Screening test for reverse-transcriptase containing virus such as non-A, non-B hepatitis, NANBH

The present invention discloses a screening test for detecting the presence of contaminating or infectious agents causing non-A, non-B hepatitis or AIDS in a blood donor setting. A kit for the detection of contaminating agents belonging to the group of retroviruses is also disclosed. Screening blood or blood related products so as to prevent spreading of infection or contamination due to retroviruses is now made possible by the present invention.

BACKGROUND OF THE INVENTION 
1. Technical Field: 
The present invention relates to a screening test for detecting the 
presence of reverse-transcriptase containing virus in blood, blood 
products or in any source containing such virus. More particularly, the 
present invention relates to the detection and diagnosis of non-A, non-B 
hepatitis in blood donors by determining the presence of reverse 
transcriptase activity in the body fluid, preferably in a blood bank 
setting. An advantage of the present invention is to prevent transmission 
of retrovirus related infection through blood donor (transfusion) program 
or through plasma-related products by identifying such blood, serum, 
plasma or products derived therefrom which may be carriers of the 
retrovirus by using the test disclosed herein. 
2. Prior Art 
Non-A, non-B hepatitis is presumed to be caused by an agent(s) which is 
serologically distinct from hepatitis A virus and hepatitis B virus. The 
diagnosis of this disease relies on the serological exclusion of hepatitis 
A, hepatitis B, cytomegalovirus, and Epstein-Barr virus. 
Non-A, non-B hepatitis infection has been reported worldwide. It accounts 
for 20% of sporadic cases of hepatitis among adults. In the United States, 
this type of hepatitis accounts for 90% of post-transfusion hepatitis. An 
alarming 50% of these cases develop chronic hepatitis, and such 
individuals remain as potential sources of infection. 
The existence of a transmissible agent in this disease has been 
demonstrated. However, presently there is no test to identify the non-A, 
non-B agent(s). The present invention for the first time demonstrates that 
non-A, non-B hepatitis is caused by a retrovirus or retrovirus-like agent 
and provides a method of screening for the same in a clinical setting, 
particularly in a blood-bank type program. 
SUMMARY OF THE INVENTION 
It is, therefore, an object of the present invention to provide a method 
for screening blood or blood donors capable of transmitting retrovirus 
related infection which may be pathogenic. 
It is a further object of the present invention to provide a method of 
detecting in the blood, serum, plasma or plasma derived products, the 
presence of virus having reverse transcriptase activity. 
It is another object of the present invention to provide a method of 
detecting the presence of an agent causing non-A, non-B hepatitis. 
It is a still further object of the present invention to provide a kit for 
the detection of pathogenic or contagious retrovirus, including an agent 
causing non-A, non-B hepatitis regardless of its epidemiology. 
Other objects and advantages will become apparent as the detailed 
description of the invention proceeds.

DETAILED DESCRIPTION OF THE INVENTION 
These and other objects and advantages of the present invention are 
achieved by a screening test for detecting the presence of reverse 
transcriptase containing viruses in serum or blood, particularly in a 
blood donor program. 
The term "blood" as used herein includes not only blood per se but also 
serum, plasma and any other products or fractions obtained or derived from 
blood or blood component. 
Although the screening test described herein detects the presence of 
reverse transcriptase of whatsoever origin, it should be noted that the 
reverse transcriptase (RT) in the retroviruses as described herein are 
particle-associated, i.e., the RT is found encapsulated within the virus. 
Hence, in order to detect the RT, a first essential step is to isolate the 
virus particles from soluble fraction of the blood. The isolated virus 
particles are then disrupted or lysed to release the RT therefrom and the 
specific enzyme activity then assayed. 
It should be clear, therefore, that the RT referred herein is specifically 
of viral origin and not a soluble protein associated with normal parts of 
the body, tissue or body fluid. 
As far as it is known, RT is found associated with all retroviruses tested. 
Hence, in this sense it may be designated a marker of such viruses. In 
particular, however, the viruses which belong to this group and which are 
clinically more significant are the human T-cell lymphocytotropic type I, 
II and III (HTLV I, II or III) and non-A, non-B hepatitis virus. It may be 
noted that the present invention is the first to show that non-A, non-B 
hepatitis is of retroviral origin; hence detectable by RT assay. 
It is noted that the term "retrovirus" as used herein includes 
retrovirus-like agents or entities which have the same density and exhibit 
RT activity as found in retroviruses mentioned atove. 
Although any suitable method of detecting RT activity can be used for the 
practice of this invention, it may be noted that the preferred methods 
include any radiolabelled, enzymatic, histologic, radioimmuno, 
fluorescent, antigenantibody, ELISA (enzyme-linked-immunosorbent assay) 
and the like. Monoclonal or polyclonal antibodies against purified RT or 
cleavage products of RT are particularly preferred. Such assay techniques 
are well known and should be obvious to those of ordinary skill in the 
art. All references or publications cited hereunder are incorporated 
herein by reference. Preferred methods and materials are now described. 
The abbreviations used herein are as follows: NANBH, non-A, non-B 
hepatitis; RT, reverse transcriptase; ALT, alanine aminotransferase; AST, 
aspartate aminotransferase; PEG, polyethylene glycol 6000; HAV, hepatitis 
A virus; HBV, hepatitis B virus; RSV, Rous sarcoma virus; CMV, 
cytomegalovirus; EBV, Epstein-Barr virus; HTLV III, human T-cell 
lymphocytotropic virus, type III; AIDS, Acquired Immune Deficiency 
Syndrome. 
MATERIALS AND METHODS 
Infectious Sera and Plasma-Derived Products 
Four serum inocula and two plasma-derived products were studied. Each of 
them had previously been shown to transmit NANBH to humans and/or to 
chimpanzees and to be free of HAV, HBV, CMV, and EBV. 
Four inocula were sera from patients with NANBH. Inculum I was a 
well-defined serum obtained from a patient with chronic NANBH acquired 
following blood transfusion. The inoculum was known to have transmitted 
NANBH to another human by accidental inoculation and to chimpanzees. 
Inocula SE and RP are sera obtained during the acute phase of NANBH from 
two patients who both developed chronic NANBH. Inoculum SE is from a 
patient with blood transfusion-associated NANBH, and inoculum RP is from a 
male homosexual. Each of these sera transmitted NANBH to chimpanzees which 
are valid human surrogates for testing purposes. The fourth inoculum 
(inoculum H) was a well-defined serum obtained from a patient with chronic 
NANBH which was shown to transmit NANBH to chimpanzees. 
Two inocula were plasma-derived products manufactured in the U.S., 
antihemophilic factor and fibrinogen, respectively. Each of them had been 
implicated in the transmission of NANBH to patients, and both were shown 
to transmit NANBH to chimpanzees. 
NANBH Patients and Health Controls 
Serum specimens from 12 patients, each with clinically, histologically, and 
(by exclusion) serologically confirmed NANBH were studied. These patients 
included recipients of blood transfusion (3 patients, all of whom 
developed chronic NANBH), intravenous drug users (4 patients, 3 of whom 
developed chronic NANBH), and sporadic cases without any known exposure to 
hepatitis agents (5 patients, 2 of whom developed chronic NANBH). The 
diagnosis of NANBH was made in each of these patients based upon serum ALT 
activity (at least 5 times the upper limit of normal, 40 IU/ml), liver 
histology consistent with viral hepatitis and the absence of serologic 
markers for HAV (anti-HAV antibodies of the IgM-type), HBV (HBsAg in serum 
or anti-HBc alone in serum), CMV, or EBV. 
Control sera were obtained from 49 healthy individuals, of whom 13 were 
workers in a plasma derivatives research and regulatory laboratory and 36 
were paid plasmapheresis donors. Both of these groups are known to be at 
somewhat higher risk of NANBH than the general population. 
Chimpanzees 
Two chimpanzees (Pan troglodytes), 1278 and 1284, were obtained from a 
breeding colony as described by Tabor et al in Lancet, 1978, 1, 463 and 
Tabor et al in N. Engl. J. Med, 1980, 303, 140. 
Detection of Reverse Transcriptase (RT) Activity 
RT Assay. Serum samples (100 .mu.l), negative control material (fetal 
bovine serum), and positive control material (Rouse sarcoma virus, 
1.times.10.sup.10 particles) were all initially centrifuged in 5 ml of 35% 
glycerol in 0.5 M Tris-HCl, pH 7.0, at 77,000 .times.g for 1 hour at 
4.degree. C. to pellet viral particles from serum proteins as described by 
Sarngadharan et al in Methods Cancer Res., 1976, 12, 3. The pellet was 
treated with 0.25% octylphenol ethylene, oxide condensate (Nonidet P40) to 
disrupt viral particles. Each treated sample was then added to 40 .mu.l of 
solution consisting of 60 mM Tris-HCl, pH 8.3, 8 mM MgCl , 80 mM KCl, 20 
mM dithiothreitol, 0.1 .mu.g actinomycin D, 80 .mu.M each of unlabeled 
deoxyribonucleoside triphosphate, 20 .mu.M [.sup.3 H]TTP (thymidine 
triphosphate, specific activity 1.2 Ci/mmol), and 1 .mu.g poly(rA) 
p(dT).sub.10 as template-primer. The reaction was incubated at 37.degree. 
C. for 1 hour and terminated by adding 40 .mu.l of 1 mg/ml yeast tRNA and 
5 ml 10% TCA containing 0.2 mM sodium phrophosphate. After 30 minutes, the 
precipitable radioactivity was collected on glass fiber filters, dried, 
and the radioactivity determined by liquid scintillation. The sample was 
considered positive if the sample cpm were 2 times the negative control 
cpm (mean 0.35.times.10.sup.3). This positive cut-off (0.7.times.10.sup.3 
cpm), based on a titration of RSV particles, represents the RT activity 
associated with 1.times.10.sup.4 particles of RSV. A gross estimate of 
virus titer in a given inoculum can, therefore, be obtained by relating 
the RT activity in cpm associated with various numbers of RSV particles, 
and comparing the cpm obtained with those for the inoculum. The product 
assayed is the radiolabelled DNA. 
Localization of RT in Sucrose Gradient Ultracentrifugation Fractions of 
Infectious Sera 
One ml each of inoculum I and inoculum SE were centrifuged separately in an 
SW41 rotor by layering on 11 ml of a 10% to 60% (by weight) sucrose 
gradient made in 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 1 mM EDTA, and 
centrifuged at 30,000 rpm for 19 hours at 4.degree. C. in a Beckman LB-70 
ultracentrifuge. Fractions (0.7 ml each) were collected from the bottom of 
the gradient and the absorbance at 260 nm determined with an LKB UVcord. 
After removal of sucrose by centrifugation, the fractions were assayed for 
RT activity as described. 
Inoculation of RT Positive Ultracentrifugation Fractions into Chimpanzees 
Fractions from each individual gradient which contained RT activity were 
pooled (fractions 12-15 in 2.8 ml), filter-sterilized (0.22 .mu.M filter), 
and injected intravenously into chimpanzees 1278 (inoculum I) and 1280 
(inoculum SE). Both chimpanzees were bled weekly to monitor serum enzyme 
activities (ALT and AST) and serological markers of hepatitis. In 
addition, liver biopsies were obtained biweekly and examined by light 
microscopy for evidence of hepatitis and by electron microscopy for 
specific ultrastructural changes in NANBH. 
Biophysical and Biochemical Characterization of RT 
In three serum specimens (inocula I, SE, and RP), the RT activity was 
assayed following treatment with 6.5% polyethylene glycol 6000 (PEG) 
according to Welsh et al in Nucleic Acids Res., 1980, 8, 2349 and, in a 
separate analysis, in the presence of 5 .mu.g RNAse A and two different 
exogenous template-primers as described by Goodman, et al in Proc. Natl. 
Acad. Sci. U.S.A., 1971, 68, 2203 and Milstein, et al in J. Clin. 
Microbiology, 1975, 1, 353. 
RESULTS 
Particle-associated RT activity was detected in all 6 infectious NANBH 
materials and in all 12 sera from patients with acute or chronic NANBH. 
The RT activity ranged from 0.85.times.10.sup.3 cpm to 16.6.times.10.sup.3 
cpm. In contrast, 47 of 49 sera (96%) from healthy controls lacked enzyme 
activity (CPM ranged from 0.2.times.10.sup.3 to 0.56.times.10.sup.3). Sera 
from two healthy controls yielded 1.2.times.10.sup.3 and 
1.0.times.10.sup.3 cpm, respectively, and were regarded as low positives 
(cut-off 0.7.times.10.sup.3 cpm). Table I shows the data from these 
studies. 
TABLE I 
__________________________________________________________________________ 
REVERSE TRANSCRIPTASE (RT) ACTIVITY 
RT activity 
No. No. (.times. 10.sup.3 cpm) 
Material studied tested 
positive (%) 
(range and mean) 
__________________________________________________________________________ 
Sera proven infectious in 
4 4 (100%) 
1.40.sup.a -16.6 
previous NANBH studies mean: 5.95 
Plasma-derived products proven 
2 2 (100%) 
0.85-1.30 
infectious in previous NANBH studies 
mean: 1.08 
Serum samples from NANBH patients 
12 12 (100%) 
0.86-2.70 
obtained during the acute phase of 
mean: 1.49 
infection 
Serum samples from healthy laboratory 
49 2 (4%) 1.0-1.20.sup.b 
workes and paid plasmapheresis donors 
mean: 1.1 
__________________________________________________________________________ 
.sup.a This value represents the RT activity in 100 .mu.l of a 10.sup.-4 
dilution of inoculum H. 
.sup.b These values represent the RT activity in the two positive samples 
The negative samples have values ranging from 0.21 .times. 10.sup.3 to 
0.56 .times. 10.sup.3 cpm. 
As shown in FIG. 1 peak RT activity for inoculum I banded at 1.14 g/ml in a 
sucrose gradient. Similar banding patterns were obtained with two other 
serum inocula. Peak RT activity for inocula SE and RP are also banded at 
1.14 g/ml. 
Following inoculation of chimpanzees with RT-positive sucrose gradient 
fractions from either inoculum I (chimpanzee 1278) or inoculum SE 
(chimpanzee 1284), both animals developed NANBH as confirmed by elevations 
of serum ALT activity (at least 3 times the baseline level), histologic 
evidence of hepatitis by light microscopy, and specific ultrastructural 
cytoplasmic alterations (type C-III tubules) by electron microscopy. 
The biophysical and biochemical characteristics of the RT activity detected 
by the assay desribed here appear in Table II. 
TABLE II 
______________________________________ 
NATURE OF THE REVERSE TRANSCRIPTASE 
(RT) ACTIVITY 
Addition.sup.b or RT activity 
Treatment (.times. 10.sup.3 cpm/assay) 
______________________________________ 
Complete.sup.a 4.09 
Actinimycin D, 100.mu.g 
4.01 
6.5% PEG pellet.sup.c 4.11 
poly(rA).p(dT).sub.10, 1 .mu.g 
7.29 
poly(dA).p(dT).sub.10, 1 .mu.g 
3.64 
poly(rA).p(dT).sub.10, 1 .mu.g, and RNAse A, 5 .mu.g 
4.34 
______________________________________ 
.sup.a The complete system represents the sandard RT reaction described i 
Materials and Methods with the exception of the exogenous template. The 
activity represents synthesis using the endogenous template only. 
.sup.b Addition to the complete reaction described above. 
.sup.c PEG precipitation of viral particles prior to addition to complete 
system. 
Besides banding at a discrete density, RT activity is associated with viral 
particles since it was completely recovered in the 6.5% PEG precipitate. 
The RT activity showed a preference for poly(rA) p(dT).sub.10 over 
poly(dA) p(dT).sub.10 as template-primer, a feature which distinguishes 
the viral enzyme from cellular DNA polymerases. Additionally, the 
incorporation of [.sup.3 H]TTP was insensitive to actinomycin D, which 
inhibits DNA-dependent DNA synthesis. The viral RT activity with 
endogenous template-primer was sensitive to RNAse A digestion, whereas the 
reaction with exogenous template-primer, [poly(rA) p(dT).sub.10 ]was 
unaffected. 
The finding of particle-associated RT activity in 4 infectious sera and in 
2 infectious plasma-derived products, as well as in 12 serum samples from 
the acute or chronic phase of NANBH, demonstrated that this disease is 
caused by a virus or a virus-like agent possessing this enzyme. The 
location of this RT activity in sucrose gradient fractions (peak activity 
at 1.14 g/ml) and the transmission of typical NANBH to chimpanzees by 
inoculating RT-positive sucrose gradient fractions provide evidence that 
the NANBH agent in the sera and plasma-derived products studied (as well 
as in the patient sera examined) is a retrovirus or retrovirus-like 
agent(s). The finding of RT activity in the sera of 2 out of 49 controls 
which banded at a density consistent with that of retrovirus strongly 
suggested that these individuals are infected with a retrovirus or 
retrovirus-like agent. Indeed, all 49 of the controls were at a higher 
risk for NANBH than the general population. 
The finding of RT activity in all 12 sera from patients with different 
epidemiological types of NANBH indicated that one or more retrovirus or 
retrovirus-like agent(s) caused all 12 cases of NANBH. The absence of RT 
activity in 47 of 49 sera from healthy laboratory workers and paid plasma 
donors, all of whom are at a higher risk for acquiring NANBH than the 
general population, support the specificity of the RT assay employed here. 
Additional evidence for this specificity is the preference shown for 
poly(rA) p(dt).sub.10 as template-primer, the precipitation of RT by PEG, 
the susceptibility of the endogenous template to inactivation by RNAse A, 
resistance of the reaction product to alkali hydrolysis, and the 
localization of the RT activity and infectivity in sucrose gradients at a 
density consistent with that reported for retrovirus as cited in 
Sarngadharan, et al, supra. 
Known characteristics of NANBH and of the agent(s) causing this disease 
appear to be consistent with the etiology being a retrovirus or 
retrovirus-like agent. Inactivation of NANBH agents has been accomplished 
by formalin, heat, or chloroform, also consistent with their being 
retroviruses. Chronic infections are common following infection with the 
NANBH agent(s), especially those acquired via blood transfusion. 
Retroviruses characteristically cause chronic infections. Antigen-antibody 
systems described in association with NANBH are consistent with the 
development of antibodies to both the external and internal antigens of 
retroviruses, all of which appear to coexist with infectious virus in 
serum. At least one antigen detected by counterelectrophoresis and 
purified from inoculum I appears to be a glyco-protein similar to one 
described by Schupbach et al in Science 1984, 224, 503, and present on the 
surface of the HTLV III retrovirus. 
Specific cytoplasmic ultrastructural changes have been consistently seen 
during NANBH in chimpanzees. Similar alterations have been reported in the 
lymphocytes of patients with the Acquired Immune Deficiency Syndrome 
(AIDS), a syndrome which is associated with chronic infection with the 
retrovirus HTLV III as described by Schaff et al in Lancet 1983, 1, 1336. 
Additionally, a known amount of human T-cell lymphocytotropic virus, Type 
III (HTLV III) when added to human plasma was detected by reverse 
transcriptase activity. Three products derived from plasma containing HTLV 
III, anti-hemophilic factor concentrate, fibrinogen and plasma-protein 
fraction were also found to contain reverse transcriptase activity. 
Reverse transcriptase activity was directly related to virus titer, 
indicating the utility of using reverse transcriptase activity to estimate 
the HTLV-III virus titer in plasma. 
It is clear from the above that the present invention now makes it possible 
for blood banks and producers of blood related products to screen all 
blood donors and blood products and identify those capable of transmitting 
retrovirus related pathogenic conditions including NANBH and AIDS. A 
single screening test utilizing the present invention enables the 
detection, diagnosis and elimination of retrovirus related contagious or 
infectious conditions. 
A kit comprising a container containing a suitable RT assay system selected 
from the group consisting of an enzymatic assay, an antigen-antibody titer 
assay including mono- or polyclonal antibodies and the like would be most 
valuable for clinical and laboratory use in accordance with the present 
disclosure. Such accessories as micro-titer plates, radiolabelled 
substrates, pipettes, buffers, coenzymes and the like which are routinely 
common and well known in the art are included in the kit in accordance 
with the present invention. 
It is understood that the examples and embodiments described herein are for 
illustrative purposes only and that various modifications or changes in 
light thereof will be suggested to persons skilled in the art and are to 
be included within the spirit and purview of this application and the 
scope of the appended claims.