Analysis of hepatitis C virus quasispecies transmission and evolution in patients infected through blood transfusion
2004; Elsevier BV; Volume: 127; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2004.06.005
ISSN1528-0012
AutoresTomasz Laskus, Jeffrey Wilkinson, Juan F. Gallegos‐Orozco, Marek Radkowski, Debra Adair, Marek Nowicki, Eva Operskalski, Zelma Buskell, Leonard B. Seeff, Hugo E. Vargas, Jorge Rakela,
Tópico(s)HIV Research and Treatment
ResumoBackground & Aims: Studies on hepatitis C virus (HCV) quasispecies dynamics in the natural course of infection are rare owing to difficulties in obtaining samples from the early phase of infection. Methods: We studied 15 patients from the Transfusion-Transmitted Viruses Study who seroconverted to anti-HCV after receiving infected blood. Follow-up serum samples were collected every 2–3 weeks for 6 months, at 10 months, and at 11–16 years. Viral quasispecies in the second envelope hypervariable region 1 (E2/HVR1) and 5′ untranslated region (5′UTR) were analyzed with single-strand conformation polymorphism (SSCP) and heteroduplex mobility assay (HMA). Results: Seven patients cleared infection within 7–24 weeks (mean, 14.0 wk) and 3 patients eventually became anti-HCV negative. In 6 patients with resolving hepatitis the SSCP band pattern remained stable, whereas in one patient minor changes appeared before clearance. In contrast, in all 8 patients progressing to chronicity, major changes in the E2/HVR1 quasispecies developed at 8–22 weeks (mean, 13.1 wk). Shannon entropy and medium mobility shift values derived from HMA gels remained stable in patients with resolving hepatitis but changed in those who developed chronic infection. Only 2 patients showed minor changes in 5′UTR. A decrease in E2/HVR1 complexity at the time of transmission (bottleneck) was found in 5 patients altogether. Conclusions: Changes in E2/HVR1 quasispecies 8–22 weeks after infection, likely caused by mounting immune pressure, were predictive of ensuing chronic infection, whereas stability was associated with resolution. Our study also showed that composition of HCV quasispecies may be preserved during transmission from host to host. Background & Aims: Studies on hepatitis C virus (HCV) quasispecies dynamics in the natural course of infection are rare owing to difficulties in obtaining samples from the early phase of infection. Methods: We studied 15 patients from the Transfusion-Transmitted Viruses Study who seroconverted to anti-HCV after receiving infected blood. Follow-up serum samples were collected every 2–3 weeks for 6 months, at 10 months, and at 11–16 years. Viral quasispecies in the second envelope hypervariable region 1 (E2/HVR1) and 5′ untranslated region (5′UTR) were analyzed with single-strand conformation polymorphism (SSCP) and heteroduplex mobility assay (HMA). Results: Seven patients cleared infection within 7–24 weeks (mean, 14.0 wk) and 3 patients eventually became anti-HCV negative. In 6 patients with resolving hepatitis the SSCP band pattern remained stable, whereas in one patient minor changes appeared before clearance. In contrast, in all 8 patients progressing to chronicity, major changes in the E2/HVR1 quasispecies developed at 8–22 weeks (mean, 13.1 wk). Shannon entropy and medium mobility shift values derived from HMA gels remained stable in patients with resolving hepatitis but changed in those who developed chronic infection. Only 2 patients showed minor changes in 5′UTR. A decrease in E2/HVR1 complexity at the time of transmission (bottleneck) was found in 5 patients altogether. Conclusions: Changes in E2/HVR1 quasispecies 8–22 weeks after infection, likely caused by mounting immune pressure, were predictive of ensuing chronic infection, whereas stability was associated with resolution. Our study also showed that composition of HCV quasispecies may be preserved during transmission from host to host. Hepatitis C virus (HCV) persists indefinitely in the majority of infected patients leading to chronic hepatitis, cirrhosis, and hepatocellular carcinoma.1Alter M.J. Margolis H.S. Krawczynski K. Judson F.N. Mares A. Alexander W.J. Hu P.Y. Miller J.K. Gerber M.A. Sampliner R.E. Meeks E.L. Beach M.J. The Sentinel Counties Chronic non-A, non-B Hepatitis Study TeamThe natural history of community-acquired hepatitis C in the United States.N Engl J Med. 1992; 327: 1899-1905Crossref PubMed Scopus (1646) Google Scholar, 2Kiyosawa K. Sodeyama T. Tanaka E. Gibo Y. Yoshizawa K. Nakano Y. Furuta S. Akahane Y. Nishioka K. Purcell R.H. Alter H.J. Interrelationship of blood transfusion, non-A, non-B hepatitis and hepatocellular carcinoma analysis by detection of antibody to hepatitis C virus.Hepatology. 1990; 12: 671-675Crossref PubMed Scopus (1199) Google Scholar, 3Simonetti R.G. Camma C. Fiorello F. Cottone M. Rapicetta M. Marino L. Fiorentino G. Craxi A. Ciccaglione A. Giuseppetti R. Stroffolini T. Pagliaro L. Hepatitis C virus infection as a risk factor for hepatocellular carcinoma in patients with cirrhosis A case-control study.Ann Intern Med. 1992; 116: 97-102Crossref PubMed Scopus (364) Google Scholar The overall prevalence of anti-HCV in the United States population is 1.8%, and it is estimated that 2.7 million Americans carry the virus.4Alter M.J. Kruszon-Moran D. Nainan O.V. McQuillan G.M. Gao F. Moyer L.A. Kaslow R.A. Margolis H.S. The prevalence of hepatitis C virus infection in the United States, 1988 through 1994.N Engl J Med. 1999; 341: 556-562Crossref PubMed Scopus (2463) Google Scholar Despite significant progress, the mechanisms behind HCV persistence still are unclear. Similar to many other RNA viruses, HCV is characterized by a high degree of genetic heterogeneity resulting from the fact that viral RNA polymerases have low fidelity owing to the lack of proofreading 3′-5′ exonuclease activity.5Choo Q.L. Richman K.H. Han J.H. Berger K. Lee C. Dong C. Gallegos C. Coit D. Medina-Selby R. Barr P.J. Weiner A.J. Bradley D.W. Kuo G. Houghton M. Genetic organization and diversity of the hepatitis C virus.Proc Natl Acad Sci U S A. 1991; 88: 2451-2455Crossref PubMed Scopus (1577) Google Scholar, 6Domingo E. Martinez-Salas E. Sobrino F. de la Torre J.C. Portela A. Ortin J. Lopez-Galindez C. Perez-Brena P. Villanueva N. Najera R. VandePol S. Steinhauer D. Holland J. The quasispecies (extremely heterogeneous) nature of viral RNA genome populations biological relevance—a review.Gene. 1985; 40: 1-8Crossref PubMed Scopus (427) Google Scholar As a consequence of high mutation and rapid turnover rates, HCV circulates as a population of closely related but nonidentical genomes, referred to as quasispecies.6Domingo E. Martinez-Salas E. Sobrino F. de la Torre J.C. Portela A. Ortin J. Lopez-Galindez C. Perez-Brena P. Villanueva N. Najera R. VandePol S. Steinhauer D. Holland J. The quasispecies (extremely heterogeneous) nature of viral RNA genome populations biological relevance—a review.Gene. 1985; 40: 1-8Crossref PubMed Scopus (427) Google Scholar, 7Martell M. Esteban J.I. Quer J. Genesca J. Weiner A. Esteban R. Guardia J. Gomez J. Hepatitis C virus (HCV) circulates as a population of different but closely related genomes quasispecies nature of HCV genome distribution.J Virol. 1992; 66: 3225-3229Crossref PubMed Google Scholar, 8Steinhauer D.A. Holland J.J. Rapid evolution of RNA viruses.Annu Rev Microbiol. 1987; 41: 409-433Crossref PubMed Google Scholar However, the genetic heterogeneity is not uniform throughout the genome. The most highly conserved are the 5′untranslated region (5′UTR) and the terminal part of the 3′ untranslated region (3′UTR) because their variability is constrained by the requirement for specific secondary structures, whereas the genes encoding the 2 envelope proteins E1 and E2 are the most heterogenous parts of viral genome.5Choo Q.L. Richman K.H. Han J.H. Berger K. Lee C. Dong C. Gallegos C. Coit D. Medina-Selby R. Barr P.J. Weiner A.J. Bradley D.W. Kuo G. Houghton M. Genetic organization and diversity of the hepatitis C virus.Proc Natl Acad Sci U S A. 1991; 88: 2451-2455Crossref PubMed Scopus (1577) Google Scholar, 9Ogata N. Alter H.J. Miller R.H. Purcell R.H. Nucleotide sequence and mutation rate of the H strain of hepatitis C virus.Proc Natl Acad Sci U S A. 1991; 88: 3392-3396Crossref PubMed Scopus (539) Google Scholar, 10Okamoto H. Kojima M. Okada S. Yoshizawa H. Iizuka H. Tanaka T. Muchmore E.E. Peterson D.A. Ito Y. Mishiro S. Genetic drift of hepatitis C virus during an 8.2-year infection in a chimpanzee variability and stability.Virology. 1992; 190: 894-899Crossref PubMed Scopus (375) Google Scholar The 5′ end of the E2 contains the most variable region of the entire genome and is referred to as hypervariable region 1 (HVR1).11Weiner A.J. Brauer M.J. Rosenblatt J. Richman K.H. Tung J. Crawford K. Bonino F. Saracco G. Choo Q.L. Houghton M. Han J.H. Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelope and NS1 proteins and the pestivirus envelope glycoproteins.Virology. 1991; 180: 842-848Crossref PubMed Scopus (543) Google Scholar, 12Hijikata M. Kato N. Ootsuyama Y. Nakagawa M. Ohkoshi S. Shimotohno K. Hypervariable regions in the putative glycoprotein of hepatitis C virus.Biochem Biophys Res Commun. 1991; 175: 220-228Crossref PubMed Scopus (308) Google Scholar The latter encodes a prominent B-cell epitope on the E2 envelope protein,13Shirai M. Arichi T. Chen M. Masaki T. Nishioka M. Ikeda K. Takahashi H. Enomoto N. Saito T. Major M.E. Nakazawa T. Akatsuka T. Feinstone S.M. Berzofsky J.A. T cell recognition of hypervariable region-1 from hepatitis C virus envelope protein with multiple class II MHC molecules in mice and humans preferential help for induction of antibodies to the hypervariable region.J Immunol. 1999; 162: 568-576PubMed Google Scholar and its high mutation rate may contribute to the evasion of the host immune response.11Weiner A.J. Brauer M.J. Rosenblatt J. Richman K.H. Tung J. Crawford K. Bonino F. Saracco G. Choo Q.L. Houghton M. Han J.H. Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelope and NS1 proteins and the pestivirus envelope glycoproteins.Virology. 1991; 180: 842-848Crossref PubMed Scopus (543) Google Scholar, 14Scarselli E. Cerino A. Esposito G. Silini E. Mondelli M.U. Traboni C. Occurrence of antibodies reactive with more than one variant of the putative envelope glycoprotein (gp70) hypervariable region 1 in viremic hepatitis C virus-infected patients.J Virol. 1995; 69: 4407-4412PubMed Google Scholar, 15Weiner A.J. Geysen H.M. Christopherson C. Hall J.E. Mason T.J. Saracco G. Bonino F. Crawford K. Marion C.D. Crawford K.A. Brunetto M. Barr P.J. Miyamura T. McHutchinson J. Houghton M. Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants potential role in chronic HCV infections.Proc Natl Acad Sci U S A. 1992; 89: 3468-3472Crossref PubMed Scopus (663) Google Scholar The existence of multiple HVR1 variants within quasispecies could be directly responsible for the lack of protective immunity. Studies in chimpanzees have shown that persistent infection does not provide protection against subsequent infection with heterologous and even homologous strains.16Farci P. Alter H.J. Govindarajan S. Wong D.C. Engle R. Lesniewski R.R. Mushahwar I.K. Desai S.M. Miller R.H. Ogata N. Purcell R.H. Lack of protective immunity against reinfection with hepatitis C virus.Science. 1992; 258: 135-140Crossref PubMed Scopus (714) Google Scholar, 17Okamoto H. Mishiro S. Tokita H. Tsuda F. Miyakawa Y. Mayumi M. Superinfection of chimpanzees carrying hepatitis C virus of genotype II/1b with that of genotype III/2a or I/1a.Hepatology. 1994; 20: 1131-1136Crossref PubMed Scopus (146) Google Scholar, 18Prince A.M. Brotman B. Huima T. Pascual D. Jaffery M. Inchauspe G. Immunity in hepatitis C infection.J Infect Dis. 1992; 165: 438-443Crossref PubMed Scopus (207) Google Scholar Similarly, chronically infected humans can be superinfected with new HCV strains in the settings of blood transfusion19Laskus T. Wang L.F. Radkowski M. Vargas H. Nowicki M. Wilkinson J. Rakela J. Exposure of hepatitis C virus (HCV) RNA-positive recipients to HCV RNA-positive blood donors results in rapid predominance of a single donor strain and exclusion and/or suppression of the recipient strain.J Virol. 2001; 75: 2059-2066Crossref PubMed Scopus (52) Google Scholar and liver transplantation.20Laskus T. Wang L.F. Rakela J. Vargas H. Pinna A.D. Tsamandas A.C. Demetris A.J. Fung J. Dynamic behavior of hepatitis C virus in chronically infected patients receiving liver graft from infected donors.Virology. 1996; 220: 171-176Crossref PubMed Scopus (77) Google Scholar Because the neutralizing antibodies are isolate-specific, they may be ineffective against other variants present in the complex of quasispecies—in a chimpanzee challenge experiment a hyperimmune anti-HVR1 serum against the predominant strain present in the inoculum was able to neutralize the predominant clone but was ineffective against minor variants.21Farci P. Alter H.J. Wong D.C. Miller R.H. Govindarajan S. Engle R. Shapiro M. Purcell R.H. Prevention of hepatitis C virus infection in chimpanzees after antibody-mediated in vitro neutralization.Proc Natl Acad Sci U S A. 1994; 91: 7792-7796Crossref PubMed Scopus (445) Google Scholar A minority of patients can mount an effective immune response and clear the virus in the early phase of infection.22Purcell R. The hepatitis C virus overview.Hepatology. 1997; 26: 11S-14SCrossref PubMed Scopus (112) Google Scholar, 23Kenny-Walsh E. Irish Hepatology Research GroupClinical outcomes after hepatitis C infection from contaminated anti-D immune globulin.N Engl J Med. 1999; 340: 1228-1233Crossref PubMed Scopus (861) Google Scholar However, studies on the dynamics of HCV quasispecies in transmission and the natural course of infection from acute hepatitis to resolution or chronicity are extremely limited, mainly owing to difficulties in obtaining samples from patients during the early phase of primary infection and the lack of subsequent long-term follow-up evaluation. Securing samples from subjects who are the source of infection is even more difficult because most new infections currently are associated either with intravenous drug use or sexual exposure.24Alter M.J. Hepatitis C virus infection in the United States.J Hepatol. 1999; 31: 88-91Abstract Full Text PDF PubMed Google Scholar A unique opportunity to study natural HCV infection is provided by the Transfusion-Transmitted Viruses Study/National Heart, Lung, and Blood Institute Repository. This prospective study of posttransfusion hepatitis was conducted in the years from 1974 to 1980, when HCV was much more prevalent in the blood donor population. Here we analyze 15 patients from the Transfusion-Transmitted Viruses Study, who were HCV negative at the time they received blood transfusions from HCV-infected donors. These cases are unique because the parental genomes from the actual infecting units of blood were defined and multiple follow-up samples were available from the infected recipient. Furthermore, late-term follow-up serum samples were collected from each infected patient 11–16 years after the original study.25Seeff L.B. Hollinger F.B. Alter H.J. Wright E.C. Cain C.M. Buskell Z.J. Ishak K.G. Iber F.L. Toro D. Samanta A. Koretz R.L. Perrillo R.P. Goodman Z.D. Knodell R.G. Gitnick G. Morgan T.R. Schiff E.R. Lasky S. Stevens C. Vlahcevic R.Z. Weinshel E. Tanwandee T. Lin H.J. Barbosa L. Long-term mortality and morbidity of transfusion-associated non-A, non-B, and type C hepatitis a National Heart, Lung, and Blood Institute collaborative study.Hepatology. 2001; 33: 455-463Crossref PubMed Scopus (303) Google Scholar The current analysis was confined to the E2/HVR1 and 5′UTR. Although the E2 region has been studied extensively and changes during transmission and acute and chronic infection have been reported,26Honda M. Kaneko S. Sakai A. Unoura M. Murakami S. Kobayashi K. Degree of diversity of hepatitis C virus quasispecies and progression of liver disease.Hepatology. 1994; 20: 1144-1151Crossref PubMed Scopus (165) Google Scholar, 27Koizumi K. Enomoto N. Kurosaki M. Murakami T. Izumi N. Marumo F. Sato C. Diversity of quasispecies in various disease stages of chronic hepatitis C virus infection and its significance in interferon treatment.Hepatology. 1995; 22: 30-35PubMed Google Scholar, 28Yuki N. Hayashi N. Moribe T. Matsushita Y. Tabata T. Inoue T. Kanazawa Y. Ohkawa K. Kasahara A. Fusamoto H. Kamada T. Relation of disease activity during chronic hepatitis C infection to complexity of hypervariable region 1 quasispecies.Hepatology. 1997; 25: 439-444Crossref PubMed Scopus (48) Google Scholar, 29Ray S.C. Wang Y.M. Laeyendecker O. Ticehurst J.R. Villano S.A. Thomas D.L. Acute hepatitis C virus structural gene sequences as predictors of persistent viremia hypervariable region 1 as a decoy.J Virol. 1999; 73: 2938-2946PubMed Google Scholar, 30Farci P. Purcell R.H. Clinical significance of hepatitis C virus genotypes and quasispecies.Semin Liver Dis. 2000; 20: 103-126PubMed Google Scholar it is unclear whether any changes would be present in the 5′UTR. The Transfusion-Transmitted Viruses Study was performed from 1974 through 1980.31Aach R.D. Lander J.J. Sherman L.A. Miller W.V. Kahn R.A. Gitnick G.L. Hollinger F.B. Werch J. Szmuness W. Stevens C.E. Kellner A. Weiner J.M. Mosley J.W. Transfusion-transmitted viruses interim analysis of hepatitis among transfused and non-transfused patients.in: Vyas G.N. Cohen S.N. Schmid R. Viral hepatitis a contemporary assessment of etiology, epidemiology, pathogenesis and prevention. Franklin Institute Press, Philadelphia1978: 383-396Google Scholar, 32Aach R.D. Szmuness W. Mosley J.W. Hollinger F.B. Kahn R.A. Stevens C.E. Edwards V.M. Werch J. Serum alanine aminotransferase of donors in relation to the risk of non-A, non-B hepatitis in recipients the transfusion-transmitted viruses study.N Engl J Med. 1981; 304: 989-994Crossref PubMed Scopus (398) Google Scholar It had as its major purpose the collection of optimally stored, serially collected specimens from transfusion recipients, and, most importantly, sera for all donors to each Transfusion-Transmitted Viruses Study recipient. Collections from recipients included a specimen before their first transfusion, and frequent specimens obtained during follow-up evaluation. Follow-up specimens from recipients were obtained for testing and storage at least every 2–3 weeks for 6 months, and again at 10 months. Recipient collections often began during the first 8 days after transfusion while the patient still was hospitalized. The current analysis was confined to 15 patients who seroconverted after having received blood from at least one anti-HCV-positive donor and who had follow-up samples collected 11–16 years after the original study. Among the 9 female subjects, 3 received transfusions at the time of genitourinary surgery, 4 underwent cardiac bypass surgery, and 2 received blood because of gastrointestinal bleeding unrelated to liver disease. The 6 male subjects were hospitalized either for a cardiosurgical procedure (3 patients) or prostate surgery (3 patients). Before transfusion all subjects were negative for both anti-HCV by second-generation enzyme immunoassay (Ortho Laboratories, Raritan, NJ) and were negative for HCV RNA by reverse-transcription polymerase chain reaction (RT-PCR). The patients received 1 U of whole blood from each of their respective donors and the number of donors for each recipient ranged from 1 to 7. Three patients received blood from more than one infected donor. However, as previously reported, only one strain will establish infection in a recipient.33Laskus T. Wang L.F. Radkowski M. Nowicki M. Wilkinson J. Rakela J. Exposure of hepatitis C virus-negative recipients to ≥ 2 infected blood donors.J Infect Dis. 2001; 183: 666-669Crossref PubMed Scopus (14) Google Scholar After transfusion all patients developed increased alanine transaminase activity and seroconverted to anti-HCV. However, the exact timing of seroconversion could not be determined because of the passive transfusion of antibodies from the donor. Eleven-16 years after the original study, 7 of the 15 patients were HCV-RNA negative in serum and 3 of these also were anti-HCV negative. These patients were considered to have cleared the infection spontaneously. The remaining 8 patients remained HCV-RNA positive throughout the follow-up period. All serum samples were kept at −70°C until the current analysis; to avoid repeat freeze-thawing they originally were aliquotted into 250-uL vials. RNA was extracted from 100 μL of serum by means of a modified guanidinium thiocyanate-phenol/chloroform technique using a commercially available kit (Triazol; Invitrogen, Carlsbad, CA) and dissolved in 20 μL of water. Five microliters of this RNA solution was reverse transcribed with Moloney murine leukemia virus (MMLV) and 5′UTR and E2/HVR1 sequences were PCR amplified as previously described.34Laskus T. Radkowski M. Wang L.F. Nowicki M. Rakela J. Uneven distribution of hepatitis C virus quasispecies in tissues from subjects with end-stage liver disease confounding effect of viral adsorption and mounting evidence for the presence of low-level extrahepatic replication.J Virol. 2000; 74: 1014-1017Crossref PubMed Scopus (106) Google Scholar Extensive measures were used to prevent and detect carry over contamination.20Laskus T. Wang L.F. Rakela J. Vargas H. Pinna A.D. Tsamandas A.C. Demetris A.J. Fung J. Dynamic behavior of hepatitis C virus in chronically infected patients receiving liver graft from infected donors.Virology. 1996; 220: 171-176Crossref PubMed Scopus (77) Google Scholar All RT-PCR runs included positive controls consisting of end-point dilutions of synthetic RNA strand and negative controls included uninfected sera. Quantification of donor sera for HCV RNA was performed with Bayer (Tarrytown, NY) HCV Quantiplex 2.0 assay. The infecting HCV genotype was determined in recipients by INNO-LiPA HCV II (Innogenetics, Ghent, Belgium). The assay was run as described previously.34Laskus T. Radkowski M. Wang L.F. Nowicki M. Rakela J. Uneven distribution of hepatitis C virus quasispecies in tissues from subjects with end-stage liver disease confounding effect of viral adsorption and mounting evidence for the presence of low-level extrahepatic replication.J Virol. 2000; 74: 1014-1017Crossref PubMed Scopus (106) Google Scholar In brief, PCR products were purified with a DNA-binding resin system (Wizard PCR; Promega, Madison, WI) and resuspended in 50 μL of water. Next, 2–4 μL of the purified product were diluted in 15 μL of low ionic strength solution (10% sucrose, 0.5% bromophenol blue, 0.5% xylene cyanol), denatured by heating at 97°C for 3 minutes, immediately cooled on ice, and subjected to nondenaturing 8% polyacrylamide gel electrophoresis in 1× Tris-borate-ethylenediaminetetraacetic acid buffer with 400 V applied for 4–5 hours at a constant temperature of 25°C. The bands were visualized with silver staining (Silver Stain; Promega). To decrease the risk for artefactual polymorphism each analysis was duplicated in an independent experiment using new RNA template. Although the single-strand conformation polymorphism (SSCP) does not provide the measurement of the genetic distance among viral variants it is highly sensitive because we routinely were able to detect minor variants representing 3% of the whole population.20Laskus T. Wang L.F. Rakela J. Vargas H. Pinna A.D. Tsamandas A.C. Demetris A.J. Fung J. Dynamic behavior of hepatitis C virus in chronically infected patients receiving liver graft from infected donors.Virology. 1996; 220: 171-176Crossref PubMed Scopus (77) Google Scholar Importantly, it is impervious to artifactual polymorphism related to cloning artifacts owing to preferential selection of genomes by Escherichia coli.35Smith D.B. McAllister J. Casino C. Simmonds P. Virus 'quasispecies' making a mountain out of a molehill?.J Gen Virol. 1997; 78: 1511-1519PubMed Google Scholar, 36Forns X. Bukh J. Purcell R.H. Emerson S.U. How Escherichia coli can bias the results of molecular cloning preferential selection of defective genomes of hepatitis C virus during the cloning procedure.Proc Natl Acad Sci U S A. 1997; 94: 13909-13914Crossref PubMed Scopus (52) Google Scholar Furthermore, it can detect a single nucleotide substitution difference between amplified products. Quasispecies complexity was measured by calculating the number of bands on the SSCP gels. To prevent bias from manual counting, the images first were recorded as high-resolution TIFF files (Epson Expression 1680; Epson, Long Beach, CA) and analyzed by Quantity One software (Bio-Rad Laboratories, Hercules, CA). To provide information about genetic distances (diversity) within the E2/HVR1 quasispecies population, RT-PCR products were analyzed as originally described by Delwart et al.37Delwart E.L. Pan H. Sheppard H.W. Wolpert D. Neumann A.U. Korber B. Mullins J.I. Slower evolution of human immunodeficiency virus type 1 quasispecies during progression to AIDS.J Virol. 1997; 71: 7498-7508PubMed Google Scholar with minor modifications. The analyzed RT-PCR products were column purified (Wizard; Promega), heated for 4 minutes at 100°C in 1 × PCR buffer II (Perkin Elmer; Foster City, CA), and snap-cooled on ice. Heteroduplexes were separated on 10% polyacrylamide gels (acrylamide/bis ratio 37:1) and run at 250 V at a constant temperature of 25°C for 3 hours. The gels were stained with Silver Stain (Promega) because this provided for higher sensitivity and better resolution of individual bands compared with standard ethidium bromide staining. The images were recorded as TIFF files with a high-resolution scanner (Epson Expression 1680; Epson) and subsequent analysis was performed using Quantity One software (Bio-Rad). Each lane was recorded from the position immediately below the homoduplex to the bottom of the gel well. To simplify calculations and to allow for unbiased comparison between various gels, each lane was standardized by division into 414 equal compartments and the signal intensity at each division along the lane was transferred to an Excel workbook (Microsoft; Redmond, CA) containing formulas for data analysis. Shannon entropy has been defined by the equation H = −Σ pilog pi where H is the entropy of the set of probabilities pi to pn.38Shannon C.E. The mathematical theory of communication. University of Illinois Press, Chicago1949Google Scholar The entropy value is 0 if all the pi but 1 are 0 and is maximum and equal to log n when all the pi are equal (i.e., 1/n). For our analysis, pi was defined as the fraction of the total signal in data point i whereas n was constant as the number of data points in each lane (n = 414). Thus, the minimum entropy value of 0 would represent the situation in which the entire signal is present in one compartment, whereas the maximum value of 6.026 would indicate that the signal is distributed evenly among all 414 compartments. To facilitate data presentation, the entropy values were normalized as H/log(n), providing convenient values within the range of 0–1. Shannon entropy calculations have been used previously by others to characterize diversity of human immunodeficiency virus quasispecies populations.37Delwart E.L. Pan H. Sheppard H.W. Wolpert D. Neumann A.U. Korber B. Mullins J.I. Slower evolution of human immunodeficiency virus type 1 quasispecies during progression to AIDS.J Virol. 1997; 71: 7498-7508PubMed Google Scholar, 39Wolinsky S.M. Korber B.T. Neumann A.U. Daniels M. Kunstman K.J. Whetsell A.J. Furtado M.R. Cao Y. Ho D.D. Safrit J.T. Adaptive evolution of human immunodeficiency virus-type 1 during the natural course of infection.Science. 1996; 272: 537-542Crossref PubMed Scopus (497) Google Scholar Median mobility shift (MMS) was measured as proposed by Delwart et al.37Delwart E.L. Pan H. Sheppard H.W. Wolpert D. Neumann A.U. Korber B. Mullins J.I. Slower evolution of human immunodeficiency virus type 1 quasispecies during progression to AIDS.J Virol. 1997; 71: 7498-7508PubMed Google Scholar The signal intensity along the lane was added starting from the bottom until reaching 50% of the total value. The MMS value was calculated by dividing the position of the data point compartment most closely matching 50% of total lane intensity by 414 (the standardized number of total compartments in each lane). An MMS value of 0.5 would indicate that the signal is distributed evenly in the top half and bottom half of the lane, whereas lower and higher values would represent bottom-weighted and top-weighted signals, respectively. RT-PCR products were cloned into the TA cloning vector (Invitrogen; Carlsbad, CA) and individual clones then were sequenced directly using Perkin Elmer ABI 377 automatic sequencer. Multiple sequence alignments were performed with MegAlign (DNASTAR, Inc; Madison, WI). Phylogenic reconstruction was performed by the neighbor-joining method with the Kimura 2-parameter distance matrix.40Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences.J Mol Evol. 1980; 16: 111-120Crossref PubMed Scopus (24705) Google Scholar Positions where gaps were inserted to preserve alignment were discounted. A bootstrap analysis using 100 bootstrap replicates was performed to assess the reliability of tree construction. Mean distances within viral sequences for each time point were calculated according to the Kimura 2-parameter model. The number of synonymous substitutions per potential synonymous site and the number of nonsynonymous substitutions per potential nonsynonymous site were calculated by the method of Nei and Gojobori.41Nei M. Gojobori T. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions.Mol Biol Evol. 1986; 3: 418-426PubMed Google Scholar The earlier-described analysis was performed using MEGA 2.1 software package.42Kumar S. Tamura K. Jakobsen I.B. Nei M. MEGA2 molecular evolutionary genetics analysis software.Bioinformatics. 2001; 17: 1244-1245Crossref PubMed Scopus (4574) Google Scholar Statistical analysis was perform
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