Generation of infectious and transmissible virions from a GB virus B full-length consensus clone in tamarins

Andrea Sbardellati1, Elisa Scarselli1, Ernst Verschoor2, Amedeo De Tomassi1, Domenico Lazzaro1 and Cinzia Traboni1

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


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The strong similarity between GB virus B (GBV-B) and hepatitis C virus (HCV) makes tamarins infected by GBV-B an acceptable surrogate animal model for HCV infection. Even more attractive, for drug discovery purposes, is the idea of constructing chimeric viruses by inserting HCV genes of interest into a GBV-B genome frame. To accomplish this, infectious cDNA clones of both viruses must be available. The characterization of several HCV molecular clones capable of infecting chimpanzees has been published, whereas only one infectious GBV-B clone inducing hepatitis in tamarins has been reported so far. Here we describe the infection of tamarins by intrahepatic injection of RNA transcribed from a genomic GBV-B clone (FL-3) and transmission of the disease from infected to naive tamarins via serum inoculation. The disease resulting from both direct and secondary infection was characterized for viral RNA titre and hepatitis parameters as well as for viral RNA distribution in the hepatic tissue. Host humoral immune response to GBV-B antigens was also monitored. The progression of the disease was compared to that induced by intravenous injection of different amounts of the non-recombinant virus.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
For many years progress in the search for effective drugs against hepatitis C virus (HCV; Rice, 1996 ; De Francesco, 1999 ) has been hampered by the lack of reliable cell infection systems and suitable animal models. This problem was in part overcome by the development of a replicon-based cellular system (Lohmann et al., 1999 , 2001 ; Blight et al., 2000 ; Pietschmann et al., 2001 ). This system could prove extremely useful for preclinical tests on antiviral compounds whose mechanisms involve interactions with the viral non-structural proteins. It is, however, unsuitable for studying HCV life-cycle phases related to cell binding, internalization and signal transmission, which presumably involve the structural proteins of the virion.

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.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus stock.
Serum of a GBV-B experimentally infected Saguinus fuscicollis tamarin (kindly provided by A. Kekulé, Halle University, Germany) was passaged in S. oedipus tamarins B234 and B242 (250 µl of 1:25 dilution in pyrogen-free PBS). Dilutions of acute phase serum (2 weeks post-infection) of B242 were used to infect other tamarins, including B131 and B132.

{blacksquare} 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 Institute’s 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.

{blacksquare} 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 RT–PCR, 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 RT–PCR, 1 µl aliquots (2·5–3·5 µg) were used.

{blacksquare} Oligonucleotides.
Oligodeoxyribonucleotides were purchased from PRIMM (Italy), Genset (France) and MWG (Germany).

{blacksquare} RT–PCR.
Serum or liver RNA was used for first strand cDNA synthesis by Superscript II reverse transcriptase (Gibco-BRL) under the manufacturer’s conditions. PCR amplification was performed in standard conditions using Elongase enzyme mix (Gibco-BRL). Non-quantitative RT–PCR 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 RT–PCR with sense primer 98149 (AGCRTCWGWCGTTAAACGGAG) and antisense primer 98150 (GATTTTTCCYCTTGCCGC).

{blacksquare} Cloning and mutagenesis.
RT–PCR 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.

{blacksquare} 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 RT–PCR 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.

{blacksquare} 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 RT–PCR. At the end of the study serum samples were subjected to quantitative PCR (qPCR), ELISA and Western blot (WB).

{blacksquare} 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.

{blacksquare} 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 RT–PCR 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, phenol–chloroform 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).

{blacksquare} 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.

{blacksquare} 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 8800–9068) was used for in situ hybridization. Digoxigenin labelling and in situ hybridization protocols were performed essentially as described in Braissant & Whali (1998) .


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Infection of tamarins by intravenous injection of GBV-B inoculum
Pairs of tamarins were intravenously injected with nine serial dilutions of GBV-B stock [serum of infected B242 tamarin, qPCR titre 1·5x108 genome equivalents (GE)/ml]. Doses containing up to 0·375x102 GE did not produce infection, whereas inocula containing from 3·75x102 to 7·5x106 GE showed clear signs of infection, as judged by monitoring ALT and viral RNA. Fig. 1 presents data on progression of the disease developed by tamarins B131 and B132, infected with the highest dose used, which did not differ significantly from that observed for animals infected with lower doses. Moderate variations in appearance and duration of viral RNA and ALT levels were observed among the different pairs (data not shown) as well as between the elements of each pair (Fig. 1).



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Fig. 1. Time-course of infection of tamarins with GBV-B. Fifty µl of GBV-B-positive B242 tamarin serum (containing 7·5x106 GE) was used for intravenous inoculation of B131 and B132 tamarins. Euthanasia was performed at week 28 p.i. ALT levels (shaded area) are reported for weekly bleeds till week 16, data about subsequent negative bleeds are not plotted. Viral RNA titre was determined by qPCR (bars) on all the bleeds except those labelled N. T., because of limited serum volume; qPCR data are reported for weekly bleeds till week 16, data about subsequent negative bleeds are not plotted.

 
Construction of a GBV-B genomic cDNA
We constructed a GBV-B genomic cDNA (FL-1) by joining RT–PCR amplification products obtained from serum and liver RNA from infected cotton-top tamarins (S. oedipus). PCR primers were designed on the basis of the unique sequence published at that time (accession no. U22304). The resulting construct was transcribed and the RNA injected intrahepatically in two tamarins but no sign of infection was detectable. We then discovered that the GBV-B genome published sequence was truncated and lacked the 3'Y region (Sbardellati et al., 1999 ; Bukh et al., 1999 ). We constructed further versions of the full-length clone by adding the 3'Y region and correcting the FL-1 sequence according to the consensus sequence of a GBV-B virus source (see below). An intermediate, not fully consensus clone, FL-2, was unsuccessfully tested in tamarins. We then completed the mutagenesis to get a clone (FL-3) bearing the consensus deduced amino acidic sequence in the ORF, and the consensus nucleotide sequence in the UTRs. In Table 1 a comparison of both nucleotide and amino acid sequences among the different GBV-B sequences is presented.


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Table 1. Comparison of GBV-B genomic sequences

 
Determination of a GBV-B genome consensus sequence
A GBV-B consensus sequence was derived by comparing individually cloned independent RT–PCR products obtained from RNA of the liver and serum of several infected animals. The consensus sequence was identical to that of a GBV-B isolate, determined by RT–PCR and direct sequencing of the viral RNA extracted from serum of an individual infected tamarin (B234). The B234/consensus sequence (Table 1) presents four amino acids substitutions with respect to the GBV-B sequence published by Bukh et al. (1999) (accession no. AF179612) and eight differences from the originally published sequence (accession no. U22304) (Simons et al., 1995 ), also lacking the 3'Y sequence.

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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Fig. 2. Time-course of infection of tamarins inoculated with FL-3 RNA (95-59 and B223) and serum of animals infected with FL-3 RNA (97-19 and 97-22). Euthanasia was performed for tamarins 95-59 and B223 at week 22 p.i., for 97-19 at week 5 p.i., and for 97-22 at week 11 p.i. ALT levels (shaded area) and viral RNA titre (bars) are reported for weekly bleeds. Data about tamarins 97-19 and 97-22 are reported till euthanasia. Data about tamarins 95-59 and B223 are reported till week 16, subsequent negative data are not plotted. N. T. indicates a sample not tested by qPCR.

 
The identity of the GBV-B sequence responsible for infection was confirmed as corresponding to FL-3 by sequencing the regions spanning two silent mutations introduced in FL-3 to remove internal SapI restriction sites (nt 2890 and 8233 respectively). The RT–PCR fragments also proved to be resistant to SapI digestions, in contrast to the corresponding fragments obtained from RNA of the non-recombinant virus-infected B234 tamarin.

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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Fig. 3. Seroconversion of tamarins infected by non-recombinant GBV-B (B131 and B132), FL-3 RNA (95-59 and B223) and monoclonal GBV-B (97-19 and 97-22). Bars represent antibody response to GBV-B NS5B measured by ELISA in serum samples. Black and white triangles represent positive and negative response respectively to GBV-B NS3 detected by WB in individual serum samples tested.

 
GBV-B RNA quantification and localization in tamarin liver
RNA was extracted from liver samples prepared after euthanasia of tamarins B239 and B243 (non-infected), B242 (infected with non-recombinant GBV-B, euthanasia in acute phase), 95-59 (infected with GBV-B RNA, euthanasia 9 weeks after serum RNA clearance), B223 (infected with GBV-B RNA, euthanasia 11 weeks after clearance), 97-19 (infected with clonal GBV-B from 95-59, euthanasia in acute phase), 97-22 (infected with clonal GBV-B from B233, euthanasia 2 weeks after clearance). Virus titre quantification obtained by qPCR, expressed as genome equivalents per mg of total RNA, is reported in Table 2 and shows a correlation between time from infection and virus titre.


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Table 2. Detection of GBV-B RNA in the liver of infected tamarins

 
A digoxigenin-labelled RNA probe corresponding to 300 bp in the 3'-end region of the GBV-B genome was used for in situ hybridization to monitor the GBV-B RNA localization in the liver of tamarins infected with non-recombinant virus (Fig. 4 b), recombinant RNA transcript (Fig. 4c) and clonal virus (Fig. 4d). The B242 and 97-19 samples corresponded to the acute phase stage of infection, whereas the 97-22 sample was dissected after the acute phase. Negative controls were made with the liver of the non-infected tamarins B239 (Fig. 4a) and B243 (data not shown). In both acute phase infected sample B242 (2 weeks p.i. with non-cloned virus) and 97-19 (5 weeks p.i. with clonal virus) a strong cytoplasmic signal was depicted. In B242 the signal was restricted to the hepatocytes and seemed preferentially distributed in those located in the perivascular areas (Fig. 4b). In the other acute phase sample, 97-19, a cytoplasmic hepatocyte-specific signal was again depicted in the perivascular areas and was slightly more distributed in the rest of the parenchyma (Fig. 4c). No major pathological modifications of the liver parenchyma were observed in any of the samples. Interestingly, in the liver of tamarin 97-22, euthanized 2 weeks after apparent remission diagnosed upon serum RNA qPCR measurement, viral RNA was still detectable by qPCR (Table 2) and by in situ hybridization, with a similar distribution to the other infected samples (Fig. 4d). qPCR was also positive for the liver of tamarin 95–59 up to 9 weeks after serum clearance.



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Fig. 4. In situ hybridization with a GBV-B digoxigenin-labelled probe of tamarin liver specimens. (a) Liver of non-infected tamarin B239 (negative control); (b) liver of tamarin B242 infected with non-recombinant virus (positive control); (c) liver of tamarin 97-19 infected with clonal GBV-B (serum from tamarin 95-59); (d) liver of tamarin 97-22 infected with clonal GBV-B (serum from tamarin B223). All liver samples were prepared upon euthanasia. The magnification bar represents 80 µm for (a) and (b) samples and 35 µm for (c) and (d) samples.

 
GBV-B RNA in tamarin PBMC
Attempts to detect GBV-B RNA by qPCR performed on Fycoll-purified peripheral blood mononuclear cells gave negative results (data not shown), suggesting that the virus is not localized in peripheral lymphocytes, in contrast to what occurs in infections sustained by HCV (Giuberti et al., 1994 ) and possibly GB virus C (Fogeda et al., 1999 ). A border-line signal was obtained with bone marrow preparation, but blood contamination of the sample cannot be excluded (data not shown).


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
The natural history and characteristics of GBV-B infection, in spite of a number of published studies, are still unclear. In the early 1960s an experimental infection of tamarins was performed to identify the aetiological agent of an unclassified hepatitis affecting a patient whose initials were G.B. (Deinhardt et al., 1967 ). Some 28 years later, the viruses GBV-A and GBV-B were identified in the eleventh serial passage of serum in tamarins (Simons et al., 1995 ; Schlauder et al., 1995 a, b ). GBV-A was also detected in wild-caught tamarins (genus Saguinus) and other New World monkeys such as marmosets (genus Callithrix) and owl monkeys (genus Aotus). GBV-B has never been identified in the serum of non-human primates or in serum of humans, possibly due to the fact that GBV-B produces an acute self-limiting disease lasting only a few months. The co-presence of GBV-A with GBV-B in experimental infections led to the hypothesis that GBV-A is a contributing factor to the severity of the GBV-B-dependent hepatitis (Schlauder et al., 1995a ; Karayiannis & McGarvey, 1995 ). Interestingly, no report has ever been published about attempts to identify GB viruses in the original G.B. serum specimen. Moreover, only one paper has been published so far reporting the infection of tamarins by cloned genomic RNA (Bukh et al., 1999 ).

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, 1–2 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 RT–PCR 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.


   Acknowledgments
 
We thank Riccardo Cortese, Raffaele De Francesco, Gloria Taliani and Giovanni Galfrè for helpful discussions, Janet Clench for revising the manuscript, Paola Gallinari for gift of GBV-B NS3 and Ernesta Dammassa for help with in situ hybridization.


   Footnotes
 
The EMBL accession number of the sequence of clone FL-3 described in this paper is AJ277947.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 19 April 2001; accepted 22 June 2001.