CA2077709A1 - Oligonucleotides and determination system of hcv genotypes - Google Patents
Oligonucleotides and determination system of hcv genotypesInfo
- Publication number
- CA2077709A1 CA2077709A1 CA002077709A CA2077709A CA2077709A1 CA 2077709 A1 CA2077709 A1 CA 2077709A1 CA 002077709 A CA002077709 A CA 002077709A CA 2077709 A CA2077709 A CA 2077709A CA 2077709 A1 CA2077709 A1 CA 2077709A1
- Authority
- CA
- Canada
- Prior art keywords
- hcv
- oligonucleotide
- genotypes
- type
- oligonucleotides
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Abstract
Abstract Hepatitis C virus oligonucleotides and a system to determine hepatitis C virus genotypes with polymerase chain reaction utilizing those oligonucleotides as primers.
Description
OLIGON~CL~OTID~8 A~D D~ER~I~A~IO~
8Y8~BM OF ~CV G~NOTYPB8 Backgrouna o~ th~ ~nve~tion The present invention concerns hepatitis C virus (hereinafter called HCV) oligonucleotides and a system to determine HCV genotypes with polymerase chain reaction (PCR) utilizing those oligonucleotides as primers.
Viral hepatitis of which DN~ and RN~ of the causative viruses have been elucidated, and their diagnosis and even prevention in some cases have been established, are hepatitis A
and hepatitis B. The general name NANB (non-A, non-B) hepatitis was given to the other forms of viral hepatitis.
Post-trans~usion hepatitis was remarkably reduced after introduction of diagno~tic system~ for screening hepatitis B virus in transfusion bloods. However, there are still an estimated 280, ooa annual cases of post~transfusion hepatitis caused by NANB hepatitis virus in Japan.
NANB hepatitis viruses were recently named C, D and E
according to their types, and scientists started a world wide effort to conduct research for the causative viruses.
In 1988, Chiron Corp. claimed that it had succeeded in cloning RNA virus genome, which it termed hepatitis C virus (HCV), as the causative agent of NAMB hepatitis and reported on its nucleotide sequence (British Patent 2,212,511 which is the equivalent o~ European Patent Application 0,318,216). HCV
(c100-3~ antibody detection systems based on the sequence are 2~ ~7 d~ ~ r9 now being introduced for screening of transfusion bloods and for diagnosis of patients in Japan and in many other countries.
The detection systems for the c100-3 antibody have proven their partial association wikh NANB hepatitis; however, they capture only about 70% of carriers and chronic hepatitis patients, or they fail to detect the antibody in acute phase infection, thus leaving problems yet to be solved even after development of the c100-3 antibody by Chiron Corp.
The genome structure of HCV, or NANB hepatitis having relation to c100-3 antibody~ has single stranded RNA which suggests some relationship to Flaviviruses and Pestiviruses.
From the comparison of those genome structures, regions coding core protein (C), envelope protein (E) and non-structure protein (NS) were found in HCV. c100-3 antibody detected by Chiron's ELISA kit is thought to recognize a part of the border region of NS3 /NS4 .
The course of NANB h~patitis is troublesome and many cases of horizontal transmission are considered to become carriers and then to develop chronic hepatitis. In addition, most patients with chronic hepatitis develop liver cirrhosis, then hepatocellular carcinoma. It is therefore very imp~rative to isolate the virus itself and to develop effective diagnostic reagents enabling earlier diagnosis.
The presence of a number of NAN~ hepatitis which can not be diagnosed by Chiron's HCV (c100-3) antibody detection kits suggests the importance of detection of antibody to core ~ ~ ~J~
protein instead of antibody to non-struotural protein. From that view point, the genome of the core region and the protein coded for by this region have been studied world wide.
From the two-by-~wo comparison o~ nucleotide sequences s o~ various HCV isola~es, homologies ~or the core region, which is rather conservative, were found to be less than 90% in some cases. In addition, more variability was found in the envelope region (which is thought to be important for development of vaccine3; less than 60% homology was found between some isolates. The sequence diversity between strains reflects the ~eatures of HCV as RNA virus.
A rapid and simple method of classifying of HCV
genomes into groups, therafore, would be very useful for diagnosis, therapy and prevention of NANB hepatitis.
Determination of HCV qenotypes could be useful in the following medical and social applications:
(1) identification of infectiou~ sources in vertical (mother-to-baby) transmission and horizontal transmission;
(2) clarification of genotype-speci~ic features in the course, condition and prediction of recuperation of NANB
liver diseases and making a guide of therapy theref~re:
8Y8~BM OF ~CV G~NOTYPB8 Backgrouna o~ th~ ~nve~tion The present invention concerns hepatitis C virus (hereinafter called HCV) oligonucleotides and a system to determine HCV genotypes with polymerase chain reaction (PCR) utilizing those oligonucleotides as primers.
Viral hepatitis of which DN~ and RN~ of the causative viruses have been elucidated, and their diagnosis and even prevention in some cases have been established, are hepatitis A
and hepatitis B. The general name NANB (non-A, non-B) hepatitis was given to the other forms of viral hepatitis.
Post-trans~usion hepatitis was remarkably reduced after introduction of diagno~tic system~ for screening hepatitis B virus in transfusion bloods. However, there are still an estimated 280, ooa annual cases of post~transfusion hepatitis caused by NANB hepatitis virus in Japan.
NANB hepatitis viruses were recently named C, D and E
according to their types, and scientists started a world wide effort to conduct research for the causative viruses.
In 1988, Chiron Corp. claimed that it had succeeded in cloning RNA virus genome, which it termed hepatitis C virus (HCV), as the causative agent of NAMB hepatitis and reported on its nucleotide sequence (British Patent 2,212,511 which is the equivalent o~ European Patent Application 0,318,216). HCV
(c100-3~ antibody detection systems based on the sequence are 2~ ~7 d~ ~ r9 now being introduced for screening of transfusion bloods and for diagnosis of patients in Japan and in many other countries.
The detection systems for the c100-3 antibody have proven their partial association wikh NANB hepatitis; however, they capture only about 70% of carriers and chronic hepatitis patients, or they fail to detect the antibody in acute phase infection, thus leaving problems yet to be solved even after development of the c100-3 antibody by Chiron Corp.
The genome structure of HCV, or NANB hepatitis having relation to c100-3 antibody~ has single stranded RNA which suggests some relationship to Flaviviruses and Pestiviruses.
From the comparison of those genome structures, regions coding core protein (C), envelope protein (E) and non-structure protein (NS) were found in HCV. c100-3 antibody detected by Chiron's ELISA kit is thought to recognize a part of the border region of NS3 /NS4 .
The course of NANB h~patitis is troublesome and many cases of horizontal transmission are considered to become carriers and then to develop chronic hepatitis. In addition, most patients with chronic hepatitis develop liver cirrhosis, then hepatocellular carcinoma. It is therefore very imp~rative to isolate the virus itself and to develop effective diagnostic reagents enabling earlier diagnosis.
The presence of a number of NAN~ hepatitis which can not be diagnosed by Chiron's HCV (c100-3) antibody detection kits suggests the importance of detection of antibody to core ~ ~ ~J~
protein instead of antibody to non-struotural protein. From that view point, the genome of the core region and the protein coded for by this region have been studied world wide.
From the two-by-~wo comparison o~ nucleotide sequences s o~ various HCV isola~es, homologies ~or the core region, which is rather conservative, were found to be less than 90% in some cases. In addition, more variability was found in the envelope region (which is thought to be important for development of vaccine3; less than 60% homology was found between some isolates. The sequence diversity between strains reflects the ~eatures of HCV as RNA virus.
A rapid and simple method of classifying of HCV
genomes into groups, therafore, would be very useful for diagnosis, therapy and prevention of NANB hepatitis.
Determination of HCV qenotypes could be useful in the following medical and social applications:
(1) identification of infectiou~ sources in vertical (mother-to-baby) transmission and horizontal transmission;
(2) clarification of genotype-speci~ic features in the course, condition and prediction of recuperation of NANB
liver diseases and making a guide of therapy theref~re:
(3) increasing the detection sensitivity of HCV by nucleic acid detection system or antibody detection system based on the study of diversity in nucleotide or amino acid sequences among HCV isolates o~ distinct genotypes;
t^1 ':~r ~
t^1 ':~r ~
(4) production of type-specific immunoglobulin (IgG) and vaccine based on the study of type-specific conservative regions in the envelope gene of various genotypes; and (5) rapid and effective prevention by selecting an appropriate IgG or vaccine for each genotype.
The inventors have obtained various HCV-RNAs from sera of humans and chimpanzees, and cloned cDNAs of HCV genomes in non-coding regions and coding regions for core and envelope proteins, followed by determination of nucleotide sequ~nces (Japanese Patent Applications 196175/91, ~7402/91 and 360441/91~, and sought to find a method to determine HCV
genotypes and oligonucleotide primers to be used in saicl method, which would be useful in diagnosis, therapy, prevention, identification of infPcticus source, and epidemiological study on HCV. As a result, variation of nucleotide sequence characteristics in some groups in the core region, which was thought to be rather conservative, was found, and much more variation ~as found in the envelope region.
~um~ary of th~ In~e~tion It has been found as a result of study of a great number of nucleotide sequences of HCV isolates that HCVs could be divided into four genotypes through the nucleotide sequence of the putative C gene and based on the homology of nucleotides.
Based on this finding, universal oligonucleotides and type-specific oligonucleotides were identified and synthesized.
d . 3 In accordance with one aspect of the invention, there is provided universal oligonucleotides ~104, #186, and ~256 and type-specific oligonucleotides #132, #133, ~134, #135 and #296.
A further feature of the invention resides in a method of determininy HCV genotypes by polymerase chain reaction (PCR) amplification of cDNA using one or more of said oligonucleotide as primers. The method of determining HCV genotypes could be carried out by measuring lengths of amplification products obtained by PCR described above.
Thus, in accordance with a further aspect of the invention, there is provided a method of determining HCV
genotypes. The method involves two stage PCR, wherein the second stage PCR comprises utilizing one or more of oligonucleotides #132, ~133, ~134, ~135 and #296 as antisense primer and utilizing one or more of oligonucleotides ~104, ~23 and #256 as sense primer. The method further involves first stage PCR utilizing one or both of oligonucleotides #23 and #256 as sense primer and #186 as antisense primer.
The method to determine genotypes can be used in a method of predicting response of a patient with type C hepatitis to drug therapy.
Brie~ De3~ription o~ the Dra~i~g~
Figure 1 i5 a table which shows the results of classification of genotypes and nucleotide sequences corresponding to primers on 39 samples.
i.
Figura 2 is a diagram which shows the structure of the coding region of NANB hepatitis virus genome and PCR products using primers of tha invention.
Figure 3 is a photo which shows electrophoresis pattern of genotypes I to IV. Indicat~d on the left lane are migration positions of molecular size markers (~X174-RF DNA
HaeIII digest; Pharmacia LKB~.
Figure 4 is a photo which shows lelectrophoresis pattern of double infection cases. Migration positions o PCR
products of the four different ~CV types are indicated on the right.
Figure 5 is a photo which shows electrophoresis pattern o~ identification of infectious sources. Migration positions o~ PCR products of the four di~ferent HCV types are indicated on the right.
Figure ~ i5 a table which shows nucleotide sequence of HCV in #256 and #296 region in 39 samples.
Figure 7 is a p~oto which shows electrophoresis pattern of 6 HCV samples. Indicated on the left lane are migration positions of molecular size markers (~X174-RF DNA
~aeIII digest; Pharmacia LKB).
Figure 8 is a photo which shows electrophoresis pattern of four HCV samples. Molecular markers in the left lane are lOObp-ladder (Gibco, BRL~.
In Figures 3 - 5, 7 and 8, I, II, IIX and IV show genotypes I, II, III and IV of HCV. 1, 2, 3, 4, 5 and 6 are lanes in electrophoresis pattern.
~etaile~ De~arip~io~ o~ the Inve~tio~
In accordance with the present inventionl there is provided a group of new oligonucleotidesj in particular, new oligonucleotide primers #132, #133, #134, ~135, #10~, #23, ~256, #186 and #296, which were obtained by chemical synthesis. These primers have the sequences shown in the attached seguence listing.
The novel oligonucleotides oP the invention were determined as follows:
In a first step, HCV-cDNAs spanning the putative C
gene were isolated from 39 serum or plasma samples from humans or chimpanzees, and nucleotide sequence~ o~ the cDNAs were determined as shown herein.
Four regions consisting of ? O successive nucleotides in common with all the cDNAs were selected at nt 148-167, nt 126-145, nt 139-158 and nt 391-410.
In the next step, regions were selected which consisted of 20 successive nucleotides which had high homology (basically containing less than 3 substitutions) in some cDNAs but showed low homology (basically containing more than 2 substitutions, and many substitutions observed in 5' terminus) in other cDNAs at nt 1~5-204, nt ~72-291, nt 302-321 and nt 251-270.
It was confirmed by the present invention that all the HCV-cDNAs could be classified into four groups when classified by th!e nucleotide sequence of five regions nt 185-204, nt 272-291, nt 302-321, nt 251-270 and nt 177-196 wi~hout an overlap.
Four nucleotides corresponding to regions having conservative sequences above were synthesized with Cyclone Plus DNA Synthesizer as ~104 (nt 148-167), #23 (nt 126-145), #256 (nt 139-158) and #186 (nt 391-410). In the same manner, five nucleotides having complementary sequences to type-specific regions of HCV genomes above were synthesized a~ ~13~ (nt 185-204), ~133 (nt 272-291~, #134 (nt 302-321), ~135 (nt 251-2703 and ~296 (nt 177-1963.
The method of classification of HCV genotypes by amplifying cDNA using oligonucleotide primers ~132, ~133, ~134, ~135 and #296 having a complementary sequence to each region of HCV genomes as antisense primers, was completed.
Oligonucleotide primers #23 (nt 148-167), #104 (nt 126-145), #256 (nt 139-158) and #186 (nt 391-410) are well-conserved among different genotypes, and can be utilized as universal sense primers or antisense primers.
A preferred way to carry out the method of detlermining HCV genotypes is as follows: C-region cDNA is obtained :by ~5 reverse transcription with primer #186, and the cDNA is amplified using oligonucleotide primer #23 or #256 as a sense ~ /J~
primer and #186 as an antisense primer in the first stage PCR
followed ~y amplification using #104 as a sense primer and #132, #133, #134 and #135 as antisense primers in the second stage PCR.
Since PCR products can be separated by electrophoresis and observed under u.v. light, the genotypes can be identified according to the length of the ampli~ication products of the second stage PCR.
When previously isolated HCV genomes were classified io by the method of the invPntion, they were clivided into each one of four groups of type I, II, III and IV.
Names o~ genotypes were defined as follows; Type I:
HCV group which can be amplified using antisense primer #132 and/or #296, and in the same manner, type II: #133, type III:
#134, and type IV: #135.
HCV clones HC-J1, HC-J4/ HC-J5, HC-J6, HC-J7 and HC-J8, which were isolated by the inventors and disclosed in the above-identified Japanese patent applications, were classified into type I, II, III, III, IV and IV respectively using the method o~ determining HCV genotypes.
As shown in the Examples, 39 HCVcDNA clones obtained from serum or plasma of humans or chimpanzees were classified into each one of four genotypes I-IV. The homology of the nucleotide sequence of 39 samples were examined at the putative C gene nt 146-486. As a result, the homology was calculated as 95.1-97.5% among isolates classified into a genotype, while it } d~
was calculated as 77.9-91.0~ among isolates classified into different genotypes. The results show that the method of the invention can iden~ify HCV genotypes e~ficiently. (s~e Table l;
values represent mean + S.D. percentage identity) Table 1. Degree of sequence identity in a part of the putative C gene within and among four diff,erent types o~ HCV
Number Type of isolates I II III IV
4 . I 97.4+0.3_ _ 20 II 91.0+1.0 95.3~1.2 _ _ III 79.5+0.8 79.4~1.1 95.1+1.0 IV 79.1+0.8 _77.9+0. 7 85 . 5+0 e 8 96 . 1+0 . 6 In the specification, i'nt?' was numbered from the start of the single open reading frame, and "nc-nt" was numbered from putative 5' terminus o~ 5' non-coding region of the HCV genome.
Genotypes of Japanese sera from patients with ~lCV
infection were examined by the method of the invention. As a result, as much as 82% were classified into type II, than 10%, 4% and 4% were respectively classified into type III, I and IV, among asymptomatic (healthy) carriers. In contrast, 60~ were classified into type II, and 23% into type III among patients with chronic NANB liver disease. (Table 2) This result V )t~ 3 suggested that type III has higher possibility to cause an outbreak of chronic hepatitis than type II~
As described above, the method of the invention was shown to be useful in prevention and prediction of prognosis of 5 HCV infection and type C chronic liver disea6es.
T~ble 2. Prevalence of four HCV types in blood donors and patients with HCV infection Donor orSample Genotype patient number I II III IV Mixed Blood 159 6 131 16 6 0 donors L~82%L_ _ ~10%) (4~) Acute 12 0 9 3 0 0 hepatatis _ (o%)(75%l ~25%~ (0%) Chronic 27 a 16 7 3 hepatatis - . . . L~L_ _ (59%! __( 26%) (ll~L__ _ (I/II
Liver 24 2 15 5 2 0 cirrhosis . __L~L f 63~ (21%) Hepatocellular 17 2 8 3 3 carcinoma (11%~ ~47~ (18%~
Hemophiliac ll 3 3 2 1 2 _ (27%) 127~1 ~18%~ (9%) ~rI/II7I/III~
The method of the invention enables identification of ~CV double infection and tracing of the sourcc of infection.
In addition, as described in the Examples, significance was found in responses to interferon (hereinafter IFN) between types II and III among patients with chronic hepatitis C. This finding made it clear that the method to 9f~ ~J.~td,~
determine HCV genotypes of the invention is very useful in predicting the efficiency of drug therapy against hepatitis C.
The invention provides a method to definit~ly determine HCV genotypes, and it will be useful in many applications e.g~ development of HCV IgG and vaccine, and establishment of the manner to administer them.
The invention provides a method to predict response of a patient with type C hepatitis to drug therapy.
The invention also provides oligonucleotides which can be utilized as sense primers or antisense primers in the method of determining HCV genotypes.
Examples of applications of this invention are shown below, however, the invention shall in no way be limited to those examples.
E:~calmple~8 Example 1 Oligonucleotide #132, ~133, #134, #135, #104, #23, ~256, ~186 and #296 were determined in the following way.
~12 Isolation of RNA
RNA of serum or plasma samples from Japanese b:Lood donors testing positive for HCV antibody (c100-3 antibody; by Ortho HCV Ab ELISA, Ortho Diagnostic System, Tokyo), and that from the chimpanzee challenged with NANB hepatitis for ;~ J .~
infectivity and negative for HCV antibody, were isolated in the following method:
~50 ml of each plasma sample was added tv 150 ml of Tris chloride buffer (50 mM, pH 8.0, containing 150 ml NaCl, lO
mM EDTA) and centrifuged at 90 x 103 rpm for 15 minutes. The precipitate was suspended in Tris-chloride buffer (50 mM, pH
8.0, containing 200 mM NaCl, 10 mM EDT~, 2% (w/v~ sodium dodecyl sul~ate (SDS~ and proteinase X 1 mg/ml1, and incubated at 60 for 1 hour. Then the nllcleic acids were extracted by phenol/chloroform and precipitated by ethanol to obtain RNAs.
(2l Detectlon of 5' non-codin~ reqion RNA and core ~ PCR) The isolated RNA were heated at 70 for 1 minute;
using this as a template, cDNA was synthesi3ed using oligonucleotide primer #36 (5' ~ A A C A C T A C T C G G C T A G
C A G T - 3', nc-nt 246 265: antisense) and reverse transcriptase (Superscript, BRL, U.S.A.), and was provided to PCR as follows.
~0 cDNA was amplified according to Saiki's method (Science (1988), volume 239, pages 487-491) using Gene Amp DNA
Amplification Reagent (Perkin-Elmer Cetus~ on a DNA Thermal Cycler ~Perkin-Elmer Cetus). PCR was performed in two stages using 5' non-coding specific primer #32A (5' - C T G T G A G G A
A C T A C T G T C T T - 3', nc nt 45-64: sense) and #36 in the first stage; and #33 (5' - T T C A C G C A G A A A G C G T C T A
y r' ~ g G - 3', nc-nt 63-82: sense) and X48 (5' - G T T G A T C C A A G
A A A & G A C C C - 3', nc-nt 188-207: antisense) locating inside of #32~ and #36, in the second stage.
28 samples obtained ~rom sera of ~apanese blood donors, 10 from patients with N~NB liver disease, and one from chimpanzee observed to be infected with NAN~ hepatitis, all testing positive for 5' non-coding region RNA by said PCR in two stages, were provided to amplification of the C gene.
cDNA was synthesized for each sample using oligonucleotide #122 (5' - A G G T T C C C T G T T G C A T A A T
T - 3', nt 487-506: antisense) as a primer, followed by generation of a product of 38~bp representing nt 126-50~
corresponding to two third of C gene by PCR using ~23 and #122.
PCR was carried out by the following reaction cycle:
denaturation at 94C for 1 minute, annealing of primers at 55C
for 1.5 minutes, and extension at 72C for 2 minutes. The products were cloned into the HincII site of M13mpl9 phage vector. cDNA clones were sequenced in both directions by dideoxynucleotide chain termination method by the use of Sequenase version 2.0 (United States Biochemical, U.S.A.).
(3) Determination of universal primers and type-specific primers In the nucleotide sequence of the obtained clones, some regions consisting of 20 successive nucleotides having conservative sequence (having substitution of less than 3 based ~ r, ~ 1 ` r r~
in 20 bases) were selected. Nucleotide sequence in col~mon with all the cDNAs were observed in regions nt 148-167, nt 126-145, nt 13g-158 and nt 391-410. On the other hand, regions nt 177-196, nt 185-204, nt 272-291, nt 302 321 and nt 251-270 were recognized conservative in some isolates but not in common with others, which were thought to be genotype specific. (Figure 1, Figure 6).
Based on consensus sequence of each region, oligonucleotides #104 (sequence list 5, nt 148-1~7: sense), #23 (sequence list 6, nt 126-145: sense), #25~ (sequence list 7, nt 139-158: sense) and #186 (sequence list 8, nt 391-410:
antisense) were specified as universal primers.
In the same manner, oligonucleotides #132 (sequence list 1, nt 185-204: antisense), #133 ~sequence list 2, nt 272-291: antisense~, #134 (sequence list 3, nt 302-321:
antisense), #135 (sequsnce list 4, nt 251-270: antisense) and #296 (sequence list 9, nt 177-196: antisense) were specified as type-specific primers.
Said oligonucleotides #104, ~23, ~256, #186, #132, ~133, #134, ~135 and #296 were synth~sized with Cyclone Plus DNA
Synthesizer (MilliGen/Biosearch, Division of Millipore) in the usual way.
2~l J)7 ~
Example 2-Genotype determination system of the invention (1)~ Typing_of HCV-RNA positive samples Genotypes were determined using primers o~tained in Example 1.
cDNAs were obtained using primer #186 from 39 sera used in Example 1. The reaction cycle was :repeated 35 times with denaturation at 94C for 1 minute, primer annealing at 55C
for 1.5 minutes, and primer extension at 72C for 2 minutes.
In the first stage PC~, 272 nucleotides of nt 139-410 were amplified with #256 and #186 for 35 cycles with a reaction cycle as described ahove.
A 1/50 amount of the products was subjected to second PCR for 30 cycles using a universal primer #104 and a mixture of equivalant amount of four type-speci~ic primers, #132, ~133, #134 and #135, with denaturation at 94C for 1 minute, primer annealing at 60C for 1 minute, and primer extension at 72~C for 1.5 minut~s. The products of the second PCR were subjected to electrophoresis on a composite agarose gel made from 1.5%
NuSieve and 1.5% SeaKem (FMC BioProducts, U.S.A.), stained with '0 ethidium bromide, and observed under u.v. light.
The products were identified by molecular sizes. That is, since a product amplified with #132 Gonsists of 57bp, and that with #133, #134 and #135 consist respectively of 144bp, 174bp and 123bp, the position of a product shows its molecular ~5 size, and the primer used for amplification therefor. (Figure 3).
2 ~ 7 . ~
Genotypes of 39 sera were completely identified as same result in Example I-(3~ (Figure 1), showing excellent specificity of type-specific primers of the invention.
Genotypes Il II, III and IV show types amplified with primer #13~, #133, ~134 and ~135.
The same procedure with primer #~3 in place of #256 in the ~irst stage PCR gave the same result as (1) above.
(2) The same procedure with primer #23 in place of #104 in the second stage PCR gave same result as ~1) above.
The same procedure with primer #256 in place of #104 in the second stage PCR gave the same result as (1) ~bove.
(3) Variability o~ nucleotide sequence in nt 146 486 of C gene were examined on 39 sera classified into genotypes in (1) ahove. The clones classifiPd into a genotype, which were 15 ampli~ied by one o~ the mixed antisense primers, showed homology as high as 95.1-97.5% while those classified into the other genotypes showed homology as low as 77.9-91.0~u (Table 1~
cDNAs of HC-J1, HC-J4, HC-J5~ HC~J6 and HC-J7 or HCV
isolates disclosed in previous Japanese applications were 20 subjected to the method of determining genotypes using #132, #133, #134 and #135 as mixed antisense primers. As a result, HC-J1 was classi~ied into type I, and HC-J4, HC-J5, HC-J6 and HC-J7 were respectively classified into types II, III, III and IV.
Example 3 Test sera from 96 patients, diagnosed histopathologically by biopsy as chronic hepatitis C and testing positive for HCV-RNA by PCR amplifying 5' non-coding gene, were sub~ected to classification of HCV genotypes in the same manner described in Example 2-(l). There was found 69 as type II
(72%), 21 as type III (22%) and 6 as type IV (6%), where none was found as type I.
Drug therapy were carried out for said 96 patients with interf,_ron ~. Recombinant interferon-~a (Rofersn A, Nippon Roche, Tokyo) was administered subcutaneously 6MU/day for sevPn successive days, followed by 3~U/day for three days a week for 23 successive weeXs.
Just after the ~4-week IFN treatment, HCV-RNA was not found in 14 patients (20%) of type II, 14 (67%) of type III and 2 (33%) of type IV, from the result of PCR amplifying 5' non-coding gene. The HCV-RNA clearance rata of type II was found to be significantly higher than that of gene type III ~p<0.001, Wilcoxon test). Moreover, type III showed very low HCV-RNA
~0 level after IFN treatment compared with types II and IV as shown in Table 3. For the level of HCV-RNA in Table 3, values of ~., 2, 3, etc. represent 10, 101, 102, etc. CIU (Chimpanzee Infectious Unit)/ml.
2 ~ ., 9 ~ble 3. HCV g~notypes and response ~ chronic hepatitis C patients to interferon therapy.
GenotypesPatients HCV-RNA quantity HCV-RNA
number before IFN after IFN ~_eliminated cases II 69 5.2+1.2 2.7+2.014 (20.3%) ~0 III 21 4.7~1.1 0.8+1.314 (66.7%3 IV 6 4.8+1.6 3O3+2.8 ~ (33.3%) Total g6 5.1+1.2 2.3+2.130 (31.3%) As a result, some difference in response to interferon among genotypes was recognized among genotypes (which were classified by the method of determining genotypes). It became clear, therefore, that the invention i5 very useful in predicting the efficiency Q~ drug therapy against hepatitis C.
A study similar to that above can be conducted using other drugs or modalities of treatment. Differences in response of the four types of HCV to the treatment can thus be determined. Such data can then be used to predict the response to treatment of a patient in~ected with a strain of HCV. After the genotype of the patient's strain has been identified, the data from such a study will indicate the response to treatment o~ that strain which belongs to a particular type. For example, tabls 3 indicates that a patient infected with HCV type III would respond better to treatment with IFN than a patient infected with HCV type II.
Example 4 (l~ HCV genotypina of Ja~anese cases For the epidemiological study on ~ICV genotypes in Japan, samples were obtained from 159 blood donors, 80 patients with N~NB liver disease and 11 hemophiliacs. HCV-RNAs were isolated from those sera, followed by cDN~ synthesis and PCR to determine genotypes a~cording to the procedure described in Example 2.
As the result, more than 80% of blood donors were typed as II as shown in Table 2. On the other hand, 48 of 80 patients with NANB liver diseases ~60%) were typed as II, a significant difference ~p<0.01) was recognized. Among those with hepatocellular carcinoma, only 48~ were typed as II, while I (11%~, III (18~), IV (18~) wers found more often than among blood donors.
When blood donors and patients with NANB liver disease were compared on type III, 10~ (16/1593 were found in the former group, and 23% (18/80) in the latter group, a significant difference (p<OoOl) was recoynized. Significance was evaluated by Chi-sq~ared analysis.
Five samples were typed as I and 4 as II among hemophiliacs, including cases of suspected double infection.
The results suggested that some coagulation factor concentrates imported from the United States in the distant past, which had not been heat-inactivated, were possibly contaminated with HCV.
2~
From the above, the method of the invention provides easy classification of HCV genotypes. In addition, the existence among genotypes of differences in the clinical course of hepatitis after infection with ~CV suggests the usefulness of the method in the therapy and protection of NANB liver disease.
As shown above, the results suggest that type III has higher possibility to cause an outbreak of chronic hepatitis than type II.
(2) Identification of double infection Four cases of double infection were observed as shown in the electrophoresis pattern in Figure 4. Two hemophiliacs (types I and II in lane 3; types I and III in lane 4) described in Example 3-(l), a patient with chronic hepatitis (types I and II in lana 1) and a patient with hepatoma (types II and III in lane 2~ were included.
Dcuble infection with HCV of different types in a case of a hemophiliac (lane 3~ was confirmed by cloning of some cDNAs and sequence analysis within 341bp in the C gene (nt 146-486);
an identlty of more than 99% was observed between isolates of clones assumed to be the same type, while only B8.8% between those assumed to be distinct types. In the same manner, cDNAs obtained from another hemophiliac (lane 4~, a patient with chronic hepatitis and that with hepatocellular carcinoma were also classified into two types, whsre identities of 79.4%, 90.1%
and 80.3~ were respectively observed in distinct types. Those 2 ~ ~ 1 7 91 identities can be confirmed to show distinct types when compared with results in Example 2-(3)(Table 1).
Compared with conventional techniques to study genotypes, that is to isolate some clones from a sample, in order to determine those nucl~otide sequences and then to calculate identities between sequences, the method of the invention provides an expeditious method of datection of double infection by HCV of distinct types.
(3~ Identification of infectious source in vertical and horizontal transmission Test sera were obtained from 2 cases suspected of vertical (perinatal) transmission, and from a case of suspected horizontal transmission by needle stick exposure, and then subjected to determination of genotype~.
Figure 5 shows the result of electrophoresis of PC~
products following cDNA synthesis and PCR of typing according to the method described in Example 2. In Figure 5, lane 1-~ were from two mother-to-baby pairs of perinatal transmission. Lane 1 and 2 were PCR products from a mother and baby of a fami:Ly where genotypes of mother and baby were identified as type II; lanes 3 and 4 were from those of another family where genotypes of the mother and baby were identified as type III~ ~ane 5 and 6 were from a case of accidental HCV infection, where two genotypes from both donor (lane 5) and recipient (lane 6) were identical (type IV).
2~
As the result, it became clear that the method of the invention is useful in tracing infectious sources of NANB
hepatitis .
Example 5 HCV genomes HC-J2, HC-J3, (Japan J. Exp. ~ed. 60: 167-177, 1990) and HC-J8 (Japanese Patent Application No.
360441~91), which the inventors isolated and reported previously, were subjected to the determination of genotypes.
In the first stage PCR, ~256 was used as a sense primer, and #186 as an antisense primer. In the second stage PCR, #104 was used as a sanse primer, and mixed primer of ~13~, #133, ~134 and #135 was used as antisense primer according to the method des~ribed in Example ~-(1). HC-J1, HC-J4 and HC-J6 dsscribed above were subjected to the test for comparison.
Figure 7 shows the result of electrophoresis, where lanes 1-6 shows respectively HC-J1, ~C-J2, HC-J3, HC-J4, HC-J6 and HC-J8. As the result, HC-J2 and HC-J3 were typed as II (the same as HC-J4), while HC-J8 was typed as IV.
In Examples above, primers were used selecting T for Y
and G for R in #132; G for R, T for W and C fox Y in #133; G for R in position five and A for R in position 16 in #134; A for R
in positions one and 19, G for R in position 13 and C for Y in #135. With regard to universal primers, they were used selecting G for R in positions three, six, and 18, A ~or R in position seven and C for S in ~104; A for M, T for N, G for R in r~r~ J~
position 15 and A for R in position 15 in ~256; T for Y and G
for R in ~186.
The method of invention using primers selecting the other possible nucleotides gave same results as described above.
Example 6 Test sera No. 1, 2, 3 and 4, which were respectively typed as I, II, III and IV by the method described in Example 2-(1), were subjected to PCR for typing using equivalent mixture of primers #296, ~133, #134 and #135 as antisense primer. cDNA
priming and two stage PCR were carried out by the method described in Example 2 ~1) except primer #296 was used for ~132.
PCR products were observad under u.v. light (after electrophoresis :in composite agarose gel of 2% NuSieve and 2%
SeaKem and stained with ethidium bromide). (Figure 8) As a result, the produc~ generated by #296 (49bp) was observed for sample No. 1 (lane 1), and products generated by #133, #134 and #135 were observed for sample No. 2 4. It became clear that the method of the invention using ~296 instead of ~132 gives the same result in determination of HCV genotypes.
The invention provides an expeditious method to classi~y HCV genomes into four types (genotypes).
The invention ha~ many effects and applications, such as development of HC IgG and vaccine with high specificity and establishment of manner to admi.nister them.
2 .' ~ ~ ~ 3 Moreover, the invention provides a method to idantify HCV double infection and ~o track down the infectious source o~
HCV, and provides an excellent mathod in the study of HCV and in diagnosis, prevention and therapy of hepatitis C.
Furthar variations and modi~icat:ions of the in~ention will become apparent to those skilled in the art from the foregoing and are intended to be encompassed by the claims appended hereto.
Japanese Patent Application No. 307296/91 filed on September 9, 1991; 93960/92 filed on February 28, 1992; and a Japanese Patent Application filed on September 2, 1992, are all relied on and incorporated by reference; Hiroaki Okamoto and Tetsuo Nakamura are the inventors oE all three Japanese applications.
Copending U.S. Patent Application Serial No.
07/712,875 filed on June 11, 1991 and 07/873,126 filed on April 24, l9g2, are incorporated by reference in their entirety.
Okamoto et al., "Typing hepatitis C virus by polymerase chain reaction with type~specific primers:
application to clinical surveys and tracing infectious sources", Journal of General Viroloqy (1992), volume 73, pages 673-679, is incorporated by ~eference in its entirety.
8equ~e li~t:
SEQ ID LIST 1 (#132) tA)L~NGTH: 20 base pairs (B)TYPE: nucleic acid ~C)STRANDEDNESS: single (D)TOPOLOGY: linear (E)MOLECULE TYPE: cDNA to genomic RNA
(y = T/C; ~ = G/A) SEQ ID LIST 2 (#133) (A)LENGTH: 20 hase pairs (B)TYPE: nucleic acid (C)STRANDEDNESS: single (D)TOPOLOGY: linear (E)MOLECULE TYPE: cDNA to genomic RNA
(~ = G/A; W = T/A; Y = C/T) SEQ ID LIST 3 (#134) ~A)LENGTH: 20 base pairs (B)TYPE: nucleic acid (C)STRANDEDNESS: ~ingle (D)TOPOLOGY: linear (E)MOLECULE TYPE: cDNA to genomic RNA
(R = G/A) ~0 SEQ ID LIST 4 (#135) (A)LENGTH: 20 base pairs (B)TYPE: nucleir- acid (C)5TRANDEDNESS: single (D)TOPOLOGY: linear (E)MOLECULE TYPE: cDNA to genomic RNA
(R = G/A; Y = C/T) . .
f~ r, ~
SEQ ID LIST 5 (~104) (A)LENGTH: 20 base pairs (B)TYPE: nucleic acid (C)STRANDEDNESS: single (D)TOPOLOGY: linear (E)MOLECULE TYPE: cDN~ to genomic RNA
(R = G/A; S = O/G) SEQ ID LIST 6 (#23) ~5 (A)LENGTH: 20 base pairs (B)TYP~: nucleic acid (C~STRANDEDNESS: single (D)TOPOLOGY: linear (E)MOLECULE TYPE: cDNA to genomic RNA
~0 ~5 SEQ ID LIST 7 (#256) ~A)LENGTH: 20 base pairs (B)TYPE: nucleic acid (C)STRANDEDNESS: single (D)TOPOLOGY: linear (E)MOLECULE TYPE: cDNA to genomic RNA
(M = A/C; N = A/T/C/G; R = A/G) SEQ ID LIST 8 (#186) ~0 (A)LENGTH 20 base pairs (R)TYPE: nucleic acid (C)STRANDEDNESS: single (D~TOPOLOGY: linear (E)MOLEC~E TYPE: cDNA to genomic RNA
~5 (Y = T/C; R = G/A) ;~g~ t^,1'-`~
SEQ ID LIST 9 (#296) (A)LENGTH: 20 base pairs (B~TYPE: nucleic acid (C)STRANDEDNESS: single (D~TOPOLOGY: linear (E)MOLECULE TYPE: cDNA to genomic RNA
The inventors have obtained various HCV-RNAs from sera of humans and chimpanzees, and cloned cDNAs of HCV genomes in non-coding regions and coding regions for core and envelope proteins, followed by determination of nucleotide sequ~nces (Japanese Patent Applications 196175/91, ~7402/91 and 360441/91~, and sought to find a method to determine HCV
genotypes and oligonucleotide primers to be used in saicl method, which would be useful in diagnosis, therapy, prevention, identification of infPcticus source, and epidemiological study on HCV. As a result, variation of nucleotide sequence characteristics in some groups in the core region, which was thought to be rather conservative, was found, and much more variation ~as found in the envelope region.
~um~ary of th~ In~e~tion It has been found as a result of study of a great number of nucleotide sequences of HCV isolates that HCVs could be divided into four genotypes through the nucleotide sequence of the putative C gene and based on the homology of nucleotides.
Based on this finding, universal oligonucleotides and type-specific oligonucleotides were identified and synthesized.
d . 3 In accordance with one aspect of the invention, there is provided universal oligonucleotides ~104, #186, and ~256 and type-specific oligonucleotides #132, #133, ~134, #135 and #296.
A further feature of the invention resides in a method of determininy HCV genotypes by polymerase chain reaction (PCR) amplification of cDNA using one or more of said oligonucleotide as primers. The method of determining HCV genotypes could be carried out by measuring lengths of amplification products obtained by PCR described above.
Thus, in accordance with a further aspect of the invention, there is provided a method of determining HCV
genotypes. The method involves two stage PCR, wherein the second stage PCR comprises utilizing one or more of oligonucleotides #132, ~133, ~134, ~135 and #296 as antisense primer and utilizing one or more of oligonucleotides ~104, ~23 and #256 as sense primer. The method further involves first stage PCR utilizing one or both of oligonucleotides #23 and #256 as sense primer and #186 as antisense primer.
The method to determine genotypes can be used in a method of predicting response of a patient with type C hepatitis to drug therapy.
Brie~ De3~ription o~ the Dra~i~g~
Figure 1 i5 a table which shows the results of classification of genotypes and nucleotide sequences corresponding to primers on 39 samples.
i.
Figura 2 is a diagram which shows the structure of the coding region of NANB hepatitis virus genome and PCR products using primers of tha invention.
Figure 3 is a photo which shows electrophoresis pattern of genotypes I to IV. Indicat~d on the left lane are migration positions of molecular size markers (~X174-RF DNA
HaeIII digest; Pharmacia LKB~.
Figure 4 is a photo which shows lelectrophoresis pattern of double infection cases. Migration positions o PCR
products of the four different ~CV types are indicated on the right.
Figure 5 is a photo which shows electrophoresis pattern o~ identification of infectious sources. Migration positions o~ PCR products of the four di~ferent HCV types are indicated on the right.
Figure ~ i5 a table which shows nucleotide sequence of HCV in #256 and #296 region in 39 samples.
Figure 7 is a p~oto which shows electrophoresis pattern of 6 HCV samples. Indicated on the left lane are migration positions of molecular size markers (~X174-RF DNA
~aeIII digest; Pharmacia LKB).
Figure 8 is a photo which shows electrophoresis pattern of four HCV samples. Molecular markers in the left lane are lOObp-ladder (Gibco, BRL~.
In Figures 3 - 5, 7 and 8, I, II, IIX and IV show genotypes I, II, III and IV of HCV. 1, 2, 3, 4, 5 and 6 are lanes in electrophoresis pattern.
~etaile~ De~arip~io~ o~ the Inve~tio~
In accordance with the present inventionl there is provided a group of new oligonucleotidesj in particular, new oligonucleotide primers #132, #133, #134, ~135, #10~, #23, ~256, #186 and #296, which were obtained by chemical synthesis. These primers have the sequences shown in the attached seguence listing.
The novel oligonucleotides oP the invention were determined as follows:
In a first step, HCV-cDNAs spanning the putative C
gene were isolated from 39 serum or plasma samples from humans or chimpanzees, and nucleotide sequence~ o~ the cDNAs were determined as shown herein.
Four regions consisting of ? O successive nucleotides in common with all the cDNAs were selected at nt 148-167, nt 126-145, nt 139-158 and nt 391-410.
In the next step, regions were selected which consisted of 20 successive nucleotides which had high homology (basically containing less than 3 substitutions) in some cDNAs but showed low homology (basically containing more than 2 substitutions, and many substitutions observed in 5' terminus) in other cDNAs at nt 1~5-204, nt ~72-291, nt 302-321 and nt 251-270.
It was confirmed by the present invention that all the HCV-cDNAs could be classified into four groups when classified by th!e nucleotide sequence of five regions nt 185-204, nt 272-291, nt 302-321, nt 251-270 and nt 177-196 wi~hout an overlap.
Four nucleotides corresponding to regions having conservative sequences above were synthesized with Cyclone Plus DNA Synthesizer as ~104 (nt 148-167), #23 (nt 126-145), #256 (nt 139-158) and #186 (nt 391-410). In the same manner, five nucleotides having complementary sequences to type-specific regions of HCV genomes above were synthesized a~ ~13~ (nt 185-204), ~133 (nt 272-291~, #134 (nt 302-321), ~135 (nt 251-2703 and ~296 (nt 177-1963.
The method of classification of HCV genotypes by amplifying cDNA using oligonucleotide primers ~132, ~133, ~134, ~135 and #296 having a complementary sequence to each region of HCV genomes as antisense primers, was completed.
Oligonucleotide primers #23 (nt 148-167), #104 (nt 126-145), #256 (nt 139-158) and #186 (nt 391-410) are well-conserved among different genotypes, and can be utilized as universal sense primers or antisense primers.
A preferred way to carry out the method of detlermining HCV genotypes is as follows: C-region cDNA is obtained :by ~5 reverse transcription with primer #186, and the cDNA is amplified using oligonucleotide primer #23 or #256 as a sense ~ /J~
primer and #186 as an antisense primer in the first stage PCR
followed ~y amplification using #104 as a sense primer and #132, #133, #134 and #135 as antisense primers in the second stage PCR.
Since PCR products can be separated by electrophoresis and observed under u.v. light, the genotypes can be identified according to the length of the ampli~ication products of the second stage PCR.
When previously isolated HCV genomes were classified io by the method of the invPntion, they were clivided into each one of four groups of type I, II, III and IV.
Names o~ genotypes were defined as follows; Type I:
HCV group which can be amplified using antisense primer #132 and/or #296, and in the same manner, type II: #133, type III:
#134, and type IV: #135.
HCV clones HC-J1, HC-J4/ HC-J5, HC-J6, HC-J7 and HC-J8, which were isolated by the inventors and disclosed in the above-identified Japanese patent applications, were classified into type I, II, III, III, IV and IV respectively using the method o~ determining HCV genotypes.
As shown in the Examples, 39 HCVcDNA clones obtained from serum or plasma of humans or chimpanzees were classified into each one of four genotypes I-IV. The homology of the nucleotide sequence of 39 samples were examined at the putative C gene nt 146-486. As a result, the homology was calculated as 95.1-97.5% among isolates classified into a genotype, while it } d~
was calculated as 77.9-91.0~ among isolates classified into different genotypes. The results show that the method of the invention can iden~ify HCV genotypes e~ficiently. (s~e Table l;
values represent mean + S.D. percentage identity) Table 1. Degree of sequence identity in a part of the putative C gene within and among four diff,erent types o~ HCV
Number Type of isolates I II III IV
4 . I 97.4+0.3_ _ 20 II 91.0+1.0 95.3~1.2 _ _ III 79.5+0.8 79.4~1.1 95.1+1.0 IV 79.1+0.8 _77.9+0. 7 85 . 5+0 e 8 96 . 1+0 . 6 In the specification, i'nt?' was numbered from the start of the single open reading frame, and "nc-nt" was numbered from putative 5' terminus o~ 5' non-coding region of the HCV genome.
Genotypes of Japanese sera from patients with ~lCV
infection were examined by the method of the invention. As a result, as much as 82% were classified into type II, than 10%, 4% and 4% were respectively classified into type III, I and IV, among asymptomatic (healthy) carriers. In contrast, 60~ were classified into type II, and 23% into type III among patients with chronic NANB liver disease. (Table 2) This result V )t~ 3 suggested that type III has higher possibility to cause an outbreak of chronic hepatitis than type II~
As described above, the method of the invention was shown to be useful in prevention and prediction of prognosis of 5 HCV infection and type C chronic liver disea6es.
T~ble 2. Prevalence of four HCV types in blood donors and patients with HCV infection Donor orSample Genotype patient number I II III IV Mixed Blood 159 6 131 16 6 0 donors L~82%L_ _ ~10%) (4~) Acute 12 0 9 3 0 0 hepatatis _ (o%)(75%l ~25%~ (0%) Chronic 27 a 16 7 3 hepatatis - . . . L~L_ _ (59%! __( 26%) (ll~L__ _ (I/II
Liver 24 2 15 5 2 0 cirrhosis . __L~L f 63~ (21%) Hepatocellular 17 2 8 3 3 carcinoma (11%~ ~47~ (18%~
Hemophiliac ll 3 3 2 1 2 _ (27%) 127~1 ~18%~ (9%) ~rI/II7I/III~
The method of the invention enables identification of ~CV double infection and tracing of the sourcc of infection.
In addition, as described in the Examples, significance was found in responses to interferon (hereinafter IFN) between types II and III among patients with chronic hepatitis C. This finding made it clear that the method to 9f~ ~J.~td,~
determine HCV genotypes of the invention is very useful in predicting the efficiency of drug therapy against hepatitis C.
The invention provides a method to definit~ly determine HCV genotypes, and it will be useful in many applications e.g~ development of HCV IgG and vaccine, and establishment of the manner to administer them.
The invention provides a method to predict response of a patient with type C hepatitis to drug therapy.
The invention also provides oligonucleotides which can be utilized as sense primers or antisense primers in the method of determining HCV genotypes.
Examples of applications of this invention are shown below, however, the invention shall in no way be limited to those examples.
E:~calmple~8 Example 1 Oligonucleotide #132, ~133, #134, #135, #104, #23, ~256, ~186 and #296 were determined in the following way.
~12 Isolation of RNA
RNA of serum or plasma samples from Japanese b:Lood donors testing positive for HCV antibody (c100-3 antibody; by Ortho HCV Ab ELISA, Ortho Diagnostic System, Tokyo), and that from the chimpanzee challenged with NANB hepatitis for ;~ J .~
infectivity and negative for HCV antibody, were isolated in the following method:
~50 ml of each plasma sample was added tv 150 ml of Tris chloride buffer (50 mM, pH 8.0, containing 150 ml NaCl, lO
mM EDTA) and centrifuged at 90 x 103 rpm for 15 minutes. The precipitate was suspended in Tris-chloride buffer (50 mM, pH
8.0, containing 200 mM NaCl, 10 mM EDT~, 2% (w/v~ sodium dodecyl sul~ate (SDS~ and proteinase X 1 mg/ml1, and incubated at 60 for 1 hour. Then the nllcleic acids were extracted by phenol/chloroform and precipitated by ethanol to obtain RNAs.
(2l Detectlon of 5' non-codin~ reqion RNA and core ~ PCR) The isolated RNA were heated at 70 for 1 minute;
using this as a template, cDNA was synthesi3ed using oligonucleotide primer #36 (5' ~ A A C A C T A C T C G G C T A G
C A G T - 3', nc-nt 246 265: antisense) and reverse transcriptase (Superscript, BRL, U.S.A.), and was provided to PCR as follows.
~0 cDNA was amplified according to Saiki's method (Science (1988), volume 239, pages 487-491) using Gene Amp DNA
Amplification Reagent (Perkin-Elmer Cetus~ on a DNA Thermal Cycler ~Perkin-Elmer Cetus). PCR was performed in two stages using 5' non-coding specific primer #32A (5' - C T G T G A G G A
A C T A C T G T C T T - 3', nc nt 45-64: sense) and #36 in the first stage; and #33 (5' - T T C A C G C A G A A A G C G T C T A
y r' ~ g G - 3', nc-nt 63-82: sense) and X48 (5' - G T T G A T C C A A G
A A A & G A C C C - 3', nc-nt 188-207: antisense) locating inside of #32~ and #36, in the second stage.
28 samples obtained ~rom sera of ~apanese blood donors, 10 from patients with N~NB liver disease, and one from chimpanzee observed to be infected with NAN~ hepatitis, all testing positive for 5' non-coding region RNA by said PCR in two stages, were provided to amplification of the C gene.
cDNA was synthesized for each sample using oligonucleotide #122 (5' - A G G T T C C C T G T T G C A T A A T
T - 3', nt 487-506: antisense) as a primer, followed by generation of a product of 38~bp representing nt 126-50~
corresponding to two third of C gene by PCR using ~23 and #122.
PCR was carried out by the following reaction cycle:
denaturation at 94C for 1 minute, annealing of primers at 55C
for 1.5 minutes, and extension at 72C for 2 minutes. The products were cloned into the HincII site of M13mpl9 phage vector. cDNA clones were sequenced in both directions by dideoxynucleotide chain termination method by the use of Sequenase version 2.0 (United States Biochemical, U.S.A.).
(3) Determination of universal primers and type-specific primers In the nucleotide sequence of the obtained clones, some regions consisting of 20 successive nucleotides having conservative sequence (having substitution of less than 3 based ~ r, ~ 1 ` r r~
in 20 bases) were selected. Nucleotide sequence in col~mon with all the cDNAs were observed in regions nt 148-167, nt 126-145, nt 13g-158 and nt 391-410. On the other hand, regions nt 177-196, nt 185-204, nt 272-291, nt 302 321 and nt 251-270 were recognized conservative in some isolates but not in common with others, which were thought to be genotype specific. (Figure 1, Figure 6).
Based on consensus sequence of each region, oligonucleotides #104 (sequence list 5, nt 148-1~7: sense), #23 (sequence list 6, nt 126-145: sense), #25~ (sequence list 7, nt 139-158: sense) and #186 (sequence list 8, nt 391-410:
antisense) were specified as universal primers.
In the same manner, oligonucleotides #132 (sequence list 1, nt 185-204: antisense), #133 ~sequence list 2, nt 272-291: antisense~, #134 (sequence list 3, nt 302-321:
antisense), #135 (sequsnce list 4, nt 251-270: antisense) and #296 (sequence list 9, nt 177-196: antisense) were specified as type-specific primers.
Said oligonucleotides #104, ~23, ~256, #186, #132, ~133, #134, ~135 and #296 were synth~sized with Cyclone Plus DNA
Synthesizer (MilliGen/Biosearch, Division of Millipore) in the usual way.
2~l J)7 ~
Example 2-Genotype determination system of the invention (1)~ Typing_of HCV-RNA positive samples Genotypes were determined using primers o~tained in Example 1.
cDNAs were obtained using primer #186 from 39 sera used in Example 1. The reaction cycle was :repeated 35 times with denaturation at 94C for 1 minute, primer annealing at 55C
for 1.5 minutes, and primer extension at 72C for 2 minutes.
In the first stage PC~, 272 nucleotides of nt 139-410 were amplified with #256 and #186 for 35 cycles with a reaction cycle as described ahove.
A 1/50 amount of the products was subjected to second PCR for 30 cycles using a universal primer #104 and a mixture of equivalant amount of four type-speci~ic primers, #132, ~133, #134 and #135, with denaturation at 94C for 1 minute, primer annealing at 60C for 1 minute, and primer extension at 72~C for 1.5 minut~s. The products of the second PCR were subjected to electrophoresis on a composite agarose gel made from 1.5%
NuSieve and 1.5% SeaKem (FMC BioProducts, U.S.A.), stained with '0 ethidium bromide, and observed under u.v. light.
The products were identified by molecular sizes. That is, since a product amplified with #132 Gonsists of 57bp, and that with #133, #134 and #135 consist respectively of 144bp, 174bp and 123bp, the position of a product shows its molecular ~5 size, and the primer used for amplification therefor. (Figure 3).
2 ~ 7 . ~
Genotypes of 39 sera were completely identified as same result in Example I-(3~ (Figure 1), showing excellent specificity of type-specific primers of the invention.
Genotypes Il II, III and IV show types amplified with primer #13~, #133, ~134 and ~135.
The same procedure with primer #~3 in place of #256 in the ~irst stage PCR gave the same result as (1) above.
(2) The same procedure with primer #23 in place of #104 in the second stage PCR gave same result as ~1) above.
The same procedure with primer #256 in place of #104 in the second stage PCR gave the same result as (1) ~bove.
(3) Variability o~ nucleotide sequence in nt 146 486 of C gene were examined on 39 sera classified into genotypes in (1) ahove. The clones classifiPd into a genotype, which were 15 ampli~ied by one o~ the mixed antisense primers, showed homology as high as 95.1-97.5% while those classified into the other genotypes showed homology as low as 77.9-91.0~u (Table 1~
cDNAs of HC-J1, HC-J4, HC-J5~ HC~J6 and HC-J7 or HCV
isolates disclosed in previous Japanese applications were 20 subjected to the method of determining genotypes using #132, #133, #134 and #135 as mixed antisense primers. As a result, HC-J1 was classi~ied into type I, and HC-J4, HC-J5, HC-J6 and HC-J7 were respectively classified into types II, III, III and IV.
Example 3 Test sera from 96 patients, diagnosed histopathologically by biopsy as chronic hepatitis C and testing positive for HCV-RNA by PCR amplifying 5' non-coding gene, were sub~ected to classification of HCV genotypes in the same manner described in Example 2-(l). There was found 69 as type II
(72%), 21 as type III (22%) and 6 as type IV (6%), where none was found as type I.
Drug therapy were carried out for said 96 patients with interf,_ron ~. Recombinant interferon-~a (Rofersn A, Nippon Roche, Tokyo) was administered subcutaneously 6MU/day for sevPn successive days, followed by 3~U/day for three days a week for 23 successive weeXs.
Just after the ~4-week IFN treatment, HCV-RNA was not found in 14 patients (20%) of type II, 14 (67%) of type III and 2 (33%) of type IV, from the result of PCR amplifying 5' non-coding gene. The HCV-RNA clearance rata of type II was found to be significantly higher than that of gene type III ~p<0.001, Wilcoxon test). Moreover, type III showed very low HCV-RNA
~0 level after IFN treatment compared with types II and IV as shown in Table 3. For the level of HCV-RNA in Table 3, values of ~., 2, 3, etc. represent 10, 101, 102, etc. CIU (Chimpanzee Infectious Unit)/ml.
2 ~ ., 9 ~ble 3. HCV g~notypes and response ~ chronic hepatitis C patients to interferon therapy.
GenotypesPatients HCV-RNA quantity HCV-RNA
number before IFN after IFN ~_eliminated cases II 69 5.2+1.2 2.7+2.014 (20.3%) ~0 III 21 4.7~1.1 0.8+1.314 (66.7%3 IV 6 4.8+1.6 3O3+2.8 ~ (33.3%) Total g6 5.1+1.2 2.3+2.130 (31.3%) As a result, some difference in response to interferon among genotypes was recognized among genotypes (which were classified by the method of determining genotypes). It became clear, therefore, that the invention i5 very useful in predicting the efficiency Q~ drug therapy against hepatitis C.
A study similar to that above can be conducted using other drugs or modalities of treatment. Differences in response of the four types of HCV to the treatment can thus be determined. Such data can then be used to predict the response to treatment of a patient in~ected with a strain of HCV. After the genotype of the patient's strain has been identified, the data from such a study will indicate the response to treatment o~ that strain which belongs to a particular type. For example, tabls 3 indicates that a patient infected with HCV type III would respond better to treatment with IFN than a patient infected with HCV type II.
Example 4 (l~ HCV genotypina of Ja~anese cases For the epidemiological study on ~ICV genotypes in Japan, samples were obtained from 159 blood donors, 80 patients with N~NB liver disease and 11 hemophiliacs. HCV-RNAs were isolated from those sera, followed by cDN~ synthesis and PCR to determine genotypes a~cording to the procedure described in Example 2.
As the result, more than 80% of blood donors were typed as II as shown in Table 2. On the other hand, 48 of 80 patients with NANB liver diseases ~60%) were typed as II, a significant difference ~p<0.01) was recognized. Among those with hepatocellular carcinoma, only 48~ were typed as II, while I (11%~, III (18~), IV (18~) wers found more often than among blood donors.
When blood donors and patients with NANB liver disease were compared on type III, 10~ (16/1593 were found in the former group, and 23% (18/80) in the latter group, a significant difference (p<OoOl) was recoynized. Significance was evaluated by Chi-sq~ared analysis.
Five samples were typed as I and 4 as II among hemophiliacs, including cases of suspected double infection.
The results suggested that some coagulation factor concentrates imported from the United States in the distant past, which had not been heat-inactivated, were possibly contaminated with HCV.
2~
From the above, the method of the invention provides easy classification of HCV genotypes. In addition, the existence among genotypes of differences in the clinical course of hepatitis after infection with ~CV suggests the usefulness of the method in the therapy and protection of NANB liver disease.
As shown above, the results suggest that type III has higher possibility to cause an outbreak of chronic hepatitis than type II.
(2) Identification of double infection Four cases of double infection were observed as shown in the electrophoresis pattern in Figure 4. Two hemophiliacs (types I and II in lane 3; types I and III in lane 4) described in Example 3-(l), a patient with chronic hepatitis (types I and II in lana 1) and a patient with hepatoma (types II and III in lane 2~ were included.
Dcuble infection with HCV of different types in a case of a hemophiliac (lane 3~ was confirmed by cloning of some cDNAs and sequence analysis within 341bp in the C gene (nt 146-486);
an identlty of more than 99% was observed between isolates of clones assumed to be the same type, while only B8.8% between those assumed to be distinct types. In the same manner, cDNAs obtained from another hemophiliac (lane 4~, a patient with chronic hepatitis and that with hepatocellular carcinoma were also classified into two types, whsre identities of 79.4%, 90.1%
and 80.3~ were respectively observed in distinct types. Those 2 ~ ~ 1 7 91 identities can be confirmed to show distinct types when compared with results in Example 2-(3)(Table 1).
Compared with conventional techniques to study genotypes, that is to isolate some clones from a sample, in order to determine those nucl~otide sequences and then to calculate identities between sequences, the method of the invention provides an expeditious method of datection of double infection by HCV of distinct types.
(3~ Identification of infectious source in vertical and horizontal transmission Test sera were obtained from 2 cases suspected of vertical (perinatal) transmission, and from a case of suspected horizontal transmission by needle stick exposure, and then subjected to determination of genotype~.
Figure 5 shows the result of electrophoresis of PC~
products following cDNA synthesis and PCR of typing according to the method described in Example 2. In Figure 5, lane 1-~ were from two mother-to-baby pairs of perinatal transmission. Lane 1 and 2 were PCR products from a mother and baby of a fami:Ly where genotypes of mother and baby were identified as type II; lanes 3 and 4 were from those of another family where genotypes of the mother and baby were identified as type III~ ~ane 5 and 6 were from a case of accidental HCV infection, where two genotypes from both donor (lane 5) and recipient (lane 6) were identical (type IV).
2~
As the result, it became clear that the method of the invention is useful in tracing infectious sources of NANB
hepatitis .
Example 5 HCV genomes HC-J2, HC-J3, (Japan J. Exp. ~ed. 60: 167-177, 1990) and HC-J8 (Japanese Patent Application No.
360441~91), which the inventors isolated and reported previously, were subjected to the determination of genotypes.
In the first stage PCR, ~256 was used as a sense primer, and #186 as an antisense primer. In the second stage PCR, #104 was used as a sanse primer, and mixed primer of ~13~, #133, ~134 and #135 was used as antisense primer according to the method des~ribed in Example ~-(1). HC-J1, HC-J4 and HC-J6 dsscribed above were subjected to the test for comparison.
Figure 7 shows the result of electrophoresis, where lanes 1-6 shows respectively HC-J1, ~C-J2, HC-J3, HC-J4, HC-J6 and HC-J8. As the result, HC-J2 and HC-J3 were typed as II (the same as HC-J4), while HC-J8 was typed as IV.
In Examples above, primers were used selecting T for Y
and G for R in #132; G for R, T for W and C fox Y in #133; G for R in position five and A for R in position 16 in #134; A for R
in positions one and 19, G for R in position 13 and C for Y in #135. With regard to universal primers, they were used selecting G for R in positions three, six, and 18, A ~or R in position seven and C for S in ~104; A for M, T for N, G for R in r~r~ J~
position 15 and A for R in position 15 in ~256; T for Y and G
for R in ~186.
The method of invention using primers selecting the other possible nucleotides gave same results as described above.
Example 6 Test sera No. 1, 2, 3 and 4, which were respectively typed as I, II, III and IV by the method described in Example 2-(1), were subjected to PCR for typing using equivalent mixture of primers #296, ~133, #134 and #135 as antisense primer. cDNA
priming and two stage PCR were carried out by the method described in Example 2 ~1) except primer #296 was used for ~132.
PCR products were observad under u.v. light (after electrophoresis :in composite agarose gel of 2% NuSieve and 2%
SeaKem and stained with ethidium bromide). (Figure 8) As a result, the produc~ generated by #296 (49bp) was observed for sample No. 1 (lane 1), and products generated by #133, #134 and #135 were observed for sample No. 2 4. It became clear that the method of the invention using ~296 instead of ~132 gives the same result in determination of HCV genotypes.
The invention provides an expeditious method to classi~y HCV genomes into four types (genotypes).
The invention ha~ many effects and applications, such as development of HC IgG and vaccine with high specificity and establishment of manner to admi.nister them.
2 .' ~ ~ ~ 3 Moreover, the invention provides a method to idantify HCV double infection and ~o track down the infectious source o~
HCV, and provides an excellent mathod in the study of HCV and in diagnosis, prevention and therapy of hepatitis C.
Furthar variations and modi~icat:ions of the in~ention will become apparent to those skilled in the art from the foregoing and are intended to be encompassed by the claims appended hereto.
Japanese Patent Application No. 307296/91 filed on September 9, 1991; 93960/92 filed on February 28, 1992; and a Japanese Patent Application filed on September 2, 1992, are all relied on and incorporated by reference; Hiroaki Okamoto and Tetsuo Nakamura are the inventors oE all three Japanese applications.
Copending U.S. Patent Application Serial No.
07/712,875 filed on June 11, 1991 and 07/873,126 filed on April 24, l9g2, are incorporated by reference in their entirety.
Okamoto et al., "Typing hepatitis C virus by polymerase chain reaction with type~specific primers:
application to clinical surveys and tracing infectious sources", Journal of General Viroloqy (1992), volume 73, pages 673-679, is incorporated by ~eference in its entirety.
8equ~e li~t:
SEQ ID LIST 1 (#132) tA)L~NGTH: 20 base pairs (B)TYPE: nucleic acid ~C)STRANDEDNESS: single (D)TOPOLOGY: linear (E)MOLECULE TYPE: cDNA to genomic RNA
(y = T/C; ~ = G/A) SEQ ID LIST 2 (#133) (A)LENGTH: 20 hase pairs (B)TYPE: nucleic acid (C)STRANDEDNESS: single (D)TOPOLOGY: linear (E)MOLECULE TYPE: cDNA to genomic RNA
(~ = G/A; W = T/A; Y = C/T) SEQ ID LIST 3 (#134) ~A)LENGTH: 20 base pairs (B)TYPE: nucleic acid (C)STRANDEDNESS: ~ingle (D)TOPOLOGY: linear (E)MOLECULE TYPE: cDNA to genomic RNA
(R = G/A) ~0 SEQ ID LIST 4 (#135) (A)LENGTH: 20 base pairs (B)TYPE: nucleir- acid (C)5TRANDEDNESS: single (D)TOPOLOGY: linear (E)MOLECULE TYPE: cDNA to genomic RNA
(R = G/A; Y = C/T) . .
f~ r, ~
SEQ ID LIST 5 (~104) (A)LENGTH: 20 base pairs (B)TYPE: nucleic acid (C)STRANDEDNESS: single (D)TOPOLOGY: linear (E)MOLECULE TYPE: cDN~ to genomic RNA
(R = G/A; S = O/G) SEQ ID LIST 6 (#23) ~5 (A)LENGTH: 20 base pairs (B)TYP~: nucleic acid (C~STRANDEDNESS: single (D)TOPOLOGY: linear (E)MOLECULE TYPE: cDNA to genomic RNA
~0 ~5 SEQ ID LIST 7 (#256) ~A)LENGTH: 20 base pairs (B)TYPE: nucleic acid (C)STRANDEDNESS: single (D)TOPOLOGY: linear (E)MOLECULE TYPE: cDNA to genomic RNA
(M = A/C; N = A/T/C/G; R = A/G) SEQ ID LIST 8 (#186) ~0 (A)LENGTH 20 base pairs (R)TYPE: nucleic acid (C)STRANDEDNESS: single (D~TOPOLOGY: linear (E)MOLEC~E TYPE: cDNA to genomic RNA
~5 (Y = T/C; R = G/A) ;~g~ t^,1'-`~
SEQ ID LIST 9 (#296) (A)LENGTH: 20 base pairs (B~TYPE: nucleic acid (C)STRANDEDNESS: single (D~TOPOLOGY: linear (E)MOLECULE TYPE: cDNA to genomic RNA
Claims (19)
1. Oligonucleotide #132 comprising the nucleotide sequence of sequence list 1.
2. The oligonucleotide according to claim 1, wherein Y is T and R is G.
3. Oligonucleotide #133 comprising the nucleotide sequence of sequence list 2.
4. The oligonucleotide according to claim 3, wherein R is G, W is T, and Y is C.
5. Oligonucleotide #134 comprising the nucleotide sequence of sequence list 3.
6. The oligonucleotide according to claim 5, wherein the first occurrence of R is G and the second occurrence of R is A.
7. Oligonucleotide #135 comprising the nucleotide sequence of sequence list 4.
8. The oligonucleotide according to claim 7, wherein Y is C, the first occurrence of R is A, the second occurrence of R is G, and the third occurrence of R is A.
9. Oligonucleotide #104 comprising the nucleotide sequence of sequence list 5.
10. The oligonucleotide according to claim 9, wherein S is C; the first, second and fourth occurrences of R is G, and the third occurrence of R is A.
11. Oligonucleotide #256 comprising the nucleotide sequence of sequence list 7.
12. The oligonucleotide according to claim 11, wherein M is A, N is T, and the first occurrence of R is G and the second occurrence of R is A.
13. Oligonucleotide #186 comprising the nucleotide sequence of sequence list 8.
14. The oligonucleotide according to claim 13, wherein Y is T and R is G.
15. Oligonucleotide #296 comprising the nucleotide sequence of sequence list 9.
16. A method of determining HCV genotypes, said method comprising two stage PCR, wherein the second stage PCR
comprises utilizing one or more of oligonucleotides #132, #133, #134, #135 and #296 as antisense primer and utilizing one or more of oligonucleotides #104, #23 and #256 as sense primer.
comprises utilizing one or more of oligonucleotides #132, #133, #134, #135 and #296 as antisense primer and utilizing one or more of oligonucleotides #104, #23 and #256 as sense primer.
17. The method according to claim 16, wherein first stage PCR comprises utilizing one or both of oligonucleotides #23 and #256 as sense primer and #186 as antisense primer.
18. The method according to claim 16, further comprising measuring the molecular length of the resulting amplified cDNA.
19. A method to predict the response to drug therapy of a patient infected with a strain of HCV, said method comprising determining the sensitivity of HCV types I, II, III
and IV to said drug therapy, determining the HCV genotype of said strain of HCV by the method according to claim 16, and comparing said HCV genotype of said strain with said sensitivity of HCV types I, II, III and IV to said drug therapy.
and IV to said drug therapy, determining the HCV genotype of said strain of HCV by the method according to claim 16, and comparing said HCV genotype of said strain with said sensitivity of HCV types I, II, III and IV to said drug therapy.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP307296/91 | 1991-09-09 | ||
| JP30729691 | 1991-09-09 | ||
| JP9396092 | 1992-02-28 | ||
| JP93960/92 | 1992-02-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2077709A1 true CA2077709A1 (en) | 1993-03-10 |
Family
ID=26435220
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002077709A Abandoned CA2077709A1 (en) | 1991-09-09 | 1992-09-08 | Oligonucleotides and determination system of hcv genotypes |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP2542994B2 (en) |
| CA (1) | CA2077709A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5216721B2 (en) * | 1995-02-01 | 2013-06-19 | 株式会社エスアールエル | Method for distinguishing genotype of hepatitis C virus |
| JP4689968B2 (en) * | 2004-03-25 | 2011-06-01 | 株式会社エスアールエル | Method of distinguishing genotype A subtype of hepatitis B virus |
| JP5813702B2 (en) | 2012-12-12 | 2015-11-17 | 株式会社国盛化学 | container |
-
1992
- 1992-09-02 JP JP4276502A patent/JP2542994B2/en not_active Expired - Lifetime
- 1992-09-08 CA CA002077709A patent/CA2077709A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| JP2542994B2 (en) | 1996-10-09 |
| JPH05301887A (en) | 1993-11-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5427909A (en) | Oligonucleotides and determination system of HCV genotypes | |
| Smuts et al. | Genotyping of hepatitis C virus in South Africa | |
| Takada et al. | Differences in the hepatitis C virus genotypes in different countries | |
| Fiordalisi et al. | High prevalence of GB virus C infection in a group of Italian patients with hepatitis of unknown etiology | |
| JP2656996B2 (en) | NANBV diagnostics | |
| JP6026401B2 (en) | Diagnostic assay for parvovirus B19 | |
| Mita et al. | Hepatitis C virus genotype and RNA titer in the progression of type C chronic liver disease | |
| Pistello et al. | Prevalence of hepatitis C virus genotypes in Italy | |
| JP2005040145A (en) | HCV-related oligonucleotides and methods for amplifying specific region genes of HCV isolates | |
| EP0633321A1 (en) | HCV related oligonucleotides and uses thereof in methods for detecting and genotyping of HCV, and kits used in these methods | |
| Mathet et al. | Hepatitis B virus S gene mutants in a patient with chronic active hepatitis with circulating Anti‐HBs antibodies | |
| JP2012080890A (en) | Heteroduplex tracking assay (hta) for genotyping hcv | |
| JPH06503707A (en) | NANBV diagnostic method: polynucleotide useful for screening hepatitis C virus | |
| Giannini et al. | Prevalence of mixed infection by different hepatitis C virus genotypes in patients with hepatitis C virus–related chronic liver disease | |
| EP0633320A1 (en) | Oligonucleotides of HCV, primers ND probes therefrom, method of determining HCV genotypes and method of detecting HVC in samples | |
| Frangeul et al. | Mutations in NS5A region of hepatitis C virus genome correlate with presence of NS5A antibodies and response to interferon therapy for most common European hepatitis C virus genotypes | |
| US5290677A (en) | Rapid and sensitive test for detecting hepatitis A virus | |
| CA2077709A1 (en) | Oligonucleotides and determination system of hcv genotypes | |
| Calvo et al. | Hepatitis C virus heteroduplex tracking assay for genotype determination reveals diverging genotype 2 isolates in Italian hemodialysis patients | |
| EP1010759B1 (en) | Non-b non-c non-g hepatitis virus gene, polynucleotide, polypeptide, virion, method for separating virion, and method for detecting virus | |
| Toniutto et al. | Discordant results from hepatitis C virus genotyping by procedures based on amplification of different genomic regions | |
| JP3110437B2 (en) | Epidemic non-A non-B hepatitis virus antigen peptide and nucleic acid fragment encoding the same | |
| KR0126107B1 (en) | DNA and component polypeptides derived from non-A non-hepatitis B virus genes | |
| Yoon et al. | Molecular typing of hepatitis C virus genome from sera and liver tissues of patients with anti-HCV positive chronic liver disease | |
| Holmes et al. | Derivation of a rational nomenclature for hepatitis C virus by phylogenetic analysis of the NS-5 region |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued | ||
| FZDE | Discontinued |
Effective date: 20010910 |