Transmissible gastroenteritis virus (TGEV) is a member of the
Coronaviridae family, composed of enveloped viruses of medical and veterinary importance causing disease in humans and animals. The
Coronaviridae,
Arteriviridae, and
Ronaviridae families are included in the
Nidovirales order and, despite significant differences in their genome size, have the same polycistronic genome organization and regulation of gene expression, leading to a nested set of subgenomic mRNAs (sgmRNAs) (
7,
8,
15). The TGEV genome is a single-stranded, positive-sense 28.5-kb RNA. About two-thirds of the entire RNA comprises open reading frames (ORFs) 1a and 1b, encoding the replicase. The 3′ one-third of the genome comprises the genes encoding the structural and nonstructural proteins (
13). Sequences preceding each gene represent signals for the discontinuous transcription of sgmRNAs (
26,
44). These are the transcription-regulating sequences (TRSs), which include a highly conserved core sequence, 5′-CUAAAC-3′, that is identical in all TGEV genes, and the core sequence 5′ upstream (5′ TRS) and 3′ downstream (3′ TRS) flanking sequences (
3). The high conservation of the core sequence suggests that this sequence may be particularly relevant in the virus life cycle.
The recent construction of a full-length cDNA clone of TGEV (
2,
54) has created the possibility of specifically engineering the TGEV genome to study fundamental viral processes and to develop expression vectors. Coronaviruses have several advantages over other viral expression systems as vectors (for a review, see reference
14). For instance, these viruses have the largest RNA virus genome and, in principle, have room for the insertion of large foreign genes (
14,
32). Since coronaviruses, in general, infect the respiratory and enteric mucosal surfaces, they may be used to target the antigen to these areas to induce a pleiotropic secretory immune response that includes lactogenic immunity (
16).
Two types of coronavirus-derived expression systems have been developed. One type, the helper-dependent expression systems, permits the production of significant levels of heterologous genes (2 to 8 μg/10
6 cells), although with limited stability (
4). Another corresponds to single-genome coronavirus vectors, which were obtained first for murine hepatitis virus (MHV) by targeted recombination (
17,
21,
32) and, recently, for TGEV and human coronavirus 229E (HCoV-229E) by the construction of an infectious cDNA clone (
9,
45). However, the wide potential of coronaviruses as vectors has not yet been systematically investigated.
In this report, the infectious cDNA clone obtained for TGEV as a bacterial artificial chromosome (
2) has been engineered as a vector to express high levels of the heterologous green fluorescent protein (GFP) gene with transcription-regulating sequences of the nonessential gene 3a (
9,
16,
27,
34,
38,
52) or regulating sequences engineered from the N gene TRS. The TGEV-derived virus vector elicited an efficient lactogenic immune response in swine against both the vector and the heterologous gene, showing its potential to provide protection to piglets against mucosal infections.
MATERIALS AND METHODS
Cells and viruses.
The TGEV PUR46-MAD strain (
43) was grown and titrated as previously described (
24). Baby hamster kidney cells (BHK-21) stably transformed with the gene coding for porcine aminopeptidase N (BHK-APN) (
11) were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum and geneticin (G418; 1.5 mg/ml) as a selection agent. Viruses were grown in swine testis cells (
33).
Plasmid constructs.
To delete the nonessential genes 3a and 3b from the TGEV genome (GenBank accession number AJ271965 ), the 872-bp
PpuMI-
BlpI fragment comprising nucleotides 24822 to 25693 was removed from the intermediate plasmid pSL-SC11-3EMN7C8-BGH (
19), which was subsequently blunt-ended with T4 DNA polymerase and religated, generating plasmid pSL-SC11-Δ3EMN7C8-BGH. The 2,029-bp
AvrII-
AvrII fragment, including the ORF 3a and 3b deletion, was inserted into the corresponding sites (nucleotides 22967 and 25867 of the TGEV genome) of plasmid pBAC-TGEV
ΔCla I (
2), leading to plasmid pBAC-TGEV-Δ3
ΔCla I. Insertion of the
ClaI-
ClaI restriction fragment (nucleotides 4417 to 9615) into
ClaI-linearized pBAC-TGEV-Δ3
ΔCla I led to plasmid pBAC-TGEV-Δ3.
The mammalian codon-optimized version of the GFP gene was amplified from plasmid pGL-1 (Gibco-BRL) with a forward oligonucleotide (5′-GCCAGGTCCTGTATGAGCAAGGGCGAGG-3′) and a reverse oligonucleotide (5′-GGCGCTAAGC TCACTTGTACAGCTCG-3′), which included PpuMI and BlpI restriction endonuclease sites, respectively (bold nucleotides). The GFP initiation and stop codons are underlined. The GFP gene was cloned at the PpuMI and BlpI sites of plasmid pSL-SC11-3EMN7C8-BGH, replacing the dispensable TGEV ORFs 3a and 3b. Transfer of the 2,752-bp AvrII-AvrII fragment including the GFP gene into the corresponding sites of plasmid pBAC-TGEV ΔCla I and subsequent insertion of the ClaI-ClaI fragment led to plasmid pBAC-TGEV-Δ3-TRS3a-GFP, encoding the GFP gene downstream of the transcription-regulatory sequences of ORF 3a.
The transcription efficacy of an engineered TRS including the 5′ TRS from the N gene (TRSn) at the position of the deleted ORFs 3a and 3b was also analyzed. The TRSn preceding the GFP gene was synthesized by overlap extension PCR. A forward oligonucleotide (5′-GCACTTGGTGGAGGCGCCGTGGCTATACCTTTTGC-3′) including the restriction site NarI (bold nucleotides) and a reverse oligonucleotide (5′-CCGAGGACCT GTTTAG TTATACCATATGTAATAATAATTTTAAATTTAATGGACGTGCACTTTTTCAATTGG-3′) containing the restriction site PpuMI (bold nucleotides), the core sequence (underlined nucleotides), and 22 nucleotides from the 5′-flanking sequences of the N gene (italic nucleotides) were used to amplify a PCR product comprised of nucleotides 23443 to 24712 of the TGEV genome. The GFP gene was independently PCR amplified from plasmid pGL-1 with a forward oligonucleotide (5′-GGCAGGTCCTGCCATGAGCAAGGGCGAGGAAC-3′) and a reverse oligonucleotide (5′-GGCGCTAAGC TCACTTGTACAGCTCG-3′) including restriction sites for PpuMI and BlpI, respectively (bold nucleotides). The GFP initiation and stop codons are underlined.
Both PCR products were gel purified and used as templates for a third PCR, performed with the two outer oligonucleotides as primers. The final PCR product was digested with restriction endonucleases NarI and BlpI and cloned into the corresponding sites of plasmid pSL-SC11-3EMN7C8-BGH. Transfer of the AvrII-AvrII fragment to the corresponding sites of plasmid pBAC-TGEV ΔCla I and subsequent insertion of the ClaI-ClaI fragment, as described above, led to plasmid pBAC-TGEV-Δ3-TRSn-GFP, encoding the GFP gene downstream of the N gene 5′ TRS at the position of deleted ORFs 3a and 3b.
The effect on transcription of the insertion site within the TGEV genome was analyzed by introducing an expression cassette containing the 5′ TRS from the N gene and the GFP gene either at the position of deleted ORFs 3a and 3b (see above) or between the N and 7 genes. The TRSn preceding the GFP gene was synthesized by PCR-directed mutagenesis with the plasmid pSL-SC11-Δ3EMN-AscI-7C8-BGH, including a unique AscI restriction site separating the N and 7 genes, as the template. The forward oligonucleotide (5′-CAGAGCAAGATGTGGTACCTGATGC-3′) including the KpnI restriction site (bold nucleotides) and the reverse oligonucleotide (5′-GCCTTGGCGCGCC GTTTAG TTATACCATATGTAATAATTTTTTAGTTCGTTACCTCATCAATTATCTCAACCTGTGT-3′) containing the AscI restriction site (bold nucleotides), the core sequence (underlined nucleotides), and 22 nucleotides from the 5′-flanking sequences of the N gene (italic nucleotides) were used to amplify a PCR product comprised of nucleotides 27971 to 28028 of the TGEV genome. The PCR product was digested with restriction endonucleases KpnI and AscI and cloned into the corresponding sites of plasmid pSL-SC11-Δ3EMN-AscI-7C8-BGH, leading to pSL-SC11-Δ3EMN-TRSn-AscI-7C8-BGH.
The GFP gene was amplified from plasmid pGL-1 with a forward oligonucleotide (5′-TTGGCGCGCC ATGAGCAAGGGCGAG-3′) and a reverse oligonucleotide (5′-TTGGCGCGCC TCACTTGTACAGCTCG-3′) including the AscI restriction site (bold nucleotides). The GFP initiation and stop codons are underlined. The PCR product was digested with restriction endonuclease AscI and cloned into the corresponding site of plasmid pSL-SC11-Δ3EMN-TRSn-AscI-7C8-BGH, leading to pSL-SC11-Δ3EMN-TRSn-GFP-7C8-BGH. Transfer of the 5,018-bp NarI-BamHI fragment to the corresponding sites of plasmid pBAC-TGEV ΔCla I and subsequent insertion of the ClaI-ClaI fragment, as described above, led to plasmid pBAC-TGEV-Δ3-N-TRSn-GFP-7, encoding the GFP gene downstream of the 5′ TRS from the N gene inserted between the N and 7 genes. To ensure that the expected plasmids were generated, the constructs were sequenced at the cloning junctions with an Applied Biosystems 373 DNA sequencer.
Transfection and recovery of infectious TGEV from cDNA clones.
BHK-APN cells were grown to 60% confluence in 35-mm-diameter plates and transfected with 10 μg of either pBAC-TGEV-Δ3, pBAC-TGEV-Δ3-TRS3a-GFP, pBAC-TGEV-Δ3-TRSn-GFP, or pBAC-TGEV-Δ3-N-TRSn-GFP-7 plasmid and 15 μg of Lipofectin (Life Technologies, Gibco) according to the manufacturer's specifications. The efficiency of this transfection system was 8%. Cells were incubated at 37°C for 6 h, and then the transfection medium was replaced with fresh Dulbecco's modified Eagle's medium containing 10% (vol/vol) fetal bovine serum. After an incubation period of 2 days, the cell supernatants (referred to as passage 0) were harvested and passaged four times on fresh swine testis cell monolayers. Viruses present in the cell supernatant were quantified by plaque titration. After four passages, viruses were cloned by three plaque purification steps. Unstable recombinant TGEV (rTGEV) viruses were cloned from passage 0 supernatant.
RNA analysis by Northern blotting.
Total intracellular RNA was extracted at 16 h postinfection from virus-infected swine testis cells with the Ultraspec RNA isolation system (Biotecx) according to the manufacturer's instructions. RNAs were separated in denaturing 1% agarose-2.2 M formaldehyde gels and blotted onto nylon membranes (Duralon-UV; Stratagene) as described before (
3). Northern hybridizations were performed with hybridization buffer containing [α-
32P]dATP-labeled probe synthesized with a random-priming procedure (Strip-EZpec DNA; Ambion) according to the manufacturer's instructions. The 3′ untranslated region-specific single-stranded DNA probe was complementary to nucleotides 28300 to 28544 of the TGEV strain PUR46-MAD genome (
40). After hybridization, RNA was analyzed with a Personal FX molecular imager (Bio-Rad).
RT-PCR.
Detection of GFP and 7 sgmRNAs was performed by reverse transcription (RT)-PCR. Total intracellular RNA was extracted (see above) at 16 h postinfection from swine testis cells infected with rTGEV viruses. cDNAs were synthesized at 42°C for 1 h with Moloney murine leukemia virus reverse transcriptase (Ambion) and antisense primers GFP2 (5′-GGCGCTAAGCTCACTTGTACAGCTCG-3′), complementary to nucleotides 702 to 717 of the GFP gene; GFP-AscI (5′-TTGGCGCGCCTTACTTGTACAGCTCG-3′), complementary to nucleotides 705 to 720 of the GFP gene; or X3-136 (5′-TCTGGTTTCTGCTAAACTCC-3′), complementary to nucleotides 136 to 155 of gene 7. The cDNAs generated were used as templates for sgmRNA-specific PCRs (leader-body PCR). A virus sense primer, leader 15+ (5′-GTGAGTGTAGCGTGGCTATATCTCTTC-3′), complementary to nucleotides 15 to 41 of the TGEV leader sequence, and the reverse-sense primers described for the RT reaction were used for the PCR.
Optimization of RT-PCRs for semiquantitative analysis was carried out by normalization to the viral genomic RNA (gRNA) in each sample and the use of serial dilutions of the cDNAs. To determine the relative amount of gRNA, the forward oligonucleotide Orf1a 4310 (5′-CTTTTATCAGGGTGCTTTGG-3′) and the reverse oligonucleotide Orf1a 4829 (5′-AACAGACACACGTTCATGG-3′), complementary to TGEV nucleotides 4310 to 4329 and 4811 to 4829, respectively, were used. PCR was performed with a GeneAmp PCR system 9600 thermocycler (Perkin-Elmer) for 35 cycles. Each cycle comprised 30 s of denaturation at 94°C, 45 s of annealing at 53°C, and 1.5 min of extension at 72°C. The 35 cycles were followed by a 10-min incubation at 72°C. RT-PCR products were separated by electrophoresis in 0.8% agarose gels, purified, and used for direct sequencing with the oligonucleotide leader 15+ and the same reverse oligonucleotide used for PCR.
Western blot analysis.
Protein expression in cells infected with rTGEV viruses was analyzed at different passages by Western blot. Cell lysates were separated by gradient sodium dodecyl sulfate-polyacrylamide (5 to 20%) gel electrophoresis (SDS-PAGE). Proteins were transferred to a nitrocellulose membrane with a Bio-Rad Mini Protean II electroblotting apparatus at 150 mA for 2 h in 25 mM Tris-192 mM glycine buffer (pH 8.3) containing 20% methanol. Membranes were blocked for 1 h with 5% dried skimmed milk in TBS (20 mM Tris-HCl [pH 7.5], 150 mM NaCl). The membranes were then incubated with monoclonal antibodies specific for the GFP protein (Roche) or the TGEV N protein (3D.C10), diluted 1:1,000 in TTBS buffer (TBS with 0.1% Tween 20). Bound antibody was detected with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin diluted 1:3,000 in TTBS buffer and the ECL detection system (Amersham Pharmacia Biotech). The amount of GFP protein expressed was determined by Western blot, with standard calibration curves generated by using purified GFP (Roche). TGEV N protein was used as an internal standard.
Flow cytometry analysis.
Swine testis cells grown in 35-mm-diameter dishes were infected with the recombinant virus rTGEV-Δ3-TRS3a-GFP, rTGEV-Δ3-TRSn-GFP, or rTGEV-Δ3-N-TRSn-GFP (multiplicity of infection, 1). At 16 h postinfection, cells were washed and resuspended in 500 μl of phosphate-buffered saline. GFP expression in mock-infected and infected cells was analyzed on a flow cytometry system (Epics XL-MCL; Coulter).
Virulence assay.
The in vivo growth and virulence of TGEV recombinant viruses were determined as described before (
42). Briefly, groups of 14 2- to 4-day-old swine obtained by crossing Large White and Belgium Landrace pigs were oronasally and intragastrically inoculated with 10
8 PFU per route of rPUR-MAD-SC11, rTGEV-Δ3, or rTGEV-Δ3-TRS3a-GFP virus in a biosafety level 3 containment facility. The virus titers in lung, jejunum, and ileum were determined 2 days after infection.
Immunization of swine.
Two pregnant sows seronegative for TGEV, as tested by enzyme-linked immunosorbent assay (ELISA), were immunized with the recombinant virus rTGEV-Δ3-TRS3a-GFP by the nasal and intramuscular routes (108 PFU in 5 ml of Dulbecco's modified Eagle's medium per route) at days 35 and 75 of gestation. Serum from the sows and their progeny (nine and seven piglets) was collected at days 2, 7, 19, and 27 after delivery. Colostrum was collected from the sows on the day of delivery. The antibody response against TGEV and GFP was evaluated by ELISA in serum and colostrum.
ELISA.
Antibodies generated against GFP and TGEV were detected by ELISA as described before (
41). ELISA was performed with purified TGEV (0.2 μg per well) or GFP protein (75 ng per well) as the antigen, bound to 96-well microplates, saturated with 5% bovine serum albumin in phosphate-buffered saline for 2 h at 37°C, and incubated with serial dilutions of the serum sample in phosphate-buffered saline-0.1% bovine serum albumin for 3 h at room temperature. Microplates were washed six times with 0.1% bovine serum albumin and 0.1% Tween 20 in phosphate-buffered saline, and bound antibodies were detected by incubation with peroxidase-conjugated protein A diluted 1:2,000 in phosphate-buffered saline with 0.1% bovine serum albumin. ELISAs were developed with phenylenediamine dihydrochloride (Sigma FAST) as the peroxidase substrate for 15 min at room temperature. Reactions were stopped with 1.5 M H
2SO
4, and the absorbance was read at 492 nm.
DISCUSSION
With a TGEV infectious cDNA clone, we have shown the engineering of a TGEV-derived single genome vector that stably expresses high levels of the heterologous gene GFP from the ORF 3a TRS. GFP expression with a recombinant TGEV has been reported (
9). In this paper we have made relevant progress over this initial observation, with a coronavirus-derived vector engineered as a bacterial artificial chromosome, by showing that (i) the TGEV single genome led to high expression levels of the heterologous protein (40 μg/10
6 cells), (ii) the virus vector stability is very high, (iii) vector tropism and virulence are present in swine, as is the induction of a lactogenic immune response, (iv) different TRSs and insertion sites have different efficacies, and (v) transcription may take place from TRSs with a noncanonical core sequence, demonstrating that complementarity between core sequence-flanking sequences and the leader TRS can compensate for the absence of a canonical core sequence.
TGEV ORFs 3a and 3b are nonessential for virus growth (
9,
16,
27,
34,
53). Therefore, it was tempting to assume that insertion of heterologous genes into the TGEV genome replacing these genes would not affect the behavior of the virus either in cell culture or in vivo. In fact, we have shown in this paper that rTGEV-Δ3 virus kept the infectivity, replication efficiency, and tropism of the wild-type virus with a very small reduction in its virulence, demonstrating that these properties were influenced very little by genes 3a and 3b. Expression levels obtained with this TGEV-derived vector are of the same order as those described for vectors derived from other positive-strand RNA viruses such as Sindbis virus (50 μg/10
6 cells) (
1,
18) or DNA expression systems such as adenovirus type 5 (10 to 100 μg/10
6 cells) (
31,
36).
With this virus vector, a good antibody response specific for viral antigens and for the heterologous GFP protein has been demonstrated in the serum of immunized animals. Interestingly, the TGEV-derived vector elicited lactogenic immunity against the virus and the GFP by the production of specific antibodies in the colostrum of sows and the transfer of maternal antibodies to the piglets. The limited antibody response against the heterologous GFP protein compared with the high titers obtained against the TGEV vector is probably due to the known low immunogenicity of GFP (
48,
49). The induction of lactogenic immunity indicates that TGEV-derived vectors are very promising for the development of vaccines to protect against mucosal infections. In fact, other antigens relevant in protection against viral infections, such as the porcine respiratory and reproductive syndrome virus ORF 5, have been efficiently expressed with a virus vector and elicited partial protection against porcine respiratory and reproductive syndrome virus-induced disease (S. Alonso, I. Sola, S. Zuñiga, J. Plana-Durán, and L. Enjuanes, submitted for publication).
A correlation has been observed between the proximity to the 3′ end of the genome and the relative efficiency of sgRNA synthesis from a given TRS in several viral systems, including coronaviruses, such as MHV and TGEV (
3,
20,
47), and the
Mononegavirales (
22,
50). In order to increase heterologous gene expression levels, an expression cassette was inserted at the 3′ end of the genome. Unfortunately, insertion of the expression cassette between TGEV genes N and 7 in the recombinant virus rTGEV-Δ3-N-TRS
n-GFP-7 resulted in an unstable virus, leading to complete deletion of the additional transcriptional unit TRS
n-GFP. Since we have shown above that TGEV ORFs 3a and 3b can be stably replaced by the GFP gene, the location of the insertion, not the nature of the gene, was most likely responsible for the instability. The instability of expression cassettes inserted at the 3′ end of the genome, between genes N and 7, seems a more general phenomenon, since, in addition to the results presented in this paper with the GFP gene, we have shown (S. Alonso, I. Sola, S. Zuñiga, J. Plana-Durán, and L. Enjuanes, submitted for publication) that several expression cassettes with the reporter gene β-glucuronidase were also unstable at this position of the genome but not at the ORF 3a site. Furthermore, in another coronavirus (MHV), insertion of other sequence fragments (i.e., 3′-end 141 nucleotides of the N gene or 717 nucleotides of GFP) between the N gene and the 3′ untranslated region also produced genomic instability (
21).
In some TGEV recombinant viruses, such as clone 12 of TGEV-Δ3-N-TRS
n-GFP-7, the origin of the instability was the homologous recombination promoted by the presence of duplicated viral sequences, while in other viruses (clone 3 of TGEV-Δ3-N-TRS
n-GFP-7) the instability was due to nonhomologous recombination yielding a virus that had lost the GFP gene and also the 5′ end of gene 7. Therefore, in addition to similarity-essential recombination, similarity-nonessential recombination (
37) may also lead to instability in these viruses. The insertion of pBAC sequences in the viral genome most likely occurred during the passages in bacteria. Possibly, this process was irrelevant for the mammalian system that we are interested in and not related to the virus life cycle except to confirm the flexibility of the virus in accepting unrelated sequences in this region.
A novel transcriptional unit with a noncanonical core sequence (5′-CUAAAA-3′) that led to a major unexpected sgmRNA species was found downstream of the N gene. Along the TGEV wild-type genome, eight noncanonical core sequences (5′-CUAAAA-3′) identical to the novel transcriptional motif that did not drive transcription of detectable sgmRNAs were found. Interestingly, the noncanonical core sequence active in transcription was the only one with the 5′-flanking sequence 5′-CGAA-3′ that increased the complementarity with the leader sequence, suggesting that the extended leader-body homology upstream of the core sequence compensates for the incomplete complementarity between the leader and the core sequence and determines which core sequence-like sequence leads to RNA transcription.
Junction of the leader to another noncanonical core sequence (5′-CUAAAGA-3′) located at the 3′ end of the GFP gene, upstream of a sequence providing extended complementarity to the leader RNA, was observed during infection with rTGEV-Δ3-TRS3a-GFP. Comparison between anomalous sgmRNAs generated from the GFP gene in the TGEV vector and those documented in the coronavirus MHV (
17) and the arterivirus equine arthritis virus (
10) showed no coincidence at the junction sites except for their location at the 3′ half of the gene. In MHV, anomalous junction sites within the GFP gene are explained by long-range RNA or ribonucleoprotein interactions independent of the core sequence-like motif, whereas in equine arthritis virus they are attributed to the presence of core sequence-like sequences.
In TGEV, in addition to the presence of a core sequence-like sequence (5′-CUAAAG-3′) just upstream of the junction site, we observed the presence of potential extra leader-body base pairing immediately downstream of the leader core sequence. Therefore, we believe that this extension of the complementarity between the leader TRS and the sequence complementary to the TRS that precedes each gene is an important determinant for sgRNA synthesis. The effect of sequences flanking the core sequence in a TRS is in agreement with previous results with MHV (
5,
21,
23,
25,
28,
29,
46,
47) showing that flanking sequences may contribute through base pairing in similarity-assisted homologous recombination that leads to the production of coronavirus mRNAs (
6).
The occurrence of sgmRNA heterogeneity in the region of the leader-body junction site, leading to the detection of minor sgmRNA species synthesized from noncanonical core sequences during infection, has been described for the coronavirus MHV (
17,
30,
55) and in arteriviruses (
12,
35). Transcription in TGEV apparently behaved with extraordinary accuracy, since no heterogeneity has been detected in the production of sgmRNAs (
40). Furthermore, no mRNA was detected even from ORF 3b in viral strains with a mutated core sequence (5′-CUAAAU-3′) (
3,
51). In contrast, we report in this paper the synthesis of abundant novel sgmRNA species from a noncanonical core sequence as a response to modification of the virus genomes. The existence of alternatives to the canonical core sequence might be an evolutionary resource to maintain virus viability.
Acknowledgments
We thank F. Almazán and J. Ortego for critically reading the manuscript.
This work was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (CICYT), La Consejería de Educación y Cultura de la Comunidad de Madrid, Fort Dodge Veterinaria, and the European Communities (Frame V, Key Action 2, Control of Infectious Disease Projects). I.S., S.A., and S.Z. received postdoctoral fellowships from the Community of Madrid and the European Union (Frame V, Key Action 2, Control of Infectious Disease Projects QLRT-1999-00002, QLRT-1999-30739, and QLRT-2000-00874).