DE3705512C2 - - Google Patents

Description

The present invention relates to that in claims 1, 2 and 5 indicated non-A, non-B hepatitis associated DNA and Fragments, according to claims 3 and 4, the method for their Production and their use according to claims 6 to 8.

Non-A, Non-B hepatitis diseases are in the literature described in great detail. Are also known but the difficulties of detecting the NANBH virus and Evidence of the disease.

It was now out of the stool of Non-A, Non-B hepatitis patient particles isolated and their molecular weight and their characteristics characterized as well a verification procedure for the existence of such Particles found and the detection of these particles Diagnosing the presence of non-A, non-B hepatitis liver sick patients applied.

DNA was isolated from the particles isolated from the stool and cloned using conventional methods. The so obtained Cloned DNA strands were used to detect the presence homologous or very similar DNA in turn in stool, serum, liver tissue be and body fluids used by liver sick patients those who have a non-A, non-B hepatitis with sufficient Probability can be diagnosed.

In summary, the invention relates to insulation Non-A, Non-B hepatitis-associated particles and that Cloning DNA and using both of these particles as well as the DNA clones obtained to narrow the liver diseases for the presence of non-A, non-B hepatitis. Further aspects of the invention lie in the development of advanced detection methods and finally also vaccines based on those to be obtained Antibodies and with synthesis of synthetic peptides the sequence of the particles that ent this virus proteins speaks and which results from the sequence of the cloned DNA lets predict.

From the stool samples of non-A, non-B hepatitis of sick patients a substance was isolated that accumulated significantly  in the stool of such non-a-non-b hepatitis patients finds, however, in healthy as well as in patients with liver diseases other genesis to a significantly lesser extent finds. A method for the detection of this substance was developed developed in which polystyrene beads with diluted serum coated by non-A, non-B hepatitis convalescent. The washed beads are then washed with a 10% Incubated stool suspension from a subject to be examined, possibly associated non-A-non-B hepatitis Substance to those contained in the convalescent serum antibodies bound to the polystyrene balls against them Substance binds and is immobilized. Binding the Non-A, Non-B hepatitis-associated particles can be detected are in turn made by binding human IgG Convalescent serum from non-a-non-b hepatitis patients, which is radioactively labeled with iodine 125. If the Non-A, Non-B hepatitis-associated substance in the stool of the Subjects are bound to radioactive immunoglobulin and the corresponding signal is used for detection.

When using this detection method for hepatitis Non-A- Non-B-associated substance in large groups of Patients with liver disease of different origins it turned out that this substance was highly significant in patients with confirmed hepatitis Non-A, Non-B im Stool is detectable (see Table 1). In patients with others Liver disease in which non-A, non-B hepatitis is rather unlikely, partly in their clinical Image of an acute or expired non-A, Non-B hepatitis can resemble, especially in patients with an acute, ongoing hepatitis A or B. In contrast, the non-A, non-B Hepatitis-associated substance (see Tables 2 and 3). Out of this it follows that the presence of this substance clear indication of the etiology of an initially unknown,  inflammatory liver disease can deliver, and that the detection of this substance therefore an investigation by ho hen diagnostic value to prove or exclude the project a non-A, non-B hepatitis.

The substance associated with the non-A, non-B thus points a high affinity for human immunoglobulins and fibronectin and its non-collagen-binding gap products on. Binding to Fab-2- Fragments from the IgG of healthy and hepatitis Non-A, Non-B convalescent people speak against an unspecific Binding of the associated substance to the Fc part of the immune globuline. The high affinity for fibronectin and its non-collagen binding fission products is a property which this substance with antigenic proteins of other viruses ge together. Treatment with organic solvents (chloroform, ether) and with heat (70 ° C, 10 min) destroys the binding affinity the non-A, non-B associated substance. While the Digestion with chymotrypsin, trypsin, elastase and neuraminidase no influence on the binding properties of the substance shows, digestion with papain leads to complete and rapid loss of binding affinity.

After centrifugation at 150,000 g, 2 hours, it can be detected in the sediment and adjusts itself 72-hour runtime at a density of 1.3 (1.29-1.32) g / ml cesium chloride. After separation of the 1.30 g / ml Cesium chloride banding fraction over a gradient poly Acrylamide gel electrophoresis are used in silver staining several bands of different molecular weights are shown. After transfer to nitrocellulose, it is possible to use a Western blot with radioactively labeled IgG and Fab-2 fragments from Non-A, Non-B hepatitis patients and healthy volunteers are gang places that do not when extracting healthy control chairs occur. A total of 4 bands are shown, two good  visible main bands with a molecular weight of approx. 64,000 and 56,000 and two weaker bonds with one Molecular weight of 51,000 and 43,000. The bands with Fab-2 Fragments are weaker and show a higher background. The separation of raw chair concentrates without Cesium chloride purification after SDS-Page and blotting showed in positive stools the two main bands with an approximate one Molecular weight around 60,000.

Upon further analysis of non-A, non-B hepatitis-associated Particles were isolated using DNA and using conventional methods clone in fragments. These cloned DNA fragments from the stool of non-a, non-b hepatitis patients could as DNA probes for the examination of stool, serum, liver tissue and body fluids, as well as blood supplies and plasma derivatives other patients suspected of having one Non-A, Non-B hepatitis can be used. With the classic hybridization processes can be shown whether in the unknown DNA is stool, the sequence of which is non-A, non-B hepatitis associated DNA of the clones obtained is so similar that a hybridization signal appears. With this further Detection method for hepatitis non-A, non-B-associated DNA sequences could then be used to examine samples from test subjects are on the presence of non-A, non-B hepatitis associated DNA. The results so far suggest that it is the non-A, non-B hepatitis-associated substance is a virus particle and that it is the isolated DNA sequence is a sequence of virus DNA, such as the digestion process on the one hand and the sequence on the other show yourself.

The following examples illustrate the invention.

Example 1 Isolation of the non-A-non-B hepatitis associated substance from patient chair Buffer used: Tris-HCl, pH 7.4; 0.05 N in all work steps a) Refurbishment

0.5 g of stool are suspended in 10 ml of buffer at 8000 g centrifuged and the supernatant collected. The backlog will still be Washed out once with 10 ml and then with 5 ml of buffer. The The supernatants collected are centrifuged (30 min, 10,000 rpm) and filtered through a bacteria-proof filter (Millipore 0.22 µm). The supernatant is precipitated with PEG 6000 (final concentration 10% or 0.4 mol). After at least 2 and at most 12 hours, the precipitate centrifuged and dissolved in 10 ml of buffer. This solution comes with 10 ml Freon shaken out, the phases separated by centrifugation, the Freon phase washed again with 5 ml buffer. Precipitation with PEG and NaCl (final concentration 10% or 0.4 mol) is repeated Precipitate taken up in approx. 800 µl buffer.

The solution thus obtained is treated with RNase and DNase for 1 hour at 37 ° C digested (800 µl PEG precipitate in Tris-HCl buffer, pH 7.4; 0.05 M; 2800 U RNase and 1500 U DNase, protease-free preparations). Isolations that are not used to extract DNA can be used without it Step.

After digestion with DNase and RNase, the incubate is put on one Brought cesium chloride gradient.

b) Isolation of the non-A, non-B associated substance via a cesium chloride gradient

The centrifuge tubes are filled with 1 ml of cesium chloride solution Given density of 1.4 g / ml and with 3 ml of cesium chloride of one Densities of 1.3 g, 1.25 g and 1.2 g / ml were overlaid. All cesium chloride Solutions are made with the above buffer, the gradient is overlaid with 800 µl stool extract, the centrifugation time is 65-72 hours. Temperature + 10 ° C, rpm 31 000. After completion the gradient is in the lower range (density 1.2-1.4) collected in fractions of approx. 200 µl, density 1.1- in the upper area 1,2 larger fractions can be removed. The density of everyone Fraction is determined by measuring the refractive index. All factions are dialyzed extensively against buffer and 50 ul of each fraction in  NANB assay examined for non-A, non-B associating substance. From the positive fractions will be the Lowry protein concentration Right.

Verification and characterization of the substance c) Creation of a gradient page

Two solutions with different acrylamide concentrations will be layered by a gradient mixer to form a linear gradient. The solution with the higher AA concentration also contains 15% Sucrose to prevent turbulence. Otherwise there are the solutions composed as described in LAEMMLI (1970). this applies also for the comb gel.

The dialyzed and possibly narrowed fractions of the cesium chloride Gradients are beta-coated with 10 µl SDS, 10 µl glycerol and 5 µl Mercaptoethanol, 5 µl bromophenol blue per 100 µl sample and 3 Min. Boiled in a water bath. Then they are mixed with 5 µl of 0.1% pyronine added and applied to the gel. Running time 3½ hours, excitement 160-300 V, current 40-25 A, power 20 W.

Approx. 5 µl of pyronine is applied again 5 minutes before the end of the run and the run ends as soon as it goes through pyronine and comb gel. The gel is either stained with silver (WRAY and co-workers 1981) or Western blotting was carried out (TOWBIN et al. 1979). Blotting is carried out overnight at 0.5 A, then 1 hour at 1 A carried out.

d) treatment of Western blots

After the blotting is complete, the different strips (on the Pyronine markings) and cut out molecular weight standards dyed with amido black. Sample strips will be Shaken for 24-72 hours in 1% gelatin PBS solution. The IgG Fraction of a patient or the Fab-2 fragments of this IgG fraction are labeled with iodine 125 (chloramine T): 0.5 mCi per 100 µg protein.  

About 70% of the activity is incorporated. The tracer, whose Volume should correspond to about 2 µg protein, is in 20 ml gelatin / PBS diluted and the strips incubated for 12 hours with shaking. Then the strips are 3 times with gelatin PBS, 2 times with 1 hour PBS-Tween 0.5% and washed 1 × with water. After drying the These are placed on a sensitive X-ray film and strips Exposed at -70 ° C for 2-7 days.

2. Detection method for HNANB-associated substance in the stool

Polystyrene beads were made with Serum from convalescent patients with non-A, non-B hepatitis in a dilution of 1: 200 in carbonate buffer, pH 9.2, 0.01 mol / l, 12 hours at room temperature incubated. The tubes coated in this way were also used phosphate buffered saline (PBS) washed extensively. The stool samples were in the form of a 10% stool suspension (w / v) for 2 hours at 37 ° C with the incubated as layered polystyrene beads, then extensively with PBS containing 0.5% Tween 20 washed and then washed out with human IgG for 1 hour the serum of convalescents from non-A, non-B hepatitis, which was labeled with iodine 125, incubated at 37 ° C. After renewed, thorough washing with distilled The radioactivity bound to the beads was in water counted a gamma counter. Stool samples in which the bound radioactivity three times the value of the negative control were considered positive for the presence of the non-A, non-B hepatitis-associated substance.

Each of Tables 1 through 4 was described as above created and evaluated according to the specified criteria. It was found that according to Table 1 in patients with confirmed non-A, non-B hepatitis found this substance in a large percentage of cases has been, that according to Table 2 when using this essay for this Non-A, Non-B hepatitis-associated substance in one Patient group with a variety of very different  Liver disease but nothing to do with a Non-A, Non-B hepatitis have to do with the substance, though at all, only found in very low percentages.

Table 3 shows that during the investigation of Patient groups with hepatitis of a different genesis, that is either hepatitis A or hepatitis B to a very low Percentage that finds Non-A, Non-B-associated substance.

It should be noted that the non-A, non-B hepatitis Cases almost always, i.e. almost 30% positive results, whereas with the suspected hepatitis A, suspected hepatitis A, Hepatitis B and posthepatitis B patients' numbers increased significantly lie lower.

The relatively high positive number for the substance chronic hepatitis B patients suggest that it is a double infection with Non-A, Non-B and hepatitis B acts.

Table 4 shows the following: An investigation on recipients with a blood transfusion that is principally considered a risk patient have to apply because common in blood transfusions a non-A, non-B hepatitis infection occurs. It deals is a prospective study.

This clearly shows that depending on the severity the consequences of transfusion, on the one hand, patients nothing has happened, so where these substances are only in very has been eliminated to a small extent, on the other hand at Patients in whom the disease is recognizable and in  to those who have overt hepatitis in over 70% of the time this substance is detectable.

Example 3 Isolation of stool DNA and cloning of DNA sequences and their incorporation into corresponding vectors. Incorporation of this DNA into cloning vectors

From the stool of patients with non-A, non-B hepatitis were, as in example 1, non-A, non-B hepatitis-associated particles isolated and, as described there, treated with RNAs and DNAs and via a Cesium chloride gradient cleaned and dialyzed extensively. The dialyzed Fractions were presence in the manner described above of NANB-associated substance examined. The faction the density of 1.3 g / ml in the cesium chloride gradient was determined with 50 micrograms proteinase K, 1% SDS and EDTA at a final concentration of 10 mM / l digested for 6 hours at 37 ° C. Proteins were extracted by extraction with 80% phenol (Weight / volume) and chloroform removed, and then the DNA dissolved in the aqueous phase in the presence of 0.3 mol / l sodium acetate with a volume two and a half times of ethanol precipitated for 60 hours at -70 ° C. After that was centrifuged and the sediment once with 70% ethanol washed and then in TE buffer consisting of 10 mmol / l Tris, pH 8.0, and 1 mmol / l EDTA, in a volume ratio from a microliter / 5 mg of processed stool, added.

15 microliters of this DNA stock solution were in a total reaction volume of 50 microliters with 6 units of Klenow Polymerase (DNA polymerase I, large fragment), 1 microliter a solution of dATP, dTTP and dGTP, each in a concentration of 1 mM / l and 5 microliters of alpha 32P-dCTP (3000 Ci / mmol, 10 mCi / ml) in the incubation buffer for Klenow polymerase according to the manufacturer's instructions Incubated for 1 hour at room temperature.  

Then 2.5 microliters of a 1 mmol / l dCTP solution were added and the sample for another hour at room temperature incubated. After inactivating the enzyme by heating at 68 ° C for 10 minutes the reaction solution was on Cooled to 0 ° C. After that, the reaction mixture became together with 2 units of T4 DNA ligase, 1 microgram phosphorylated EcoR-I linker and 6 microliters of 10 mM ATP solution for 16 hours incubated at 16 ° C. The reaction mixture was then adjusted to a final concentration of 150 mmol / l NaCl and with 240 units of EcoR-I restriction endonuclease (80th Units / microliter) digested for 3 hours at 37 ° C. The reaction was by adding 5 microliters of 80% phenol and 1 microliter of 20% SDS stopped. The radioactively marked and linked DNA was from salt and unalloyed linkers on a Sepharose 4B-CL column (3 ml column volume, 25 cm length) separated and with 2.5 Volumes of ethanol precipitated at -70 ° C for 16 hours. The precipitated DNA was centrifuged at 14,000 g for 10 min and the Sediment washed at 150 microliters of 70% alcohol, dried and taken up in 10 microliters of TE buffer.

Example 4 Incorporation of the isolated DNA in lambda phages and transfection on bacteria

For the ligation with the vector DNA, 2 micrograms of DNA from the Phage lambda 1149 with 2 units of EcoR-I (4 U / microliter) in a total reaction volume of 6 microliters (incubation buffer according to the enzyme manufacturer (Boehringer, Mannheim) incubated for 1 hour at 37 ° C. Then the enzyme inactivated by heating to 68 ° C for 10 minutes. This solution was labeled with 3 microliters of DNA, as in the previous Example described, was isolated, 1 microliter 5 mM ATP and 1 unit T4 DNA ligase ligated at room temperature. 4 microliters of this reaction mixture were in lambda phage casings packed (using a finished packaging extracts, vector-cloning systems). With these packaged phages, the Escherichia Coli strain became  NM 514 infected. For screening for the presence of built-in DNA was about 10⁵ "plaque forming units" on 22 × 22 cm screening plates plated and over Incubated at 37 ° C overnight. Phage DNA was on nitrocellulose filter transferred by imitation, and the so obtained Contact filters were used with 1 microliter of that described above radioactive DNA stock solution according to Maniatis et al. (1982) hybridized, washed and autoradiographed for 6 hours at 70 ° C.

Of several positive ones Hybridization signals were assigned to 17 phage colonies placed in a density of approx. 5 plaques per cm². Out of 70 signals on these sample plates Plaque could be assigned, 17 were followed up through plaques purification according to BENTON and DAVIS (1978) and out of the 16 positive ones finally received Lysates were prepared for plaques. Several of these phages contained DNA inserts between 0.3 and about a length 1.6 KB with a sample of the original DNA stock solution hybridized.

Example 5 Characterization of a DNA fragment of 0.5 KB and subcloning of this fragment in a plasmid PUC 19 in Escherichia coli

The DNA fragment was digested with appropriate Restriction endonucleases (EcoR-I) among those already above described conditions removed from that of the phage DNA, the fragment then on agarose gel from the lambda phage DNA separated and the DNA band corresponding to the insert from the Gel eluted. The insert DNA thus isolated is then exactly as described above, ligated into vector DNA PUC 19 and then transfected to Escherichia coli strain DH 1. To Selection of bacterial strains that this plasmid with the built-in hepatitis Non-A, Non-B DNA insert added have and multiply, the breeding was then carried out and as in T. Maniatis et al. 1982, in "Molecular Cloning, a  laboratory manual ", described, processed.

The same insert was labeled radioactively and with Southern analysis for hybridization with the DNA starting material, DNA extractions from stools of control subjects, hepatitis B virus DNA and Escherichia coli plasmid PBR 322 tested.

Example 6 Detection of Non-A, Non-B-associated DNA in the serum

2-5 ml of serum are centrifuged at 150,000 g for 2 hours. The sediment is dissolved in 200 µl Proteinase K solution (0.5 mg / ml + 10 mmol EDTA) digested at 37 ° C for 1 hour. After that, too this solution 165 µl of a hot saturated sodium iodine solution (2.5 g sodium iodide / ml H₂O, 75 ° C) added (final concentration 12.5 M). This solution is heated to 90 ° C for 10 minutes and directly through nitrocellulose filter using vacuum filtered (dot blot apparatus) After filtration, the nitrocellulose sheet Washed 3 × with 70% ethanol and then at 10 min Room temperature in 100 ml acetic anhydride (100 ml 0.1 M Triethanolamine + 250 ul acetic anhydride) incubated. To The incubation is done in vacuum at 80 ° C for 1-2 hours baked. The dry film is in pre-hybridization solution brought and incubated for 3 hours at 65 ° C (6 × SSC, 1 × Denhardt, 0.5% SDS, 20 µg / ml heat denatured herring sperm DNS according to the information in: Maniatis, Molecular Cloning, 1982). Using nick translation (see Maniatis, Molecular Cloning, 1982) with 32P-labeled sample overnight under pre-hybridization conditions incubated. Then the nitrocellulose film Washed 3 × (1). 6 × SSC, 1 × Denhardt, 0.5% SDS, 20 µg / ml herring sperm DNA, 2). 1 × SSC, 0.5% SDS, 1 × Denhardt, 3). 0.1 × SSC + 0.05% SDS). After drying the film is placed on an X-ray film hung up (autoradiography time 6 hours to 2 days at -70 ° C).  

In the case of sera with a high content of HNANB viruses, the concentration can be reduced of 2-5 ml of serum by centrifugation are omitted and the sera after Proteinase K digestion according to the method (Seelig et al., clinician 2/1985, page 86 ff.) directly on nitrocellulose be applied (see Example 7).

Example 7 HNANB DNA detection in serum

200 ul serum with 75 ul proteinase K solution (4 mg / ml) and 25 µl 0.5 M EDTA incubated at 37 ° C for 1 hour. The incubate is mixed with 100 µl 1 N Incubate NaOH for 10 minutes at room temperature and with 250 µl 2 M NH₄OAc neutralized. 500 µl of the mixture (corresponding to 181 µl serum) are applied Nitrocellulose applied (Dott-Blott apparatus). To neutralization again with 250 µl 2 M NaH₄OAc Vacuum dried at 80 ° C for 45 minutes. The filters are in 6 × SSC (20 × SSC (standard saline citrate) corresponds to 3 M NaCl, 1.5 M sodium citrate), 0.5% SDS (SDS sodium dodecyl sulfate), 1 × Denhardt's solution (100 × Denhardt's solution corresponds to 2% Ficoll; 2% polyvinylpyrolidone, 2% Bovin serum albumin) and 20 µl / ml heat denatured (5 minutes, 100 ° C) Pre-hybridized herring sperm DNA at 65 ° C for 3 hours. For hybridization, that takes place overnight under prehybridization conditions will by nick translation with 32P-labeled phage clone inserts used (specific activity approx. 1-2 × 10⁹ cpm / µg DNA). The filters are then once each 20 minutes at 65 ° C with 6 × SSC, 1 × Denhardt Solution, 0.5% SDS, 20 µg / ml denatured herring sperm DNA, then with 1 × SSC, 0.5% SDS, 1 x Denhardt's solution and 0.1 x SSC, 0.05% SDS and dried at 80 ° C. The autoradiography is carried out using an X-ray film and intensifying screen with an autoradiography time from 6-48 hours at -70 ° C.  

The sequencing of the attached sequence was carried out after the Sanger method after cloning PUC 8 into M 13.

The DNA sequence found is shown in FIGS. 1 to 3.

Fig. 1 shows the minus strand,

Fig. 2 the plus strand and

Fig. 3 shows the interfaces with the enzymes also specified.

In summary, the following can be said: The non-A, non-B-associated substance is characterized by significant binding to immunoglobulins and binding IgGs purified on Fab2 fragments by binding to not collagen-binding fibronectin cleavage products and by Infectiousness to human cell culture lines that are morphologically representable through virus-typical changes the same (bottles) and molecular genetically by the Evidence of a specifically hybridizing, approximately 3.2 KB in size DNA within these cell cultures. Those in the infected Cells as well as in the serum of non-A, non-B hepatitis patients Detectable DNA hybridizes with that from non-A, non-B substance isolated, approx. 3.2 KB large DNA or the DNA from it Material produced cloned DNA.

The Non-A, Non-B hepatitis-associated DNA is approximately 3.2 KB circular, partially double-stranded and can be Fill in polymerase 1.

With the DNA, the expression of virus systems is suitable Vectors in E.coli and yeast (Saccharomyces cereviciae) as well as cell cultures for diagnostics and the production of immune reagents and vaccines possible. When diagnosing are particular the examination for infectiousness (blood test) and to mention the exclusion diagnosis.  

A partial sequence of approximately 300 base pairs of the cloned DNA was determined and with known human DNA sequences, Phage DNA sequences, plasmid DNA sequences and known published Virus DNA sequences compared. It corresponds after this data no sequence described so far. To conclusions can be drawn from the open reading frame of the sequence the encoded and expressed virus-specific proteins pull. This allows you to see the hydrophilic and hydrophobic regions identify within the peptides, and thus the Production of synthetic peptides from the possible antigens Epitopes possible in the hydrophilic regions. This allows antibody detection in vivo and antibody synthesis.  

Table 1

HNANB-associated substance in the stool in patients with sporadic and post-transfusion hepatitis Non-A, Non-B

Table 2

Elimination of HNANB-associated substance in patients with various diseases affecting the liver

Table 3

Hepatitis Non-A, non-B associated substance for hepatitis A and hepatitis B virus infections

Table 4

Prospective study in transfusion recipients

Claims (8)

1. Non-A, Non-B hepatitis-associated DNA and fragments of which, obtainable by forming a Cesium chloride gradient of the commonly cleaned Stool preparation, isolating the cesium chloride fraction with a density of 1.3 (1.29-1.32) g / ml cesium chloride, this fraction after electrophoresis and transfer Nitrocellulose in the form of four bands with one Molecular weights of 64,000, 56,000, 51,000 and 43,000 is present, and digestion of the addressed fraction with Proteinase, extraction with phenol and chloroform and subsequent precipitation with ethanol and centrifugation, where the DNA is 3.2 KB, circular and partial is double-stranded and deals with Klenow polymerase 1 can fill up. 2. Non-A, Non-B hepatitis associated DNA and fragments thereof according to claim 1, characterized in that the Cleaning the stool specimen a digestion with RNase and DNase. 3. Process for the production of non-A, non-B hepatitis associated DNA and fragments thereof according to claim 1, characterized in that by forming a Cesium chloride gradient of the cleaned in the usual way Stool preparation, isolating the cesium chloride fraction with a density of 1.3 (1.29 to 1.32) g / ml cesium chloride, this fraction after electrophoresis and transfer Nitrocellulose in the form of four bands with one Molecular weights of 64,000, 56,000, 51,000 and 43,000 is present or after separation of raw stool concentrates  without cesium chloride purification according to SDS Page and blotting in Form of two main bands with a molecular weight of 60,000 is present, and subsequent digestion of the addressed fraction with proteinase, extraction with Phenol and chloroform and subsequent precipitation with Ethanol and centrifugation, the DNA being 3.2 KB, circular and is partially double-stranded and deals with Klenow polymerase 1 can be filled. 4. The method according to claim 3, characterized in that cleaning the stool with digestion RNase and DNase included. 5. DNA partial sequence according to claim 1 6. Use of the DNA and fragments thereof according to claim 1 and the sequence of claim 5 as DNA probes Diagnosis of non-A, non-B hepatitis according to classic Hybridization process. 7. Use of the sequence according to claim 5 for the preparation synthetic peptides. 8. Use of the DNA and fragments thereof according to claim 1 and the sequence of claim 5 for the production of Vaccines and immune reagents.