Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Via Pontina Km 30.600, 00040 Pomezia (Roma), Italy1
Biomedical Primate Research Centre (BPRC), PO Box 3306, 2280 GH Rijswijk, The Netherlands2
Author for correspondence: Cinzia Traboni. Fax +39 06 91093225. e-mail cinzia_traboni{at}merck.com
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Abstract |
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Introduction |
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The limitations in animal models depend on the extremely restricted host range of HCV, which only infects humans and chimpanzees (Farci et al., 1992a , b
; Abe et al., 1993
). The use of chimps for research purposes is becoming ever more restricted, so that planning a drug discovery programme involving these animals is inconceivable. It is therefore crucial to identify alternative models to study virus infection in the context of an entire organism and to address issues related to the early phases of infection.
In the absence of suitable animal models for HCV, a few years ago the idea of a surrogate animal model based on tamarins began to make its way in the scientific community. Tamarins (Saguinus species) are small New World monkeys susceptible to hepatitis caused by GB virus B (GBV-B) (Simons et al., 1995 ; Schlauder et al., 1995a
, b
). This virus was originally indicated as a valid candidate for a surrogate infection system because of its genome similarity to HCV (Muerhoff et al., 1995
). The virus is now fairly well characterized not only in terms of its genome organization, but also of several aspects of its enzymology and pathogenicity. All the studies performed in the last few years on virus (Scarselli et al., 1997
; Grace et al., 1999
; Bukh et al., 1999
; Traboni et al., 1999
; Zhong et al., 1999
, 2000
; Sbardellati et al., 1999
, 2000
; Butkiewicz et al., 2000
) and host relevant molecules (Meola et al., 2000
; Aurisicchio et al., 2001
) indicate the feasibility of using the tamarin/GBV-B model in place of a direct HCV/chimp model, whilst taking into account the differences that do exist between the two systems.
The most important goal, however, would be the development of infectious chimeric viruses bearing HCV genes of interest inserted into a GBV-B genomic scaffold. To construct such chimeras, infectious molecular clones of both partner viruses are required. Several infectious clones have been reported belonging to different HCV strains (Kolykhalov et al., 1997 ; Yanagi et al., 1997
, 1998
, 1999a
, b
; Beard et al., 1999
; Hong et al., 1999
, Major et al., 1999
; Forns et al., 2000
). In this study we describe the construction of a genomic clone of GBV-B, the characterization of the infection produced by intrahepatic injection of the corresponding RNA, and the transmission of the infection to naive animals.
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Methods |
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Animals.
Captive-outbred S. oedipus tamarins were individually housed at the Biomedical Primate Research Center, Rijswijk, The Netherlands. During the course of the study they were checked twice daily for appetite and behaviour changes. Protocols were approved by the Institutes Animal Care and Use Committee (IACUC) according to international ethical and scientific standards and guidelines. This work meets all requirements of The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and the regulations set out by Dutch law. Animals negative for GBV-B RNA and anti-GBV-B antibodies were chosen for this study. Tamarins B234, B242, B243 and 97-19 were positive for GB virus A (GBV-A) RNA, whereas tamarins B131, B132, B239, B223, 95-59 and 97-22 were negative.
RNA preparation.
RNA was prepared from serum samples (140 µl) using the QIAamp Viral RNA kit (Qiagen) and eluted in 50 µl sterile water. For first strand cDNA synthesis in RTPCR, 10 µl aliquots were used. Total RNA was prepared from liver specimens using the Ultraspec II RNA isolation system (Biotecx). For first strand cDNA synthesis in RTPCR, 1 µl aliquots (2·53·5 µg) were used.
Oligonucleotides.
Oligodeoxyribonucleotides were purchased from PRIMM (Italy), Genset (France) and MWG (Germany).
RTPCR.
Serum or liver RNA was used for first strand cDNA synthesis by Superscript II reverse transcriptase (Gibco-BRL) under the manufacturers conditions. PCR amplification was performed in standard conditions using Elongase enzyme mix (Gibco-BRL). Non-quantitative RTPCR detection of GBV-B RNA in serum of tamarins was obtained with antisense primer GBB-N2 (CACATATAGGTGGGCTTGC) and sense primer GBB-N1 (TCATGACGCTCGCGTGATG); nested set was performed using sense primer GBB-N3 (CAAGCTTGACTTGGATGGC) and antisense primer GBB-N4 (GCGTCCTTGGTAGTGACCG). GBV-A RNA was detected by RTPCR with sense primer 98149 (AGCRTCWGWCGTTAAACGGAG) and antisense primer 98150 (GATTTTTCCYCTTGCCGC).
Cloning and mutagenesis.
RTPCR products were purified by routine methodologies and joined together stepwise to get intermediate clones. Final full-length genomic clones include the GBV-B genome cDNA downstream of the T7 polymerase promoter in the pACYC177 vector. Non-consensus mutations were removed in the FL-3 clone by oligonucleotide-based mutagenesis. Two internal SapI sites were also mutagenized to leave a unique SapI site at the 3'-end of the GBV-B coding sequence.
Sequencing and sequence analysis.
Sequencing was performed by the Big Dye Terminator Cycle sequencing kit with AmplyTaq (Applied Biosystems) and run with an Applied Biosystems model 373A sequencer. To obtain accurate determination of GBV-B genomic sequences, primers were used spanning the entire sequence in such a way that each nucleotide (nt) position was read multiple times in both directions. A GBV-B consensus sequence was obtained by comparing the sequences of at least three independently produced and cloned GBV-B fragments from various sources. The sequence of infectious RNA from tamarin B234 was obtained by directly sequencing three independent RTPCR products for each of nine overlapping fragments spanning the whole genome. Sequence homology analysis was performed using FastA and Blast programs, Wisconsin Package version 9.1, Genetics Computer Group (GCG), Madison, Wisconsin, USA.
Infection of tamarins by intravenous injection of GBV-B-infected serum.
Tamarins were inoculated intravenously with acute phase serum of a tamarin previously infected with GBV-B (tamarin B242). The progression of the disease was monitored weekly for 28 weeks by determining alanine aminotransferase (ALT) levels using standard methodologies for human specimens and by viral RNA detection with qualitative RTPCR. At the end of the study serum samples were subjected to quantitative PCR (qPCR), ELISA and Western blot (WB).
In vitro transcription and RNA intrahepatic transfection of tamarins.
SapI-linearized plasmid (10 µg) encoding GBV-B full-length cDNA was in vitro-transcribed by T7 RNA polymerase into a final volume of 100 µl using an Ambion Megascript kit under sterile conditions. The reaction was terminated by adding 400 µl of sterile PBS without calcium and magnesium. RNA was frozen in dry ice and kept at -80 °C until liver injection. Laparotomy was performed and three aliquots (150 µl each) of diluted transcription mixture were injected into different lobes of the liver of individual tamarins. The amount of RNA injected was about 300 µg per animal. ALT, viral RNA and antibodies were measured as described in the specific sections.
TaqMan quantification of GBV-B RNA.
GBV-B RNA was quantified by a real-time, 5' exonuclease PCR (TaqMan) assay using a primer/probe set that recognized a portion of the GBV-B 5' untranslated region (5'UTR). The primers (GBV-B-F3, GTAGGCGGCGGGACTCAT, and GBV-B-R3, TCAGGGCCATCCAAGTCAA) and probe (GBV-B-P3, 6-carboxyfluorescein-TCGCGTGATGACAAGCGCCAAG-N,N,N,N-tetramethyl-6-carboxyrhodamine) were selected using the Primer Express software (PE Applied Biosystems). The fluorescent probe was obtained from PE Applied Biosystems. The primers were used at 10 pmol/50 µl reaction, and the probe was used at 5 pmol/50 µl reaction. The reactions were performed using a TaqMan Gold RTPCR kit (PE Applied Biosystems) and included a 30 min reverse transcription step at 48 °C, followed by 10 min at 95 °C and by 40 cycles of amplification using the universal TaqMan standardized conditions (15 s 95 °C denaturation step followed by a 1 min 60 °C annealing/extension step). RNA transcribed from a plasmid containing the first 2000 nt of the GBV-B genome was used as a standard to establish genome equivalents. Synthetic RNA was prepared using a T7 Megascript kit (Ambion) and was purified by DNase treatment, phenolchloroform extraction, Sephadex-G50 filtration and ethanol precipitation. RNA was quantified by absorbance at 260 nm, and stored at -80 °C. All reactions were run in duplicate by using the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems).
Immune response analysis.
Individual tamarin serum samples were tested for the presence of antibodies to the GBV-B proteins NS3 and NS5B by WB and ELISA, respectively. GBV-B NS3 protein was a gift from P. Gallinari, IRBM; 10 ng per slot was used in WB. A truncated form of GBV-B NS5B lacking 23 C-terminal residues (L. Tomei & C. Traboni, unpublished) was produced in E. coli and purified as described for HCV NS5B (Tomei et al., 2000 ); 6 µg were used in ELISA. WB and ELISA were performed according to routine protocols using 1:50 and 1:2000 dilutions of tamarin sera respectively. Alkaline phosphatase-conjugated anti-monkey IgG (Sigma) was used as a secondary antibody in both systems.
In situ hybridization.
A cRNA probe obtained by in vitro transcription (T7 Megascript kit, Ambion) and corresponding to a fragment of the GBV-B RNA negative strand (nt 88009068) was used for in situ hybridization. Digoxigenin labelling and in situ hybridization protocols were performed essentially as described in Braissant & Whali (1998) .
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Results |
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Intrahepatic transfection of tamarins with RNA transcribed from the FL-3 clone
RNA was in vitro-transcribed from plasmid FL-3 and injected into the liver of two cotton-top tamarins (B95-59 and B223). A hepatitis marker (ALT) and the viral RNA titre, monitored weekly in the serum (Fig. 2), showed that the injected RNA was able to replicate and induce hepatitis very early after injection with a progression similar to that observed in tamarins infected by a virus inoculum (Fig. 1
).
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Transmission of RNA-induced GBV-B infection to naive tamarins
Fifty µl acute phase serum (1·7x107 and 1·1x107 GE/ml respectively) of each animal injected with GBV-B RNA (95-59 and B223) was used to intravenously inject each of two naive animals (97-19 and 97-22 respectively). Results shown in Fig. 2 indicate that these tamarins also developed hepatitis with similar parameters to the infection induced by a non-recombinant GBV-B inoculum (Fig. 1
). Follow-up of the disease course was carried out for tamarin 97-22 until the normalization of ALT and disappearance of detectable viral RNA in serum, whereas tamarin 97-19 was euthanized during the acute phase. The identity of the infectious agent responsible for this secondary experimental infection was confirmed as described above for tamarins B223 and 95-59.
Host humoral immune response to GBV-B antigens
Humoral immune response against GBV-B NS3 and NS5B proteins was also analysed in the two tamarins injected with GBV-B RNA (95-59 and B223) as well as in those in which the recombinant virus was passaged (97-19 and 97-22) and compared to the response detected in two tamarins injected with the non-recombinant virus (B131 and B132). In Fig. 3 the results are shown for each tamarin, indicating that seroconversion for one or both antigens occurred following a rise in ALT, although with individual variations, regardless of the type of infectious agent. A reduced timespan of response to NS3 and no detectable response to NS5B were observed for tamarin B223. In tamarin 97-19, euthanized in the early acute phase, no response was observed.
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Discussion |
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Here we describe the characterization of tamarin infection by GBV-B and of some aspects of the host immune response. The infection with genomic RNA from a molecular clone and that produced by the clonal virus formed in the RNA-dependent infection event were compared to the infection with the non-recombinant virus.
We first performed an experimental infection of cotton-top tamarins (S. oedipus) with a characterized GBV-B inoculum to acquire direct information about development of hepatitis in this species and the relation to features such as the virus load of the inoculum and the co-infection with GBV-A. GBV-B replication and hepatitis development occurred with similar parameters in all animals that were infected, independent of the infectious dose used (see the example in Fig. 1). The appearance of GBV-B RNA preceded or coincided with the ALT increase, 12 weeks p.i.; the rise in RNA titre was very rapid, reaching a plateau and declining several weeks after, usually before the normalization of ALT levels. Slight variations in the measured parameters showed no evident correlation with GBV-A infection or GBV-B virus load, but seemed rather to reflect individual variations. The lack of graduality in infection parameters suggests that a threshold has to be reached to start the infection process, beyond which it progresses with standard parameters. Since only some of the liver cells are infected, possibly a balance between initial replication levels and efficiency of early immune response against the virus is crucial to determine the fate of the infection.
The humoral immune response against the GBV-B NS5B polymerase was monitored by ELISA in pre- and post-infection bleeds from tamarins B131 and B132 (Fig. 3). Both animals seroconverted with a rapid rise in antibody response coinciding with virus clearance and ALT normalization. The antibodies against NS5B decreased slowly, so that at the time of euthanasia the response to this antigen was still detectable. Due to the limited volume of blood we withdrew for ethical reasons, it was not possible to check antibodies to NS3 in all the bleeds from these two animals. WB analysis showed, however, that seroconversion also occurred to this protein.
After the characterization of reference parameters of the disease provoked by non-recombinant virus we tested a GBV-B molecular clone in vivo. Due to initial unsuccessful results we decided to derive a consensus sequence and construct a GBV-B genomic clone accordingly. We compared the sequences of a number of independent GBV-B subgenomic clones that we had previously constructed for different purposes (Scarselli et al., 1997 ; Sbardellati et al., 2000
) and those of other fragments cloned and sequenced for this purpose. The source of this information was notably heterogeneous, since different individual tamarins belonging to both S. oedipus and S. fuscicollis and different tissues (blood and liver) were used to collect data. The result of this analysis was a sequence that we define as consensus, but that might not correspond to an existing virus genome, as the information was derived from fragments which might not be arranged in cis in a unique molecule. Nonetheless, direct sequencing of RTPCR fragments of a bona fide homogeneous and infectious GBV-B RNA source, tamarin B234, confirmed the previously determined consensus sequence in every detail, so that we can refer indifferently to B234 or to consensus sequence. These results also suggest that the sequence variability of GBV-B is very limited.
The consensus clone, FL-3, proved to be infectious in vivo. To demonstrate that the hepatitis provoked by FL-3 RNA was not due to transient local effects but was sustained by replicating virions generated by the injected RNA, an aliquot of serum from each of the infected tamarins was used to infect naive animals. The result of this experiment confirmed that transmissible infectious virions were formed upon injection of GBV-B RNA.
The duration and levels of ALT alteration and of viral RNA were not significantly different from those detected during the infection with non-recombinant virus (see Figs 1 and 2
), indicating that both modes of infection produce identical disease and virus replication profiles. None of the tamarins injected with FL-3 RNA and only one recipient of the secondary infection was GBV-A-positive, but the progression of infection did not show appreciable differences. Observation of the distribution of viral RNA molecules in the liver of animals 97-19 and 97-22, euthanized in acute and post-acute phases respectively, revealed the same situation as in infection produced by the normal virus inoculum. Moreover, no histopathological modification was detectable in any sample, as already reported for HCV infection in chimpanzees (Negro et al., 1992
). From data summarized in Table 2, it appears that the virus titre is higher in the liver of animals euthanized during the acute phase than in those euthanized after that phase. In the case of tamarin B223, the RNA was actually below detection limits. Interestingly, in tamarin 97-22 and 95-59 the RNA was still detectable in the liver a few weeks after clearance of virus from serum. This might indicate a slower clearance in the liver, producing sub-minimal virus secretion into the bloodstream or a new burst of virus replication following apparent remission diagnosed on the basis of serum RNA levels. The humoral immune response to GBV-B antigens in these tamarins (Fig. 3
) did not corroborate the hypothesis of a virus replication rebound, since antibodies to both NS3 and NS5B were present until euthanasia. It is not known, however, whether humoral immune response to these non-structural antigens plays a role in limiting virus replication; moreover, a correlation between seroconversion and clearance was not observed in any animals of this study. Attempts to detect antibodies against GBV-B structural antigens (core protein) gave negative results (data not shown), in spite of expectations based on the known HCV core antigenicity (Pujol et al., 1996
; Tafi et al., 1997
) and on some data published about GBV-B (Pilot-Matias et al., 1996
).
In summary, we here report the construction of a functional molecular clone of GBV-B which produces hepatitis in tamarins with features that cannot be distinguished from those of the disease caused by the non-recombinant virus. This study is also the first formal demonstration that a GBV-B molecular clone is able to produce pathogenic and transmissible virus particles and, hence, that it is a suitable scaffold to construct viable HCV/GBV-B chimeric viruses.
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Acknowledgments |
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Footnotes |
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References |
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Received 19 April 2001;
accepted 22 June 2001.