Animal model for hepatitis virus infection

Chimeric mouse and rat models useful as models for human hepatitis virus (HV) infection are disclosed. These chimeras are made by substantially destroying the hematopoietic cells of a host mouse or rat and then transplanting into the resultant animal hematopoietic cells from SCID mice. The resultant chimera is then used as a host for transplantation of xenogeneic liver tissue, including liver tissue from humans. The liver tissue may be infected with HV either prior to or after transplantation.

FIELD OF INVENTION 
The present invention concerns an animal model for hepatitis virus (HV) 
infection in humans, particularly hepatitis B virus (HBV) and hepatitis C 
virus (HCV) infection. 
PRIOR ART 
The following is a list of prior art and references considered to be 
pertinent for the description below: 
1. Choo, Q-L, Kuo G, Weiner, A. J. Overby L. R., Bradley D. W., Houghton, 
M. Isolation of CDNA clone derived from a blood-borne non-A, non-B viral 
hepatitis genome. 1989. Science 244:359-362. 
2. Kuo G, Choo Q-L, Alter H. J., Gitnick, G. L., Redeker A. G., Purcell R. 
H., Miyamura T, Dienstag J. L., Alter H. J. Stevenes C. E., Tegtmeier G. 
E., Bonnino F., Colombo M, Lee W-S, Kuo C, Berger K, Shuster J. R., Overby 
L. R., Bradley D. W., Houghton M. 1989. An essay for circulating 
antibodies to major etiologic virus of human non-A, non-B hepatitis. 
Science 244:362-344. 
3. Prince, A. M., Brotman B., Huima T., Pascual D., Jaffery M. Inchauspe G. 
1992. Immunity in hepatitis C infection. J. Infec. Dis. 165:438-443. 
4. Shimizu Y. K., Weiner, .J. Rosenblatt J., Wong D. C., Shapiro M., Popkin 
T, Houghton M, Alter H. J., Purcell R. H. 1990. Early events in hepatitis 
C virus infection in chimpanzees. Proc. Natl. Acad. Sci. (USA) 
87:6441-6444. 
5. Shimizu, Y. K., Iwamoto A., Hijikata M., Purcell R. H., Yoshikura H. 
1992. Evidence for in vitro replication of hepatitis C virus genome in a 
human T cell line. Proc. Natl. Acad. Sci. (USA) 89:5477-5481. 
6. Shimizu Y. K., Purcell R. H., Yoshikura H. 1993. Correlation between the 
infectivity of hepatitis C virus in vivo and its infectivity in vitro. 
Proc. Natl. Acad. Sci. (USA) 90:6037-6041. 
7. Nakamura T., Good R.A., Yasumizu R., Inoue S., Oo M. M., Hamashima Y, 
Ikehara S. 1986. Successful liver allografts in mice by combination with 
allogeneic bone marrow transplantation. Proc. Natl. Acad. Sci. (USA) 
83:4529-45326. 
8. Bosma, M. J. Carroll, A. M. 1991. The SCID mouse mutant: Definition, 
characterization, and potential uses. Annu. Rev. Immunol. 9:323-350. 
9. Soriano, H. E., Adams, R. M., Darlington G., Finegold M. Steffen D. L., 
Ledley F. D. 1992. Retroviral transduction of human hepatocytes and 
orthotopic engraftment in SCID mice after hepatocellular transplantation. 
Trans. Proc. 24:3020-3021. 
10. Aldrovandi G. M., Feurer G., Gao L., Jamieson B., Kristeva M., Chen I. 
S. Y., Zack J. A., 1993. The SCID-hu mouse as a model for HIV-1 infection. 
Nature 363:732-736. 
11. European laid open Patent Application, Publication No. 438053. 
12. European laid open Patent Application, Publication No. 517199. 
The citation herein of the above publications is given to allow an 
appreciation of the prior art. This citation should not, however, be 
construed as an indication that this art is in any way relevant to the 
patentability of the invention, as defined in the appended claims. 
The above publications will be acknowledged herein by indicating their 
number from the above list. 
BACKGROUND OF THE INVENTION 
Five different viruses have been identified as causes of viral hepatitis. 
These include hepatitis A, B, C, D and E viruses. Of these, the viruses 
which cause the most serious infections are hepatitis B virus (HBV) and 
hepatitis C virus (HCV). 
Hepatitis A virus has a single serotype and causes a self-limited acute 
infection. A large percentage of the population, approaching 50%, has 
hepatitis A antibodies in serum and is probably immune to disease. 
Infection with hepatitis A does not progress to chronic disease. 
HBV is implicated in both acute and chronic hepatitis. The disease is 
endemic in Asia, is increasing in prevalence in the U.S. and Europe. 
Chronic liver disease, resulting in significant morbidity and increased 
mortality, is sequela of infection in 1-10% of infected individuals. HBV 
infection is also correlated with the development of primary liver cancer. 
HCV was recently shown to be the major causative agent of parenterally 
transmitted non-A, non-B hepatitis.sup.(1). It is estimated that 0.5-1% of 
the world population is infected with HCV, and in some developing 
countries the prevalence rate is up to 40%. Moreover, 40-60% of newly 
infected patients develop persistent HCV infections.sup.(2) and are at 
risk of developing acute, fulminant hepatitis and various chronic liver 
diseases (including cirrhosis, chronic active hepatitis and in some cases 
hepatocellular carcinoma). 
Hepatitis D virus ("Delta Virus") is a defective RNA virus that can only 
infect the liver in the presence of an active HBV infection. Hepatitis E 
virus appears to be a single-stranded RNA virus. Infection with hepatitis 
E virus is not known to progress to chronic liver disease. 
Although HBV and HCV have been identified and characterized, the 
development of new anti-viral strategies has been greatly hampered by the 
lack of adequate, simple and low cost animal model systems. 
Currently, biological assays for HBV and HCV have been limited to the 
experimental inoculation of chimpanzees.sup.(3,4), which are expensive and 
limited in numbers. In addition, an in vitro system for the propagation of 
HCV was developed in the murine retrovirus infected human T cell lines, 
HPB-Ma.sup.(5) and Molt4-Ma.sup.(6), in which replication of HCV is 
achieved. 
It has recently been demonstrated in several studies that human solid 
organs such as fetal thymus or fetal liver as well as several types of 
tumors were successfully grafted into SCID mice under the kidney 
capsule.sup.(7). In addition, transplantation of other organs such as 
lymph nodes and bone marrow spicules and engraftment of organs to other 
sites (i.e. subcutane and peritoneum) have also been reported. 
A SCID mouse mutant was reported to support human cell implantation, i.e. 
single hepatocyte transplantation.sup.(8,9), and was also used as a model 
for human infectious diseases, i.e. HIV-1 infection.sup.(10). 
It has been disclosed that lethally irradiated mice, radio-protected with 
bone marrow from SCID mice, developed marked immune-deficiency and 
supported engraftment of human peripheral blood lymphocytes (PBL) for a 
long period of time.sup.(11). It was also disclosed that human implants of 
non-hematopoietic origin were accepted and maintained for prolonged 
periods of time after transplantation under the kidney capsules of these 
chimeras.sup.(12). 
GENERAL DESCRIPTION OF THE INVENTION 
It is an object of the present invention to provide a convenient non-human 
animal model for HV infection. 
It is further an object of the present invention to provide a method for 
evaluation of preventive and therapeutic agents for the treatment and 
prophylaxis of HV infections using the above non-human animal model. 
It is still another object of the present invention to provide methods for 
production of anti-HV xenogeneic antibodies or T cells, and particularly 
human monoclonal antibodies and cytotoxic T cells, using chimeric 
non-human mammals transplanted with human hematopoietic cells and human 
liver tissue infected by HV either pre- or post-transplant. 
The present invention provides, by its first aspect, a non-human chimeric 
animal useful as a model for human HV infection, comprising a mammal M5 
having xenogeneic cells; mammal M5 being derived from a mammal M1 treated 
to substantially destroy its hematopoietic cells and then transplanted 
with hematopoietic cells derived from one or more mammals M2 and 
transplanted with liver tissue from a mammal M3, the one or more mammals 
M2 and mammal M3 being from the same or from a different species; the 
transplanted hematopoietic cells from the one or more mammals M2 being 
either one or both of a hematopoietic cell preparation from a T cell 
deficient mammal or of a T cell depleted mammalian stem cell or bone 
marrow preparation; the transplanted liver tissue from mammal M3 being 
either a human liver tissue preparation or a liver tissue preparation from 
a non-human mammal capable of being infected by HV; the liver tissue 
preparation in the M5 mammal being infected by HV. 
The M1 mammal may typically be a mouse or a rat, although the M1 mammal may 
also be a non-human mammal of a higher order such as a primate, e.g. 
marmoset monkeys. 
For the obtaining of an M5 mammal from said M1 mammal, the M1 mammal is 
first treated in a manner so as to substantially destroy its hematopoietic 
system. The term "substantially destroyed" should be understood as meaning 
that the number of hematopoetic cells which survive following the 
treatment are insufficient to immune-protect the animal in the absence of 
the transplant from the M2 mammal. Following treatment intended to 
substantially destroy the hematopoietic cells, some such cells survive but 
the number is small such that the animal could not survive under normal 
laboratory conditions. 
A treatment intended to substantially destroy the hematopoietic cells may, 
for example, be a split dose total body irradiation, (TBI). A TBI 
effective in destroying the hematopoietic system requires typically an 
accumulative dosage of 4-50 Gy (1 Gy=100 rad). In the case of a mouse, the 
irradiation may, for example, be a 4 Gy on day 1 and 9-15 Gy three days 
later. A similar irradiation dose was found to be effective in destroying 
the hematopoietic cells also in rats and marmoset monkeys. 
The M2 mammal may be from the same or a different species than the M1 
mammal. In principle, any mammal with a T cell deficiency may serve as a 
donor for the transplanted hematopoietic cells. An example of an M2 donor 
is a severe combined immuno-deficient (SCID) mouse or a SCID animal from 
another mammalian species or genera. The transplanted hematopoietic cell 
preparation in this case is suitably a bone marrow preparation. 
The transplanted hematopoietic cells derived from the M2 mammal may also be 
a T cell depleted hematopoietic stem cell preparation obtained from a 
donor M2 mammal, such as a primate, e.g. a monkey or a human. In the case 
of humans, a stem cell enriched preparation may, for example, be obtained 
from peripheral blood of donors pretreated with a granulocyte colony 
stimulating factor (G-CSF) or from cancer patients undergoing chemotherapy 
known to cause migration of stem cells to the periphery. After withdrawal 
of the blood preparation from such donors, the preparation is typically 
treated to remove various blood components and to deplete the T cells 
therefrom. For T cell depletion, the M2 derived D hematopoietic cell 
preparation may be subjected to treatment intended for enrichment with 
cells displaying the CD34 antigen (CD34.sup.+ cells). Each of the above 
stem cell enriched, T cell depleted preparations can either be used 
directly after their withdrawal from the donor, or may be a 
cell-preparation which underwent one or a plurality of passages in vitro. 
The transplanted hematopoietic cells may also be a T cell depleted bone 
marrow preparation. 
The M1 mammal may also be transplanted with both a hematopoietic cell 
preparation from a T cell deficient mammal and a T cell depleted mammalian 
stem preparation. A specific example is a combined transplantation of bone 
marrow from a SCID mammal, e.g. a SCID mouse, and a T cell depleted human 
bone marrow preparation. 
In order to obtain the M5 mammal, the M1 mammal may be transplanted with an 
HV infected liver tissue. Such an HV infected liver tissue may be obtained 
from an M3 mammal infected with HV, e.g. a liver biopsy from an HV 
infected human. Furthermore, an HV infected liver tissue preparation may 
also be obtained by in vitro infection of an a priori non-HV infected 
liver tissue preparation obtained from a non-HV infected M3 donor mammal. 
Alternatively, rather than transplanting the M1 mammal with an HV infected 
liver tissue, the M1 mammal may first be transplanted with liver tissue 
not infected by HV, thus obtaining an M4 mammal, and then inoculating the 
M4 mammal with HV leading to infection of the transplanted liver tissue. 
An M4 mammal may thus serve as a model for testing the efficacy of an agent 
in the prophylaxis of HV. In such a model, the putative prophylactic agent 
is administered to the M4 mammal either prior or together with the HV and 
its ability to inhibit HV infection can then be determined. 
In addition to human liver tissue preparation, it is also possible to use 
liver tissue preparations from non-human M3 mammals susceptible to HV 
infections such as chimpanzees or other non-human primates. 
The animal model of the invention is particularly suitable for the study of 
the pathology for HBV and HCV infections and the development of therapies 
therefor. Models for both HBV and HCV are particularly preferred in 
accordance with the invention, as no simple and low cost models for these 
viral infections are currently available. 
The invention further provides, by a second of its aspects, a method for 
evaluating the potential of an agent or a combination of agents in the 
therapy of an HV infection, comprising: 
(a) obtaining an M5 mammal as defined above; 
(b) administering said agent or said combination of agents to said M5 
mammal; and 
(c) evaluating the effectiveness of said agent or said combination of 
agents in preventing spread of HV infection, reducing its physiological 
symptoms or reducing the evidence of active infection in said M5 mammal. 
The present invention still further provides, by a third of its aspects, a 
method for evaluating the potential of an agent or a combination 5 of 
agents, in the prevention of an HV infection, comprising: 
(a) obtaining said mammal M4; 
(b) administering said agent or said combination of agents to said M4 
mammal; 
(c) infecting said M4 mammal with HV; and 
(d) evaluating the effectiveness of said agent or said combination of 
agents in preventing primary HV infection of the liver tissue of said M4 
mammal. 
By a modification thereof, the methods according to the second or third 
aspects, may also be applied in determining the effective dose of said 
agent or said combination of agents in therapy or prevention, as the case 
may be. 
By a fourth of its aspects, the present invention provides a method of 
obtaining anti-HV immune cells or antibodies, comprising: 
(a) obtaining a mammal M5, as defined above, wherein at least one of said 
one or more M2 mammals is human; 
(b) recovering immune cells or antibodies from the blood of said M5 mammal; 
and 
(c) selecting for the immune cells or antibodies having an anti-HV 
reactivity. 
Optionally, in accordance with the fourth aspect, the M5 mammal, is treated 
so as to increase the immune response against HV, such as for example by 
vaccination. 
The selected immune cells may be cytotoxic T cells reactive against HV 
infected liver cells. Such a cytotoxic T cell preparation may be obtained 
by growing lines of T cells obtained from the M5 mammal and then selecting 
those lines which develop a cytotoxic cell response against cells 
expressing HV antigens. Such a cytotoxic T cell preparation may be 
injected to HV patients within the framework of an anti-HV therapy. 
The selected immune cells may also be antibody producing B cells 
immortalized and selected for those producing anti-HV antibodies. The 
antibodies produced by these B cells may then be used as therapeutic 
agents in anti-HV therapies of hepatitis patients. 
The manner of growing cytotoxic T cell lines, the manner of immortalization 
of B cells to produce B cell lines, as well as the manner of selecting 
specific cytotoxic T cells or immortalized B cell lines to obtain those 
having the desired reactivity, is generally known per se and the full 
explanation of such methods goes beyond the present writing.

EXAMPLES 
Example 1 
Engraftment of Human Liver Segment from non-HCV Patients 
BNX mice (6-10 weeks old, female) were purchased from Harlam Sprague-Dawley 
(Indianapolis, Ind.) and CB17/SCID mice were from the Animal Breeding 
Center, Weizmann Institute, Rehovot, Israel. Mice were kept in small cages 
(5 animals in each cage) and fed sterile food and acid water containing 
cyprofloxacin (20 mg/ml). Prior to transplantation, the BNX mice were 
conditioned with 12 Gy TBI and radioprotected the following day with 
2-3.times.10.sup.6 T cell depleted SCID bone marrow cells. TBI was 
administered from a gamma beam 150-A .sup.60 Co source (Atomic Energy of 
Canada, Kanata, Ontario) with F.S.D. of 75 cm and a dose rate of 0.7 
Cy/min. Bone marrow cells obtained from SCID mice (4-10 weeks old) were 
fractionated by differential agglutination with soybean agglutinin (to 
remove T lymphocytes that may be present in occasional "leaky" SCID mice) 
as previously described.sup.(11). One day after bone marrow 
transplantation, human, rat or mouse liver fragments were grafted under 
the kidney capsule. 
Rat and mouse liver tissue fragments were collected through laparotomy, in 
which a wedge biopsy was cut from the animal liver and kept under sterile 
conditions at 4.degree. C., in Dulbecco modified Eagle medium containing 
10% fetal calf serum or ViaSpan (Belzer UW solution, Du Pont 
Pharmaceuticals, Hertogenbosch, The Netherlands). 
Human liver segments were obtained during hepatic segmentectomy when 
performed for primary or secondary liver tumors. In all cases, the 
non-tumor tissue was non-cirrhotic, as confirmed by hematoxylin and eosin 
(H&E) staining. The liver segments were kept, for up to 2 hours in UW 
solution prior to transplantation. For engraftment of liver tissue, BNX 
mice were anesthetized with Nembutol or Avertin. An incision of approx. 1 
cm was then made in the right or left flank, the kidney was exposed and 
liver tissue (cut into 1 mm.sup.2 pieces) was placed under the renal 
capsule using fine forceps. One suture was placed to close the wound. 
Kidneys, with the attached transplanted tissue, were removed at various 
time intervals (from 8 days to 3 months), fixed in Bouin's liquid, 
embedded in paraffin, and 4 .mu.m sections were stained with H&E. 
A summary of the transplantations of liver fragments in the SCID.fwdarw.BNX 
chimeric mice is shown in the following Table 1. 
TABLE 1 
______________________________________ 
Number of Mice Follow-up 
Transplanted 
Surviving 
Engrafted Source of liver 
(weeks) 
______________________________________ 
10 9 4 Mouse 10 
20 12 8 Mouse 3 
10 7 4 Mouse 1.4 
10 10 8 Rat 10 
20 8 5 Rat 8 
20 7 2 Rat 2 
10 5 2 Rat 1.4 
20 11 5 Human 14 
20 11 5 Human 14 
20 11 5 Human 12 
20 10 2 Human 6 
20 9 3 Human 4 
20 16 12 Human 2 
20 15 14 Human 2 
16 12 9 Human 2 
______________________________________ 
As seen from the above table, the survival rate of the chimeric mice 
receiving the human liver graft was at the order of about 50-60%. 
Histological examination of the transplanted liver fragments at this 
subcapsular area of the transplanted SCID.fwdarw.BNX chimera showed that 
following transplantation, the typical liver cell architecture disappeared 
and, in most transplants, central ischemia occurred while the peripheral 
tissue of the transplant was markedly fibrotic (see FIG. 1A-1C). In most 
cases, hepatocytes were recognized in addition to proliferating epithelial 
cells (FIG. 2A-2C) and the engrafted tissue mainly resembled the 
morphological characteristics of biliary epithelium (FIG. 2B and 2C), A 
very mild inflammatory reaction was occasionally observed consisting a few 
polymorphonuclear and plasma cells. 
Evaluation of liver engraftment rate showed that among the mice receiving 
the human liver grafts, 15 out of 33 retained the graft for more than 12 
weeks while 40 out of 62 were stably engrafted at six weeks or less (Table 
1). 
Example 2 
Transplantation of Liver Tissue from a human infected with hepatitis B 
virus (HBV) 
A liver biopsy from a patient infected with hepatitis B virus (HBV) was 
obtained and transplanted under the kidney capsule of SCID.fwdarw.BNX 
chimera mice as described in Example 1. Immmunohistology of the liver 
segment prior to transplantation showed that the hepatocytes stained 
positively for the HBV surface protein (HBsAg) (FIG. 3B). After 
transplantation, evidence was provided for the human origin of the 
transplanted cells by using staining with periodic acid-Schiff reaction 
which identifies glycogen in human hepatocytes (FIG. 3C). 19 days after 
transplantations the subcapsular area of the SCID.fwdarw.BNX chimera 
kidney was immunohistologically stained and HBsAg was detected in discrete 
areas of the cytoplasm in the engrafted tissue but not in the neighboring 
mouse kidney cells (FIG. 3D). 
Example 3 
Transplantation of Liver Fragments Infected in vitro with HBV 
Liver fragments were obtained from non HBV infected humans as described in 
Example 1. The non infected human liver fragments were incubated in vitro 
with HBV resulting in their infection by HBV. The in vitro HBV infected 
liver fragments were transplanted under the kidney capsule of 
SCID.fwdarw.BNX mice as described in Example 1 and the detection of HBV in 
the transplanted mice was assessed either by immunohistology of the 
hepatocytes as described in Example 2 or by testing the level of HBV in 
the serum of the mice by PCR. 
The results are shown in the following Table 2. 
TABLE 2 
__________________________________________________________________________ 
Transplantation Results of in vitro Viral Hepatitis B Infected Human 
Livers in SCID.fwdarw.BNX Mice 
No. of Animals, 
No. of Animals Infection Indicators 
No. of Animals 
Survived 
Engrafted 
Route of Infection* 
and comments** 
__________________________________________________________________________ 
10 5 5 Pre-transplantation in vitro 
PCR +ve (3/5) 
incubation of liver fragments 
with HBV-DNA, +ve sera 
10 7 ND*** Pre-transplantation in vitro 
PCR +ve 1/7 at day 11 
incubation of liver fragments 
PCR +ve 4/7 at day 30 
with HBV-DNA, +ve sera 
10 5 ND Pre-transplantation in vitro 
PCR +ve 1/5 at day 11 
incubation of liver fragments 
with HBV-DNA, +ve sera 
__________________________________________________________________________ 
*+ve sera = sera positive for viremia 
**PCR +ve = PCR test positive for hepatitis virus DNA 
***ND = No Data: Mice have not yet been sacrificed for histological 
examination. 
As seen in the above table, HBV sequences were observed in the sera of some 
of the transplanted mice about ten days after transplantation and with 
progression of time after transplantation HBV sequences were observed in a 
larger number of transplanted mice. 
Example 4 
Detection of HCV in sera of mice transplanted with liver fragments from HCV 
infected patients 
Liver fragments from three patients with chronic HCV infection were 
obtained transplanted under the kidney capsule of SCID.fwdarw.BNX mice as 
described in Example 1. The presence and level of HCV in the serum of the 
transplanted mice was assessed by reverse transcriptase-nested-polymerase 
chain reaction (RT-PCR) as follows. 
RNA was dissolved in 10 .mu.l RNAase-free water. cDNA was synthesized using 
50 ng of the antisense primer ASI in a reaction mixture containing 
2.times.Taq polymerase buffer (Promega Corp., Madison, Wis.) 0.5 mM dNTP, 
20 units RNAsin (Promega), 10 mM dithiothreitol and 30 units avian 
myeloblastosis virus reverse transcriptase (Life Sciences, Bethesda, Md.) 
for 60 min. at 42.degree. C. PCR was performed in reaction mixture volume 
of 50 .mu.l containing Taq Polymerase buffer (Promega), 2 mM dNTP, 1.5 mM 
MgCl.sub.2, 20 ng of sense primer SI and 2.5 units Taq Polymerase 
(Promega). The reaction was carried out by 35 cycles of PCR consisting of 
94.degree. C. for 1.5 min. 55.degree. C. for 1.5 min. and 72.degree. C. 
for 3 min. The second PCR reaction was performed as before, with 5 .mu.l 
of the first PCR reaction mixture and the nested set of primers SII 
(sense) and ASII (antisense). The two sets of primers used are from the 
highly conserved 5' untranslated region (5' UTR). 
The following primers were used: 
SI 7-26: 5'-CAC-TCC-ACC-ATA-GAT-CAT-CCC-3' (SEQ ID NO:1). 
ASI 248-222: 5'-ACC-ACT-ACT-CGG-CTA-GCA-GT-3' (SEQ ID NO:2). 
SII 46-65: 5'-TTC-ACG-CAG-AAA-GCG-TCT-AG-3' (SEQ ID NO:3). 
ASII 190-171: 5'-GTT-GAT-CCA-AGA-AAG-GAC-CC-3' (SEQ ID NO:4). 
The results obtained following transplantation of HCV infected liver 
fragments is shown in the following Table 3: 
TABLE 3 
______________________________________ 
Transplantation results of liver fragments 
from HCV infected human patients in SCID.fwdarw.BNX mice 
Number of Mice 
Transplanted with HCV Stably Viremia 
infected liver fragments 
Surviving Engrafted 
positive 
______________________________________ 
20 17 15 8 
17 14 10 7 
13 11 6 5 
12 10 ND* 6 
______________________________________ 
*ND = No Data: mice have not yet been sacrificed for histological 
examination 
HCV sequences were first observed in the sera of the transplants mice two 
weeks post transplantation and contiiiued to be detected intermittently 
for about two months after transplantation, at which time animals were 
sacrificed (a typical experiment is shown in FIG. 4A and 4B). Similar 
fluctuations in detection of the HCV RNA have been previously observed in 
chimpanzees experimentally infected with HCV and in chronically infected 
patients, probably resulting from the very low levels of the virus in the 
serum. 
Example 5 
Transplantation of Liver Fragments Infected in vitro with HCV to C3H Mice 
C3H mice, which are not of an immune deficient strain, were irradiated by 
split total body irradiation (TBI) (a first dose of 400 rads and a second 
dose of 1,200 rads) and radioprotected the following day with 
3.times.10.sup.6 SCID bone marrow cells (as described in Example 1 above). 
Liver fragments were obtained from non HCV infected humans as described in 
Example 1 and the non infected human liver fragments were incubated in 
vitro with HCV resulting in their infection by HCV. The in vitro HCV 
infected liver fragments were transplanted under the kidney capsule of the 
C3H mice as described above and the detection of HCV in the transplanted 
mice was assessed by testing the level of HCV in the serum of the mice by 
RT-PCR as described in Example 4 above. 
The results are shown in the following Table 4: 
TABLE 4 
__________________________________________________________________________ 
Transplantation Results of in vitro Viral Hepatitis C Infected Human 
Livers in C3H Mice 
No. of Animals 
No. of Animals Infection Indicators 
No. of Animals 
Survived 
Engrafted 
Route of Infection 
and comments*** 
__________________________________________________________________________ 
10 10 ND* Pre-transplantation in vitro 
PCR +HCV 2/10 
incubation of liver fragments 
at day 14 
with HCV-DNA, 
+HCV sera** 
__________________________________________________________________________ 
*ND = No Data: Mice have no yet been sacrificed for histological 
examination 
**+HCV sera = sera positive for HCV 
***PCR +HCV = PCR test positive for hepatitis C virus DNA. 
The results shown in the table above, demonstrate for the first time, that 
human liver fragments infected in vitro with HCV may be engrafted and 
result in the infection of the transplanted mice with HCV as assessed by 
the detection of HCV sequences in the sera of the transplanted mice. 
In addition, the above results, for the first time, show that human HCV 
infected livers may be transplanted under the kidney capsules of C3H mice, 
which are not of an immune deficient strain. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 4 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CACTCCACCATAGATCATCCC21 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ACCACTACTCGGCTAGCAGT20 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
TTCACGCAGAAAGCGTCTAG20 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GTTGATCCAAGAAAGGACCC20 
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