hepatitis G virus RNA in serum

Journal of Virological Methods 74 (1998) 185 – 191

Multicyclic reverse transcription-polymerase chain reaction assay system for quantitation of GB virus-C/hepatitis G virus RNA in serum Tsuyoshi Saito a,*, Shinji Matsumoto a, Osamu Nojiri a, Tsutomu Kageyama a, Shuetsu Fukushi a, Narihiro Ishiyama b, Chie Kurihara b, Kazuhiko Katayama a a b

Basic Research Di6ision, BioMedical Laboratories, 1361 -1 Matoba Kawagoe-shi, Saitama 350 -1101, Japan Second Department of Internal Medicine, National Defense Medical College, 3 -2 Namiki Tokorozawa-shi, Saitama 359 -8513, Japan Received 7 April 1998; received in revised form 10 June 1998; accepted 10 June 1998

Abstract A new quantitative reverse transcription-polymerase chain reaction (RT-PCR) method is described for analyzing the amount of GB virus-C (GBV-C)/hepatitis G virus (HGV) RNA in serum. This multicyclic RT-PCR (MRT-PCR) method used oligonucleotide primers deduced from the 3% noncoding region (3%NCR) that is highly conserved among GBV-C/HGV isolates. Quantitation of GBV-C/HGV RNA using MRT-PCR ranged between 102 and 1010 copies/ml when PCR cycle number was regulated at exponential amplification of the products. Competitive RT-PCR (CRT-PCR) was carried out with mutant RNA and sample that had been measured by MRT-PCR. Quantitation of GBV-C/HGV RNA using both methods agreed. MRT-PCR detected viral RNA in a single step PCR, and demonstrated a high degree of sensitivity that was equal to that of the RT-PCR procedure, which used nested primers deduced from the non-structural (NS) 3 region. The MRT-PCR method for quantitation of GBV-C/HGV RNA in serum may prove useful for diagnosis. © 1998 Elsevier Science B.V. All rights reserved. Keywords: GB virus-C; Hepatitis G virus; Multicyclic RT-PCR; 3% noncoding region

1. Introduction A novel Flaviviridae-like genome was isolated recently from the plasma of several patients with * Corresponding author. Tel.: +81-492-320440; Fax: +81492-325480; E-mail: [email protected]

cryptogenic hepatitis. This virus was designated provisionally GB virus-C (GBV-C) (Simons et al., 1995) or hepatitis G virus (HGV) (Linnen et al., 1996). GBV-C/HGV infection is found not only in acute and chronic hepatitis but also in : 1–2% of healthy blood donors worldwide (Fiordalisi et al., 1996; Masuko et al., 1996; Wang et al., 1997).

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Early experiments suggested that GBV-C/HGV was a newly identified causative agent of hepatitis, but the relationship of hepatitis and GBV-C/HGV infection is not clear at present (Alter, 1996; Wang et al., 1996). The RNA genomes of GBV-C/HGV are 9.4 kb in size and contain a single long open reading frame, a 5% noncoding region (5%NCR), and a 3% noncoding region (3%NCR). The nucleotide sequence of individual GBV-C/HGV isolates from nine infected Japanese patients was described previously. These isolates are clearly distinguishable from prototypic viruses by comparison of the 5%NCR sequence, which indicates that at least three distinct genomic variants of GBV-C/HGV exist (Fukushi et al., 1996; Katayama et al., 1997). That study also suggested that genotyping of GBV-C/HGV may be possible using a reverse transcription-polymerase chain reaction (RTPCR) method based on the sequence of the variable region in the 5%NCR. On the other hand, conservation of nucleotide sequences was found highest in the 3%NCR from previous study (Katayama et al., 1998). Therefore this region was ideal for the optimum amplification of PCR target region, for GBV-C/HGV RNA detection. A new quantitative RT-PCR-based method is described. This multicyclic RT-PCR (MRT-PCR) method (Ishiyama et al., 1993) used oligonucleotide primers deduced from the 3%NCR for analyzing the amount of GBV-C/HGV RNA in serum. The accuracy of MRT-PCR was compared with competitive RT-PCR (CRT-PCR) (Gilliland et al., 1990) and nested RT-PCR methods.

2. Materials and methods

2.1. Synthesis of GB 6irus-C/hepatitis G 6irus RNA standard Sera from individuals shown previously to be infected with GBV-C/HGV were used as the source of viral RNA. GBV-C/HGV RNA was extracted from a Japanese patient’s serum using SMITEST R KIT (Sumitomokinzoku, Japan). To obtain cDNA of GBV-C/HGV 3% noncoding region, 486 nt from the 3% end of the genome were

synthesized using a 3% RACE method (Tanaka et al., 1996; Katayama et al., 1998). To create a new EcoRI site, a mutation was introduced into the cDNA of GBV-C/HGV 3%NCR at nucleotide position 9239 using a site-directed mutagenesis kit (Promega, USA) according to the manufacturer’s instructions. The mutant GBV-C/HGV cDNA fragment was cloned into the pT7 Blue vector (Novagen, USA) to yield clone pT7HGm. RNA was transcribed from BamHI-digested pT7HGm using T7 RNA polymerase (Promega). Synthesized RNA was purified with NucTrap® probe purification columns (Stratagene, USA) and brought to a concentration of 1013 copies/ml based on optical density, and then diluted to concentrations ranging from 1010 to 102 copies/ml. These diluted solutions were used as GBV-C/ HGV RNA standards for MRT-PCR or as competitor RNAs for CRT-PCR.

2.2. Multicyclic re6erse transcription-polymerase chain reaction GBV-C/HGV RNA was extracted from 100 ml of patient sera using SMITEST R KIT (Sumitomokinzoku). This RNA served as a template for cDNA synthesis in a reaction with four units of AMV-XL reverse transcriptase (Life Science, USA) and antisense primer HGJ9342A (5%-AGA TCA CAG TGC ACT GTG C-3%) and buffer [50 mM Tris–HCl (pH 8.3), 50 mM KCl, 8 mM MgCl2, 1 mM dNTP, 4 mM DTT]. A 20 ml volume from the RT reaction was incubated with 24 units of RNasin (Toyobo, Japan) at 52°C for 1.5 h. Then, 20 ml of the cDNA sample was pre-incubated for 10 min at 95°C with sense primer HG9155S (5%-GGG TCT CCC CGC TGG GTA-3%) and 2.5 units of Amplitaq Gold DNA polymerase (Perkin-Elmer, USA) in a final 75 ml reaction mixture [10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2], followed by incubation at 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min. The reaction mixture was sampled, and 20 ml aliquots were denatured with 180 ml of 0.4 N NaOH after 25 and 40 cycles. A total of 75 ml of the denatured sample was blotted onto a nylon membrane. Membranes were pre-hybridized for 4 h followed by hybridization overnight with 32P-5%-

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end-labeled internal probe HG9192S (5%-AGG CAT GGT GGT TAC TAA CCC CCT GGC A-3%) at 42°C. Filters were washed in 1× SSC – 0.1% SDS for 15 min at room temperature, followed by a 42°C wash with 1× SSC – 0.1% SDS for 15 min. Membrane radioactivity was measured using a BAS1000 Phosphor Imager (Fuji Photo Film, Japan). The copy number of the samples was determined by comparing the intensity of the sample dot against the GBV-C/HGV RNA standards.

2.3. Competiti6e re6erse transcription-polymerase chain reaction The CRT-PCR was carried out using RNA that was extracted from patient samples 16 and 31. To synthesize GBV-C/HGV cDNA, 100 ml of reaction mixture was incubated containing an unknown concentration of GBV-C/HGV RNA from 50 ml of serum samples 16 and 31, a known concentration of mutant GBV-C/HGV RNA, and four units of AMV-XL reverse transcriptase (Life Science), anti sense primer HGJ9353A 5%-TGC ACC CCT TCA GAT CAC AG-3%), RT mixture [50 mM Tris–HCl (pH8.3), 50 mM KCl, 8 mM MgCl2, 1 mM dNTP, 4 mM DTT] and 24 units of RNasin (Toyobo, Japan) for 42°C overnight. For amplification of cDNA, a set of HG9156S (5%GGT CTC CCC GCT GGG TAA AA-3%) and HGJ9353A primers, located in the 3%NCR of the GBV-C/HGV genome, were prepared. After 10 min pre-incubation at 95°C, 75 ml of a PCR mixture [10 mM Tris – HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2] containing 20 ml cDNA sample plus 2.5 units of Amplitaq Gold DNA polymerase (Perkin-Elmer) was amplified for 45 cycles at 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min. A 5 ml sample of the PCR product was mixed with EcoRI and incubated at 37°C for 1 h. EcoRI-digested PCR products were electrophoresed and stained with ethidium bromide.

2.4. Nested re6erse transcription-polymerase chain reaction The non-structural 3 region (NS3) nested RT-

187

PCR was carried out using a method described previously (Yoshiba et al., 1995).

3. Results and discussion Primer choice often centers on highly conserved nucleotide target sequences that are expected to bind with optimum sensitivity in RT-PCR. Preliminary results indicated that the sensitivity of RTPCR was higher using the 3%NCR primers than 5%NCR primers (data not shown). The 3%NCR sequence is highly conserved in the GBV-C/HGV genome (Katayama et al., 1998), therefore, primer pairs deduced from the 3%NCR were selected for quantitative detection of the GBV-C/HGV genome by the MRT-PCR method. Nucleotide sequence homology of the amplified fragment was 96% among the GBV-C/HGV isolates (data not shown). The RT-PCR products amplified from GBV-C/ HGV RNA were dot-blotted and hybridized with 32 P-labeled internal probe. As shown in Fig. 1, the signal from the dot blot hybridization increased with increasing amounts of standard GBV-C/HGV RNA. A linear increase in the signal was obtained at 106 –1010 copies/ml for 25 cycles and 102 –107 copies/ml for 40 cycles. Thus, quantitation of GBV-C/HGV RNA by the MRT-PCR was possible between 102 and 1010 copies/ml by restricting PCR cycle number to 25 and 40 by comparing the sample signal with the standards containing known copy number of RNA on a dot blot format. The dot signal of the blotted sample 16 on the membrane (IV-B in Fig. 1) had a similar dot signal with one of the series GBV-C/HGV RNA standards. A comparison was made with the dot signals at the end of 25 and 40 cycles and it was found that the titer for sample 16 of GBV-C/HGV RNA was 106 copies/ml. Similarly, the titer of GBV-C/HGV RNA in sample 31 was determined on the membrane (VI-C in Fig. 1) to be 108copies/ml. Serial 10-fold dilutions were made of mutant GBV-C/HGV 3%NCR RNA, ranging from 105 to 109 copies/ml. CRT-PCR was carried out with mutant RNA and sample 16 or 31 had been measured by MRT-PCR to compare the quantitation of GBV-C/HGV RNA by these two methods. The amount of GBV-C/HGV RNA was quanti-

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tated from the relative ratio of the undigested GBV-C/HGV RNA-PCR product (wild type) to the EcoRI-digested mutant RNA. EcoRI-digested PCR products were electrophored and stained with ethidium bromide (Fig. 2). The 198-bp fragment that resisted EcoRI digestion represents the RT-PCR products of the wild type RNA. Faster migrating bands represent two fragments generated by digestion of the competitor’s PCR product with EcoRI. The GBV-C/HGV RNA titers in samples 16 and 31 were determined using CRT-PCR to be 106 and 108 copies/ml, respec-

tively, as shown in Fig. 2. These results were in good agreement with those from MRT-PCR. The sensitivity of the MRT-PCR was compared with that of RT-PCR with nested primers deduced from the NS3 region using five viremic serum samples, named A, B, C, D, and E, by limiting dilution (10-fold dilution, 105-fold limited). As shown in Table 1, GBV-C/HGV RNA was not detected in the 105 dilution after ethidium bromide staining of the nested RT-PCR products when the NS3 primer pairs were used (A, D, and E). On the other hand, when the samples were

Fig. 1. Dot blot hybridization analysis of PCR products from MRT-PCR. Upper left panel: dot blot signals of PCR products after 25 cycles. RNA standards in vertical rows I-B to I-G contain 105, 106, 107, 108, 109, and 1010 copies/ml of GBV-C/HGV RNA, respectively. Row I-A: no serum. Rows II–VI: serum samples 1 – 35. Bottom left panel: PCR products after 40 cycles. RNA standards in vertical rows I-B to I-G contain 102, 103, 104, 105, 106, and 107 copies/ml of GBV-C/HGV RNA, respectively. Row I-A: no serum. Rows II – VI: serum samples as above. Upper and bottom right panels: dot blot format.

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GBV-C/HGV RNA. MRT-PCR was able to detect GBV-C/HGV RNA by single step PCR. Therefore, the risk of contamination by aerosol in MRT-PCR was lower than that for nested RTPCR. A total of 68 sera with GBV-C/HGV RNA were titrated. Using MRT-PCR, these samples were estimated to have 102 –109 copies/ml of GBV-C/HGV RNA, as shown in Fig. 3(A), and the histogram of the viral titer showed a normal distribution. The highest RNA titer of 107 copies/ ml was found in the GBV-C/HGV positive group. On the other hand, an RNA titer of 106 –108 copies ml was seen in the majority of sera that were positive for the hepatitis C virus (HCV) (Fig. 3B). The average RNA titer of GBV-C/HGV was slightly lower than that of HCV. Although the same distribution of RNA titer was seen in those genetically related viruses, a correlation between GBV-C/HGV infection and clinical course of hepatitis remains unclear. Whether GBV-C/HGV infection influences genomic replication of HCV is not known. The relative amounts of GBV-C/ HGV and HCV RNA in patient sera may be one indicator for resolving this question. Although persistent infection with GBV-C/HGV does not seem to be associated with significant hepatic injuries (Masuko et al., 1996), Colombatto et al. (1997) reported that elevated serum g-glutamyl

Fig. 2. Polyacrylamide gel electrophoresis of competitive RTPCR (CRT-PCR) products. CRT-PCR was carried out using serum samples 16 and 31. These samples contained 106 and 108 copies/ml, respectively, as measured by MRT-PCR. (A) 106 copies from serum sample 16. (B) 108 copies form serum sample 31. A 198-bp fragment resistant to EcoRI digestion represents the RT-PCR products of the wild type RNA. Faster migrating bands represent two fragments generated by digestion with EcoRI of the PCR product of the competitor. M: molecular weight marker (øx174/HincII digest). Samples 16 and 31 had 106 and 108 copies/ml, respectively.

diluted to 105-fold, GBV-C/HGV RNA could not been detected in sample A, D, and E by MRTPCR. MRT-PCR and nested RT-PCR was possible to detect each GBV-C/HGV RNA from 105-fold diluted serum sample. These results suggested that MRT-PCR method has a high sensitivity equal to nested RT-PCR for detection of Table 1 The sensitivity of NS3 nested RT-PCR and MRT-PCR Dilution 1

10

102

103

104

105

Nested RT-PCR (NS3)

A B C D E

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

− + + − −

MRT-PCRa (3%NCR)

A B C D E

7 8 8 7 7

6 7 7 6 7

5 6 6 5 5

5 4 5 5 5

4 4 5 4 3

n.d.b 2 4 n.d. n.d.

a b

Numbers are logarithms. n.d., not detected.

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References

Fig. 3. The histogram of GBV-C/HGV RNA and HCV RNA titer using MRT-PCR. (A) The frequency distribution of GBV-C/HGV infection. Using MRT-PCR, 68 samples were estimated to have 102 – 109 copies/ml of GBV-C/HGV RNA. (B) The frequency distribution of HCV infection. Using MRTPCR, 100 samples were estimated to have 102 –1010 copies/ml of HCV RNA.

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