PEN.017.

H-/
24. A. J. Weinnr a a!., J. Med. Vint. 2 1 , 239 (1987). Mulknbach, W. Rmtcr, and R . Halkwell for critical (13-15). I n this w a y , a S O D / H C V polypep­
25. I . M. Chi/guirt, A. H. Prxybytt, R_ J. MacDonald, review of the manuscript and T . White for typing.
W. J. Rutter, BlWtnntr/ry 38, 5294 (1979). Supported by- Chiton Corporation, Ortho Diagnos­ tide ( C 1 0 0 - 3 ) c o n t a i n i n g 3 6 3 viral a m i n o
26. P. S. Thomas, Pm. /Vjrf, Acad. Sti. U-S.A. 7 7 , tic Systems Ltd., and Ciba-Geigy. acids w a s synthesized a t h i g h levels (—4%
5201 (1980). total p r o t e i n ) in r e c o m b i n a n t yeast. A f t e r
27. We think R_ Spaetc, E. Penhoet, P. Valenaucla, G, 24 January1989; accepted 10 March 1989
solubilization a n d purification, C 1 0 0 - 3 w a s
used t o c o a t t h e wells o f microliter plates s o
t h a t circulating H C V antibodies i n b l o o d
samples could b e c a pture d a n d measured.
An Assay for Circulating Antibodies to a Major Detection o f b o u n d a n t i b o d y was achieved
Etiologic Virus o f Human Non-A, Non-B Hepatitis w i t h a radioactive second antibody.
Initially, t o test t h e specificity a n d sensi­
tivity o f t h i s assay, sera o f k n o w n N A N B H
G . K u o , Q . - L . C H O O ,H . J . A L T E R , G . L . G I T N I C K , A . G . R £ D E K E K ,
infectivety w a s assayed i n a blind f a s h i o n
R . H . P U R C E L L , T . M I Y A M U R A , J . L . D I E N S T A G ,M . J . ALTER, C S E . S T E V E N S ,
(Table 1). T h i s p a n e l o f well-pedigreed a n d
G . E . T E G T M E I E R , F . B O N I N O ,M . C O L O M B O , W . - S . L E E , C . K U O ] K . B E R G E R ,
well-characterized samples has been accept­
J . R . S H U S T E R , L . R . O V E R B Y ,D . W . BRADLEY, M . H O U G H T O N
e d widely as a crucial test o f t h e validity o f
putative specific assays f o r N A N B H (16).
A specific assay h a s b e e n d e v e l o p e d f o r a b l o o d - b o r n e n o n - A , n o n - B hepatitis O f seven N A N B H s c r u m samples s h o w n t o
( N A N B H ) v i m s in w h i c h a polypeptide synthesized i n recombinant yeast c l o n e so f t h e b e infectious in chimpanzees, all b u t o n e
hepatitis C virus ( H C V ) is u s e d t o capture circulating viral antibodies.H C V antibodies gave very high signals in t h e assay a s c o m ­
w e r e detected i n six o f seven h u m a n sera that w e r e s h o w n previously t o transmit pared t o t h e results o b t a i n e d w i t h sera f r o m
N A N B H t o chimpanzees. A s s a y s o f t e n b l o o d transfusions i n t h e U n i t e d S t a t e s that t w o control patients w i t h alcoholic hepatitis
resulted i n chronicN A N B H revealed t h a t there w a s a t least o n e pos itive b l o o d d o n o r o r primary biliary cirrhosis a n d five n o n ­
i n n i n e o f these cases a n d t h a t all ten recipients seroconverted d u r i n g their illnesses. infectious normal blood d o n o r s . T h e s e re­
A b o u t 8 0 percent o f chronic, post-transfusion N A N B H ( P T - N A N B H ) patients from sults w e r e reproducible i n quadruplicate
Italy a n d Japan h a d circulating H C V antibody; a m u c h l o w e r frequency ( 1 5 percent) analysis (Table 1 ) . T h e o n l y proven infec­
w a s observed i n acute, resolving infections. I n a d d i t i o n , 5 8 percent o f N A N B H tious s a mple t h a t w a s negative in t h e assay
patients from the U n i t e d States w i t h n o identifiable source o f parenteral e x p o s u r et o w a s o b t a i n e d f r o m a n individual i n t h e acute
t h e virus w e r e a lso positive for H C V antibody. T h e s e data indicate t h a t H C V i s a m a j o r phase o f post-transfusion N A N B H ( P T -
cause o f N A N B H t h r o u g h o u t t h e w o r l d . N A N B H ) , a l t h o u g h a n o t h e r acute-phase se­
r u m o f u n p r o v e n infcctivity w a s similarly

V IRAL HEPATITIS COMMONLY O C ­

CUR in t h e absencc o f serologic
markers f o r s u c h k n o w n h e p a t o t r o -
pic agents as hepatitis A virus ( H A V ) , h e p a ­
titis B virus ( H B V ) , cytomegalovirus ( C M V ) ,
a n d Epstein-Ban- virus ( E B V ) ( / ~ 4 ) . T e r m e d
non-A, non-B hepatitis ( N A N B H ) , diis entity
represents greater than 9 0 % o f transfusion-
associated hepatitis cases i n t h e U n i t e d
States, a n d u p t o 1 0 % o f transfusions h a v e
been estimated t o result in N A N B H (5, 6).
M o r e recent!)', t h e f r e q u e n t occurrencc o f
N A N B H i n t h e absence o f an)' o b v i o u s
parenteral exposure h a s b ee n well d o c u ­
mented ( 7 - 9 ) . Whereas acute disease is o f t e n
subclinical, a t least h a l f o f N A N B H infec­
tions result i n chronic hepatitis, w h i c h m a y
result in cirrhosis in approximately 2 0 % o f
eases (10). A potential association w i t h he­
patocellular carcinoma h a s also b e e n p r o ­
posed (11). Because o f t h e frequency a n d
G, Kuo, Q.-L Choo, W.-S. Lee; C . Kuo, K. Berger, J.
R, Shuster, L R. Ovetby, M . I loughton, Chiron Corpo­
ration, 4560 Horton Street, Emeryville, CA 94608.
H . J. Alter, Department of Transition Medicine Clini­
cal Center, National Institutes of Health, Bcthesdi,M O
20205.
G. L Gitnick, Department of Medicine, UCLA School
of Medicine, Los Angeles, CA 90024.
A. G. Rcdckcr, Department of Medicine, University of
Southern California, Liver Unit, Ranclio Los Anugos
Medical Center, Do*vney, CA 90242.
R. 1L Purcell, Laboratory o f Infectious Diseases, Na­
tional Institute of Allergy and Infectious Disease, Na­
tional Institutes of Health, Bcthesda, M D 20205.
T . Miyamura, National Institute of Health, 10-35, 2-
severity o f N A N B H , t h e r e is a n u r g e n t need
t o d e v e l o p a direct diagnostic test f o r t h e
causativc a g e n t o r agents. W e h a v e recently
cloned t h e g e n o m e o f a N A N B H a g e n t
(12), designated t h e hepatitis C virus ( H C V ) ,
and n o w r e p o r t t h e d e v e l o p m e n t a n d u s e
o f a recombinant-based assay f o r H C V
antibodies.
T h r e e overlapping clones w e r e isolated b y
means o f t h e c D N A in H C V c l o n e 5 - 1 - 1 ,
which w a s used a s a hybridization p r o b e t o
t h e original c D N A library (12). T h e s e clones
have o n e c o m m o n o p e n re a ding f r a m e
( O R F ) extending t h r o u g h o u t t h e m t h a t e n ­
codes part o f a viral antigen associated w i t h
N A N B H (12), T h i s c o n t i n u o u s O R F w a s
reconstructed f r o m these clones a n d t h e n
expressed in yeast (13) a s a f u s i o n polypep­
tide w i t h h u m a n superoxide dismutasc
( S O D ) , w h i c h facilitates t h e efficient expres­
sion o f foreign proteins i n yeast a n d bacteria
Glome, Kamicaiki, Shinagiua-Ku, Tokyo 141, Japan.
J. L Dienstag, Gastrointestinal Unit, Massachusetts
General I lospiul, Boston, MA 02114.
M. J. Alter and D . W. Bradley, Centers for Disease
Control, 1600 Clifton Road NE, Atlanta, GA 30333.
C E. Stevens, Laboratory of Epidemiology, New York
Blood Ccnte^310 East 6 7 Street, New \ o r k 10021.
G- E. Tcpmcitt, Community Blood Gcnrcr of Greater
Kjums City, KjibSj-Cjiv, H O M U L
F. Bonino, Division? i3i~ Gastrocntcrobgia, Ospcdalc
Maggiore di S. Giovanni Battiua, Molinette, Toono,
Way.
M. Colombo, Instituto di Mcdicina Interna, Oinita
Medics 3, Univcnita di Mitino, Via Tact, 9, 20122,
Milan, Italy.
negative. A b l o o d d o n o r implicated i n trans­
mission o f N A N B H b u t w h o s e s c r u m w a s
o f equivocal infcctivity in chimpanzees w a s
also f o u n d negative in t h i s assay. T h u s , t h e
data f r o m this panel o f sera indicates a h i g h
sensitivity and specificity' o f t h e a n t i b o d y
assay f o r b l o o d - b o r n e N A N B H .N o o t h e r
assay evaluated b y this panel has achieved
this d e g r e e o f specificity a n d sensitivity (16).
N e x t , w c assayed m a tc he d b l o o d d o n o r
a n d prospectively o b t a i n e d recipient sera
f r o m t e n well-characterized cases o f c h r o n i c
P T - N A N B H in t h e U n i t e d States. T h e re­
sults o f t h e H C V a n t i b o d y assayso f s e q u e n ­
tial samples taken a t 3 - m o n t h intervals f r o m
each recipient d u r i n g t h e d e v e l o p m e n t o f
N A N B H a n d i n s t o r e d samples f r o m t h e
corresponding d o n o r s a r e s h o w n (Table 2 ) .
E a c h o f t h e t e n recipients seroconvcrted
against H C V d u r i n g t h e course o f disease,
a l t h o u g h seroconversion i n ease 4 w a s m a r ­
ginal a n d n o t a p p a r e n t until 12 m o n t h s a f t e r
transfusion. I n contrast, seroconversion
against H C V w a s n o t observed in prospec­
tively s tudie d individuals infcctcd w i t h o t h e r
viral hepatitis agents. A n t i b o d y seroconver­
s i o n w a s generally detectable w i t h i n 6
m o n t h s o f transfusion. T h e p r o l o n g e d inter­
val t o a n t i b o d y de ve lopme nt m a y explain
t h e observed abscnce o f H C V antibodies i n
t h e acute-phase samples assayed in T a b i c 1 .
W i t h o n e exception, significant levels o f
H C V antibody w e r e detected in a t least o n e

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 PEN.017.2765

T Table 1 . Detection of H C V antibodies in proven infectious Wood samples. Avsays were performed (2Z)
undercodc and in quadruplicateo n a panel (16) containing sera from three patients with biopsy-proven
chronic PT-NANBH, three implicated blood donors, and one patient with acute P T N A N B H , all of
d o n o r t o each o f t h e t e n recipients w i t h
N A N B H (Table 2). C a s e n u m b e r 4 h a d n o
which had been proven t o transmit N A N B H t o chimpanzees. Also included were sera from a patient positive d o n o r s a n d represented t h e recipi- '
with acute NANBH and a donor thrice implicated in N A N B H , each of which were equivocally c n t w i t h t h e weakest seroconversion o b ­
infectious in the chimpanzec. Control sera were assayed from five normal blood donors who had each served. S o m e o f t h e positive d o n o r s h a d n o
donated blood o n at least ten occasions without the development o f N A N K H in the recipients, from a surrogate markers for N A N B H [elevated
patient with alcoholic hepatitis, and from a n individual with primary biliary cirrhosis. Seta scoring
s e r u m alanine aminotransferase ( A L T ) con­
positive in these assays were negative when purified S O D was used to coat wells instead of C100-3.
Such samples were also positive in immunoblot analyses containing recombinant H C V polypeptides, centrationso r t h e prcscncc o f a n t i b o d y t o
but not S O D alone (J2). t h e hepatitis B c o r e antigen ( H B e A g ) , o r
b o t h [6, 17-19)]. T h e prevalence o f H C V
Serum Counts per minute a n t i b o d y in voluntary b l o o d d o n o r s from
I'.i .ij 1 irijiiiiuM tn ihiinp N e w York w i t h n o r m a l A L T levels ( < 4 S
Chronic N A N R H patients international u n i t s p e r liter) a n d n o anti­
1 (PT-NANBH) 31,962 32,107 32,121 28,584 b o d yt o H B c A g w a s a b o u t 0 . 5 % ( 2 o f 4 1 2 } .
2 (PT-NANBH) 22,871 17,483 21,623 19,863
T h i s frequency increased t o 4 4 % ( 1 6 o f 3 6 )
3 (PT-NANBH) 25,381 20,983 21,039 20,047
Acute PT-NANBH patient 909 726 767 580 in d o n o r s w i t h b o t h elevatedA L T levels a n d
Implicated blood donor; a n t i b o d y t o H B c A g (20).
1 40,883 33,521 35,870 34,526 T h e s e da ta f r o m characterized N A N B H
2 25,812 23,512 26,476 23,723
panels c o m b i n e d w i t h previous d a t a ( f 2 )
3 31,495 30,907 33,723 33,043
indicate a specific association between H C V
Unprovni bifntivity tu chimp
Acute PT-NANBH patient 1,207 740 1,786 1,489 a n t i b o d y a n d b l o o d - b o r n e N A N B H . This
Implicated blood donor 590 469 477 461 conclusion w a s also s u p p o r t e d f r o m assays
Pedigreed normal controls o f o t h e r chronic P T - N A N B H patients (Ta­
Blood donors b i c 3 ) . T h e s e cases differ f r o m t h e N A N B H
J 998 775 647 584 eases citcd i n Tables 1 a n d 2 i n t h a t ' y
2 887 632 561 469
w e r e n o t prospectively m o n i t o r e d f r o n .
3 591 446 459 327
t i m e o f transfusion a n d , in m a n y eases, o n l y
4 634 533 758 649
5 5S4 531 553 429 o n e s c r u m sample w a s assayed. T h i s m a y
Distajt controls a c c ount f o r t h e o b s e r v e d lower prevalence o f
Alcoholic hepatitis 842 571 586 566 H C V antibody.
Primary biliary cirrhosis 915 1,US 741 750

Table 3 . HCV antibody in N A N B H patients

from the United Slates.
Table 2 . Detection o f H C V antibodies in t h e blood donors and recipients of ten cases of chronic PT-
NANBH from tlje United States. There were 138 blood donations of apparent negativity that closely Total Percent
followed a normal distribution with a mean o f 3536 cpm (range, 187 t o 3 0 9 7 cpm) and a standard Transmission
patients positive
deration (SD) of 6 7 1 cpm. Samples > 3 5 4 9 epm (mean + 3 5 D ) are considered positive. All
prospectively studied blood recipients developed chronic N A N B H as diagnosed by the persistent Blood transfusion 24 71*
elevation o f scrum ALT levels ( > 6 months) in the absence o f immunoglobulin M antibody t o HAV, N o identifiable source 59 58+
HBV surface antigen (HBsAg), antibody to HBsAg and HBcAg, and serologic markers for C M V and (community-acquired)
EBV infection. Biopsies from all ten patients confirmed the diagnosis of chronic ity. Recipient sera were
assayed at 3-month intervals ( 0 represents a sample obtained immediately before transfusion). Control "Between one and three serum samples assayed Irom
patients who had received transfusions and vdto were
samples consisted o f sera from a prospective study o f male homosexuals (23} that were assayed foru p t o diagnosed with chronic N A N B H on the bans o f dtnkal
1 year after the onset ofhqistitis as a result o f infection with eirhet HAV (18 cases), HBV (20 cases), or symptoms, elevations of serum ALT ft* >6 months,
CMV (S cases). None of these disease controls showed positive seroconversion t o ann-HCV. The serologic eiclusion o f infection with other agents (Table
results of every positive donor unit arc shown. 2), and t h t cidusion o r other apparent causes erf' liver
injury. {Sequential serum samples obtained prospec­
tively u p t o 3 years after t h e onset of clinical hepatitis
Num­ associated a-ith elevated scrum ALT in die absr of
ber Anti-HCV assay (cpm) serologic m a r t e n f o r other agents (Tabic 2) am, jrr
of identifiable causa o f liver injury.
Case donors Recipients (months)
per Positive
trans­ donors Table 4 . HCV antibody in PT-NANBH cases
fusion 0 3 6 12
from Italy and Japan.
I 18 3,910 1,870 3,220 13,120 26,780 Court- Number of Percent
2 18 4,590 2,530 1,170 11,400 20,750 Disease
try patients positive
3,800
3 13 6,140 1,800 1,850 14,990 4,720 Italy 32 Chronic 84*
4 18 None 1,430 1,370 750 4.260 Japan 23 Chronic 78+
5 16 24,420 2,230 790 13,960 22,020 Japan 13 Acute, 15+
6 11 6,080 2,100 10,160* 21,490 24,900 resolving
25,600
7 15 15,970 2,120 2,090 10,470 16,140 'Serum samples (about three) assayed from each patient
S 20 13,240 1,920 2,860 8,160 22,510 wiih transfusion - related chronic NANBH (diagnosed 11
8+ 32,790 3,370 5,800* 4,700 11,380 in Tables 2 and 3). * A prospective study in which
9 sequential serum samples were assayed foe at least 6
10 15 20,430 1,530 5,830* 19,960 20,580 months after t h e onset o f acute N A N B H (diagnosed as
19,760 in Tables 2 and 3). The serum A L T of acute, resolving
patients returned t o normal and stable levels, whereas
'These moderately high counts per minute were shown in additional studies t o be due t o passiw: transfer of antibody chronic patients displayed abnormal levels for at least 6
from donors who had high antibody titers. f Oi i h ' six o f t h e eight donors were assayed. months.

21 APRIL 1989 REPORTS 363

Supplied by The British Library - "The world's knowledge"


Assays were also performed on a group o f
patients with well-defined clinical NANBH
who were prospectively monitored for up t o
3 years after onset o f illness but who had no
identifiable source of infection (9). More
than 50% of these individuals were either
positive for HCV antibody at the time o f the
initial consultation with the physician or
seroconverted subsequently (Table 3).
Thus, it appears that HCV is a major cause
of community-acquired NANBH as well as
PT-NANBH!
To initiate investigations into the contri­
bution of HCV to global NANBH, a collec­
tion o f sera from NANBH patients from
Italy and Japan was assayed for HCV anti­
body. The results indicate that 84% o f Ital­
ian patients diagnosed with chronic PT-
NANBH contained HCV antibody (Table
4). A similar frequency was observed in
prospectively studied chronic PT-NANBH
cases from Japan, but a much lower preva­
lence was seen in Japanese patients with
NANBH that had resolved their acute infec-
"on without progression to chronic hepati­
tis (Table 4). The lower incidence o f anti­
body to HCV in acute, resolving NANBH
has also been observed in other human
studies (21) and may reflect a lower stimula­
364
Supplied by The British Library - "The world's knowledge"
tion of the immune system in these cases as
compared with chronic, persistent infec­
tions.
These data suggest that HCV is a major
cause of chronic NANBH throughout die
world. The advent of the specific, sensitive
test for HCV antibody described here
should improve the safety of the world's
blood supply as well as provide an important
clinical diagnostic tool. With this assay and
the availability o f HCV hybridization
probes (12), it should also be possible to
address the issue o f whether other parenteral
NANBH agents exist.
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21. J. Moslcy and M. I. Alter, personal communications.
22. CI00-3 was purified from recombinant yeast by
breaking the cells in 2 0 mM tris-HCI,pH 8 . 0 , 1 mM
EDTA, ImAf dithiothreitol (DTT), and 1 m M
phcnylmethylsulfonyl fluoride with glass beads and
extracting the insoluble Traction with SOS before
chromatography on successive Q-Sepharose and Se-
phacryl S-300 (Pharmacia) columns. The final puri­
ty o f C100-3 was >90%. Wells o f microtitcr plates
(Immolon 2 ) were coated with 0.1 jig o f purified
C100-3 before incubation for 1 hour at 37^C with
100 |il o f serum (diluted 1:100). Wells were then
washed and bound antibody was detected by further
incubation for 1 hour at 37°C with 100 (il o f , , J I -
labelcd sheep antibody t o human immunoglobulin
( 1 |iCi/ml; Amasham).
23. W. Szmuncss et at., N. Engl. J. Med. 3 0 3 , 8 3 3
(1980); W. Szmuncss et al., ibid. 307,1481 (1982);
C. E. Stevens et al., ibid. 3 1 1 , 4 9 6 (1984).
24. We thank R. Spaete, A . Wcincr, G. Mullenbach, R .
Hallcwcll, and P. Valenzuela for critical reviews o f
the manuscript and Peter Anderson for word pro­
cessing. Supported by Chiron Corporation, Ortho
Diagnostic Systems Ltd., and Ciba Gcigy.
2 4 January 1989; accepted 1 0 March 1989
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