Molecular cloning and disease association of hepatitis G virus: A transfusion-transmissible agent

Science

Volumn 271, Issue 5248, 1996, Pages 505-508

  Linnen, Jeff ^a   Wages Jr , John ^a   Zhang Keck, Zhen Yong ^a   Fry, Kirk E ^a   Krawczynski, Krzysztof Z ^b   Alter, Harvey ^c   Koonin, Eugene ^d   Gallagher, Margaret ^b   Alter, Miriam ^b   Hadziyannis, Stephanos ^e   Karayiannis, Peter ^f   Fung, Kevin ^a   Nakatsuji, Yoshiyuki ^c   Shin, J Wai Kuo ^c   Young, Lavonne ^a   Piatak Jr , Michael ^a   Hoover, Cameron ^a   Fernandez, John ^a   Chen, Stacie ^a   Zou, Jian Chao ^a   Morris, Timothy ^b   Hyams, Kenneth C ^g   Ismay, Susan ^h   Lifson, Jeffrey D ^a   Hess, Georg ⁱ   Foung, Steven K H ^a,k   Thomas, Howard ^f   Bradley, Daniel ^j   Margolis, Harold ^b   Kim, Jungsuh P ^a   more..

^a Genelabs Incorporated   (United States)

^b CENTERS FOR DISEASE CONTROL AND PREVENTION   (United States)

^c NATIONAL INSTITUTES OF HEALTH   (United States)

^d NATIONAL INSTITUTES OF HEALTH   (United States)

^e HIPPOKRATION HOSPITAL   (Greece)

^f IMPERIAL COLLEGE LONDON   (United Kingdom)

^g NAVAL MEDICAL RESEARCH INSTITUTE   (United States)

^h New South Wales Red Cross Blood Bank   (Australia)

ⁱ F HOFFMANN LA ROCHE LTD   (Switzerland)

^j NONE   (United States)

^k STANFORD UNIVERSITY SCHOOL OF MEDICINE   (United States)

more..

Author keywords

[No Author keywords available]

Indexed keywords

COMPLEMENTARY DNA; VIRUS PROTEIN;

ARTICLE; BLOOD DONOR; BLOOD TRANSFUSION; CHRONIC HEPATITIS; CLINICAL ARTICLE; DISEASE ASSOCIATION; HEPATITIS C VIRUS; HEPATITIS VIRUS; HUMAN; MOLECULAR CLONING; NONHUMAN; PRIORITY JOURNAL; RNA VIRUS; UNITED STATES; VIREMIA; VIRUS GENOME; VIRUS TRANSMISSION;

GB VIRUS C/HEPATITIS G VIRUS; HEPATITIS C VIRUS; HEPATITIS GB VIRUS A; RNA VIRUSES;

EID: 13244300646     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5248.505     Document Type: Article

References (38)

1. R. H. Purcell et al., J Immunol. 116, 349 (1976); R H Decker, in Viral Hepatitis and Liver Disease, F B. Hollinger, S. M Lemon, H. S. Margolis, Eds. (Williams and Wilkins, Baltimore, MD, 1991). pp. 795-798, G. Kuo et al. , Science 244, 362 (1989), P O. Yarbough et al , J. Virol. 65, 5790 (1991); M Rizetto, R. H. Purcell, J. L. Gerin, Lancet i, 1215 (1980).
2. R. H. Purcell et al., J Immunol. 116, 349 (1976); R H Decker, in Viral Hepatitis and Liver Disease, F B. Hollinger, S. M Lemon, H. S. Margolis, Eds. (Williams and Wilkins, Baltimore, MD, 1991). pp. 795-798, G. Kuo et al. , Science 244, 362 (1989), P O. Yarbough et al , J. Virol. 65, 5790 (1991); M Rizetto, R. H. Purcell, J. L. Gerin, Lancet i, 1215 (1980).
3. R. H. Purcell et al., J Immunol. 116, 349 (1976); R H Decker, in Viral Hepatitis and Liver Disease, F B. Hollinger, S. M Lemon, H. S. Margolis, Eds. (Williams and Wilkins, Baltimore, MD, 1991). pp. 795-798, G. Kuo et al. , Science 244, 362 (1989), P O. Yarbough et al , J. Virol. 65, 5790 (1991); M Rizetto, R. H. Purcell, J. L. Gerin, Lancet i, 1215 (1980).
4. R. H. Purcell et al., J Immunol. 116, 349 (1976); R H Decker, in Viral Hepatitis and Liver Disease, F B. Hollinger, S. M Lemon, H. S. Margolis, Eds. (Williams and Wilkins, Baltimore, MD, 1991). pp. 795-798, G. Kuo et al. , Science 244, 362 (1989), P O. Yarbough et al , J. Virol. 65, 5790 (1991); M Rizetto, R. H. Purcell, J. L. Gerin, Lancet i, 1215 (1980).
5. R. H. Purcell et al., J Immunol. 116, 349 (1976); R H Decker, in Viral Hepatitis and Liver Disease, F B. Hollinger, S. M Lemon, H. S. Margolis, Eds. (Williams and Wilkins, Baltimore, MD, 1991). pp. 795-798, G. Kuo et al. , Science 244, 362 (1989), P O. Yarbough et al , J. Virol. 65, 5790 (1991); M Rizetto, R. H. Purcell, J. L. Gerin, Lancet i, 1215 (1980).
6. H. J Alter et al. , N. Engl. J. Med. 321, 1494 (1989)
7. M J. Alter et al. , ibid. 327, 1899 (1989).
8. We designated the virus hepatitis G virus (HGV) because an agent called hepatitis F virus (HFV) has been described [N. Deka, M. D. Sharma, R. Mukerjee, J. Virol 68, 7810 (1994)]
9. RT-PCR analysis was performed for detection of HCV RNA sequences essentially as described by J. H. Han et al. [Proc. Natl. Acad. Sci. U.S.A 88, 1711 (1991)] with the following primer sequences: primer UTR-1: 5′-TTCACGCAGAAAGCGTCTAGCCAT-3′ and primer UTR-2: 5′-TCGTCCTGGCAATTCCG-GTGTACT-3′.
10. For isolation of RNA used in both the construction of the λ gt11 cDNA library [R. A. Young and R W. Davis, Proc. Natl. Acad. Sci U.S.A. 80, 1194 (1983)] and the cloning of the HGV genome by anchored PCR, viral particles present in 1 ml of undiluted PNF2161 plasma were precipitated by the addition of polyethylene glycol (PEG, molecular weight 6000) to 8% (v/v) and were centrifuged at 10,000g for about 15 min at 4°C. RNA was extracted from the resulting pellet essentially as described by P. Chomczynski [Biotechniques 15, 532 (1993)] The cDNA was synthesized according to standard methods [U. Gubler and B. J. Hoffmann, Gene 25, 263 (1983)]. After synthesis, the cDNA was amplified with the use of sequence-independent single primer amplification (SISPA) as previously described [G. R. Reyes and J. P. Kim, Mol. Cell. Probes 5, 473 (1991)]
11. For isolation of RNA used in both the construction of the λ gt11 cDNA library [R. A. Young and R W. Davis, Proc. Natl. Acad. Sci U.S.A. 80, 1194 (1983)] and the cloning of the HGV genome by anchored PCR, viral particles present in 1 ml of undiluted PNF2161 plasma were precipitated by the addition of polyethylene glycol (PEG, molecular weight 6000) to 8% (v/v) and were centrifuged at 10,000g for about 15 min at 4°C. RNA was extracted from the resulting pellet essentially as described by P. Chomczynski [Biotechniques 15, 532 (1993)] The cDNA was synthesized according to standard methods [U. Gubler and B. J. Hoffmann, Gene 25, 263 (1983)]. After synthesis, the cDNA was amplified with the use of sequence-independent single primer amplification (SISPA) as previously described [G. R. Reyes and J. P. Kim, Mol. Cell. Probes 5, 473 (1991)]
12. For isolation of RNA used in both the construction of the λ gt11 cDNA library [R. A. Young and R W. Davis, Proc. Natl. Acad. Sci U.S.A. 80, 1194 (1983)] and the cloning of the HGV genome by anchored PCR, viral particles present in 1 ml of undiluted PNF2161 plasma were precipitated by the addition of polyethylene glycol (PEG, molecular weight 6000) to 8% (v/v) and were centrifuged at 10,000g for about 15 min at 4°C. RNA was extracted from the resulting pellet essentially as described by P. Chomczynski [Biotechniques 15, 532 (1993)] The cDNA was synthesized according to standard methods [U. Gubler and B. J. Hoffmann, Gene 25, 263 (1983)]. After synthesis, the cDNA was amplified with the use of sequence-independent single primer amplification (SISPA) as previously described [G. R. Reyes and J. P. Kim, Mol. Cell. Probes 5, 473 (1991)]
13. For isolation of RNA used in both the construction of the λ gt11 cDNA library [R. A. Young and R W. Davis, Proc. Natl. Acad. Sci U.S.A. 80, 1194 (1983)] and the cloning of the HGV genome by anchored PCR, viral particles present in 1 ml of undiluted PNF2161 plasma were precipitated by the addition of polyethylene glycol (PEG, molecular weight 6000) to 8% (v/v) and were centrifuged at 10,000g for about 15 min at 4°C. RNA was extracted from the resulting pellet essentially as described by P. Chomczynski [Biotechniques 15, 532 (1993)] The cDNA was synthesized according to standard methods [U. Gubler and B. J. Hoffmann, Gene 25, 263 (1983)]. After synthesis, the cDNA was amplified with the use of sequence-independent single primer amplification (SISPA) as previously described [G. R. Reyes and J. P. Kim, Mol. Cell. Probes 5, 473 (1991)]
14. D. A. Benson, M. Boguski, D. J Lipman, J. Ostell. Nucleic Acids Res 22, 3441 (1994).
15. 32P-labeled oligonucleotide probe (152F: 5′-TCGGTTACTGAGAGCAGCTCAGATGAG-3′) used in solution hybridization, followed by polyacrylamide gel electrophoresis and autoradiography. Results from PCR were confirmed by Southern (DNA) blot analysis.
16. To determine the presence of the 470-20-1 sequence in the cloning source, 125 μl of plasma from PNF2161 was ultracentrifuged (Beckman 70 1 Ti rotor at 40,000 K for 1 hour). RNA was extracted from the pellet (6) and subjected to RI-PCR analysis with the use of RNA corresponding to the equivalent of 50 μl of plasma per reaction. Reverse transcription was performed essentially as described (5), except that only random hexamers were used for priming. Fortyfive cycles of PCR amplification followed by solution hybridization were performed essentially as described above (8) with the primers 77F and 211R and hybridization probe 152F. No product was obtained from PNF2161-derived plasma if the PCR reactions were performed without prior reverse transcription, which indicated that the 470-20-1 sequence was of RNA origin.
17. J. Wages Jr. et al. , unpublished results
18. Extension cDNA clones were generated by anchored PCR with a ligation reaction containing SISPA-amplified PNF2161 cDNA (6) and λ gt11 DNA as template for PCR amplification. A λ gt11 reverse primer (5′-TGGTAATGGTAGCGACCGGCGCTCA-GC-3′) was used in combination with HGV-specific primers for 35 to 40 cycles of PCR amplification. All primer concentrations for PCR were 0.2 μM Amplification products were agarose gel-purified, identified by hybridization to an HGV-specific oligonucleotide probe. and cloned into the plasmid vector pCR II (invitrogen, San Diego, CA).
19. The cDNAs derived from the 5′ end of the HGV genome were obtained by a RACE method modified from that originally described [M A Frohman, M K Dush, G. R. Martin, Proc Natl. Acad. Sci U. S A. 85, 8998 (1988); A Belyausky, T Vinogradova, K. Rajewsky, Nucleic Acids Res. 17, 2919 (1989)]. First-strand cDNA synthesis was primed with random hexamers and synthesis was carried out with the use of either Superscript II (Perkin-Elmer/ABI, Foster City, CA) or rTth reverse transcriptase (Perkin-Elmer). Other reagents used in the RACE procedures were obtained from Clontech (Palo Alto, CA) as part of the AmpiFinder RACE kit. The cDNA clones derived from the 3′ end of the HGV genome were obtained by a modified anchored RT-PCR method. Polyadenylate [poly(A)] polymerase (Gibco/BRL, Gaithersburg, MD) was used to catalyze the addition of a poly(A) tail to PNF2161 RNA before cDNA synthesis The poly(A) addition was performed according to the manufacturer's recommendations. After purification of the poly(A) modified RNA, reverse transcription with Superscript II (Gibco/BRL) was carried out with primer GV-5446IRT (5′-CGGTCCCTCGAACTC-CAGCGAGTCTTTTTTTTTTTTTTT-3′). The resulting cDNA was amplified by PCR with primers GV59-5446F (5′-CTGAGCGACCTCAAGCTCCC-TGGC-3′) and GV-5446IR (5′-CGGTCCCTCGA-ACTCCAGCGAGTC-3′). After amplification, the products of interest were identified by hybridization to an HGV-specific oligonucleotide probe.
20. The cDNAs derived from the 5′ end of the HGV genome were obtained by a RACE method modified from that originally described [M A Frohman, M K Dush, G. R. Martin, Proc Natl. Acad. Sci U. S A. 85, 8998 (1988); A Belyausky, T Vinogradova, K. Rajewsky, Nucleic Acids Res. 17, 2919 (1989)]. First-strand cDNA synthesis was primed with random hexamers and synthesis was carried out with the use of either Superscript II (Perkin-Elmer/ABI, Foster City, CA) or rTth reverse transcriptase (Perkin-Elmer). Other reagents used in the RACE procedures were obtained from Clontech (Palo Alto, CA) as part of the AmpiFinder RACE kit. The cDNA clones derived from the 3′ end of the HGV genome were obtained by a modified anchored RT-PCR method. Polyadenylate [poly(A)] polymerase (Gibco/BRL, Gaithersburg, MD) was used to catalyze the addition of a poly(A) tail to PNF2161 RNA before cDNA synthesis The poly(A) addition was performed according to the manufacturer's recommendations. After purification of the poly(A) modified RNA, reverse transcription with Superscript II (Gibco/BRL) was carried out with primer GV-5446IRT (5′-CGGTCCCTCGAACTC-CAGCGAGTCTTTTTTTTTTTTTTT-3′). The resulting cDNA was amplified by PCR with primers GV59-5446F (5′-CTGAGCGACCTCAAGCTCCC-TGGC-3′) and GV-5446IR (5′-CGGTCCCTCGA-ACTCCAGCGAGTC-3′). After amplification, the products of interest were identified by hybridization to an HGV-specific oligonucleotide probe.
21. M Y Lim, K. G. Hadlock, J. P Kim, unpublished results.
22. Analysis was performed with the ALIGN program [W R. Pearson and D. J. Lipman, Proc. Natl. Acad Sci U.S.A. 85, 2444 (1985)].
23. The nonredundant protein sequence database used for screening is constructed at the National Center for Biotechnology Information (NIH) by merging of nonidentical entries from the SWISS-PROT, PIR, and GenBank databases. The database comparison was performed with the BLASTP program [S. F Altschul, W. Gish, E W. Myers, D. J. Lipman, J. Mol. Biol. 215, 403 (1990), S. F. Altschul, M. S. Boguski, W Gish, J C. Wootton, Nature Genet 6, 119 (1994)].
24. The nonredundant protein sequence database used for screening is constructed at the National Center for Biotechnology Information (NIH) by merging of nonidentical entries from the SWISS-PROT, PIR, and GenBank databases. The database comparison was performed with the BLASTP program [S. F Altschul, W. Gish, E W. Myers, D. J. Lipman, J. Mol. Biol. 215, 403 (1990), S. F. Altschul, M. S. Boguski, W Gish, J C. Wootton, Nature Genet 6, 119 (1994)].
25. J N. Simons et al. , Proc. Natl. Acad. Sci. U.S A 92, 3401 (1995)
26. E V Koonin and V V Dolja, Crit. Rev. Biochem Mol. Biol. 28, 375 (1993).
27. M Houghton, A Weiner, J Han, G. Kuo, Q -L. Choo, Hepatology 14, 381 (1991); A. Grakoui, D W. McCourt, C. Wychowski, S. M. Feinstone, C. M. Rice, J. Virol. 67, 2832 (1993).
28. M Houghton, A Weiner, J Han, G. Kuo, Q -L. Choo, Hepatology 14, 381 (1991); A. Grakoui, D W. McCourt, C. Wychowski, S. M. Feinstone, C. M. Rice, J. Virol. 67, 2832 (1993).
29. J N. Simons et al. Nature Med 6, 564 (1995).
30. Analysis was performed with MACAW [G D. Schuler, S F Altschul, D. J. Lipman, Proteins Struct. Funct. Genet. 9, 180 (1991)].
31. 2-terminal 58-amino acid residues of R10291 contain 19% arginine residues and have a net positive charge of 9, as compared with 18.3% arginine and a net positive charge of 21 in the capsid protein of HCV1 that consists of 191 residues. Compositional bias analysis performed with the SEG program [J C. Wootton, Comput. Chem. 18, 269 (1994)] indicated that both proteins are likely to have a nonglobular conformation.
32. RNA was extracted as described (9) or with Purescript (Gcntra Systems, Minneapolis, MN) according to the manufacturer's instructions. PCR was carried out as described (8, 9). Initial product analysis was performed with a Perkin-Elmer QPCR 5000 system according to the manufacturer's instructions and with the use of an oligonucleotide probe (probe 152F) Analysis was performed in duplicate for each specimen. Specimens were scored as initially positive if both duplicate reactions were positive; if one of two duplicate reactions was positive, the result was considered indeterminate. Most specimens that gave indeterminate or positive results were retested in duplicate. Specimens that gave positive results with both duplicates were considered positive Most specimens scored as positive were confirmed by RT-PCR analysis with the use of a second, nonover-lapping primer set (GV57-4512MF: 5′-GGACTTCC-GGATAGCTGARAAGCT 3′ and 5′-GCRTCCACAC-AGATGGCGCA-3′) and a hybridization probe (5′-CY-CGCTGRTTTGGGGTGTACTGGAAGGC-3′) (R = A or G; Y = C or T)
33. M. J. Alter et al , JAMA 264, 2231 (1990).
34. Single-letter abbreviations for the amino acid residues are as follows A, Ala; C, Cys; D, Asp, E, Glu, F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met, N, Asn, P, Pro; Q, Gin, R, Arg, S, Ser, T, Thr; V, Val; W, Trp; and Y, Tyr.
35. GenBank accession numbers are U22303 for GBV-A(16), U22304 for GBV-B (16), and S62220 for HCV [N. Hayashi et al , J. Hepatol. 17 (suppl. 3), S94 (1993)]
36. A. Grakoui, D. W. McCourt, C Wychowski, S M. Feinstone, C. M. Rice, Proc. Natl Acad. Sci U S.A. 90, 10583 (1993).
37. Patient 1 received blood from nine donors. Stored samples were available from eight, and two of the eight were found to be HGV RNA-positive No samples were available from the two donors to case 2. All 14 donors from case 2 were tested and 1 was found to be HGV-positive All of the donors to these cases were negative for HBV and HCV markers and all had a normal ALT level.
38. We thank R. H Purcell, F. A. Murphy, K G.Hadlock. and M. Y Lim for helpful discussions and critical review of the manuscript and J. Deikman, A Hwang, D. Mo, and A. Yun for excellent technical assistance.