleva tions were id enti f ied and have beeninocula ted into hepatocyte tissue culture systems.

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BIBLIOGRAPHY OF PERTINENT PUBLICATIONS

1. Koretz RI, and Gitnick GL: Chronic sequelae of post transfusion hepatitis.Gastroenterology 66:726, 1974.

2. Krugman S, Su es JP and Hammond J: Infectious hepatitis: evidence for twodistinctive clinical epidemiological arid immunological types of infection.JAMA 200:365-373, 1967.

3. Feinstone SM, Kapikian AZ, Purcell RH, et al: Hepatitis A detection y immuneelectronmicroscopy of a virus -like antigen associated with acute illness.Science 182:1026-1028, 1973.

4. Hi lleman MR. Provos t PH, Mil ler WJ , V i llarejos VM , Ittensohn OL , McA 1~c WJ:Immune adherence and complement-fixation tests for human hepatitis A.Diagnostic and epidemiologic investigations. Presented at Internationa~Association of Biological Standardization (lABS), Symposium on Viral Hepatitis ,Mi lan , Italy, December 16-19, 1974.

5. Hi lleman HR, Provos t PJ , Miller WJ , et al: Experimental hepatitis B vaccine.4 International Association of Biological Standardization Symposium on Viral

Hepatitis. Milan , Italy, December 16-19, 1974. -

6. Watanabe M , Ohor i H, and Ishida N: British Journal of Experimental Pathology57:211 , 1976.

7. Ibid (In Press).I

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_______________________________________ Annual Report March 1 , 197~~CULTIVATION OF HEPATITIS~V IRUS IN TISSU E CULTURE’ to February 28 , 1979

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~~~~~ G.L./Gitn~~~~ M.D. ~~~~~~N 0OO i4- 76 C-i24j J —

9. PE RFo RM ING O R G A N I Z A T I ON NAME AND A O D R LS S *0 PflL ~GNA M ELEMENT . P R O J E C T , T A S K

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1$. S UPPLEMENTARY NOTES

19 KEY WORDS (Ccr.tlnue an reverse side if n.c.aaary and id.ntify by block num ber)

Hepatitis virus Hepatitis VaccineHepatitis B Antigen Non A Non B HepatitisTissue CultureCultivation of Hepatitis VirusHepatocyte cultures

20 ABSTRACT (Continu, on rever s e aid. If necessary end Identif y by Sto c k nimib.,)

Since hepatitis virus has the propensity in vivo to infect h~ ‘ai hepato-cytes it seemed appropriate to undertake attempts at hepatitis v ii :cultivation using hepatocyte cultures . Early studies led to technology for

7 the cultivation of fetal hepatocytes wi th the exclusion of fibroblastic cells.~~~~

~~~ - Siin.iJ r approaches were used to cultivate adult hepatocytes and most recentlythesë~studies have demonstrated the successful long-term cultivation of adulthepatocytes for periods in excess of 10 weeks. The conditions for growth —.>

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including temperature, serum content and appropriate antibiot ic~~~ave beendelineated and the methods —de-velope4,~ are now reproducible. —.-.,

fln the past twelve months , attempts ~~~ ~~~~~ade to use hepatocytecultures as a substrate for hepatitis B and hepatitis A cultivation. Theinocula used have been hepatitis B antigen positive sera known to be rich inDane particles , filtrates of stools from patients with hepatitis A and liverbiopsies obtained from patients with hepatitis B surface antigen positivechronic aggressive hep,~titis. Inoculation of cultures with Dane particle-rich hepatitis B seral has not resulted in cytopathogenic change (CPE) inmonolayer cultures. Efforts have been devoted at attempts at cultivationof hepatitis B virus by using *slow v i rus tec hniques 11 . By means of co—cultivation , mi xed monolayers of normal hepatocytes were co-cultivated withhepatocytes obtained from liver biopsies of patients with hepatitis B surfaceantigen positive chronic aggre.ssive hepatitis. The cell layers which -

resulted were maintained and were sub-cultured but did not yield evidence ofeither CPE or evidence of multi plication of hepatitis B surface antigen.~.Recently inoculated suspension oval cell cultures have demonstrated posit\veimmunofluorescence for HBsAg suggesting possibl e early vira l growth .

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