1164
Correspondence
Ll-Min Huang, Chin-Yun Lee, Jen-Yang Chen, Czau-Siung Yang, Jung-Der Wang, Mei-Hwei Chang, Ching-Ying Hsu, and Pel-Fen Kuo Departments 0/Pediatrics. Bacteriology. and Public Health and Internal Medicine. College 0/ Medicine. National Taiwan University. and
Lack of Polymerase Chain Reaction Amplification in the
5' Region of a Hepatitis C Virus Isolate Colleagues-Hepatitis C virus (HCY) infection can be assessed by the detection ofviral RNA by reverse transcription (RT) and nested polymerase chain reaction (PCR). The sensitivity of this technique allows early diagnosis in acute infection, evaluation of infectivity of serum positive or negative for HCY antibodies, and monitoring of antiviral therapy. Since significant variability of the HCY genome has been observed [1-3], the choice of a region for primer selection appears to be of great importance. Recent identification of a highly conserved sequence located in the 5' noncoding region of the HCY genome [4, 5] has allowed the selection of primer sets efficient for RT and PCR. Indeed, several groups have reported a correlation between detection of HCY antibodies by ELISA and the RIBA HCY test system (Chiron, Emeryville, CA) and HCY RNA using PCR primer sets located in the 5' noncoding region [6]. We previously selected such inner and outer primers to evaluate the presence of HCY RNA by RT-nested PCR [7]. With regard to these data, we thought it would be interesting to ascertain if a PCR primer set (inner and outer primers) selected in only one conserved region (5') of the HCY genome is sufficient
Reprints or correspondence: Dr. D. Larzul, Molecular Biology Department. EIBET. 48-52 rue de la Gare, 78370. Plaisir, France. The Journal of Infectious Diseases 1992;165:1164-5 © 1992 byThe University ofChicago. All rights reserved.
0022-1899/92/6506-0036$01.00
Department 0/Pediatrics. Taipei Municipal Maternal and Child Health Hospital. Taiwan. Republic of China
References I. Salahuddin SZ,Ablashi DV. Markham PD,etal. Isolation ofa new virus, HBLV. in patients with Iymphoproliferative disorders. Science 1986;234:596-60 I. 2. Saxinger C. Polesky H, Eby N, et al. Antibody reactivity with HBLV (HHV-6) in USpopulations. J Virol Methods 1988;21: 199-208. 3. OkunoT, Takahashi K. Balachandra K, et al. Seroepidemiology of human herpesvirus 6 infection in normal children and adults. J Clin Microbiol 1989;27:651-3. 4. Rin H. Psychiatry and society. Taipei: Medicine Today Publishing. 1978:54-5. 5. Yamanishi K. OkunoT, Shiraki K, et al. Identification of human herpesvirus-6 asa causal agentforroseola infantum. Lancet 1988;I:1065-7. 6. Balachandra K. Ayuthaya PIN, Auwanit W, et al. Prevalence ofantibody to human herpesvirus 6 in women and children. Microbiol Immunol 1989;33:515-8. 7. Pietroboni GR. HarnettGB. Bucens MR. Honess RW. Antibody to human herpesvirus 6 in saliva. Lancet 1988;1:1059. 8. Levy JA. Ferro F. Greenspan D. Lennette ET. Frequent isolation of HHV-6 from saliva and high seroprevalence ofthe virus in the population. Lancet 1990;335: 1047-50.
to evaluate the presence of HCY RNA. In an attempt to clarify this question, we studied an asymptomatic subject from southern Italy with community-acquired chronic non-A, non-B hepatitis who was HCY-positive by ELISA (HCY ELISA, second generation; Ortho Diagnostic Systems, Roissy, France) and indeterminate by second-generation RIBA (Chiron). RIBA results showed a reactivity towards the c22-3 recombinant antigen band and indeterminate data for 5-1-1, cI00-3, and c33c antigens. HCY RNA evaluation of this patient's serum was repeatedly negative with our 5' region primer set and positive with an NS5 region primer set (N IJN2, d94/d95) [8]. Experiments were carried out on two serum samples, independently collected at a I-week interval with each sample studied in duplicate. The amplification protocol was an RT-nested PCR done under low stringency conditions for primer hybridization; the annealing temperature was 37°e. The 5' primer set allowed the amplification of a 177-bp DNA fragment that has never been demonstrated with either ethidium bromide-stained agarose gel or hybridization in low stringency conditions with a specific oligonucleotide probe. However, with the NS5 primer set, amplified DNA fragments of the expected size were visualized directly on the agarose gel after the first (730 bp) and second (405 bp) amplifications. An internal oligonucleotide probe hybridized specifically with these two amplified fragments. To. confirm the specificity of this amplification, the 405-bpamphfied DNA fragment was cloned in pBluescript KS (-) (Stratagene/Ozyme, Paris) and partly sequenced with the dideoxy method (170 bp). Sequencing was done on two strands of three independent clones. The sequence obtained revealed a homology of 88.2%,90.6%, and 77.6% with two other published
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 21, 2015
HHY-6. Although there has been no direct proof of protection by maternal HHY-6 antibodies, several studies [3, 6, 7], including this one, have found that HHY-6 infection increases after the first few months of life, suggesting a protective role for the maternal antibody. In addition, our findings that all patients were seronegative before they became infected strongly supports the possibility that infection seldom occurs before maternal antibody disappears. However, babies could not be protected long. We found that babies usually were born with a low anti-HHY-6 titer and lost the antibody within 6 months. After the protection was lost, they were easily infected by HHY-6 since other members in the home consistently excrete HHY-6 in saliva [7, 8]. The high chance (51 %) of infection from 6 to 12 months of age may also be related to the frequent oral exploration of the environment by children ofthis age group, which allows contact with saliva or saliva-contaminated fomites.
1101992;165 (June)
JID 1992;165 (June)
Correspondence
1165
G FAY 0 T R C F 0 S T V T E GGGATTTGCATATGACACCCGCTGTTTTGACTCAACGGTCACTGA C. CT. C. III C . . T. T. C. C. IV G . . CT. G.. T.. C. C .. A .. I II
Figure 1. Nucleotide sequence of hepatitis C virus (HCV) isolate (I) from a patient positive for HCV antibodies by second generation ELISA and indeterminate by second generation RIBA. This sequence is aligned with HCV prototype (IV) and two published HCV sequences, II [2] and III [3]. Deduced amino acid sequences are on top line. Lowercase letters represent sequence of N2.
I
II ~
SOl R VEE SlY Q C C D L GAGCGATATCCGTGTGGAGGAGTCGATCTACCAATGTTGTGACTT AT. C. ACT. A . . A. T. . . A.
C..
II
A . . T.
AC
APE I
..T..
...... C A
R
G.A Q
V
C.
IRS
L
T
E
R
L
GGCCCCCGAAGCCAGACAGGTCATAAGGTCGCTCACGGAGCGGCT G. . .
C. C.
III
IV C. A. Y
I
G
A A.
C. CGT . . C.
C. G
P
L
T
A. A ..
C. A. N
S
C.. KG
C . . . AG . . . Q
III
IV
T.
T.
G.T . . . . . C . . T . . T . . C . . T . . . . GG . . . G
HCY sequences [2, 3] and the HCY prototype [9], respectively (figure 1). Finally, we examined serum from a patient who was positive for HCY antibodies by ELISA, indeterminate by RIBA, and positive by RT-nested PCR with the NS5 primer set. Partial sequence analysis of the amplified fragment showed a high homology (90.6%) with one Japanese isolate [3]. Surprisingly, serum from this patient was not amplified by a primer set located in the conserved 5' noncoding region ofthe HCY genome. The lack of amplification could result from a genetic variability ranging from a point mutation located at the 3' end of a primer to a large deletion in the 5' non coding region. Thus, we suggest that HCY RNA negativity with a 5' region primer set might be confirmed with a primer set located in another conserved region in a patient positive for HCY antibodies by ELISA and indeterminate or positive by RIBA.
Li-Zhe Xu, Michele Martinot-Peignoux, Patrick Marcellin, Jean Pierre Benhamou, and Daniel Larzul Molecular Biology Department, EIBET, Plaisir; Service d'Hepatologie and Unite de Recherches de Physiopathologie Hepatique (INSERM U 24), Hiipital Beaujon, Clichy. France
References I. Weiner AJ. Brauer MJ, Rosenblatt J. et al. Variable and hypervariable domains are found in the region of Hev corresponding to the f1avivirus envelope and NS I proteins and the pestivirus envelope glycoproteins. Virology 1991; 180:842-8. 2. Kato N. Hijikata M. Ootsuyama Y, et al. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A. non-B hepatitis. Proc Nat! Acad Sci USA 1990;87:9524-8. 3. Takamizawa A. Mori C. Fuke I. et al. Structure and organization of the hepatitis C virus genome isolated from human carrier. J Virol 1991;65:1105-13. 4. Fuchs K, Motz M, Schreier E, et al. Characterization of nucleotide sequences from European hepatitis e virus isolates. Gene 1991; I03: 163-9. 5. Okamoto H, Okada S, Sugiyama Y. et al. The 5'-terminal sequence of the hepatitis e virus genome. Jpn J Exp Med 1990;60: 167-77. 6. Okamoto H. Okada S. Sugiyama Y, et al. Detection of hepatitis C virus RNA by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5'-noncoding region. Jpn J Exp Med 1990;60:215-22. 7. Martinot-Peignoux M. Marcellin P. Xu LZ, et al. Reactivity to c33c antigen as a marker of hepatitis C virus multiplication [letter]. J Infect Dis 1992;165:595-6. 8. Garson JA, Tedder RS. Briggs M. et al. Detection of hepatitis C viral sequences in blood donations by "nested" polymerase chain reaction and prediction of infectivity. Lancet 1990;335: 1419-22. 9. Houghton M. Choo QL, Kuo G. NANBV diagnostics and vaccines. Chiron Corp. European patent application. 88310922.5. filed European Patent Office, 18 Nov 1988.
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 21, 2015
I T T A TAT C G G G G G T C C C C T G ACT A ACT C A A A A G G G C a g a a c t g c g g c t a t c g c II G. T. G . . G.
Correspondence
Ll-Min Huang, Chin-Yun Lee, Jen-Yang Chen, Czau-Siung Yang, Jung-Der Wang, Mei-Hwei Chang, Ching-Ying Hsu, and Pel-Fen Kuo Departments 0/Pediatrics. Bacteriology. and Public Health and Internal Medicine. College 0/ Medicine. National Taiwan University. and
Lack of Polymerase Chain Reaction Amplification in the
5' Region of a Hepatitis C Virus Isolate Colleagues-Hepatitis C virus (HCY) infection can be assessed by the detection ofviral RNA by reverse transcription (RT) and nested polymerase chain reaction (PCR). The sensitivity of this technique allows early diagnosis in acute infection, evaluation of infectivity of serum positive or negative for HCY antibodies, and monitoring of antiviral therapy. Since significant variability of the HCY genome has been observed [1-3], the choice of a region for primer selection appears to be of great importance. Recent identification of a highly conserved sequence located in the 5' noncoding region of the HCY genome [4, 5] has allowed the selection of primer sets efficient for RT and PCR. Indeed, several groups have reported a correlation between detection of HCY antibodies by ELISA and the RIBA HCY test system (Chiron, Emeryville, CA) and HCY RNA using PCR primer sets located in the 5' noncoding region [6]. We previously selected such inner and outer primers to evaluate the presence of HCY RNA by RT-nested PCR [7]. With regard to these data, we thought it would be interesting to ascertain if a PCR primer set (inner and outer primers) selected in only one conserved region (5') of the HCY genome is sufficient
Reprints or correspondence: Dr. D. Larzul, Molecular Biology Department. EIBET. 48-52 rue de la Gare, 78370. Plaisir, France. The Journal of Infectious Diseases 1992;165:1164-5 © 1992 byThe University ofChicago. All rights reserved.
0022-1899/92/6506-0036$01.00
Department 0/Pediatrics. Taipei Municipal Maternal and Child Health Hospital. Taiwan. Republic of China
References I. Salahuddin SZ,Ablashi DV. Markham PD,etal. Isolation ofa new virus, HBLV. in patients with Iymphoproliferative disorders. Science 1986;234:596-60 I. 2. Saxinger C. Polesky H, Eby N, et al. Antibody reactivity with HBLV (HHV-6) in USpopulations. J Virol Methods 1988;21: 199-208. 3. OkunoT, Takahashi K. Balachandra K, et al. Seroepidemiology of human herpesvirus 6 infection in normal children and adults. J Clin Microbiol 1989;27:651-3. 4. Rin H. Psychiatry and society. Taipei: Medicine Today Publishing. 1978:54-5. 5. Yamanishi K. OkunoT, Shiraki K, et al. Identification of human herpesvirus-6 asa causal agentforroseola infantum. Lancet 1988;I:1065-7. 6. Balachandra K. Ayuthaya PIN, Auwanit W, et al. Prevalence ofantibody to human herpesvirus 6 in women and children. Microbiol Immunol 1989;33:515-8. 7. Pietroboni GR. HarnettGB. Bucens MR. Honess RW. Antibody to human herpesvirus 6 in saliva. Lancet 1988;1:1059. 8. Levy JA. Ferro F. Greenspan D. Lennette ET. Frequent isolation of HHV-6 from saliva and high seroprevalence ofthe virus in the population. Lancet 1990;335: 1047-50.
to evaluate the presence of HCY RNA. In an attempt to clarify this question, we studied an asymptomatic subject from southern Italy with community-acquired chronic non-A, non-B hepatitis who was HCY-positive by ELISA (HCY ELISA, second generation; Ortho Diagnostic Systems, Roissy, France) and indeterminate by second-generation RIBA (Chiron). RIBA results showed a reactivity towards the c22-3 recombinant antigen band and indeterminate data for 5-1-1, cI00-3, and c33c antigens. HCY RNA evaluation of this patient's serum was repeatedly negative with our 5' region primer set and positive with an NS5 region primer set (N IJN2, d94/d95) [8]. Experiments were carried out on two serum samples, independently collected at a I-week interval with each sample studied in duplicate. The amplification protocol was an RT-nested PCR done under low stringency conditions for primer hybridization; the annealing temperature was 37°e. The 5' primer set allowed the amplification of a 177-bp DNA fragment that has never been demonstrated with either ethidium bromide-stained agarose gel or hybridization in low stringency conditions with a specific oligonucleotide probe. However, with the NS5 primer set, amplified DNA fragments of the expected size were visualized directly on the agarose gel after the first (730 bp) and second (405 bp) amplifications. An internal oligonucleotide probe hybridized specifically with these two amplified fragments. To. confirm the specificity of this amplification, the 405-bpamphfied DNA fragment was cloned in pBluescript KS (-) (Stratagene/Ozyme, Paris) and partly sequenced with the dideoxy method (170 bp). Sequencing was done on two strands of three independent clones. The sequence obtained revealed a homology of 88.2%,90.6%, and 77.6% with two other published
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 21, 2015
HHY-6. Although there has been no direct proof of protection by maternal HHY-6 antibodies, several studies [3, 6, 7], including this one, have found that HHY-6 infection increases after the first few months of life, suggesting a protective role for the maternal antibody. In addition, our findings that all patients were seronegative before they became infected strongly supports the possibility that infection seldom occurs before maternal antibody disappears. However, babies could not be protected long. We found that babies usually were born with a low anti-HHY-6 titer and lost the antibody within 6 months. After the protection was lost, they were easily infected by HHY-6 since other members in the home consistently excrete HHY-6 in saliva [7, 8]. The high chance (51 %) of infection from 6 to 12 months of age may also be related to the frequent oral exploration of the environment by children ofthis age group, which allows contact with saliva or saliva-contaminated fomites.
1101992;165 (June)
JID 1992;165 (June)
Correspondence
1165
G FAY 0 T R C F 0 S T V T E GGGATTTGCATATGACACCCGCTGTTTTGACTCAACGGTCACTGA C. CT. C. III C . . T. T. C. C. IV G . . CT. G.. T.. C. C .. A .. I II
Figure 1. Nucleotide sequence of hepatitis C virus (HCV) isolate (I) from a patient positive for HCV antibodies by second generation ELISA and indeterminate by second generation RIBA. This sequence is aligned with HCV prototype (IV) and two published HCV sequences, II [2] and III [3]. Deduced amino acid sequences are on top line. Lowercase letters represent sequence of N2.
I
II ~
SOl R VEE SlY Q C C D L GAGCGATATCCGTGTGGAGGAGTCGATCTACCAATGTTGTGACTT AT. C. ACT. A . . A. T. . . A.
C..
II
A . . T.
AC
APE I
..T..
...... C A
R
G.A Q
V
C.
IRS
L
T
E
R
L
GGCCCCCGAAGCCAGACAGGTCATAAGGTCGCTCACGGAGCGGCT G. . .
C. C.
III
IV C. A. Y
I
G
A A.
C. CGT . . C.
C. G
P
L
T
A. A ..
C. A. N
S
C.. KG
C . . . AG . . . Q
III
IV
T.
T.
G.T . . . . . C . . T . . T . . C . . T . . . . GG . . . G
HCY sequences [2, 3] and the HCY prototype [9], respectively (figure 1). Finally, we examined serum from a patient who was positive for HCY antibodies by ELISA, indeterminate by RIBA, and positive by RT-nested PCR with the NS5 primer set. Partial sequence analysis of the amplified fragment showed a high homology (90.6%) with one Japanese isolate [3]. Surprisingly, serum from this patient was not amplified by a primer set located in the conserved 5' noncoding region ofthe HCY genome. The lack of amplification could result from a genetic variability ranging from a point mutation located at the 3' end of a primer to a large deletion in the 5' non coding region. Thus, we suggest that HCY RNA negativity with a 5' region primer set might be confirmed with a primer set located in another conserved region in a patient positive for HCY antibodies by ELISA and indeterminate or positive by RIBA.
Li-Zhe Xu, Michele Martinot-Peignoux, Patrick Marcellin, Jean Pierre Benhamou, and Daniel Larzul Molecular Biology Department, EIBET, Plaisir; Service d'Hepatologie and Unite de Recherches de Physiopathologie Hepatique (INSERM U 24), Hiipital Beaujon, Clichy. France
References I. Weiner AJ. Brauer MJ, Rosenblatt J. et al. Variable and hypervariable domains are found in the region of Hev corresponding to the f1avivirus envelope and NS I proteins and the pestivirus envelope glycoproteins. Virology 1991; 180:842-8. 2. Kato N. Hijikata M. Ootsuyama Y, et al. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A. non-B hepatitis. Proc Nat! Acad Sci USA 1990;87:9524-8. 3. Takamizawa A. Mori C. Fuke I. et al. Structure and organization of the hepatitis C virus genome isolated from human carrier. J Virol 1991;65:1105-13. 4. Fuchs K, Motz M, Schreier E, et al. Characterization of nucleotide sequences from European hepatitis e virus isolates. Gene 1991; I03: 163-9. 5. Okamoto H, Okada S, Sugiyama Y. et al. The 5'-terminal sequence of the hepatitis e virus genome. Jpn J Exp Med 1990;60: 167-77. 6. Okamoto H. Okada S. Sugiyama Y, et al. Detection of hepatitis C virus RNA by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5'-noncoding region. Jpn J Exp Med 1990;60:215-22. 7. Martinot-Peignoux M. Marcellin P. Xu LZ, et al. Reactivity to c33c antigen as a marker of hepatitis C virus multiplication [letter]. J Infect Dis 1992;165:595-6. 8. Garson JA, Tedder RS. Briggs M. et al. Detection of hepatitis C viral sequences in blood donations by "nested" polymerase chain reaction and prediction of infectivity. Lancet 1990;335: 1419-22. 9. Houghton M. Choo QL, Kuo G. NANBV diagnostics and vaccines. Chiron Corp. European patent application. 88310922.5. filed European Patent Office, 18 Nov 1988.
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 21, 2015
I T T A TAT C G G G G G T C C C C T G ACT A ACT C A A A A G G G C a g a a c t g c g g c t a t c g c II G. T. G . . G.
