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Division of Viral Hepatitis, National Center for Infectious Diseases, Centers for Disease Control and Prevention (CDC), 1600 Clifton Road NE, MS A33, Atlanta, GA 30333, USA
Correspondence
Feng-Xiang Gao
Fgao{at}cdc.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequence data reported here are DQ249472–DQ249796.
Present address: Division of Gastroenterology/Hepatology, Department of Medicine, University of Kansas Medical Center, 4035 Delp, Kansas City, KS 66160, USA. 
Present address: International Vaccine Institute, Kwanak PO Box 14, Seoul 151-600, Korea.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Alter & Seeff, 2000). Infection is transmitted primarily by parenteral exposure to blood or blood products that have not undergone screening or viral inactivation, or by exposure to blood from shared needles and syringes during injection drug use or unsafe medical procedures (Alter, 1997).
Six major HCV genotypes and several subgenotypes have been identified (Simmonds et al., 1993; de Lamballerie et al., 1997). In addition, HCV displays a high rate of nucleotide substitutions in the hypervariable region 1 (HVR1) of the envelope gene and exists as a number of distinct quasispecies (Pawlotsky, 1998). Analysis of the genetic relatedness of HVR1 among infected persons has been used to track transmission of HCV infections (Gretch et al., 1996; Ni et al., 1997; Ross et al., 2000; Cody et al., 2002).
Both superinfections and mixed infections have been observed in patients with chronic hepatitis C (Kao et al., 1993, 1994; Widell et al., 1995; Giannini et al., 1999; Herring et al., 2004). In addition, persons can be infected with several genotypes or subgenotypes of HCV following exposure to blood or blood products (Qian et al., 2000; Bowden et al., 2005). Possible explanations for mixed infections include initial exposure to multiple viruses, such as occurred from receipt of clotting-factor concentrates prepared from multiple donors prior to HCV testing and inactivation, or from exposure to multiple viruses over time among networks of injection drug users (Jarvis et al., 1994; Eyster et al., 1999; Bowden et al., 2005). This study was designed to assess the number of HCV genotypes and quasispecies transmitted to a human and experimentally inoculated chimpanzees who received a commercially prepared factor VIII concentrate containing multiple HCV genotypes and quasispecies.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Material from lot DO56 was subsequently used to inoculate five chimpanzees experimentally. Plasma from one of these infected animals was used for molecular cloning of HCV, which was classified as subgenotype 1a (HCV-1, GenBank accession no. M62321
[GenBank]
; Bradley et al., 1979, 1990; Choo et al., 1989).
The five chimpanzees inoculated intravenously with lot DO56 factor VIII concentrate developed acute non-A, non-B hepatitis, as indicated by elevated liver-enzyme levels and liver histology (Table 1). In four animals, the hepatitis and HCV infection resolved over a 6-month period, whereas one animal (CH771) developed chronic hepatitis with persistently elevated liver enzymes and abnormal liver histology (Table 1; Beach et al., 1992). Subsequently, plasma from animal CH771 was inoculated into two chimpanzees, CH910 and CH509 (Fig. 1), and plasma from CH910 was inoculated into three other animals, CH1439, CH1483 and CH1556. Plasma from CH509 was used to inoculate chimpanzee CH508, whose plasma was inoculated into chimpanzee CH1208 (Fig. 1).
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) were used to evaluate transmission of HCV genotypes and subgenotypes. Specimens obtained from the chimpanzee that developed chronic hepatitis and HCV infection (CH771) and from chimpanzees during serial passages of DO56-derived HCV from CH771 (Fig. 1) were used to evaluate HCV transmission at the quasispecies level.
PCR amplification of HCV-genome regions.
The following primers were used to amplify selected genome regions and nested RT-PCR was performed as described previously (Alter et al., 1999): 5' untranslated region (UTR), F15 (5'-CTGTGAGGAACTACTGTCT-3'), A329 (5'-TGGTGCACGGTCTACGAGAC-3'), F13 (5'-GAAAGCGTCTAGCCATGGCGT-3') and A295 (5'-CAAGCACCCTATCAGGCAGT-3'); E1–E2 region, C5-56 (5'-YTGCGGSTGGGCRGGDTGGCTCCTGTC-3'), CE4R (5'-ATCATTGCAGTTCARGGCCGT-3'), C4-56 (5'-CGCAAYTTGGGYARRGTCATCGATACC-3') and E2-56 (5'-GCKRTTKAKGTGCCARCTGCCRTTGGTGT-3'); core, NS5B regions and HVR1, as described previously by Cody et al. (2002) and Lu et al. (2005).
Limiting-dilution ‘cloning’ PCR (LDC-PCR).
LDC-PCR was used to isolate and amplify individual HCV molecules (clones) as described previously (Cody et al., 2002). Briefly, for each specimen, serial dilutions of cDNA were amplified by nested RT-PCR to determine an end-point titre. A twofold dilution of the end-point titre dilution was used as the working dilution. For each specimen, 40 nested PCR amplifications were performed from the working dilution and positive amplicons were identified by ethidium bromide staining. Previous studies had shown that 10–15 positive amplicons from the 40 amplifications were the optimum number needed to isolate individual HCV molecules/clones (F.-X. Gao, O. V. Nainan, M. J. Alter & H. S. Margolis, unpublished data). The amplicons were purified and the nucleotide sequences were determined as described below.
Direct cloning of PCR products.
PCR amplicons were purified from low-melting-point agarose gel by digestion with agarase (Boehringer Mannheim); an extra A was added to the 3' end by incubating the purified amplicons in 1x PCR buffer, 1 U Taq DNA polymerase and 1x nucleotides in a total of 20 µl at 72 °C for 20 min, and the fragment was cloned into the pT-Adv vector (Advantage PCR cloning kit; Clontech) according to the manufacturer's protocol. A representative number of white colonies were selected and DNA from the plasmids was extracted.
Sequence analysis.
LDC-PCR products, as well as cloned plasmids, were purified (QIAquick PCR purification kit or plasmid mini kit; Qiagen) according to procedures recommended by the manufacturer. Purified DNA was sequenced in both directions by using ABI Prism dRhodamine terminators with appropriate primers on an ABI 3100 Genetic Analyser (Applied Biosystems).
Alignment of nucleotide sequences was performed by multiple comparisons with the GCG package (Wisconsin Sequence Analysis package, version 10.0; Genetics Computer Group). Phylogenetic trees were constructed by using the neighbour-joining algorithm based on distance matrices generated using the Jukes–Cantor model of nucleotide substitution with MEGA 3.0 (Kumar et al., 2004).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Of these, 40 (73 %) were classified as genotype 1, seven (13 %) as subgenotype 2a, six (11 %) as subgenotype 2b and two (4 %) as subgenotype 3a (Table 2). From the core region, 101 amplicons were obtained by LDC-PCR –43 (42·6 %) were subgenotype 1a, 48 (47·5 %) subgenotype 1b, three (3·0 %) subgenotype 2a and seven (6·9 %) subgenotype 2b (Table 2). A similar subgenotype distribution was obtained by direct cloning of the core region (Table 2). However, genotype classifications derived from direct cloning of the E1 and NS5B regions differed substantially from those obtained by LDC-PCR of the core region (Table 2).
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), the DO56 factor VIII concentrate is estimated to have 103 chimpanzee infectious doses (CID) ml–1.
HCV genotypes in the index patient
Only one HCV genotype was found in the index patient approximately 1 year after the onset of her non-A, non-B hepatitis. Although attempts were made to amplify all regions, only the 5' UTR and NS5B produced amplicons, presumably because of low HCV titre and our inability to use larger starting volumes because of a limited available serum volume. Because of the low HCV titre, LDC-PCR was not performed for the NS5B region and, of the 22 clones obtained by direct cloning, all were subgenotype 1b (Table 2).
Experimental chimpanzee infections
Genotype transmission.
By using the 5' UTR, all animals inoculated with the DO56 factor VIII concentrate were found to be infected with only HCV genotype 1 (Table 2). When analysed by direct cloning and LDC-PCR of the core region, mixed infections with subgenotypes 1a and 1b were identified in chimpanzees CH921, CH810 and CH1433, and direct cloning of the E1 region identified mixed infections with the same subgenotypes in these animals (Table 2). Infection with subgenotype 1a alone was identified in two animals (CH505 and CH771) by direct cloning and LDC-PCR of the core region (Table 2). Direct cloning of the NS5B region detected no mixed infections in all but one animal (Table 2). Taken together, the detection methods indicate that single-subgenotype infections probably occurred in chimpanzees CH505 and CH771 (subgenotype 1a) and mixed infections (subgenotypes 1a and1b) occurred in the remaining animals. Genotyping was not performed for experimental infections derived from the plasma of chimpanzee CH771, as it only contained subgenotype 1a (Fig. 1, second to fourth passages).
HVR1 quasispecies transmission.
HVR1 quasispecies analysis of 81 clones obtained by LDC-PCR from the DO56 inoculum showed 0–56 % nucleotide variation among the sequences and identified 57 unique quasispecies and several major quasispecies clusters (Table 4; Fig. 2). The nucleotide variation of these clusters ranged from 0–38 to 0–44 %.
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), produced 26 LDC-PCR clones and identified 12 unique HVR1 quasispecies with 0–52 % nucleotide variation. Ten of the 12 unique quasispecies belonged to cluster A of the DO56 quasispecies (distance, 0–26 %) and two belonged to cluster B (distance, 0–31 %) (Fig. 2; Table 3). The minimum nucleotide variation between quasispecies from the DO56 inoculum and CH771 was 2 %. Quasispecies analyses were not performed on the other animals inoculated with DO56.
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). Of 17 clones analysed from CH910, two unique quasispecies were identified, which were identical to two quasispecies found in CH771 and belonged to cluster A (nucleotide variation, 0–1 %; Fig. 2; Table 3). Of 42 clones analysed from CH509, six unique quasispecies were identified; all belonged to cluster A, with nucleotide variation from 0 to 5 %. In addition, the major quasispecies found in CH509 was identical to one found in CH771 and CH910.
Plasma from chimpanzees CH910 and CH509 obtained during the acute phase of infection was the source for a third passage series of experimental infections in four chimpanzees, and the plasma from one of these animals was used to produce a fourth-passage infection (Fig. 1, Table 1). Two to three unique HVR1 quasispecies were found in the specimens obtained from the third and fourth chimpanzee passages, with nucleotide variations of 0–3 and 0–7 %, respectively (Fig. 2; Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Transmission appeared to relate directly to HCV subgenotype concentration in the factor VIII concentrate. The predominant subgenotypes were 1a and 1b, with subgenotypes 2a, 2b and 3a present in lower relative proportions (these were not detected in the human or chimpanzee recipients).
The minor HCV subgenotypes were presumed not to have been transmitted following inoculation of the factor VIII concentrate, although they may have been missed by each of the detection methods if they remained present at a low proportion/concentration following infection. The patient and chimpanzees received 30–78 ml factor VIII concentrate (Table 1), which should have contained 
104 HCV CID based on our estimates of virus concentration. Based on genotype distribution, even the minor subgenotypes should have been present at 2x102 CID (e.g. two viral particles of subtype 3a detected from a total of 55 HCV particles), which suggests that the minor subgenotypes were present in sufficient concentrations to produce infection. In addition, LDC-PCR was used to estimate the concentrations of HCV species in the lot DO56 factor VIII concentrate. In parallel comparisons with direct cloning, we have found that LDC-PCR is more sensitive in detecting minor HCV species (F.-X. Gao, O. V. Nainan, M. J. Alter & H. S. Margolis, unpublished data).
Among the infected chimpanzees, there was some discordance in detection of subgenotypes between direct cloning and LDC-PCR of the core region and between core region and E1 analysis (Table 2). The observed differences between LDC-PCR and direct cloning occurred in only two animals and may be due to expected variation between the two methods or our observations that LDC-PCR appears more sensitive in identifying minor viral species (F.-X. Gao, O. V. Nainan, M. J. Alter & H. S. Margolis, unpublished data). However, when all core and E1 detection methods were used to classify animals with regards to mixed infections, at least two of the three methods were in agreement.
Following exposure to the factor VIII concentrate, only HCV subgenotype 1b was identified in the index patient. It is possible that subgenotype 1a was not identified because the detection scheme used for the index patient was not as robust as that used for the chimpanzees, because of limited specimen quantity and low viral titre. Another explanation could be that the PCR-primer configuration may have biased toward subgenotype 1b detection, which could not be evaluated as other regions could not be sequenced because of limited specimen volume. However, these primers and PCR conditions have been used previously and bias has not been observed in other HCV subgenotype studies (Alter et al., 1999). Subgenotype proportions derived by PCR-amplified clones would be expected to be affected by virus concentrations in the factor VIII concentrate, which could introduce a resampling bias and result in underrepresentation of the true genetic diversity in the sample. In addition, LDC-PCR could contribute bias, as the distribution of DNA molecules at the end-point dilution follows a Poisson distribution and there is a reasonable chance that some PCR products are derived from two or more cDNA molecules (Simmonds et al., 1990). However, given the almost-equal distribution of subgenotypes 1a and 1b in the factor VIII concentrate, finding only subgenotype 1b in the index patient could not be explained by these technical limitations, which, if they occurred, would not have been expected to change the observed distributions so significantly. This suggests either a transmission or replication advantage of subgenotype 1b over subgenotype 1a in the index patient.
Of the five experimentally infected chimpanzees, three had a mixed infection with subgenotypes 1a and 1b. Mixed infections with HCV genotypes among recipients of factor VIII concentrate before 1985 were common because source plasma was derived from thousands of donors (Preston et al., 1995; Franchini et al., 2002). Why exposure to an inoculum with a mixture of HCV genotypes of sufficient concentration to produce an infection resulted in a mixed-subgenotype infection in some recipients and a single-genotype infection in others is not known. Studies of human donor–recipient pairs after liver transplantation or blood transfusion indicate that frequently only one HCV strain becomes dominant after exposure to multiple strains and that mixed infections with different strains may occur temporarily (Lin et al., 2001; Fan et al., 2003). Experimental infections in chimpanzees have shown that one strain predominates after exposure to multiple strains (Okamoto et al., 1994). In addition, chimpanzees infected chronically with HCV can be reinfected with the original infecting HCV strain (Farci et al., 1992; Prince et al., 1992). Reinfection was thought to be due to the presence of minor quasispecies to which there was little or no immunity (Wyatt et al., 1998). If infection is controlled by the size of the infectious inoculum (Gordon et al., 1993; Alter, 1994), the predominant strain might outcompete the minor strain. In our study, we may have missed the dynamics of transient infections with selected strains because of long intervals between samples. Another hypothesis is that unidentified host factors, in addition to viral load, are involved in the establishment of the dominant infection (Domingo et al., 1985; Domingo & Holland, 1994).
The high number of HVR1 quasispecies observed soon after the first passage of DO56 in chimpanzees suggests transmission of multiple HCV strains. In contrast, the low observed genetic diversity of HVR1 quasispecies among the second to fourth chimpanzee passages strongly suggests a single-strain infection. The decreasing diversity of HVR1 quasispecies during serial transmission of HCV in chimpanzees supports the hypothesis that this region might undergo fewer mutations in experimentally infected chimpanzees than in humans (Ray et al., 2000).
Several other differences appear to exist between HCV infections in chimpanzees and humans. For example, chimpanzees elicit a selective humoral immune response compared with humans (Bassett et al., 1998) and humoral immune responses to HCV structural proteins are observed less frequently in chimpanzees than in humans (Bassett et al., 1999). The present study suggests that there may be differences in transmission of HCV species to humans and chimpanzees following exposure to a mixture of viruses. Whereas only HCV subgenotype 1b was identified in the index patient following inoculation with the factor VIII concentrate, three chimpanzees inoculated with the same factor VIII concentrate produced mixed infections with subgenotypes 1a and 1b, whilst two chimpanzees were infected with subgenotype 1a only.
During the second chimpanzee passage, the selective transmission of the predominant HVR1 quasispecies found in the inoculum is consistent with the findings from other studies (Sugitani & Shikata, 1998; Bassett et al., 1999; Ray et al., 2000). However, we also observed that a minor or previously undetectable quasispecies may become predominant in subsequent passages. One possible explanation for this change in quasispecies population is that the major variant was neutralized partially or fully by antibody in the inoculum and the minor variant had a selective replication advantage because it was not inhibited by neutralizing antibody (Manzin et al., 2000).
Selective transmission of predominant and minor HCV quasispecies has been shown in humans (Weiner et al., 1993; Gretch et al., 1996; Manzin et al., 2000; Lin et al., 2001; Cody et al., 2002) and experimentally infected chimpanzees (Kojima et al., 1994; Hijikata et al., 1995). However, neither the location nor the time of selection is known. The present study suggests that initially all viral strains (quasispecies) are transmitted equally and produce a primary infection, with subsequent selection of dominant strains.
Another important parameter that may affect selection of quasispecies is genetic interaction between virus and host (Domingo et al., 1985; Domingo & Holland, 1994). Several studies have described compartmentalization of different HCV genotypes in tissues of the same patient, including between serum and brain (Radkowski et al., 2002), serum and saliva (Roy et al., 1998) and serum and peripheral blood mononuclear cells (Roque-Afonso et al., 2005). These observations suggest that at least some HCV genotypes may be sensitive to the genetic, biochemical or metabolic background of the host cells. If so, this may contribute significantly to the selective transmission of HCV strains to different hosts.
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ACKNOWLEDGEMENTS |
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Received 16 June 2005;
accepted 4 October 2005.
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