1 Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
2 Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid, Spain
Correspondence
Esteban Domingo
edomingo{at}cbm.uam.es
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ABSTRACT |
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Present address: Department of Microbiology and Immunology, University of California San Francisco, 600 16th Street, San Francisco, CA 94143-2280, USA.
Present address: UCLA AIDS Institute, Microbiology, Immunology and Molecular Genetics Department, 10833 Le Conte Avenue 11-934A Factor Building, Los Angeles, CA 90095, USA.
Present address: Institut National de la Recherche Agronomique, UMR 1225, Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des Capelles, 31076 Toulouse Cedex 3, France.
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INTRODUCTION |
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It was unexpected that an expansion of host-cell tropism, rather than a more specialized use of a single cell type, occurred as a result of replication in the specific environment provided by BHK-21 cells. This observation, along with the extensive distribution of FMDV in nature and the frequent contact of humans with infected animals, prompted us to examine in greater detail the extent of the expansion of cell tropism of these variant FMDVs highly adapted to BHK-21 cells, with regard to infection of human and primate cell lines. Here we show that, as a result of extensive passage in BHK-21 cells, FMDV acquired the capacity to productively infect several human and primate cell lines. Studies with chimeric viruses have identified the virus capsid as an important region for infectivity in human cells. The results have implications for understanding of the expansion of the host range of viruses.
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METHODS |
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FMDV C-S8c1 is a plaque-purified derivative of natural isolate C1 Santa Pau-Spain 70 (Sobrino et al., 1983). FMDV C-S8c1p100c10 is a plaque-purified clone derived from a population obtained after 100 serial cytolytic passages of C-S8c1 in BHK-21 cells (C-S8c1p100), described by Martínez et al. (1997)
(Fig. 1
). FMDV RGG is a monoclonal antibody (mAb)-resistant mutant derived from C-S8c1p100, with an Asp-143
Gly change at the RGD motif of VP1 (Martínez et al., 1997
). This is the only amino acid difference between the FMDV C-S8c1p100c10 and FMDV RGG capsids. The FMDV C-S8c1 population at passage 213, termed C-S8c1p213, was used to select MARLS, a mAb-resistant mutant which includes substitution Leu-144
Ser in VP1 (Charpentier et al., 1996
; Mateu et al., 1990
). These closely related variants belong to the same evolutionary lineage derived from C-S8c1, and were chosen because they share a prolonged history of serial passages in BHK-21 cells (Fig. 1
).
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Infections with FMDV.
Procedures for infections of BHK-21 cell monolayers and plaque assays with FMDV have been described previously (Domingo et al., 1980; Sobrino et al., 1983
). The number of cells and m.o.i. are indicated for each experiment. Virus was allowed to adsorb at 37 °C for 1 h (BHK-21 cells) or 1 h 15 min (HeLa, LLC-MK2 and CHO cells); then monolayers were washed once with 0·1 M phosphate buffer (pH 6·0) to inactivate unadsorbed virions, twice with DMEM, and further incubated in 2 ml DMEM/2 % FCS. At different times after infection, samples were taken for titration of infectivity on BHK-21 cell monolayers as described previously (Sobrino et al., 1983
). For K-562 and Jurkat cells growing in suspension, virus adsorption was performed in 200 µl culture medium (2x106 cells) at m.o.i. 210 p.f.u. per cell, with gentle rocking at 37 °C for 1 h 15 min. Cells were washed with 0·1 M phosphate buffer (pH 6·0) and DMEM prior to further incubation in 2 ml culture medium. Samples of culture medium were taken for titration of infectivity on BHK-21 cell monolayers, as described previously (Sobrino et al., 1983
).
Immunofluorescence assays.
HeLa cells were grown on coverslips and infected at m.o.i. 1 p.f.u. per cell. Jurkat cells were grown in suspension and infected at m.o.i. 1 p.f.u. per cell, then added to coverslips that had previously been treated with 1 mg ml1 polyLys (Sigma). Cells were fixed with 4 % paraformaldehyde (Merck) at different times after infection. Fixation was blocked with 10 mM glycine (pH 8·5) in PBS, and membranes were permeabilized with 0·2 % Triton X-100 in PBS. Immunofluorescence testing was performed using a 1 : 500 dilution of mAb 3B2, which recognizes FMDV non-structural protein 3A, as the primary antibody. The secondary antibody was Alexas 488 anti-mouse (Molecular Probes), used at a 1 : 500 dilution. The percentage of cells positive for 3A expression was calculated after counting cells from four microscope fields.
cDNA synthesis, PCR amplification, nucleotide sequencing and RNA quantification.
Viral RNA extraction and RT-PCR amplification were performed as described previously (Escarmís et al., 1996). Consensus nucleotide sequences were determined on PCR-amplified DNA in an automated sequencer (ABI Prism 3730). The oligonucleotides used for RT-PCR and nucleotide sequencing have been described previously (Baranowski et al., 1998
). RNA quantification was performed with a LightCycler Instrument (Roche) using the LightCycler-RNA Master SYBR Green I kit (Roche), which allows a one-step RT-PCR amplification with Tth polymerase. A standard calibration curve and the minimal amounts of FMDV RNA that can be reliably quantified were determined for each experiment and are indicated in the corresponding figures. The genomic region amplified for quantification was the VP1-coding region, and the primers used were 5'-GAGCTCCGGCTACCTGTGGA-3' (sense, 5' position 3194) and 5'-GGATTGGTTGTGTTGTTAAGTGC-3' (antisense, 5' position 3518).
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RESULTS |
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DISCUSSION |
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Infection of established human cell lines does not imply that the corresponding related epithelial or lymphoid cells in humans will be permissive to the same viruses. Even if they were infected, the virus may be only mildly pathogenic due to barriers preventing completion of the infectious cycle. The same issues have been amply discussed in connection with transmission of swine viruses to humans associated with xenotransplantation (Matthews, 2001). Even if some natural variants could replicate in human cells, they are likely to remain as a minority in the mutant spectra of infected animals, and humans may never come in contact with them. Despite these barriers, the adaptive potential of FMDV and the results presented here encourage avoidance of human exposure to the virus. As additional evidence of adaptability to cross host and cell barriers, FMDV mutants with amino acid substitutions at or around the RGD in VP1, showing modifications of cell tropism, were selected in cattle that had been immunized only partially with experimental peptidic vaccines (Taboga et al., 1997
; Tami et al., 2003
). Host-range alterations are not associated exclusively with structural FMDV proteins: a single amino acid substitution in non-structural protein 3A mediated adaptation of an FDMV from swine to guinea-pig (Núñez et al., 2001
). Mutant FMDVs have been described with atypical pathogenic manifestations such as myocarditis, pancreatitis, diabetes or neurological symptoms, selected in animals infected in the laboratory, although the underlying molecular basis has not been investigated (for reviews, see Domingo et al., 1990
; Mason et al., 2003
). Perhaps the most dramatic demonstration of the adaptive potential of FMDV at the epidemiological level has been the recent expansion of the strains referred to as FMDV O PanAsia to three continents in less than a decade, displacing previously dominant FMDVs (Rowlands, 2003
; Sobrino & Domingo, 2004
). Our results of expansion of the host-cell tropism of FMDV to human cells, associated with amino acid substitutions in the virus capsid, provide further justification for considering genetic variation as a major factor in microbial disease emergence (Smolinski et al., 2003
).
In the different replicas of variant FMDVs used in parallel infections of Jurkat cells (105106 p.f.u. MARLS in each passage; Fig. 4), the repertoire of variants found in parallel mutant spectra is unlikely to be identical. This may contribute to stochastic effects in the capacity for adaptation of a small population of a genetically heterogeneous virus to a given cell type or host. Interestingly, MARLS, the variant with the most prolonged passage history in BHK-21 cells, always produced infection in Jurkat cells. Thus, the more adapted the variant, the broader the tropism. It is possible that in p100c10 and RGG populations a minority of variants could sustain infection in Jurkat cells, but this trait was not yet imposed in the quasispecies. Also noteworthy is the difference between HeLa and Jurkat cells regarding infection by the parental C-S8c1. While there is no evidence of infection of Jurkat cells, HeLa cells produced C-S8c1 progeny as shown by infectivity measurements (Figs 2 and 4
) and a significant increase in extracellular and intracellular virus RNA (Fig. 3a
). These results are explained by a small proportion of HeLa cells that can be infected (Fig. 3b
), but whether infection is supported by minority subsets of susceptible HeLa cells, by rare C-S8c1 variants, or by other influences is not known. These possibilities deserve further investigation as the initial infection did not proceed and the virus became spontaneously extinct.
From the standpoint of RNA virus evolution, it was most remarkable that the expansion of host-cell tropism occurred as a result of replication in the constant biological environment provided by cultured BHK-21 cells, and the longer the replication in the latter cells, the broader the expansion of host-cell tropism (Fig. 1). Extended replication in the same cells would be expected to render a virus showing an increased specialization to enter the same cell type. In contrast, alterations in the environment would be expected to produce disequilibria in the mutant spectra of virus quasispecies, and to expand the variant repertoire for exploration of new phenotypes (Domingo et al., 1990
). Therefore, our results indicate that, contrary to theoretical predictions, replication of an RNA virus in a constant environment may lead to trait alterations that are highly relevant biologically. One possibility is that BHK-21 cells actually express multiple potential receptors for FMDV, and that prolonged passage permitted selection of viruses able to use multiple entry pathways into BHK-21 cells; some of these routes may also be represented in established human cell lines. More work is needed to elucidate the molecular basis of cell tropism modifications, which are being documented for an increasing number of animal viruses (Baranowski et al., 2003
).
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ACKNOWLEDGEMENTS |
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Received 22 March 2004;
accepted 23 April 2004.