Enterovirus Laboratory, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland1
Transplantation Laboratory, Haartman Institute and Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland2
Author for correspondence: Merja Roivainen. Fax +358 9 4744 8406. e-mail merja.roivainen{at}ktl.fi
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Abstract |
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Introduction |
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In the present study, we investigated the kinetics and consequences of EV replication in foetal porcine -cells in order to see whether these cells could be used instead of primary human
-cells for screening human EV strains and isolates for
-cell tropism.
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Methods |
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Viruses.
Prototype strains of CVBs (CVB-1 to -6), echoviruses (E-1, -7 and -11), some CVAs (CVA-9/Griggs and CVA-16), human parechovirus 1 (HPEV-1, previously known as E-22) and poliovirus type 1/Mahoney (PV-1) were obtained either from the ATCC or from the World Health Organization Enterovirus reference laboratory. The diabetes-associated CVB-4 strain E2 was kindly provided by J.-W. Yoon (Yoon et al., 1979 ). Viruses were passaged in continuous cell lines of monkey kidney (GMK or Vero) or human lung carcinoma (A-549) origin. The identity of all virus preparations used in this study was confirmed using a plaque neutralization assay with type-specific antisera.
Replication of viruses.
Free-floating islets were infected with different virus preparations at an apparent high m.o.i. of between 30 and 100 (if not otherwise stated). After adsorption for 1 h at 36 °C, virus inoculum was removed. Cells were then washed twice with Hanks balanced salt solution supplemented with 20 mmol/l HEPES, pH 7·4. Incubation medium was added to all cultures, including uninfected controls, and the virus was allowed to replicate at 36 °C. Samples of suspended islets taken at different time-points were freezethawed three times to release virus, clarified by low-speed centrifugation and assayed for total infectivity using end-point dilutions in microwell cultures of GMK, Vero (CVA-16) or A-549 (HPEV-1) cells. TCID50 titres were calculated by the Kärber formula (Lennette, 1969 ) and expressed as the mean of two parallel series of dilutions.
Immunocytochemistry.
Samples of infected and uninfected islets were harvested 1 day after infection on glass slides using a cytocentrifuge and fixed with cold methanol for 15 min at -20 °C. After washing three times with PBS, cells were double-stained overnight at room temperature with EV-specific polyclonal rabbit antiserum (1:300, KTL-510; Hovi & Roivainen, 1993 ) and insulin-specific polyclonal sheep antiserum (30 µg/ml, PC059; The Binding Site). Visualization was achieved by FITC (711-095-152; Jackson ImmunoResearch Laboratory) and lissamine rhodamine sulfonyl chloride (LRSC) (713-085-147; Jackson ImmunoResearch Laboratory) conjugated anti-species sera. Photographs were taken using a confocal microscope (Leica TCS NT) and Paint Shop Pro software.
DNA and insulin content of cells.
Islet cells were ultrasonically homogenized in distilled water. DNA was measured from dried samples using a fluorometric method based on diaminobenzoic acid-induced fluorescence (Hinegardner, 1971 ). Insulin was measured with a commercial Solid-Phase Insulin RIA kit (DPC) after overnight extraction with acid ethanol, as described previously (Otonkoski et al., 1993
).
Cell viability.
After infection, the viability of islet cells was measured using a commercial live/dead cell assay kit (L-3224; Molecular Probes). This assay is based on the simultaneous determination of live and dead cells with two fluorescent probes. Live cells are stained green with calcein due to their esterase activity and the nuclei of dead cells are stained red with ethidium homodimer-1. Islets harvested at different time-points were incubated with the labelling solution for 30 min at room temperature in the dark, according to the manufacturers instructions, cytocentrifuged onto glass slides and analysed using confocal microscopy.
Insulin secretion.
Insulin release in response to glucose and glucose plus theophylline was studied separately by perifusion, as described previously (Otonkoski & Hayek, 1995 ; Roivainen et al., 2000
). Briefly, after taking samples for insulin and DNA content measurement, islets were loaded into perifusion chambers in KrebsRinger bicarbonate buffer supplemented with 20 mmol/l HEPES (pH 7·35) and 0·2% BSA. After a 60 min stabilization period in low glucose (1·67 mmol/l), cells were stimulated first with high glucose (16·7 mmol/l) and then with a mixture of 16·7 mmol/l glucose and 10 mmol/l theophylline (Sigma). Fractions of 1 ml were collected every 4 min. After stimulation, the basal buffer (1·67 mmol/l glucose) was used for the final five fractions. Five or six perifusion lines were run in parallel using a multi-channel perifusion apparatus (Brandel).
Statistical methods.
Differences between groups were tested with Microsoft Excel 97 software using one-way analysis of variance and taking 95% as the limit of significance.
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Results |
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The insulin response of islets infected with a genetically related virus, CVA-9, was usually enhanced rather than decreased 1 week after infection (Fig. 4B). Not surprisingly, a large variation in the time scale and degree of CVA-9-induced disturbance was seen in different islet cell preparations (Fig. 6
). At 3 weeks after infection, a clear CVA-9-induced disturbance in stimulated insulin release was seen in two of four experiments [stimulation index (SI) 33% or less of uninfected controls]. For all four experiments, the mean stimulated insulin release (SI±SD) of uninfected and CVA-9-infected islets at this time-point was 6·14±2·95 and 3·80±2·69, respectively.
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Titre of cytolytic virus needed for -cell damage
Some of the differences found between the various viruses might be based on the different virus titres used for islet cell infections. To determine how strongly the infection-induced consequences are dependent on the kinetics of virus replication, virus-induced cell damage was studied in detail after infecting foetal porcine islets with decreasing concentrations of CBV-1. As seen in Fig. 7(A), the infectivity of the virus inocula used correlated well with the eclipse phase titres found in the islets after virus adsorption. In spite of this, practically no differences were seen in virus replication in islets infected with our normal amount (1x108 TCID50) of virus or 4-, 10- or even 40-fold less (corresponding to 2·6x107, 8·3x106 or 2·6x106 TCID50, respectively) (Fig. 7A
). By using lower titres of virus (1·2x10655 TCID50), progeny virus production was gradually delayed, but even in this case, the maximal titre of progeny virus was produced within 2 days (Fig. 7B
). Islets infected with less than 55 TCID50 of CBV-1 did not produce measurable amounts of infectious virus within 3 days (data not shown).
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Discussion |
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In this study, virus replication was documented by measuring the infectivity of progeny virus in samples taken at different time-points. However, this might not be the most sensitive and accurate method to use when only marginal titres of progeny are produced. This is especially true for viruses that are sensitive to heat-induced inactivation. For example, infectivity of CVA-9, unlike that of PV, is clearly decreased after overnight incubation at 37 °C (Piirainen et al., 1998 ).
In addition to CVBs and CVA-9, several other strains of EVs were included in this study. PV-1, which is known to replicate only in cells of primate origin, was used as a negative control. No replication was seen with CVA-16, E-1, -7 and -11, or HPEV-1 in any porcine islet preparation, and, thus, they were not included in further functional analysis. Of course, we cannot exclude the possibility of marginal progeny production in the case of some viruses because no method other than regular progeny titration was used. In theory, replication of echoviruses could have been inhibited by the BSA (1%) used in our incubation medium after virus infection (Ward et al., 1999 ). However, this explanation is not plausible because the same echoviruses replicated well in primary human islets in the presence of 1% BSA (unpublished data). In conclusion, there seem to be species-specific differences in EV permissibility between human and porcine pancreatic islets.
Through our previous studies, we have used high multiplicity infections in order to confirm that all susceptible cells are infected at the beginning of the experiment. In this way, the virus-induced consequences of infection could be followed reliably, but it is not possible to study the kinetics of these effects. After extending our studies to several different serotypes, some of them more difficult to propagate at high titres than others, such a high multiplicity was not always achieved. Some of the observed differences in virus-induced consequences might have been based on differences in the infectivity of the virus inoculum used. To exclude this possibility, the kinetics of virus replication and virus-induced cell damage were studied in detail. We found that the amount of virus needed for -cell destruction was small. Even 55 infectious virus particles were enough to destroy all
-cells of one pancreas in vitro.
Assuming that EVs are indeed involved in the pathogenesis of IDDM, one of the most obvious questions would be whether all EVs are diabetogenic or whether certain serotypes or strains are particularly diabetogenic. It is possible that in a genetically susceptible individual, several different serotypes of EV, or perhaps even all EVs, could be diabetogenic. The large number of different EV serotypes makes the identification of the most pathogenic serotypes and/or strains both important and tedious. The screening process could be simplified significantly by standardized experiments with primary human islet cultures. Unfortunately, human islets are not easily available. Previous studies with isolated pancreatic islets have revealed that porcine -cells share important properties with human
-cells (Tuch & Bai, 1998
).
The aim of this study was to evaluate whether foetal porcine -cells could be used instead of the scarcely available human
-cells for monitoring the diabetogenic properties of human EVs. The responses to CV infection were quite similar in the two species, both differing clearly from previously reported rodent
-cells. The function of porcine
-cells was affected by CVBs even more severely than human
-cells. CVA-9 did not replicate in porcine islets as readily as in human islets, but when it did replicate, a clear functional destruction of
-cells was evident after prolonged incubation, suggesting that foetal porcine islets are highly sensitive to this virus-induced damage as well. Furthermore, porcine foetal
-cells, unlike human
-cells (unpublished data), were unable to support the replication of other tested EVs. Thus, species-specific differences do exist and systematic screening of various EVs for
-cell tropism should be carried out in primary human
-cells. Foetal porcine
-cells could be used in studies that address details of the interaction between CVs and
-cells.
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Acknowledgments |
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References |
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Received 6 April 2001;
accepted 23 April 2001.