Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA

Science

Volumn 277, Issue 5325, 1997, Pages 570-574

  Kolykhalov, Alexander A ^a   Agapov, Eugene V ^a   Blight, Keril J ^a   Mihalik, Kathleen ^b   Feinstone, Stephen M ^b   Rice, Charles M ^a  

^a WASHINGTON UNIVERSITY SCHOOL OF MEDICINE   (United States)

^b CENTER FOR BIOLOGICS EVALUATION AND RESEARCH   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ALANINE AMINOTRANSFERASE; COMPLEMENTARY DNA; GAMMA GLUTAMYLTRANSFERASE; VIRUS RNA;

ANIMAL EXPERIMENT; ANIMAL MODEL; ANIMAL TISSUE; ARTICLE; CHIMPANZEE; CONTROLLED STUDY; HEPATITIS C; HEPATITIS C VIRUS; INOCULATION; LIVER BIOPSY; MOLECULAR CLONING; NONHUMAN; PRIORITY JOURNAL; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; RNA SEQUENCE; RNA TRANSCRIPTION; SEROLOGY; VIREMIA; VIRUS REPLICATION; VIRUS TRANSMISSION;

ANIMALS; CLONING, MOLECULAR; CONSENSUS SEQUENCE; DNA, COMPLEMENTARY; HEPACIVIRUS; HEPATITIS C; LIVER; MOLECULAR SEQUENCE DATA; PAN TROGLODYTES; POLYMERASE CHAIN REACTION; RNA, MESSENGER; RNA, VIRAL; TRANSFECTION; VIREMIA; VIRUS REPLICATION;

ANIMALIA; HEPATITIS C VIRUS; PAN (APE); PAN TROGLODYTES; RNA VIRUSES;

EID: 0030864915     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5325.570     Document Type: Article

References (35)

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3. M. Houghton, ibid., pp. 1035-1058; M. E. Major and S. M. Feinstone, Hepatology 25, 527 (1997); K. E. Reed and C. M. Rice, in Hepatitis C Virus, H. W. Reesink, Ed. (Karger, Basel, Switzerland, 1997), vol. 61, in press.
4. M. Houghton, ibid., pp. 1035-1058; M. E. Major and S. M. Feinstone, Hepatology 25, 527 (1997); K. E. Reed and C. M. Rice, in Hepatitis C Virus, H. W. Reesink, Ed. (Karger, Basel, Switzerland, 1997), vol. 61, in press.
5. M. Houghton, ibid., pp. 1035-1058; M. E. Major and S. M. Feinstone, Hepatology 25, 527 (1997); K. E. Reed and C. M. Rice, in Hepatitis C Virus, H. W. Reesink, Ed. (Karger, Basel, Switzerland, 1997), vol. 61, in press.
6. T. Tanaka, N. Kato, M.-J. Cho, K. Shimotohno, Biochem. Biophys. Res. Comm. 215, 744 (1995); T. Tanaka, N. Kato, M.-J. Cho, K. Sugiyama, K. Shimotohno, J. Virol. 70, 3307 (1996); N. Yamada et al., Virology 223, 255 (1996).
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9. A. A. Kolykhalov, S. M. Feinstone, C. M. Rice, J. Virol. 70, 3363 (1996).
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11. H. J. Alter, R. H. Purcell, P. V. Holland, H. Popper, Lancet i, 459(1978); S. Feinstone et al., J. Infect. Dis. 144, 588 (1981); Y. K. Shimizu, R. H. Purcell, H. Yoshikura, Proc. Natl. Acad. Sci. U.S.A. 90, 6037 (1993).
12. H. J. Alter, R. H. Purcell, P. V. Holland, H. Popper, Lancet i, 459(1978); S. Feinstone et al., J. Infect. Dis. 144, 588 (1981); Y. K. Shimizu, R. H. Purcell, H. Yoshikura, Proc. Natl. Acad. Sci. U.S.A. 90, 6037 (1993).
13. We screened 41 HCV primer pairs and found 11 sets useful for amplifying overlapping 1 - to 4-kb portions of the H77 genome RNA [A. A. Kolykhalov, K. E. Reed, C. M. Rice, in Hepatitis C Protocols, J. Y. N. Lau, Ed. (Humana Press, Totowa, NJ), in press). A mixture of thermostable enzymes was used to reduce error frequency and enhance synthesis of full-length products [W. M. Barnes, Proc. Natl. Acad. Sci. U.S.A. 91, 2216 (1994 ) ; K. S. Lundberg et al., Gene 108, 1 (1991)). With sequential rounds of assembly PCR, and a limited number of amplification cycles, intermediate PCR products were combined to produce nearly full-length HCV cDNA (nt 39 to 9352), which was cloned as a Kpn 1 (580)-Not 1 (9219) fragment into a recipient plasmid. This plasmid contained the T 7 promoter, the consensus HCV-H 5′-and 3′-terminal sequences 5′ to the Kpn I site and 3′ from the Not I site, and a specific restriction site for template linearization and production of runoff RNA transcripts.
14. We screened 41 HCV primer pairs and found 11 sets useful for amplifying overlapping 1 - to 4-kb portions of the H77 genome RNA [A. A. Kolykhalov, K. E. Reed, C. M. Rice, in Hepatitis C Protocols, J. Y. N. Lau, Ed. (Humana Press, Totowa, NJ), in press). A mixture of thermostable enzymes was used to reduce error frequency and enhance synthesis of full-length products [W. M. Barnes, Proc. Natl. Acad. Sci. U.S.A. 91, 2216 (1994 ) ; K. S. Lundberg et al., Gene 108, 1 (1991)). With sequential rounds of assembly PCR, and a limited number of amplification cycles, intermediate PCR products were combined to produce nearly full-length HCV cDNA (nt 39 to 9352), which was cloned as a Kpn 1 (580)-Not 1 (9219) fragment into a recipient plasmid. This plasmid contained the T 7 promoter, the consensus HCV-H 5′-and 3′-terminal sequences 5′ to the Kpn I site and 3′ from the Not I site, and a specific restriction site for template linearization and production of runoff RNA transcripts.
15. We screened 41 HCV primer pairs and found 11 sets useful for amplifying overlapping 1 - to 4-kb portions of the H77 genome RNA [A. A. Kolykhalov, K. E. Reed, C. M. Rice, in Hepatitis C Protocols, J. Y. N. Lau, Ed. (Humana Press, Totowa, NJ), in press). A mixture of thermostable enzymes was used to reduce error frequency and enhance synthesis of full-length products [W. M. Barnes, Proc. Natl. Acad. Sci. U.S.A. 91, 2216 (1994 ) ; K. S. Lundberg et al., Gene 108, 1 (1991)). With sequential rounds of assembly PCR, and a limited number of amplification cycles, intermediate PCR products were combined to produce nearly full-length HCV cDNA (nt 39 to 9352), which was cloned as a Kpn 1 (580)-Not 1 (9219) fragment into a recipient plasmid. This plasmid contained the T 7 promoter, the consensus HCV-H 5′-and 3′-terminal sequences 5′ to the Kpn I site and 3′ from the Not I site, and a specific restriction site for template linearization and production of runoff RNA transcripts.
16. Seventeen clones were injected per animal by essentially the same procedures, which were eventually successful (16). Intrahepatic inoculation with RNA has been used successfully to initiate infection for two positive-strand RNA viruses: hepatitis A virus [S. U. Emerson et al.. J. Virol. 66, 6649 (1992)] and rabbit hemorrhagic disease virus [V. F. Ohlinger, B. Haas, G. Meyers, F. Weiland, H.-J. Thiel, ibid. 64, 3331 (1990)].
17. Seventeen clones were injected per animal by essentially the same procedures, which were eventually successful (16). Intrahepatic inoculation with RNA has been used successfully to initiate infection for two positive-strand RNA viruses: hepatitis A virus [S. U. Emerson et al.. J. Virol. 66, 6649 (1992)] and rabbit hemorrhagic disease virus [V. F. Ohlinger, B. Haas, G. Meyers, F. Weiland, H.-J. Thiel, ibid. 64, 3331 (1990)].
18. A. A. Kolykhalov et al., unpublished data.
19. As will be reported elsewhere (A. A. Kolykhalov and C. M. Rice, in preparation), the HCV H77 consensus sequence (GenBank accession number AF009606) was determined by sequencing of six clones from the H77 combinatorial library (clones 12, 209, 211, 213, 227, and 248) (7), and selected regions amplified by RT-PCR.
20. Fragments produced by restriction digestion of the sequenced clones (10) were used to assemble a full-length consensus clone by standard methods. They were derived from the following regions and clones (in parentheses): nt 580-1045 (213); 1046-1173 (248); 1174-1356 (12); 1357-1481 (209); 1482-1747 (227); 1748-1907 (209); 1908-2107 (227); 2108-2321 (12); 2322-2439 [HCV-H prototype, as described in (23)]; 2440-2525 (213); 2526-2827 (HCV-H prototype); 2828-2977 (211); 2978-3235 (209); 3236-3477 (227); 3478-3732 (209); 3733-3941 (12); 3942-4068 (211); 4069-4544 (227); 4545-4645 (248); 4646-4975 (211); 4976-5609 (227); 5610-5749 (209); 5750-6208 (HCV-H prototype); 6209-6301 (213); 6302-7528 (227); 7529-7860 (213); 7861-8204 (209); 8205-9219 (213).
21. Both the cDNA cyclization and inverse PCR and oligonucleotide-cDNA ligation (5′ rapid amplification of cDNA ends) methods were used to determine the 5′-terminal sequence of HCV RNA from high-titer H77 plasma (10). The predominant sequence, 5′-GCCAGCC . . . -3′, was identical to that determined for most other HCV isolates. Less frequently, clones with additional 5′ bases (including G, U, A, AA, GC, GG, GGG, and AAAGTCC) were recovered.
22. 2-terminal portion of E2 (HVR1) is highly variable and believed to be the target of immune selection (3). In the H77 sample, considerable variability exists in HVR1 [N. Nakajima, M. Hijikata, H. Yoshikura, Y. K. Shimizu, J. Virol. 70, 3325 (1996); N. Ogata, H. J. Alter, R. H. Miller, R. H. Purcell, Proc. Natl. Acad. Sci. U.S.A. 88, 3392 (1991); G. Inchauspe et al., ibid., p. 10292]. We sequenced multiple independent clones from this region (70) and the predominant HVR1 sequence in each position was used in our consensus clones.
23. 2-terminal portion of E2 (HVR1) is highly variable and believed to be the target of immune selection (3). In the H77 sample, considerable variability exists in HVR1 [N. Nakajima, M. Hijikata, H. Yoshikura, Y. K. Shimizu, J. Virol. 70, 3325 (1996); N. Ogata, H. J. Alter, R. H. Miller, R. H. Purcell, Proc. Natl. Acad. Sci. U.S.A. 88, 3392 (1991); G. Inchauspe et al., ibid., p. 10292]. We sequenced multiple independent clones from this region (70) and the predominant HVR1 sequence in each position was used in our consensus clones.
24. 2-terminal portion of E2 (HVR1) is highly variable and believed to be the target of immune selection (3). In the H77 sample, considerable variability exists in HVR1 [N. Nakajima, M. Hijikata, H. Yoshikura, Y. K. Shimizu, J. Virol. 70, 3325 (1996); N. Ogata, H. J. Alter, R. H. Miller, R. H. Purcell, Proc. Natl. Acad. Sci. U.S.A. 88, 3392 (1991); G. Inchauspe et al., ibid., p. 10292]. We sequenced multiple independent clones from this region (70) and the predominant HVR1 sequence in each position was used in our consensus clones.
25. 2, 5 mM dithiothreitol (DTT), 10 mM NaCl, 3 mM each NTP, 100 units of T7 RNA polymerase, and 0.02 unit of inorganic pyrophosphatase. After 1 hour of incubation at 37°C, typical yields were about 350 μg with >80% full-length RNA, as estimated by gel electrophoresis (9).
26. Two different inoculation and transfection protocols were used. For chimpanzee 1535, 100 μl of each transcription reaction mixture was diluted with 400 μl of phosphate-buffered saline (PBS) and stored frozen at -80°C until used for inoculation. These storage conditions were tested and shown to have no observable effect on the integrity of HCV RNA transcripts. Before inoculation, samples were thawed and eacn sample was injected intrahepatically at two sites (∼0.25 ml per site). Injection sites for the 10 clones were distributed in three lobes of the liver. Chimpanzee 1536 was inoculated with smaller amounts of RNA that had been mixed with lipofectin (Bethesda Research Labs). In this case, 20 μl of the same transcription reaction mixtures of the 10 full-length HCV H77 templates was treated with deoxyribonuclease (DNase I) to remove template DNA; 0.15-μg, 0.5-μg, and 1.5-μg portions were diluted to 50 μl with PBS and stored at -80°C until used for inoculation. After the mixtures were thawed, 100 μl of PBS containing 9 μg of lipofectin was added to each sample, mixed, and injected into a single site. Hence, each transcript preparation, with different RNA/lipofectin ratios, was injected at three separate sites. The chimpanzees were caged and maintained under conditions that met all relevant requirements for the use of primates in an approved facility. Animal protocols were reviewed and approved by the animal care and use committees from each institution involved with the chimpanzee studies. The chimpanzee experiments were also reviewed and approved by the Public Health Service Interagency Animal Model Committee.
27. 2-terminal portion of NS4B). Tests were performed according to the manufacturer's directions.
28. H. Popper, J. L. Dienstag, S. M. Feinstone, H. J. Alter, R. H. Purcell, Virchows Arch. A Pathol. Anat. Histol. 387, 91 (1980).
29. P. Farci et al., J. Infect. Dis. 165, 1006 (1992); M. Shindo, A. M. DiBisceglie, R. Biswas, K. Mihalik, S. M. Feinstone, ibid. 166, 424 (1992).
30. P. Farci et al., J. Infect. Dis. 165, 1006 (1992); M. Shindo, A. M. DiBisceglie, R. Biswas, K. Mihalik, S. M. Feinstone, ibid. 166, 424 (1992).
31. The small amounts of circulating HCV RNA preclude direct determination of 5′-terminal RNA sequences. Therefore, virus derived from transcripts containing the most prevalent 5′ end (5′-GCCA. . . -3′) was distinguished from that derived from transcripts with additional 5′ nucleotides by the presence or absence of the Xho I site at position 514 (Fig. 1). The region containing this marker was amplified by RT-PCR under conditions that ensured that a representative number of independent CDNAs [A. A. Kolykhalov, K. E. Reed, C. M. Rice, in Hepatitis C Protocols, J. Y. N. Lau, Ed. (Humana Press, Totowa, NJ), in press] were analyzed (>50 in this case). The resulting products were analyzed for digestion with either Xho I or, as a control, Acc I, an enzyme that should digest this fragment for all input clones. For chimpanzee 1535 (week 3 sample), the fraction of products digested by excess Xho I paralleled the input inoculum: about 20% was digested by Xho I; 80% was resistant to digestion (values were determined by scanning ethidium bromide-stained digestion patterns with an IS-1000 Digital Imaging System, Alpha Innotech Corp.). Complete digestion was observed for Ace I. In the week 4 sample analyzed for chimpanzee 1536, 45% was digested by Xho I; 55% was resistant to digestion. Again, complete digestion was observed for Acc I. Thus, in chimpanzee 1536 an advantage was observed for transcripts without additional 5′ bases (5′-GCCA . . . -3′).
32. Transcripts containing 75-base or 133-base poly(U/ UC) tracts were distinguished by the silent marker at nt 8054 in the NS5B coding region (Fig. 1). The region between nt 7955 and 8088 was amplified by RT-PCR, with enough starting material to ensure amplification of > 100 independent cDNA molecules [A. A. Kolykhalov, K. E. Reed, C. M. Rice, in Hepatitis C Protocols, J. Y. N. Lau, Ed. (Humana Press, Totowa, NJ, in press)], and molecularly cloned. Sequences of 10 and 9 independent clones were determined for chimpanzee 1535 (week 3) and chimpanzee 1536 (week 4), respectively. It was found that 90% (chimpanzee 1535) and 67% (chimpanzee 1536) of the clones contained the G at nt 8054, indicative of the 133-base poly(U/UC) tract. Thus, the 133-base tract appears to be preferred, although we cannot rule out the possibility that this preference was because of a deleterious effect of the marker mutation on the transcripts with the 75-base tract.
33. S. Falkow, Rev. Infect. Dis. 10 (Suppl. 2), S274 (1988).
34. A. Grakoui, C. Wychowski, C. Lin, S. M. Feinstone, C. M. Rice, J. Virol. 67, 1385 (1993).
35. We thank C. Read for expert technical assistance, S. Emerson for positive control hepatitis A virus cDNA clones, R. Purcell for generously making available his primate facilities, M. Shapiro for collection and analysis of chimpanzee samples, and A. DiBisceglie for bDNA analyses. We are also grateful to A. Grakoui, M. Heise, and J. A. Lemm for their participation in the early phases of this work and to S. Amberg, D. Goldberg, S. Hultgren, B. Lindenbach, M. MacDonald, J. Majors, and K. Reed for helpful discussion and comments on the manuscript. This work was supported in part by grants from the Public Health Service to C.M.R. (CA57973 and Al40034).