Martin Rose
email: m.rose@csl.gov.uk

Malcolm Baxter

Laurence Castle

Helen M Crews

Graham Lappin

Daniel Leong

Colin Garner

A SENSITIVE METHOD FOR THE DETERMINATION OF CHLORINE -36 IN FOODS USING ACCELERATOR MASS SPECTROMETRY

Keywords: Chromatography, Exposure, ICP/MS, Risk assessment Additives/Contaminants: Environmental contaminants Chlorine-36, accelerator mass spectrometry, foods, analysis

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INTRODUCTION

The radioisotope chlorine-36 ( 36 Cl) might potentially be discharged from waste incinerators on Magnox or AGR (Advance Gas Cooled) nuclear reactor sites. In a reactor, the 36 Cl is produced unintentionally as a result of neutron activation of stable 35 Cl which is present in the materials of the reactor. Nuclear power stations may emit typically up to 1 GBq of 36 Cl per annum from incineration of the power station waste streams. The exact emissions depend of course on the scale, the type and the operating licence of the power plant. 36 Cl is a low energy β-emitter with a half-life of 3 x 10 5 years. It has high mobility in many environmental conditions but also a high uptake into plants. If it is taken-up by food crops or by foods growing wild, then there is the potential for human exposure via the diet. This could include milk from cattle grazing on affected land or wild berries growing nearby. 36 Cl has the potential to give reasonably high doses of radioactivity to consumers if present in appreciable amounts in foods. Radioactivity, like any other chemical hazard present in food, needs to be monitored to ensure that food safety is not compromised and consumers are adequately protected.

Limited and provisional results [FSA 2002;[START_REF]Review of FSA research programmes on radiological protection[END_REF] have suggested that 36 Cl may be present in milk, blackberry and raspberry samples and that there might be higher than expected dietary exposure to this isotope for a susceptible group of the population such as infants. Milk is important because it is a dietary staple. Food surveys have shown that people can collect and eat large quantities of foods from the wild, including blackberries, mushrooms, nuts and elderberries and they may be stored or frozen to eat throughout the rest of the year after seasonal collection. Given the relatively large amounts of these particular foods that might be consumed, there is the potential that even small concentrations of 36 Cl might result in significant doses. However, the initial data [FSA 2002;[START_REF]Review of FSA research programmes on radiological protection[END_REF] were collected using scintillation counting method.

Interpretation of the results was limited by the poor sensitivity of the scintillation counting method that had a detection limit estimated to be about 5 to 10 Bq/kg.

A method has been more recently described for the determination of 36 Cl in nuclear waste from reactor decommissioning [START_REF] Hou | Determination of 36 Cl in nuclear waste from reactor decommissioning[END_REF]. This method uses scintillation 3 counting and although was only described for more intractable material such as steels and graphite, it would translate to a detection limit of below 100 mBq/kg if it were transferred to materials such as milk or blackberries, since it would be able to handle relatively large sample amounts. This would be roughly equivalent to the limit of detection in the method described here using AMS.

Because of the long half-life and therefore the relative infrequency of a radioactive decay event for this isotope, decay counting techniques have poor sensitivity. AMS is a nuclear physics technique that can be up to one million times more sensitive than decay counting methods for long-lived isotopes. The easiest way to view AMS is to regard the instrument as an atom counter, simply separating elemental isotopes on the basis of their mass, charge and momentum. 36 Cl has been measured by AMS in samples including meteorites, radioactive waste and Arctic ice cores [START_REF] Synal | 36 Cl measurements at the Zürich AMS facility[END_REF]. A sensitivity of 3

x 10 -15 was reported by these authors, i.e. three 36 Cl atoms in 10 15 normal chlorine atoms.

The main problem in measuring 36 Cl by AMS is the potential interference by sulfur-36 ( 36 S) which is an isobar of 36 Cl. An accurate measurement of sulfur was therefore required to allow its contribution to the mass-36 channel to be subtracted. In turn, measurement of 32 S, the most abundant isotope of sulfur, had to be resolved from interferences produced from dioxygen, O 2 , which has the same approximate mass.

In the work reported here we have simplified the analysis by reducing the inference from sulfur by analytical chemical separation prior to the AMS analysis. Also, we have used the mass resolution offered on the AMS instrument to separate 36 Cl from any low level of 36 S remaining in the silver chloride (AgCl) target material produced. This project thus aimed to confirm or refute the earlier data [FSA 2002 and[START_REF]Review of FSA research programmes on radiological protection[END_REF] and also to provide a sensitive and specific method for monitoring 36 Cl in the environment and food chain. Although there is no formal limit for 36 Cl in food, an occupational limit has been set by the US Nuclear Regulatory Commission. The annual limit on intake is 2 mCi (74 MBq) for oral ingestion and 200 µCi (7.4 MBq) for inhalation. The limit of 74 MBq per year is 0.2 MBq per day. Assuming a worst case that a person consumes 2 kg of food or beverage each day, a detection limit of 1 Bq/kg food would correspond to an exposure 100,000-times lower than the occupational limit.

With a half-life of 3 x 10 5 years the specific activity of 36 Cl is 1.22 x 10 9 Bq per gram.

One Bq is one disintegration per second. So whilst a level of interest around 1 Bq/kg or lower is clearly beyond scintillation counting methodology, it equates to a concentration of 0.8 ng/kg or about 1 ppt which should be within the sensitivity range of the AMS approach.

EXPERIMENTAL

Materials and methods

Preparation of the samples was carried out using a Colworth Stomacher Laboratory Blender 400 and a Perkin Elmer 'Multiwave' microwave digestion system fitted with 50 ml quartz vessels. Plastic materials, e.g. polypropylene, polyethylene or fluorinated ethylene-propylene were used in all experiments. Concentrated reagents were stored in fluorinated ethylene-propylene bottles. All plastic items were cleaned with dilute detergent, then soaked in dilute acid before a final thorough rinse with deionised water.

All reagents used in this study were of the highest purity commercially available. Subboiled 'Ultrapur' nitric and hydrochloric acids plus 'Aristar' ammonia, sodium chloride and silver nitrate solution were obtained from BDH/Merck, Poole, Dorset UK.

Deionised water (18 MΩ) was produced using a Millipore MilliQ Plus filtration system.

A certified reference 36 Cl standard SRM 4943 was obtained from NIST, Gaithersburg, USA.

Samples

Samples of blackberry (3) and milk (5) were taken from close to two potential sources of 36 Cl located in the UK. One was a Magnox power station and the other was a AGR power station. Control samples of blackberry and milk were obtained locally in the York area.

Preparation of samples

Blackberry samples were homogenises thoroughly using the bender. The blackberry and milk samples were then freeze-dried. Milk gave an average of 12.7% dry mass and blackberries gave an average of 18.6% dry mass.

Sample pre-treatment

Acid digestion. Prior to the analysis of food samples, the quartz vessels were thoroughly cleaned by addition of 5 ml concentrated nitric acid, then heated in the microwave oven (conditions described below). The vessels were then rinsed thoroughly with deionised water. An portion of test material (0.5 g dry wgt) was placed into the quartz digestion vessel followed by 5 mL of concentrated nitric acid and then heated in the microwave oven to produce a complete digestion. The apparatus was loaded with 6 vessels and the operating pressure was 75 bar. The heating power and times applied at fan setting #1 were; 0 to 500W over 2 min and held for 5 min; 500 to 1000W over 2 min and held for 20 min; zero W for 15 min to cool at fan setting #3. The cool digests were transferred to centrifuge tubes (30 ml, Oak Ridge, polypropylene). The quartz vessels were rinsed with a further 5 ml water and this was added to the digest. Five digests of each sample plus a reagent blank were completed in each digestion run.

A 1.095 Bq/g solution of sodium chloride in water (SRM 4943, NBS, Gaithsburg, USA) was used to prepare a set of calibration standards. From serial dilution of SRM 4943 a set of six standards covering the range 0.0000243 to 2.43 Bq at order of magnitude intervals was prepared in centrifuge tubes containing 10 ml nitric acid (50 %v/v). Each standard was prepared in triplicate.

Chloride precipitation. The natural level of chloride present in blackberries, blanks and standards was insufficient for the minimum amount of target material required. To achieve sufficient precipitate, all samples and blanks were fortified with 1 mg chloride (1 ml of 2.54 mg/ml sodium chloride solution). Standards were fortified with 5 mg of chloride (1 ml of 12.7 mg/ml sodium chloride solution).

Each tube was vortex mixed to create a swirl of the contents and silver nitrate solution was quickly added (3 ml, 10 mg/ml) before the swirl ceased, in order to create a uniform precipitate. The tubes were capped, centrifuged, and the supernatant carefully poured off. The tube was then slowly filled with pure water, which was used to wash the precipitate. Between all manipulations the tubes were stored in the dark to minimise any interaction with light.

The precipitate was re-dissolved in ammonia solution (1 ml, s.g. 0.91), water was added (10 ml) and the solution was then re-precipitated with concentrated nitric acid (1 ml) after vortex mixing. The precipitates were again centrifuged, rewashed, then dried overnight in an air circulating oven (80 °C). Finally, the precipitate was pressed into the AMS target for measurement.

AMS measurements

The AMS instrument and operation has been previously described in detail [START_REF] Lappin | Biomedical Accelerator Mass Spectrometry[END_REF] Ion Source. A caesium sputter negative ion source was used whereby a caesium ion beam sputtered negative ions from the cathodes, which were extracted at 68 kV. The ion source and injector were set up using the standard setting for 14 C. The source wheel was loaded with a selection of cathodes with a range of 36 Cl contents.

Injector magnet. To ensure that the ion source was producing the required Cl beam, the injector magnet was set-up using 35Cl beam . Prior to the injection magnet, an electrostatic voltage was applied which was "bounced" sequentially, injecting m/z 37, 36 and 35 into the injector magnet. The "bounce" times were 1 ms, 100 ms and 1 ms for 37 Cl, 36 Cl and 35 Cl respectively. With the 'bounce' voltage set to zero the field was optimised for 35 Cl by maximising the current measured on the low energy Faraday cup and centred on the low energy beam scanners. The magnet was adjusted to allow the 37 Cl beam to go into the low energy Faraday cup and to centre it on the low energy beam scanners. The values obtained were checked to ensure that they were in the correct ratio for 35 Cl/ 37 Cl. Analyser magnet and high energy beam line. The 37 Cl beam was accelerated, using foil stripping and a terminal voltage of 5 MV. The analyser magnet was set for 40 MeV 7 + 37 Cl ions and the magnet and other high-energy beam line elements were optimised to maximise and centre the beam in front of the detector. These settings were checked by scaling them for 6 + and 8 + ions and the values of the beam currents for 6 to 8 + ions measured using the Faraday cup immediately after the analyser magnet.

Initial Detector set-up

A gas ionization detector was used using 10% methane in argon as the ionization gas mixture. Ionised atoms entering the detector were counted against individual channels.

With the injector and analyser magnets set for 40 MeV 7 + 36 Cl ions the detector parameters were changed to optimise the pressure and amplifier gain to give the best separation of the 36 S and 36 Cl ions.. The change in energy divided by the total energy as recorded in the gas ionization detector was recorded as a spectrum.

Adjustment of the off-axis Faraday cups. With the injector magnet set to pass 36 Cl, the position of the low energy offline Faraday cups were adjusted as follows: the output signal from the ' 27 Al' Faraday cup was fed to an oscilloscope input, set to DC coupling and the position adjusted to just intercept the beam at one edge, the position was recorded. The cup was moved further in the same direction until the beam just moved off the other edge and this position was also recorded. The cup was then set to the midpoint of these two settings which should centre the 37 Cl beam in the Faraday cup when the 36 Cl beam was transmitted through the injector magnet. The ' 13 C' Faraday cup position was adjusted in the same way to record the 35 Cl beam. With the analyser magnet set to pass 40MeV 7 + 36 Cl ions the high-energy off-axis Faraday cups were set using the same technique as for the low energy ones.

The results of these procedures were that: a) When the 36 Cl was transmitted through the accelerator the 37 Cl was incident on the low energy ' 27 Al' cup and the 35 Cl on the low energy ' 13 C' cup, b) When 37 Cl was accelerated it would be collected in the high energy ' 27 Al' cup, c) When 35 Cl was accelerated it would be collected in the high energy ' 13 C' cup. The ' 12 C' Faraday cups were not used for the 36 Cl measurements. The ' 27 Al' Faraday cups were fed through the ' 12 C' Faraday cup electronics by swapping the leads. The software was originally configured for carbon analysis, thus the 37 Cl/ 35 Cl ratio was recorded by the software as the 12 C/ 13 C ratio.

Setting up injector bounce voltages. To enable fast sequential measurement of the 35 Cl, 36 Cl and 37 Cl content of each cathode it was necessary to optimise the low energy bounce voltages. These enable each isotope to be accelerated in turn without making changes to the magnetic elements such as the injector and analyser magnets.

For 36 Cl, the bounce sequence was that 37 Cl was injected with zero bounce voltage and the 36 Cl and 35 Cl were bounced into the accelerator using suitable +ve bounce voltages.

The injector magnet was adjusted to pass 37 Cl with the bounce voltage set to zero (i.e. VCReg0=0). To set the bounce voltage for 35 Cl the injector magnet was left set for 37 Cl and the VCReg2 voltage adjusted until 35 Cl entered the accelerator. To set the bounce voltage for 35 Cl, the injector was left set at the value for 37 Cl and the VReg2 bounce voltage increased until the 35 Cl beam entered the machine and was accelerated. This setting of VReg2 would then bounced 35 Cl into the accelerator when the injector magnet was set for 37 Cl.

As the 36 Cl beam was too weak to be measured directly with a Faraday Cup, the same procedure could not be adopted when setting its 'bounce' voltage. The following procedure was therefore adopted; the injector was scaled by the ratio (37/36) 1/2 , this moved the 37 Cl beam by the same amount as the 36 Cl beam would be moved when the 37 Cl was transmitted through the injector magnet. The VReg1 control was adjusted to bring the 37 Cl back through the accelerator. The setting for VReg1 then bounced 36 Cl through the accelerator when the injector was set for 37 Cl. Also for this bounce voltage, the 37 Cl beam would go into the " 27 Al" cup and the 35 Cl into the " 13 C"cup.

RESULTS AND DISCUSSION

The AMS required a suitable target material e.g. silver chloride to be prepared from the original foodstuff. A pre-requisite for the final target sample was that at least 2 mg AgCl was required and sulfur contamination had to be kept to less than one part per million. [START_REF] Parry | Radiochemical methods in nuclear waste management[END_REF] determined the chlorine content in reactor materials (cast iron, steel and stainless steel) by neutron activation of the metals to form 38 Cl, dissolution in nitric acid, precipitation as silver chloride with one methanol wash before gamma-ray spectrometry of the 38 Cl. Similarly, [START_REF] Chant | I-129 and Cl-36 concentrations in lichens collected in 1990 from three regions around Chernobyl[END_REF] used a simple silver chloride precipitation followed by AMS measurement to test lichen samples taken from around Chernobyl. The samples were combusted in an oxygen stream, the gases trapped in water, chloride precipitated as silver chloride and the precipitate measured by AMS by determining the ratio of 36-Cl to 35/37-CL (in the range 3 to 100 x 10 -12 ) and determining total chloride independently using IC and NAA. The lichen samples were in the range 1.6 to 82 milliBq/kg.

In method development work here, the sulfur concentration in the silver chloride target materials produced was measured using ICP-MS (data not shown). By using a double precipitation technique the sulfur concentration was reduced low enough to allow measurement by AMS without the need to correct for sulfur contamination measured independently by ICP-MS. The isolation and purification scheme used is shown in Figure 1.

Standards

The AMS resolved 36 Cl from 36 S present as an impurity. Figure 2 shows the AgCl calibration standards. The peak centred on channel number 28 is due to the 36 Cl ions and the peak centred on channel number 33 is 36 S that has not been removed completely.

The reason for the distortion at channel 36 is not known but it is of no consequence.

There is good resolution of the two peaks and at the low level of sulfur achieved it presents little interference. To minimise further any interference in determining the lowest levels of 36 Cl it was quantified as the sum of counts in channels 25 to 28. This means, not using channels 29 and 30 which although they do contain some signal from The results for the calibration standards are shown in Table 1. Also shown in this table is the current counted for the natural 37 Cl isotope. It can be seen that the AMS failed for one of the standards which no 37 Cl current was observed, most probably because the sample was not mounted into the AMS target holder adequately. Otherwise, the 37 Cl current was very consistent for the standards (Table 1). However, for the different batches of samples the current was seen to vary indicating variation in the ionisation process. Therefore the 36 Cl counts were normalised against the 37 Cl current. The calibration line for the standards is plotted in Figure 3.

Fortified Samples

No certified reference food materials with a value for 36 Cl were available. The microwave-assisted acid digestion was conducted in a sealed container, and it was unlikely that chloride would be lost during this stage of the experiment. All digests were clear solutions, which gave a good indication of the thoroughness of sample digestion.

Figure 4 shows the samples of milk and blackberry fortified with activity of 0.005 and 0.0005 Bq along with a reagent blank. The 36 Cl can be clearly seen, centred at channel 28. The level of activity is detectable at the lowest level and can be clearly distinguished from the blank. Since the target material is the silver chloride precipitate from a 4.1g (milk) or 2.8g (blackberry) fresh weight specimen of food, the fortification levels of 0.0005 and 0.005 Bq are equivalent to 0.12 and 1.2 Bq/kg food for milk and 0.17 and 1.7 Bq/kg for blackberries.

Samples

Figure 5 shows the AMS traces obtained for the unspiked milk and blackberry samples.

The spectra shows evidence of only one peak. There is no evidence of a peak centred around channel number 28 due to 36 Cl ions. The single peak centred around channel number 33 is due to 36 S. Plot of the number of counts versus the channel number recorded on the AMS Gas Ionization Detector as determined for calibration standards (0, 0.5, 1.5, 3, 5, 30 and 300 mBq in 5 mg chloride as AgCl precipitate). The peak around channel number 25 was 26 S and the peak around channel 34 was 36 Cl.

  these channels would be most prone to interference from the leading edge of the 36 S peak when measuring 36 Cl at very weak signal levels.

  Figure 1. The sample preparation procedure used

Figure 4 .

 4 Figure3. A calibration line constructed from single analyses of 36Cl standards plotting the amount of 36Cl in mBq in 5 mg AgCl precipitate against the counts recorded in the AMS Gas Ionisation Detector.

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

ACKNOWLEDGEMENTS

The authors thank the UK Food Standards Agency for funding this work (Project code R02014).

CONCLUSIONS

A method for the determination of 36 Cl in foods using AMS has been developed. The instrument used in this study was a 5 million Volt AMS. The main problem to overcome was the potential interference of 36 S and a method was developed to reduce the sulfur by chemical purification (double-precipitation as silver chloride) prior to the AMS analysis. The AMS method was much more sensitive than standard decay counting techniques, unless it is possible to transfer the method described by [START_REF] Hou | Determination of 36 Cl in nuclear waste from reactor decommissioning[END_REF] to use for foods. For a 3 to 4g fresh sample weight the detection limit is around 0.1 Bq/kg. The method was rather imprecise at levels below 1 Bq/kg. The method was tested using a few samples of milk and blackberries gathered from near two nuclear power stations as potential source. The method clearly detected samples spiked at 0.12 Bq/kg (milk) or 0.17 Bq/kg (blackberries) and there was no 36 Cl detected in the unspiked samples.


Mette Brandt Eriksen

Louise Bruun Jørgensen

Henrik Krarup

Peer Alex Lund Laursen

Peer Brehm Christensen

Axel Møller

Poul Schlichting

Carla Kuiken

Jens Bukh

Nina Weis

Danhep Group

Molecular and Epidemiological Profiles of Hepatitis C Virus Genotype 4 in Denmark

Keywords: HCV, genotype 4, intravenous drug use , core/E1 region, phylogenetic analysis HCV, genotype 4, intravenous drug use, core/E1 region, phylogenetic analysis

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche

INTRODUCTION

Hepatitis C virus (HCV) is currently one of the most widespread infectious viruses, with more than 170 million people infected worldwide [WHO, 1999]. About 65-85% of individuals with acute HCV infection develop a chronic infection, with risk increased significantly of developing liver cirrhosis and hepatocellular carcinoma [START_REF] Hoofnagle | Hepatitis C: The clinical spectrum of disease[END_REF][START_REF] Seeff | Natural history of chronic hepatitis C[END_REF]. The HCV virion contains a single-stranded, positive-sense RNA genome and has been classified into the Hepacivirus genus of the Flaviviridae family. The HCV genome is very heterogeneous, as reflected both by genome length and by the identification of seven major genotypes and more than 67 subtypes with nucleotide divergences of 31-33% and 20-25%, respectively [START_REF] Bukh | Sequence analysis of the core gene of 14 hepatitis C virus genotypes[END_REF][START_REF] Simmonds | Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes[END_REF][START_REF] Kuiken | The hepatitis C sequence database in Los Alamos[END_REF][START_REF] Kuiken | Nomenclature and numbering of the Hepatitis C virus[END_REF]. Genotypes 1, 2, and 3 are found worldwide, particularly in Western countries. Genotypes 4 and 5 display a more restricted geographic pattern with high prevalences in the Middle East and Africa. Egypt has the highest prevalence of HCV; 20% of the Egyptian population is chronically infected with HCV, and among this group, almost 90% are infected with genotype 4 [START_REF] Kamal | Hepatitis C genotype 4: What we know and what we don't yet know[END_REF]. Genotype 6 is mainly found in Southeast Asia, and genotype 7 has been found in patients of African origin living in Canada and Belgium [Murphy et al., 2007].

Although infection with all HCV genotypes can progress to chronic liver disease, genotype-specific differences in the natural history of the disease may exist. For example, HCV genotypes 1 and 3 have been found to induce steatosis [START_REF] Cross | The importance of steatosis in chronic hepatitis C infection and its management: A review[END_REF]. The efficacy of the standard treatment with ribavirin and pegylated interferon depends on the genotype, with infection with either genotype 1 or 4 leading generally to a poorer treatment response than infection with genotype 2 or 3 [START_REF] Fried | A multicenter, randomized trial of daily high-dose interferon-alfa 2b for the treatment of chronic hepatitis c: pretreatment stratification by viral burden and genotype[END_REF][START_REF] Fabiani | Hepatitis C virus: Virology, diagnosis and management of antiviral therapy[END_REF].

Within HCV genotype 4, there is a widespread genetic heterogeneity, which is reflected by the presence of at least 19 subtypes (4a-h, 4k-4u) according to the Los Alamos HCV Database [START_REF] Kuiken | The Los Alamos HCV sequence database[END_REF]. The overall prevalence of genotype 4 among the general population of patients infected with HCV in the United States and Europe is thought to be low. Two U.S. studies done 10 years apart reported a low prevalence (approximately 1%) of genotype 4 [START_REF] Lau | Distribution of hepatitis C virus genotypes determined by line probe assay in patients with chronic hepatitis C seen at tertiary referral centers in the United States. Hepatitis Interventional Therapy Group[END_REF][START_REF] Nainan | Hepatitis C virus genotypes and viral concentrations in participants of a general population survey in the United States[END_REF]. A Slovenian study found a genotype 4 prevalence of 1.4% among a large cohort of 1,504 patients infected with HCV [START_REF] Seme | Hepatitis C virus genotypes in 1,504 patients in Slovenia, 1993 -2007[END_REF]. A fourfold increase in the prevalence of genotype 4 during a five-year period was reported among older patients with sporadically acquired infection in Calabria, Southern Italy [START_REF] Matera | Changes in the prevalence of hepatitis C virus (HCV) genotype 4 in Calabria, Southern Italy[END_REF]. It is of note that the first identified subtype 4d strain was isolated from a Danish patient in 1993 [START_REF] Bukh | At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide[END_REF].

Although HCV genotype 4 seems to constitute a rather low percentage of patients infected with HCV in the Western world, it has spread throughout Europe, especially among intravenous drug users. In Greece, genotype 4 is the second most frequent genotype among intravenous drug users [START_REF] Savvas | Changes in epidemiological patterns of HCV infection and their impact on liver disease over the last 20 years in Greece[END_REF], and similar results were obtained in a study from Southern Spain [START_REF] Sánchez-Quijano | Unexpected high prevalence of hepatitis C virus genotype 4 in Southern Spain[END_REF]. In Southwestern France, a genotype 4 prevalence of 7.4% was found, with subtypes 4a and 4d identified almost exclusively among intravenous drug users (99%) [START_REF] Nicot | Heterogenity of hepatitis C virus genotype 4 strains circulating in South-Western France[END_REF]. In the Netherlands, a study using a molecular epidemiological approach identified genotype 4 in three different epidemiological profiles. One group was found to be correlated to Egypt, one to intravenous drug users, and one with men who have sex with men [START_REF] De Bruijne | Emergence of Hepatitis C Virus Genotype 4: Phylogenetic Analysis Reveals Three Distinct Epidemiological Profiles[END_REF]. A European multi-center study of intravenous drug users co-infected with HCV and human immunodeficiency virus (HIV) found genotype 4 prevalences ranging from 6.9% in Northern Europe (Holland, Scotland) to 24.4% in Southern Europe (France, Spain), with subtype 4d dominating among intravenous drug users [START_REF] Van Asten | Spread of hepatitis C virus among European injection drug users infected with HIV: a phylogenetic analysis[END_REF]]. and is similar to the prevalence found in the other Scandinavian countries [START_REF] Esteban | The changing epidemiology of hepatitis C virus infection in Europe[END_REF]. A 2003 study of 71 patients using phylogenetic analysis of C-E1 reported that the most common HCV genotypes found in Denmark are genotypes 1a (43%), 3a (39%), 1b (11%), and 2b (6%) [START_REF] Corbet | Hepatitis C virus subtyping by a core-Envelope 1-based reverse transcriptase PCR assay with sequencing and its use in determining subtype distribution among Danish patients[END_REF], but genotype 4 was not found. However, genotype 4 has a prevalence of approximately 2% among patients with hepatitis C referred to hospital departments in Denmark [DANHEP, Annual Report 2004].

The aim of the present study was to gain knowledge about patients infected with HCV genotype 4 regarding subtypes, routes of transmission, ethnic origin, and co-infection with HIV. This information may make it possible to characterize the introduction and spread of HCV genotype 4 strains among subpopulations in Denmark, which could have important implications for preventing HCV transmission and treatment.

MATERIALS AND METHODS

Study Population

The inclusion of patients into the study was based on initial HCV genotype 4 determinations carried out from 1999 to 2007 at the Department of Clinical Biochemistry, Unit for Molecular Diagnostics, Aalborg University Hospital, Denmark. Aalborg University Hospital is the primary site for HCV genotypic assay in Denmark, and no patients tested at Statens Serum Institut were found to harbor genotype 4. Patients were genotyped with genotypespecific primers located in the 5' HCV non-coding region, as described previously [START_REF] Krarup | A quantitative HCV-PCR test for routine diagnostics[END_REF][START_REF] Krarup | Haemophilic patients with hepatitis C have higher viral load compared to other welldefined patient groups[END_REF].

A total of 72 samples from patients infected chronically with HCV genotype 4 were available for analysis. Clinical data regarding the patients was received from the Danish nationwide viral hepatitis database (the Danish Database for Hepatitis B and C, DANHEP) covering all regions of Denmark.

The Danish Ethics Council and The Danish Data Protection Supervisory Authority approved this study.

HCV Reverse Transcription, Genotyping, and Phylogenetic Analysis

The envelope 1 (E1) gene of HCV was chosen because this assay is used at the Statens Serum Institut for routine patient samples for HCV genotyping. To amplify and sequence part of the E1 gene, HCV RNA was isolated from 280 µl plasma using the QIAmp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to manufacturer guidelines. This was followed by a one-step RT-PCR with 60 pmol primers (Outer C-E1 F and Outer C-E1 R) using the Qiagen OneStep RT-PCR Kit according to manufacturer guidelines (Qiagen, Hilden, Germany). The cycling conditions were as follows: (1): 50°C (30 minutes), ( 2 4): 55°C (30 seconds), and (5): 72°C (30 seconds); steps 3-5 were repeated 39 times. Final extension was carried out at 72°C for 10 minutes followed by 30 minutes at 4°C. PCR products were sequenced with the primers "Inner C-E1 F" and "Inner C-E1 R" using the BigDye Terminator v3.1 Cycle Sequencing Kit and the ABI 3130x sequencer (Applied Biosystems, Foster City, CA, USA). Primers used were from a study published previously [START_REF] Ray | Genetic epidemiology of hepatitis C virus throughout Egypt[END_REF] and have been shown previously to have equal sensitivity against isolates of the different HCV genotypes [START_REF] Corbet | Hepatitis C virus subtyping by a core-Envelope 1-based reverse transcriptase PCR assay with sequencing and its use in determining subtype distribution among Danish patients[END_REF]. Sequences were aligned and edited manually using the SeqScape Software v2.6 (Applied Biosystems, Foster City, CA, USA) and BioEdit Software [START_REF] Hall | BioEdit: A User-Friendly Biological Sequence Alignment Editor and Analysis Program for Windows 95/98/NT[END_REF]. Multiple alignments of the patient and reference sequences were carried out with ClustalX software v. 1.8 [START_REF] Thompson | The Clustal X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools[END_REF].

To ensure an equal length of all included sequences, the phylogenetic analysis was based on a subdomain of the E1 region from nucleotides 923-1202, with nucleotide positions relative to the HCV H77, genotype 1a reference strain [START_REF] Kolykhalov | Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA[END_REF]. The phylogenetic tree was inferred by the neighbor-joining (NJ) method using the Kimura 2parameter correction distances with bootstrap values based on 1,000 replicates (Fig. 1). To ensure the strength of the phylogenetic analysis, a multiple sequence comparison by logexpectation (MUSCLE) alignment with a maximum number of iterations equal to 2 was carried out [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF]. A maximum likelihood tree was built with PHYML using the HKY85 substitution model and bootstrapped with 100 replicates [START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF]. The reference strains included in the phylogenetic tree are named with genotype/subtype and country of origin when possible, otherwise with sampling country and the GenBank accession numbers (Fig. 1). Reference sequences were included for genotype 1 [START_REF] Kolykhalov | Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA[END_REF], genotype 2 [START_REF] Kato | Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient[END_REF], genotype 3 [START_REF] Sakamoto | Entire nucleotide sequence and characterization of a hepatitis C virus of genotype V/3a[END_REF], and genotype 4 [START_REF] Bukh | At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide[END_REF][START_REF] Stuyver | Classification of hepatitis C viruses based on phylogenetic analysis of the envelope 1 and nonstructural 5B regions and identification of five additional subtypes[END_REF][START_REF] Van Doorn | Analysis of hepatitis C virus genotypes by a line probe assay and correlation with antibody profiles[END_REF][START_REF] Fujimura | Genotypes and multiple infections with hepatitis C virus in patients with haemophilia A in Japan[END_REF][START_REF] Ray | Genetic epidemiology of hepatitis C virus throughout Egypt[END_REF][START_REF] Morice | Phylogenetic analyses confirm the high prevalence of hepatitis C virus (HCV) type 4 in the Seine-Saint-Denis district (France) and indicate seven different HCV-4 subtypes linked to two different epidemiological patterns[END_REF][START_REF] Lavillette | Characterization of host-range and cell entry properties of the major genotypes and subtypes of hepatitis C virus[END_REF][START_REF] Brown | Crossgenotype characterization of genetic diversity and molecular adaptation in hepatitis C virus envelope glycoprotein genes[END_REF] [START_REF] Fabiani | Hepatitis C virus: Virology, diagnosis and management of antiviral therapy[END_REF][START_REF] Hmaied | Full-length genome sequences of hepatitis C virus subtype 4f[END_REF]Murphy et al., 2007;[START_REF] Timm | Characterization of full-length hepatitis C virus genotype 4 sequences[END_REF][START_REF] Kuntzen | A set of reference sequences for the hepatitis C genotypes 4d, 4f, and 4k covering the full open reading frame[END_REF][START_REF] Demetriou | Molecular epidemiology of hepatitis C infection in Cyprus: evidence of polyphyletic infection[END_REF][START_REF] Khan | Epidemic spread of hepatitis C virus genotype 3a and relation to high incidence of hepatocellular carcinoma in Pakistan[END_REF].

The patients were defined as being infected with a particular subtype when included in a defined cluster with given reference strains and with bootstrap values > 90. Subtypes were defined as unclassified (UN) if sequences clustered with reference sequences of unknown subtype and a BLAST search did not reveal similarity with reference sequences of known subtypes.

Statistical Analysis

For statistical analysis, the Fisher's exact test and the Student's t-test were used for comparing age distribution of the patients and for comparing the HIV prevalence of the DANHEP participants and HIV prevalence among the patients in this study. A Bonferroni adjustment was used to correlate for multiple comparisons. P values of < 0.05 were considered significant.

RESULTS

Seventy-two Danish patients with genotype 4 were included in the phylogenetic and epidemiological analyses. Two patients were excluded because of negative PCR results in the C-E1 assay, and an additional two patients were excluded because of uncertainty about their identity. The consensus E1 partial sequence (nucleotides 923-1202, according to the H77 reference strain) was the same length without insertions or deletions in all 72 genotype 4 isolates, and each sequence encoded 59 amino acids. All patients except two were infected with unique HCV strains. Pairwise identity values at the nucleotide and amino acid levels were 76.6% (range 64-99%) and 81.8% (range 68-100%). Almost 70% of patients with chronic infection with HCV genotype 4 were males (Table I).

The median age was 49.5 years (interquartile range (IQR) 40-59 years) (Table I). The phylogenetic N-J tree of the partial E1 sequences (Fig. 1) defined accurately the subtypes of the 72 patients infected with HCV genotype 4 and was reproducible (maximum likelihood tree, not shown). The partial E1 region (nucleotides 924-1202) was sufficient to define and distinguish both the different HCV genotypes and the genotype 4 subtypes, as indicated by bootstrap values > 90. The subtypes detected most frequently were 4d, 4a, and 4r, infecting 23 (31.9%), 21 (29.2%), and 12 (16.7%) of 72 patients, respectively. Thus, subtypes 4a, 4d, and 4r accounted for 77.8% of the genotype 4 infections in Denmark. The infections of the remaining 16 patients with genotype 4 (22.2%) represented subtypes 4h, 4k, 4l, 4n, 4o, and unclassified subtypes (4UN). The majority of patients were ethnic Danes (17 of 72 patients; 23.6%) and Africans (28 of 72 patients; 38.9%), but the group included also patients from Southern Europe, Egypt, the Middle East, and Pakistan (Table I).

The main route identified for transmission among patients with genotype 4 was intravenous drug use, which accounted for transmission in 16 of the 72 patients (22.2%); for the majority (36 patients; 50%), however, the transmission routes were unknown. Other transmission routes included blood transfusion, sexual contact, needle injury, and vaccination (Table I). The three major subtypes (4d, 4a, and 4r) were associated with different routes of transmission. Of the 23 patients with subtype 4d, 16 were infected via intravenous drug use (69.5%, Table II). Of these patients infected with subtype 4d via intravenous drug use, 15 patients (93.8%) were ethnic Danes; the remaining patient was from Southern Europe. The phylogenetic tree indicated two monophyletic clusters with seven and eight Danish subtype 4d patients, plus one patient of unknown origin; these clusters were not supported, however, by significant bootstrap values (Fig. 1). For patients infected with other subtypes, the route of I andII). The patients infected with subtype 4a all came from Africa, Pakistan, or the Middle East. Subtype 4r infected 10 patients from Southern Africa (83.3%), one patient from Northern Africa (8.3%), and a Southern European patient (8.3%). The 4r patients were significantly older (median age: 64 years; IQR: 58-71 years, Table II) than subtype 4d (P = 0.0044) and subtype 4a (P = 0.013) patients. The median ages in the latter two groups were comparable (4d, median: 45 years, IQR: 41-51.5 years; 4a, median: 51 years, IQR: 37.75-58 years, P = 0.588).

Pairwise Identity and Similarity of Patient Sequences with Published Sequences

The similarity between the patient nucleotide sequences (subtypes 4a, 4d, 4r, and 4UN) and published nucleotide sequences was investigated using BLAST, and each closest match for 4a, 4d, 4r, and 4UN was retrieved. All of the subtype 4d patient strains were most similar to a published genotype 4d sequence isolated from an African patient (FJ462437). The average pairwise nucleotide identity within the entire group of subtype 4d patient strains was 94.2% (64-99%); within the Danish patient strains, it was 94.9% (91-99%). The different subtype 4a patient strains were most similar to various sequences: an isolate from a Pakistani patient (AB444559), an isolate from a Yemeni patient (D43681), an isolate from an Egyptian patient (DQ988079), an isolate published from the United States (DQ418787), and an isolate published from Japan (D45193).

The pairwise nucleotide identity within the entire group of subtype 4a patients was 87% (67-93%); within the Egyptian patient group, it was 85.4% (69-93%). The pairwise nucleotide identity within the subtype 4r patient strains was 91.3% (86-96%), and 11 of the 12 patients were most similar to two Yemeni isolates (D43681, D43678). In the five cases of unclassified subtype (4UN), two of the patient strains were most similar to sequences from Canada (EF115910) and Cyprus (EU684694), one was related to a strain from Canada

HCV/HIV Co-infection

Of the 47 patients who were tested for HIV, 9 were HIV positive (19.2%) and 38 (80.8%)

were HIV negative (Table I). Of the 72 patients, 25 (34.7%) had not been tested.

Among patients co-infected with HIV, the known routes of HCV transmission were intravenous drug use and sexual contact. One patient co-infected with HCV/HIV was a man having sex with men and self-reported to have been infected by sexual contact with a former partner from Germany. No single HCV genotype 4 subtype dominated among patients coinfected with HCV and HIV (Table II).

DISCUSSION

This nationwide study of patients infected with genotype 4 identified three epidemiological profiles. HCV subtype 4d was found in an ethnic Danish patient for the first time in 1993 [START_REF] Bukh | At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide[END_REF] and seems to have spread, dominating completely now among patients who are ethnic Danish intravenous drug users infected with genotype 4. Genotype 4 has been reported throughout Europe, and subtype 4d has been linked particularly in epidemiological studies to intravenous drug use in the Netherlands, Poland, and Southern Europe [START_REF] Van Asten | Spread of hepatitis C virus among European injection drug users infected with HIV: a phylogenetic analysis[END_REF][START_REF] Nicot | Heterogenity of hepatitis C virus genotype 4 strains circulating in South-Western France[END_REF][START_REF] Chlabicz | High prevalence of genotype 4 among hepatitis c virus-infected intravenous drug users in North-Eastern Poland[END_REF][START_REF] De Bruijne | Emergence of Hepatitis C Virus Genotype 4: Phylogenetic Analysis Reveals Three Distinct Epidemiological Profiles[END_REF]. Our results do not support a hypothetical single-transmission introduction of subtype 4d in Danish intravenous drug users. This hypothesis would require a distinct, single monophyletic cluster containing intravenous drug users, supported by significant bootstrap values [ [START_REF] Oliveira | HIV-1 and HCV sequences from Liyan Outbreak[END_REF]. Immigration of intravenous drug users from regions or countries with a high prevalence of subtype 4d among intravenous drug users [START_REF] Van Asten | Spread of hepatitis C virus among European injection drug users infected with HIV: a phylogenetic analysis[END_REF][START_REF] Nicot | Heterogenity of hepatitis C virus genotype 4 strains circulating in South-Western France[END_REF] could indicate a possible route of introduction of subtype 4d in the population of ethnic Danish intravenous drug users. In all, HCV genotype 4 has been introduced into the European intravenous drug-use milieu including Denmark, and there is an increasing prevalence of HCV genotype 4 throughout Europe [Sánchez-Ouijano et al., 1997;[START_REF] Savvas | Changes in epidemiological patterns of HCV infection and their impact on liver disease over the last 20 years in Greece[END_REF].

The presence of only subtype 4d among Danish intravenous drug users might reflect an effect of preventive measures such as needle-exchange and substitution programs, leading to a decrease in the frequency of HCV exposure. However, the overall prevalence of hepatitis C among intravenous drug users was not lower than in other European countries [EMCDDA;annual report, 2009]. Missing information on transmission route weakens the conclusion about introduction of subtypes other than subtype 4d in Danish intravenous drug users. The high degree of nucleotide identity among patients infected with subtype 4d who were ethnic Danish could indicate a recent introduction of subtype 4d among Danish intravenous drug users. De Bruijne et al. [2009] showed introduction of 4d among intravenous drug users in the Netherlands. The introduction and spread of subtype 4a among European intravenous drug users has been reported also [START_REF] Schröter | Epidemiological Dynamics of Hepatitis C Virus among 747 German Individuals: New Subtypes on the Advance[END_REF]Murphy et al., 2007], but according to our results, subtype 4a has not been introduced in Danish intravenous drug users. In addition, in this study, genotype 4 has not been established among men who have sex with men, as has been seen the Netherlands and in other European countries [START_REF] De Bruijne | Emergence of Hepatitis C Virus Genotype 4: Phylogenetic Analysis Reveals Three Distinct Epidemiological Profiles[END_REF][START_REF] Van De Laar | Evidence of a Large, International Network of HCV Transmission in HIV-Positive Men Who Have Sex with Men[END_REF].

A second epidemiological profile comprised patients infected with subtype 4a who originated from restricted geographic areas like Northern Africa, Egypt, Pakistan, and the Middle East, where subtype 4a is prevalent [START_REF] Djebbi | Genotypes of hepatitis C virus circulating in Tunisia[END_REF]. As expected, all of the eight Egyptians included in the study (Table II) were infected with subtype 4a [START_REF] Elkady | Genetic variability of hepatitis C virus in South Egypt and its possible clinical implication[END_REF]. The 4a sequences from these patients did not cluster, and the nucleotide divergence ranged widely, explained possibly by the early introduction and exponential spread of this subtype in Egypt [START_REF] Tanaka | Exponential spread of hepatitis C virus genotype 4a in Egypt[END_REF].

The third epidemiological profile was found for patients with subtype 4r. This subtype was found mostly among Southern African patients, and these patients were significantly older than patients infected with subtypes 4d and 4a (64 years versus 45 and 51 years, respectively, Table II). Though not supported by significant bootstrap values, nine Southern African patients and one Southern European patient along with the GenBank reference strains D43681, D43679, and D43678 seemed to form a monophyletic cluster. Subtype 4r has been described previously in patients of Central African origin: A Canadian study found a prevalence of 10.2% of subtype 4r among patients mostly of Central African origin [Murphy et al., 2007;[START_REF] Ndong-Atome | High prevalence of hepatitis C virus infection and predominance of genotype 4 in rural Gabon[END_REF]. The nucleotide variation within subtype 4r patient strains is between that of subtypes 4d and 4a. However, the molecular epidemiology of HCV in Africa is documented poorly, and the number of African 4r sequences available publicly is small [START_REF] Ndong-Atome | High prevalence of hepatitis C virus infection and predominance of genotype 4 in rural Gabon[END_REF]. A high prevalence of HCV genotype 4 has been found in the Central African Republic, but the prevalence of subtype 4r was low (5%) [START_REF] Njouom | Predominance of hepatitis C virus genotype 4 infection and rapid transmission between 1935 and 1965 in the entral African Republic[END_REF].

Five African patients were infected with unclassified subtypes (4UN). Two of these patients were men of Northern African origin and clustered with GenBank reference strains of unclassified subtypes, EU684694, and a Canadian strain, EF115910, isolated from a patient originating from Rwanda [Murphy et al., 2007] (Fig. 1). Unclassifiable genotype 4 strains have been described in African patients [START_REF] Ndong-Atome | High prevalence of hepatitis C virus infection and predominance of genotype 4 in rural Gabon[END_REF]. These new sequences could form a new subtype, genetically closely related to subtypes 4q, 4e, and 4l, but to determine this, more sequence data are needed. In addition, the unclassified strains were sequenced in NS5B and analyzed phylogenetically, with results showing a similar The subtypes infecting the patients co-infected with HCV and HIV varied, and no particular genotype 4 subtypes dominated (Table I). The HCV subtype 4d sequences from coinfected patients did not cluster together, and there was no indication of a connection between these patients. In addition, there were no significant differences between the prevalence of HCV/HIV co-infection among the genotype 4 patients (19.2%) in this study and the prevalence of HCV/HIC co-infection among the entire group of DANHEP participants, which included patients of all genotypes (17.9%), (P = 0.4153).

In conclusion, this population from Denmark seemed to display a subtype distribution pattern similar to that found earlier in other Western countries, but with a more restricted distribution of subtypes in relation to ethnic origin [START_REF] Nicot | Heterogenity of hepatitis C virus genotype 4 strains circulating in South-Western France[END_REF][START_REF] Cantaloube | Molecular characterization of genotype 2 and 4 hepatitis C virus isolates in French blood donors[END_REF]. Genotype 4 has been established and spread among men who have sex with men in Europe, an epidemiological profile not found in Denmark. The subtypes 4d, 4a, and 4r dominate among patients who are infected chronically with HCV and living in Denmark.

This study has shown that subtype 4d as a single subtype has been introduced in and spread among Danish intravenous drug users with chronic HCV. In general, the strains among Danish patients infected with chronic HCV genotype 4 are very heterogeneous genetically.

Numerous genotype 4 subtypes were found, with a distribution resembling findings from other Western countries [START_REF] Nicot | Heterogenity of hepatitis C virus genotype 4 strains circulating in South-Western France[END_REF]Murphy et al., 2007;[START_REF] Cantaloube | Molecular characterization of genotype 2 and 4 hepatitis C virus isolates in French blood donors[END_REF].

  ): 95°C (15 minutes), (3): 94°C

  mostly as unknown (Table

  , one was situated between subtypes 4a and 4n, and one was situated between 4e and 4q. Neither the BLAST investigation nor comparison against the HCV Los Alamos Database yielded further subtype information.

  sequences as found for E1 (data not shown). BLAST analysis of the remaining 4UN patient strains did not identify a subtype.

  Figure Legend

  Fig. 1 Phylogenetic tree based on E1 sequences (nucleotides 923-1202, according to the H77 Los Alamos reference sequence) from 72 patients infected with HCV genotype 4. Colors show the ethnic origin of the patients as indicated. Reference sequences were retrieved from GenBank and are indicated by their accession number and if possible, country of origin, otherwise by country of sampling. The neighbor-joining tree was based on the Kimura 2-parameter correction distances with bootstrap values based on 1,000 replicates. Bootstrap values > 90 are indicated with a dot. 298x266mm (300 x 300 DPI)

Journal of Medical Virology

Acknowledgements

We thank laboratory technicians Lone Sandbjerg Hindbaek, Statens Serum Institute, Copenhagen, Denmark, and Anne Bentzen-Pedersen, Department of Gastroenterology and Department of Clinical Chemistry, Unit for Molecular Diagnostics, Aalborg University Hospital, Denmark, for their dedicated work. The study was supported financially by The Danish Research Counsel and The Hartmann Foundation (grant support to Mette Brandt Eriksen) and the A. P. Møller Foundation for the Advancement of Medical Science (grant support to Nina Weis). Jens Bukh is the recipient of a professorship at the University of Copenhagen with external funding from the Lundbeck Foundation.

Appendix


Teemu Petteri Smura
email: teemu.smura@thl.fi

Petri Ylipaasto

Päivi Klemola

Svetlana Kaijalainen

Lauri Kyllönen

Valeria Sordi

Lorenzo Piemonti

Merja Roivainen

CELLULAR TROPISM OF HUMAN ENTEROVIRUS D SPECIES SEROTYPES EV-94, EV-70 AND EV-68 IN VITRO -IMPLICATIONS FOR PATHOGENESIS

Keywords: enterovirus, tissue tropism, leukocyte, endothelial cell, pancreatic islet enterovirus, tissue tropism, leukocyte, endothelial cell, human pancreatic islet, type 1 diabetes

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Enterovirus genus (family Picornaviridae) contains six species causing infections in humans, Human enterovirus A (HEV-A) to HEV-D, Human rhinovirus A (HRV-A) and HRV-B, classified into over 200 currently known serotypes. Although the majority of enterovirus infections are subclinical, they can lead to a variety of acute and chronic diseases including mild upper respiratory illness, febrile rash, aseptic meningitis, encephalitis, acute hemorrhagic conjunctivitis, pleurodynia, acute flaccid paralysis (AFP), myocarditis, type 1 diabetes (T1D) and neonatal sepsis-like disease [START_REF] Pallansch | Enteroviruses: polioviruses, coxsackieviruses, echoviruses and newer enteroviruses[END_REF].

The primary site of enterovirus infection is the mucosal tissue of the respiratory or gastrointestinal tract. Virus replication can continue in the intestinal mucosa for several weeks. During this period, the progeny virus is excreted in the faeces. The primary infection can occasionally be followed by a viremic phase, during which the virus spreads through the lymphatic system and circulation, and may gain access to the secondary target tissues. Most enterovirus-related diseases are consequences stemming from the viral infection of secondary target tissues. Thus the tissue tropism of a virus is a major factor influencing the incidence and severity of enterovirus-induced diseases.

Molecular typing methods have led to characterization of several new enterovirus types.

Enterovirus 94 (EV-94) was recently detected during poliovirus surveillance of waste water in Egypt and in acute flaccid paralysis patients in the Democratic Republic of Congo [START_REF] Smura | Enterovirus 94, a proposed new serotype in human enterovirus species D[END_REF], Junttila et al., 2007). EV-94 is a member of the HEV-D species. This species is known to have only two other serotypes: enterovirus 68 (EV-68), which has been associated with respiratory infections [START_REF] Schieble | A probable new human picornavirus associated with respiratory diseases[END_REF][START_REF] Oberste | Enterovirus 68 is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses[END_REF]; and enterovirus 70 (EV-70), a causative agent of acute haemorrhagic conjunctivitis [START_REF] Mirkovic | Enterovirus type 70: the etiologic agent of pandemic acute haemorrhagic conjunctivitis[END_REF]. Both EV-70 and EV-68 can 5 occasionally infect the central nervous system [START_REF] Pallansch | Enteroviruses: polioviruses, coxsackieviruses, echoviruses and newer enteroviruses[END_REF][START_REF] Khetsuriani | Enterovirus surveillance--United States, 1970-2005[END_REF].

Seroprevalence studies have revealed a high prevalence of EV-94 in the Finnish population over the past two decades [START_REF] Smura | Enterovirus 94, a proposed new serotype in human enterovirus species D[END_REF]. As yet, however, the pathogenesis and possible clinical manifestations of this virus have not been studied in detail.

EV-94, unlike EV-68 and some strains of EV-70 [START_REF] Blomqvist | Human rhinovirus 87 and enterovirus 68 represent a unique serotype with rhinovirus and enterovirus features[END_REF][START_REF] Oberste | Enterovirus 68 is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses[END_REF], is acid stable and thus probably able to use the faecal-oral route of transmission to gain access to intestinal tissues [START_REF] Smura | Enterovirus 94, a proposed new serotype in human enterovirus species D[END_REF]. If the primary infection in mucosal tissue is followed by viremia, the leukocytes may provide an additional reservoir for viral replication and/or persistence. This could increase the viral load in the circulation, thereby lengthening the viremic period.

Endothelial cells, on the other hand, act as a barrier between vascular space and tissue parenchyma.

In order to infect secondary target tissues, the virus has to pass through endothelial cells either by infecting them or by transcytosis. Alternatively, the viruses can be transported through the endothelial barrier by infected leukocytes migrating to the target tissues.

The pancreas is one of the secondary target tissues for enteroviruses [START_REF] Pallansch | Enteroviruses: polioviruses, coxsackieviruses, echoviruses and newer enteroviruses[END_REF][START_REF] Huber | Coxsackievirus-induced pancreatitis[END_REF], and enteroviruses are considered to be a major environmental factor causing predisposition to type 1 diabetes [START_REF] Varela-Calvino | Enteroviruses and type 1 diabetes[END_REF][START_REF] Knip | Environmental triggers and determinants of type 1 diabetes[END_REF][START_REF] Roivainen | Enteroviruses: new findings on the role of enteroviruses in type 1 diabetes[END_REF]). The enterovirus genome or enterovirus-specific antibodies can be detected more often from the blood samples of patients with type 1 diabetes than from blood samples of control subjects (reviewed in [START_REF] Jaidane | Role of coxsackievirus B4 in the pathogenesis of type 1 diabetes[END_REF]. Many enterovirus strains are capable of infecting pancreatic islets in vitro [START_REF] Roivainen | Mechanisms of coxsackievirus-induced damage to human pancreatic beta-cells[END_REF][START_REF] Roivainen | Functional impairment and killing of human beta cells by enteroviruses: the capacity is shared by a wide range of serotypes, but the extent is a characteristic of individual virus strains[END_REF], and enterovirus genome/proteins have been detected in the pancreatic islets of patients with type 1 diabetes [START_REF] Ylipaasto | Enterovirus infection in human pancreatic islet cells, islet tropism in vivo and receptor involvement in cultured islet beta cells[END_REF][START_REF] Dotta | Coxsackie B4 virus infection of beta cells and natural killer cell insulitis in recent-onset type 1 diabetic patients[END_REF][START_REF] Richardson | The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes[END_REF]. However, the pathogenetic processes leading to virally-induced type 1 diabetes have not been characterized in detail. This study investigated the seroprevalences of HEV-D serotypes and their abilities to infect and damage cell types that are thought to play an essential role in enterovirus-induced diseases. A high seroprevalence and wide leukocyte tropism were detected for EV-68 and EV-94. EV-94 was able to damage pancreatic islet β-cells, to infect, replicate and cause necrosis in human pancreatic islets and to induce proinflammatory and chemoattractive cytokine expression in endothelial cells. The results suggest that HEV-D viruses may be more prevalent than has previously been thought. They also provide in vitro evidence that EV-94 may be a potent pathogen and should be considered a potentially diabetogenic enterovirus type.

Viruses

The isolation and plaque purification of EV-94 strain E210 have been described in a previous study [START_REF] Smura | Enterovirus 94, a proposed new serotype in human enterovirus species D[END_REF]. The prototype strains EV-68-Fermon and EV-70-J670/71 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Cell lines

The human rhabdomyosarcoma cell line (RD) was provided by the World Health Organization (WHO) Polio Labnet. The green monkey kidney cell line (GMK) has been maintained in the laboratory since the 1960s. The leukocyte cell lines KG-1, HL-60, THP-1, U-937, Jurkat and Raji were purchased from ATCC (Manassas, VA, USA). MOLT and RC-2A leukocyte cell lines were kindly provided by Leif Andersson (University of Helsinki, Finland). The leukocyte cell lines were maintained in RPMI 1640 medium (Cat. No. R-6504, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% foetal bovine serum (FBS), except for the KG-1 cell line, which was cultured in Iscove's modified Dulbecco's Medium (IMDM) (Invitrogen, Gibco, Carlsbad, CA, USA) supplemented with 10% FBS.

Human pancreatic islets

Human pancreatic islets were isolated and purified at the Uppsala University Hospital (coordinator, Prof. Olle Korsgren) as previously described [START_REF] Johansson | Inflammatory mediators expressed in human islets of Langerhans: implications for islet transplantation[END_REF], with the consent of the ethics committee of Uppsala University. After 1-5 days of culture in Ham's F10 (Cat. No. N6013, Sigma- Aldrich) medium supplemented with 10 mmol/l HEPES and 2% FBS, the islets were sent to Helsinki. Before the experiments, the islets were maintained for 1-6 days in sterile, non-adherent culture plates in incubation medium (Ham's F10 containing 25 mmol/l HEPES, pH 7.4, 2% FBS, penicillin and streptomycin). The islet cell studies were approved by ethics committees both in Finland (The Institutional Review Board) and in Sweden (the ethical committee of Uppsala University).

Endothelial cells

Human primary endothelial cells were isolated from umbilical cords obtained from the Department of Obstetrics and Gynaecology, Helsinki University Central Hospital, Finland, with parents' permission. The umbilical cords were collected in phosphate buffered saline (PBS). The cells were isolated as described by [START_REF] Saijets | Enterovirus infection and activation of human umbilical vein endothelial cells[END_REF]. Briefly, the tissue was digested with 0.03% collagenase (Cat. No. C-5138, Sigma-Aldrich, NY, USA) in Hanks balanced salt solution supplemented with 20 mM Hepes pH 7.4, at +37° C for 10 minutes. Isolated cells were washed with growth medium (DMEM, Cat. No. D-7777, Sigma-Aldrich, NY, USA) supplemented with 50 µg/ml gentamycin, 150 µg/ml endothelial cell growth supplement (ECGS, Cat. No. E0760, Sigma-Aldrich, NY, USA), 2.5 U/ml heparin (Cat. No. H3149, Sigma-Aldrich, NY, USA) and 15% FBS, and subsequently suspended in the same medium. The cells were cultured in gelatine (Cat. No 4070, Merck, Darmstadt, Germany) coated flasks (0.2% sterile filtered gelatine in PBS) for 7 days at +36° C in a 5% CO 2 atmosphere. The medium was changed three times a week. Before the experiments, the cells were harvested and seeded (approximately 0.3 × 10 6 cells/ml) into 8 chamber cell culture slides (Cat. No 354108, Falcon, Becton Dickinson, Lincoln Park, NJ, USA) for immunofluorescence assays, or into 96-well plates for infectivity assays. The purity of endothelial

Infection and viral progeny production

The cells were infected in Hanks' balanced salt solution supplemented with 20 mM Hepes pH 7.4.

For the leukocyte cell lines, 10 6 cells/ml and a multiplicity of infection (m.o.i.) of 0.2-0.5 were used. The endothelial cells were infected at 80-95% confluency with various multiplicities (m.o.i. 0.01-10). The human pancreatic islets were infected as described previously [START_REF] Ylipaasto | Global profiling of coxsackievirusand cytokine-induced gene expression in human pancreatic islets[END_REF]. Approximately 2000 islets/ml and a high m.o.i. were used. After incubation of 1 h at +36° C in a 5% CO 2 atmosphere, the cells were washed twice with Hanks balanced salt solution and growth medium (supplemented with 20 mM Hepes pH 7.4, 20 mM MgCl, 1% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin) was added to the cultures. For pancreatic islets, a serum-free medium was used (Ham's F10 supplemented with 25mM Hepes pH 7.4, 1% BSA, 100 U/ml penicillin and 0.1 mg/ml streptomycin). The cultures were incubated at +36° C in a 5% CO 2 atmosphere.

For virus titration, the cell cultures were harvested at different intervals and freeze thawed three times. The total infectivity of each sample was determined by end-point titration in microwell cultures of RD cells. Cytopathic effects (cpe) were read on day 6 after infection by microscope, and 50% tissue culture infectious dose (TCID 50 ) titres were calculated using the Kärber formula (Lenette, 1969). The infected cells were fixed with cold methanol for 15 min at +4° C, washed three times with PBS and incubated with antiserum for 1 h at +36° C. A polyclonal rabbit antiserum produced against EAIPALTAVETGHTSQVC-peptide designed according to the VP1 region of the enterovirus genome [START_REF] Harkonen | Enterovirus infection can induce immune responses that cross-react with beta-cell autoantigen tyrosine phosphatase IA-2/IAR[END_REF], or polyclonal rabbit antiserum against EV-94 (K-HAX), was used.

The endothelial cells were identified with Factor VIII related antigen (vWF) specific antibody (Cat No. MS-722-S, Neomarkers). Insulin-producing islet β-cells were identified with insulin-specific polyclonal sheep antiserum (30 µg/ml; PC059, The Binding Site, Birmingham, UK). Unbound antibody was removed by washing three times with PBS. The conjugates for rabbit Jackson ImmunoReseach Laboratories,Inc.,West Grove,PA,USA) and mouse antibodies (antimouse REDX, Cat No. 715-295-150 Jackson ImmunoReseach Laboratories, Inc.) were incubated for 30 min at +36° C. After staining, the slides were washed five times with PBS. The slides were analysed under a confocal microscope (Leica TCS SPE, Wetzlar, Germany).

Activation of endothelial cells

The endothelial cells were either infected with viruses at m.o.i. of 1-10, incubated for 24 h with recombinant human interleukin-1β (10 ng/ml, 1500 U/ml, Becton Dickinson) or mock infected. The samples were fixed with cold methanol and stained with mouse anti-human intercellular adhesion molecule 1 (ICAM-1) (Cat. No. HM4004, HyCult Biotechnology bv., Uden, The Netherlands) or mouse anti-human E-selectin (Cat. No. HM4001 HyCult Biotechnology bv., Uden, The Netherlands) antibodies as described above. For the measurement of DNA and insulin content, the islet cells were homogenized ultrasonically in distilled water. DNA was measured using Fluoreportex Blue Fluorometric dsDNA quantitation kit (Cat. no. F2962, Molecular probes). Insulin was measured with a solid-phase insulin RIA kit (DPC, Los Angeles, CA, USA) after overnight extraction with acid ethanol as described previously [START_REF] Otonkoski | Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells[END_REF].

Cell viability assays

Serum neutralization assay

The study group for the serum neutralization assay consisted of 281 women at the end of the first trimester of pregnancy. The sera were derived from the Finnish Maternity Cohort and had been collected in 1983 (n=86), 1993 (n=99) and 2002 (n=96).

Cytokine assay

The capability of EV-94 to induce cytokine expression in endothelial cells was measured using multiplex bead-based assays based on xMAP technology (Bio-Plex; Biorad Laboratories, Hercules, CA, USA). The parallel wells of endothelial cells (~75 000 cells/well) were infected with EV-94 at the m.o.i. of 4 or 0.01, or they were mock infected. The culture media supernatant was collected 0, 6, 24 and 48 hours post infection and assayed for 48 cytokines. The limits of detection and the coefficients of variability (intra Assay % CV and inter Assay % CV) of the cytokine/chemokine assay were: IL-1α: 0.5 pg/ml (4; 4) ; IL-1β: 0.6 pg/ml (6; 8); IL-1ra: 5.5 pg/ml (9;8); IL-2: 1.6 pg/ml (7; 9); IL-2Rα: 2.1 (6;4); IL-3: 4.8 pg/ml (7;4); IL-4: 0.7 pg/ml (9;8); IL-5: 0.6 pg/ml (8;10); IL-6: 2.6 pg/ml (7;11); IL-7: 1.1 pg/ml (6;8); IL-9: 2.5 pg/ml (8;9); IL-10: 0.3 pg/ml (5;6); IL-12p40: 23.3 pg/ml (5;8); IL-12p70: 3.5 pg/ml (6;6); IL-13: 0.7 pg/ml (8;7); IL-15: 2.4 pg/ml (5;6); IL-16: 0.4 pg/ml (6; 4); IL-17: 3.3 pg/ml (8;6); IL-18: 0.2 pg/ml (4;5); INFγ: 6.4 pg/ml (15;9); TNFα: 6 pg/ml (8;6); TNFβ: 0.3 pg/ml (4;4); GM-CSF: 2.2 pg/ml (12;6); G-CSF: 1.7 pg/ml (10;5); M-CSF: 0.9 pg/ml ((4,5); CCL2/MCP-1: 1.1 pg/ml (9;7); CCL3/MIP-1α : 1.6 pg/ml (7;8); CCL4/MIP-1β: 2.4 pg/ml (8;8); CCL5/Rantes: 1.8 pg/ml (9;6);CCL7/MCP-3: 1 pg/ml (7;8); CCL11/Eotaxin: 2.5 pg/ml (8;11); CCL27/CTAK: 3.4 pg/ml (5;6); CXCL1/GRO-α: 6.3 pg/ml (5; ); CXCL9/MIG: 1.2 pg/ml (6;6); CXCL10/IP-10: 6.1 pg/ml (11; 9); CXCL12/SDF1α: 8.7 pg/ml (6;6); VEGF: 3.1 pg/ml (9;7); CXCL8/IL-8: 1.0 pg/ml (9;4); basic FGF: 1.9 pg/ml (8;8); PDGF-BB: 2.9 pg/ml (9; 8); HGF: 4.9 pg/ml (5; 6); IFN-α2: 4.3 pg/ml (7;3); LIF: 5.5 pg/ml (4;3); MIF:

1.5 pg/ml (5;8); β-NGF: 0.2 pg/ml (4;7); SCF: 1 pg/ml (5;4); SCGF-β: 45.4 pg/ml (6;8); TRAIL 2.1pg/ml (4;8).

Statistical analysis

One-way analysis of variance (ANOVA) and Kruskal-Wallis one-way analysis of variance were used for statistical evaluation of cell viabilities and serum antibody levels, respectively.

RESULTS

Seroprevalence of HEV-D serotypes in Finland

A previous study detected a high seroprevalence of EV-94 in the Finnish population [START_REF] Smura | Enterovirus 94, a proposed new serotype in human enterovirus species D[END_REF]. In this study, the prevalence of antibodies against the two other HEV-D serotypes, EV-68 and EV-70, in Finland was assessed using the serum neutralization assay. Serum samples obtained from pregnant women and collected in 1983 (n=86), 1993 (n=99) and 2002 (n=96) were studied.

Neutralizing antibodies against EV-68 were found in 100%, against EV-70 in 15.1%, 21.2 % and 13.5 %, and against EV-94 in 79.1%, 79.8% and 79.2% of the subjects in the years 1983, 1993 and 2002, respectively (Fig. 1).

The geometric means for the serum end point titres are shown in Table 1. The serum samples studied had significantly higher antibody levels against EV-68 than against EV-70 or EV-94 in all of the years for which samples were studied (Kruskal-Wallis p < 0.01). The mean antibody levels against EV-68 showed a decreasing temporal trend whereas those against EV-70 and EV-94 remained constant over the study period (Kruskal-Wallis p < 0.01).

Susceptibility of human leukocyte cell lines to EV-94 and EV-68

Replication in blood cells may provide enteroviruses an additional replication site during viremia, thus lengthening the viremia and/or increasing the viral load during viremia. The viruses may also spread to secondary target tissues within infected circulating leukocytes. Therefore, the susceptibilities of monocytic (THP-1, RC-2A and U937), granulocytic (HL-60 and KG-1), T-cell (MOLT and Jurkat) and B-cell (Raji) lineages to EV-94 and EV-68 infections were studied. or in the granulocytic cell line HL-60 (Fig. 2 c). EV-68 infection did not induce statistically significant changes in the viabilities of the leukocytes (Fig. 2 d).

Susceptibility of endothelial cells to viral infections

Replication in the endothelial cell lining of blood vessels may provide a virus with an access route to secondary target tissues. To assess the susceptibility of endothelial cells to EV-68, EV-70 and EV-94 infections, single-step growth curve experiments were carried out in primary human umbilical vein derived endothelial cells.

EV-94 and EV-70 showed a definite cytopathic effect and progeny production in endothelial cells (Fig. 3a). However, there was substantial donor-specific variation in the susceptibility of endothelial cells to HEV-D infections. Infective progeny formation was detected in the cells isolated from twelve out of fourteen donors after EV-94 infection and in seven out of eleven donors after EV-70 infection. A low level of progeny formation but no definitive cytopathic effect was detected after EV-68 infection in the cells derived from two out of six donors.

To verify that the viruses infected endothelial cells specifically, the endothelial cell specific Factor VIII related antigen (von Willebrand factor) and enterovirus capsid protein were co-stained. EV-94 16 capsid protein positive cells were detected in a subset of endothelial cells (Fig. 3b). Owing to a lack of suitable antibody against EV-68 and EV-70, such staining could not be performed for these viruses.

EV-94 induced cytokine expression in endothelial cells

The capability of EV-94 to induce cytokine expression in endothelial cells was measured using multiplex bead-based assays based on xMAP technology. The parallel wells of endothelial cells (~75 000 cells/well) were infected with EV-94 at the m.o.i. of 4 or 0.01, or they were mock infected. The culture media supernatant was collected 6, 24 and 48 hours post infection and assayed for 48 cytokines.

Ten cytokines showed over fivefold increase in EV-94 infected cell supernatants compared to mock-infected controls (Table 2). Seven of these peaked 48 h post infection and showed a stronger response to high m.o.i. infection than to low m.o.i. infection. The interferon γ (IFN-γ) levels showed a stronger response to low m.o.i. infection. CCL11/Eotaxin and VEGF peaked 6 h after high m.o.i.

infection and 24 h after low m.o.i infection, respectively.

Fifteen cytokines showed detectable expression, but no differences were detected between EV-94 and mock-infected cell cultures. The highest concentrations were detected for CXCL1/GRO-α (max 1117 pg/ml), MIF (max 2685 pg/ml), PDGF-BB (max 601 pg/ml), IL-6 (max 1497 pg/ml) and CXCL8/IL-8 (max 7722 pg/ml

Activation of endothelial cells by viruses

The capability of HEV-D viruses to induce the activation of endothelial cells was studied by immunohistochemical staining of E-selectin and ICAM-1, which are surface molecules associated with the activation of endothelial cells [START_REF] Collins | Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers[END_REF]. None of the viruses (EV-68, EV-70 or EV-94) was able to induce E-selectin or ICAM-1 expression in the endothelial cell surface 6-24 h after infection, whereas cells treated with IL-1 showed definite E-selectin and ICAM-1 expression (the number of donors studied: : n=5 for EV-94; and n= 2 for EV-68 and EV-70).

Human pancreatic islet infection with EV-94 and EV-68

The pancreas is one of the most important secondary target tissues for human enteroviruses, and enteroviruses have been considered to play a significant role in the aetiology of type 1 diabetes. The pancreatic islets of three donors were infected with both EV-94 and EV-68. Viral infection led to the detection of an infectious progeny formation with both viruses (Fig. 4a). The viability of the islets was drastically reduced 4-7 days after infection with EV-94 (n=4), whereas the mock-infected . The co-staining of viral protein and insulin indicated that both viruses were able to infect insulin-producing pancreatic β-cells (Fig. 4c). To assess the β-cell specific destruction, the insulin to cellular DNA ratios were measured at several time points after infection. The insulin to DNA ratio decreased significantly 8-14 days after infection with EV-94 (n=3). There was no change in the insulin to DNA ratio after EV-68 infection (n=2) (Fig. 4d). The results for EV-68 and EV-94 obtained in this study and in earlier studies on EV-70 [START_REF] Haddad | Binding to decay-accelerating factor is not required for infection of human leukocyte cell lines by enterovirus 70[END_REF] suggest that HEV-D viruses have an unusually wide leukocyte tropism. However, the role of leukocytes in enterovirus pathogenesis is somewhat obscure. After primary infection of the gastrointestinal tract, the viremia could be started via leukocytes if the virus were able to infect the dendritic cells and/or macrophages of Payer's patches. Leukocytes may also serve as an additional reservoir for virus replication, thereby lengthening the viremic phase and increasing the amount of virus in circulation. Accordingly, a correlation between poliovirus neurovirulence and replication efficiency in monocytes has been observed [START_REF] Freistadt | Correlation between poliovirus type 1 Mahoney replication in blood cells and neurovirulence[END_REF]. Tissue migrating leukocytes may also provide a virus with an access to secondary target tissues. Viral replication in leukocytes is also likely to affect the host immune system [START_REF] Kramer | Echovirus infection causes rapid loss-of-function and cell death in human dendritic cells[END_REF][START_REF] Kramer | Phagocytosis of picornavirus-infected cells induces an RNA-dependent antiviral state in human dendritic cells[END_REF]. At least some enteroviruses (e.g. CBV-3, CBV-4 and PV-1) are able to induce pro-inflammatory cytokine expression in human leukocytes [START_REF] Henke | Coxsackievirus B3-induced production of tumor necrosis factor-alpha, IL-1 beta, and IL-6 in human monocytes[END_REF][START_REF] Vreugdenhil | Enterovirus-induced production of pro-inflammatory and T-helper cytokines by human leukocytes[END_REF]. This can cause and/or maintain local inflammation in the secondary target tissues, thus contributing to immunemediated chronic diseases such as type 1 diabetes.

It seems there are considerable differences between the abilities of different enterovirus serotypes to infect and replicate in leukocytes. Poliovirus is able to infect the monocytic fraction of peripheral blood mononuclear cells [START_REF] Eberle | Low levels of poliovirus replication in primary human monocytes: possible interactions with lymphocytes[END_REF], monocytic and granulocytic cell lines (Kitamura et , 1985[START_REF] Okada | Poliovirus infection of established human blood cell lines: relationship between the differentiation stage and susceptibility of cell killing[END_REF][START_REF] Roivainen | Replication of poliovirus in human mononuclear phagocyte cell lines is dependent on the stage of cell differentiation[END_REF][START_REF] Freistadt | Correlation between poliovirus type 1 Mahoney replication in blood cells and neurovirulence[END_REF][START_REF] Vuorinen | Enterovirus receptors and virus replication in human leukocytes[END_REF] and monocyte-derived dendritic cells and macrophages [START_REF] Wahid | Dendritic cells and macrophages are productively infected by poliovirus[END_REF]. However, both T-cells and B-cells seem to be resistant to poliovirus infection [START_REF] Okada | Poliovirus infection of established human blood cell lines: relationship between the differentiation stage and susceptibility of cell killing[END_REF][START_REF] Vuorinen | Enterovirus receptors and virus replication in human leukocytes[END_REF]. Coxsackie B viruses, in contrast, are able to replicate and persist in T-cell and B-cell lines [START_REF] Matteucci | Group B coxsackieviruses readily establish persistent infections in human lymphoid cell lines[END_REF][START_REF] Vuorinen | Coxsackievirus B3 infection in human leukocytes and lymphoid cell lines[END_REF], 1996), but not in the granulocytic or monocytic lineages or monocyte-derived dendritic cells [START_REF] Vuorinen | Susceptibility of human bone marrow cells and hematopoietic cell lines to coxsackievirus B3 infection[END_REF][START_REF] Kramer | Echovirus infection causes rapid loss-of-function and cell death in human dendritic cells[END_REF]. The other members of HEV-B species -echoviruses -seem to have a very different leukocyte tropism. Echo 1 is able to replicate in monocytic (U937) cells but not in B-cells or T-cells (Raji or Molt-4) [START_REF] Vuorinen | Enterovirus receptors and virus replication in human leukocytes[END_REF]. Several echovirus strains are able to infect monocyte-derived dendritic cells and to form productive progeny in them, but are not able to infect PBMC-isolated monocytes [START_REF] Kramer | Echovirus infection causes rapid loss-of-function and cell death in human dendritic cells[END_REF].

Virus receptors are considered to be one of the key determinants of viral tissue tropism. Receptor usage may thus be one of the causative factors of the wide leukocyte tropism of HEV-D viruses.

The receptor for EV-94 is not known, but both EV-70 and EV-68 use DAF as a receptor in Hela cells and sialic acid-containing receptors in leukocytes [START_REF] Uncapher | The major and minor group receptor families contain all but one human rhinovirus serotype[END_REF], Karnaukhow et al., 1996[START_REF] Alexander | Sialic acid functions in enterovirus 70 binding and infection[END_REF][START_REF] Blomqvist | Human rhinovirus 87 and enterovirus 68 represent a unique serotype with rhinovirus and enterovirus features[END_REF].

Another important site in enterovirus pathogenesis is the endothelial cell lining of the circulatory In this study, EV-94 and EV-70 were capable of infecting primary human umbilical vein endothelial cells, whereas EV-68 had only marginal progeny production and did not induce cytopathic effect in these cells. It has previously been shown that many other enterovirus strains, including poliovirus 1 [START_REF] Macgregor | Virus infection of endothelial cells increases granulocyte adherence[END_REF][START_REF] Friedman | Virus infection of endothelial cells[END_REF], coxsackie B viruses [START_REF] Conaldi | Persistent infection of human vascular endothelial cells by group B coxsackieviruses[END_REF][START_REF] Zanone | Persistent infection of human microvascular endothelial cells by coxsackie B viruses induces increased expression of adhesion molecules[END_REF] and some of the coxsackie A and echoviruses (CVA-13, E-6, E-7, E-9, E-11, E-30) [START_REF] Friedman | Virus infection of endothelial cells[END_REF][START_REF] Saijets | Enterovirus infection and activation of human umbilical vein endothelial cells[END_REF] are able to infect and replicate in primary human endothelial cells. Most of the serotypes (e.g. echoviruses, PV-1 and CVA-13) induce cell lysis [START_REF] Saijets | Enterovirus infection and activation of human umbilical vein endothelial cells[END_REF], whereas persistent infection is a more prominent outcome of infection with coxsackie B viruses [START_REF] Conaldi | Persistent infection of human vascular endothelial cells by group B coxsackieviruses[END_REF][START_REF] Zanone | Persistent infection of human microvascular endothelial cells by coxsackie B viruses induces increased expression of adhesion molecules[END_REF].

An enterovirus infection can induce tissue damage either by direct cytolysis or by immunopathogenetic mechanisms (autoimmunity or virus-targeted immunity). With regard to virusinduced type 1 diabetes, the possible pathogenetic mechanisms (which are not mutually exclusive) include direct cytolysis of pancreatic β-cells after viral infection, virally-induced bystander activation of autoreactive T-cells [START_REF] Horwitz | Diabetes induced by Coxsackie virus: initiation by bystander damage and not molecular mimicry[END_REF] and molecular mimicry [START_REF] Atkinson | Cellular immunity to a determinant common to glutamate decarboxylase and coxsackie virus in insulin-dependent diabetes[END_REF].

While direct cytolysis and/or virus-targeted immunity can be a possible cause for some cases of fulminant type 1 diabetes [START_REF] Imagawa | Fulminant type 1 diabetes mellitus[END_REF][START_REF] Akatsuka | A case of fulminant type 1 diabetes with coxsackie B4 virus infection diagnosed by elevated serum levels of neutralizing antibody[END_REF], it is likely that autoimmune reactions triggered by viral infection are also involved in enterovirus-induced type 1 diabetes. In this scenario, a virus infection in the pancreas and a (limited) β-cell damage (by a virus and/or virally-induced cytokines) would result in the exposure of autoantigens in a local differentiation and suppresses Th2 differentiation [START_REF] Goodbourn | Interferons: cell signalling, immune modulation, antiviral response and virus countermeasures[END_REF]. Moreover, IFN-γ (in combination with TNF-α, IL-1 or synthetic dsRNA analogue polyinosinic-polycytidylic acic) is sufficient to induce apoptosis and to upregulate chemokine expression in pancreatic β-cells [START_REF] Eizirik | A choice of death--the signal-transduction of immunemediated beta-cell apoptosis[END_REF], Liu et al., 2001[START_REF] Ylipaasto | Global profiling of coxsackievirusand cytokine-induced gene expression in human pancreatic islets[END_REF]. Thus the endothelial cells can both provide an accession route for a virus to infect pancreatic β-cells and contribute to the necessary proinflammatory milieu for the induction of beta cell autoimmunity.

Endothelial cell activation could not be detected after EV-94 infection (data not shown), even though activation promoting cytokines -e.g. IL-1A, which has been shown to be the predominant endothelial cell activating factor after EV-70 infection [START_REF] Chang | Towards an in vitro model for acute hemorrhagic conjunctivitis: cytokine-mediated vascular endothelial cell activation triggered by enterovirus type 70 infection[END_REF]) -were detected. This may be due to the rapid lysis of endothelial cells following high m.o.i. EV-94 infection. Some other enterovirus serotypes (e.g. CBV-2, CBV-4, CBV-5, CVA-9, CVA-13, E-1, E-11, E-30, PV-1, EV-70 and EV-71) have been shown to induce endothelial cell activation [START_REF] Saijets | Enterovirus infection and activation of human umbilical vein endothelial cells[END_REF][START_REF] Chang | Towards an in vitro model for acute hemorrhagic conjunctivitis: cytokine-mediated vascular endothelial cell activation triggered by enterovirus type 70 infection[END_REF][START_REF] Liang | Human endothelial cell activation and apoptosis induced by enterovirus 71 infection[END_REF][START_REF] Zanone | Human pancreatic islet endothelial cells express coxsackievirus and adenovirus receptor and are activated by coxsackie B virus infection[END_REF]. The endothelial cells derived from different organs show distinct susceptibility to enterovirus infections, and the cellular response of macrovascular and microvascular endothelial cells to viral challenge may differ [START_REF] Huber | Functional diversity in vascular endothelial cells: role in coxsackievirus tropism[END_REF][START_REF] Conaldi | Persistent infection of human vascular endothelial cells by group B coxsackieviruses[END_REF][START_REF] Zanone | Persistent infection of human microvascular endothelial cells by coxsackie B viruses induces increased expression of adhesion molecules[END_REF], 2007, 2008).

Further studies are needed to assess the capability of different enterovirus types to replicate in microvascular endothelial cells and to determine the host cell responses after enterovirus infection.

EV-68 and EV-70 differ from most enteroviruses by infecting their primary target tissues (respiratory tract for EV-68 and conjunctiva for EV-70) directly. The primary target tissue of EV-94

is not known, but the acid stability of this virus suggests that it may use the faecal-oral route of transmission [START_REF] Smura | Enterovirus 94, a proposed new serotype in human enterovirus species D[END_REF]. Leukocyte and endothelial cell tropism of HEV-D viruses suggests that these viruses also have the potential to cause secondary target tissue infections, although the low endothelial cell tropism of EV-68 suggests that secondary target tissue infections may be less common after EV-68 than after EV-70 or EV-94 infection. Indeed, both EV-70 and, to an lesser extent, EV-68 can infect occasionally the central nervous system [START_REF] Pallansch | Enteroviruses: polioviruses, coxsackieviruses, echoviruses and newer enteroviruses[END_REF][START_REF] Khetsuriani | Enterovirus surveillance--United States, 1970-2005[END_REF], and both in vitro results in neuroblastoma cells [START_REF] Smura | Enterovirus 94, a proposed new serotype in human enterovirus species D[END_REF] and the association with acute flaccid paralysis cases [START_REF] Junttila | New enteroviruses, EV-93 and EV-94, associated with acute flaccid paralysis in the Democratic Republic of the Congo[END_REF] suggest that EV-94 may also be neurovirulent. According to the in vitro results of this study, EV-94 may also have another secondary target: the pancreatic islets.

Since an unexpectedly high seroprevalence of EV-94 was detected previously in the Finnish population [START_REF] Smura | Enterovirus 94, a proposed new serotype in human enterovirus species D[END_REF], the seroprevalences of the other two HEV-D serotypes (EV-68 and EV-70) were assessed. EV-68 seems to have an extremely high prevalence in Finland. This observation is in line with the findings of a previous study on a limited number of individuals [START_REF] Blomqvist | Human rhinovirus 87 and enterovirus 68 represent a unique serotype with rhinovirus and enterovirus features[END_REF]. The high seroprevalence of EV-68 is in an apparent contrast with the findings of enterovirus surveillance studies from clinical or environmental samples ( (d) Insulin to DNA ratios of islet preparations 8-14 days after infection with EV-68 and EV-94.

DNA was measured using a Fluoreportex Blue Fluorometric dsDNA quantitation kit (Molecular probes), and insulin was measured with a solid-phase insulin RIA kit (DPC).

TABLES

Table 1. The geometric means of the end-point titres for neutralizing antibodies against EV-68, EV-70 and EV-94 in the sera of Finnish women at the end of the first trimester of pregnancy.

Table 2. Cytokines showing more than a fivefold increase in the endothelial cell culture supernatants after infection with EV-94.

  Fig 3b 155x96mm (300 x 300 DPI)

  determined by staining von Willebrand's factor (vWF) with the immunofluorescence technique as described below.

  WST-1 (Cat. No. 1644807, Roche Diagnostics, Mannheim, Germany) and Sytox Green nucleic acid stain (Cat. No. S7020, Invitrogen) were used for quantification of leukocyte and endothelial cell viabilities, respectively. In WST-1 assay, a tetrazolium salt WST-1 is added to the cell culture medium. The tetrazolium salts are cleaved to formazan by cellular enzymes of metabolically active cells. The number of viable cells is proportional to the amount of formazan dye, which was quantified by using a spectrophotometer (Wallac 1420 Victor 2 TM, PerkinElmer, MA, USA) to measure the absorbance of the culture media.. The viability of human pancreatic islets was assessed using Live/Dead assay (Cat No L3224, Molecular Probes, Leiden, The Netherlands) and a confocal microscope.DNA and insulin content of pancreatic β β β β-cells.

  sera were inactivated at +56º C for 20 minutes and stored at +4º C. Serial fourfold dilutions of sera were mixed with an equal volume of virus (100 TCID 50 ). The mixtures were neutralized first at +36º C for 1 hour and then overnight at +4º C. The mixtures were transferred into 96-well cell culture plates with RD cells for EV-70 and EV-94, or with GMK cells for EV-68 (~0.3 million cells ml -1 ), and incubated at +36° C in a 5% CO 2 atmosphere for 6 days. The highest dilution completely inhibiting the viral cytopathic effect was regarded as the end point titre of the serum.

  94, infectious progeny production was detected in all studied cell lines 24 hours after infection (Fig.2 a). The viabilities of the cells were reduced significantly three days after EV-94 infection (ANOVA p < 0.005) (Fig.2 b). For EV-68, an increase in viral titre was detected in T-cell lines MOLT and Jurkat, in the B-cell line Raji, the granulocytic line KG-1 and in the monocytic line U-937, whereas no definitive infective virus production was detected in the monocytic RC-2A line

  the islets infected with EV-68 (n=3) remained viable during the same period of time(Fig 4b)

  the in vitro pathogenetic properties of the enterovirus serotypes of the HEV-D species (EV-68 and EV-70) were compared in cell types that are considered to be essential for enterovirusinduced diseases. The cell types studied included human leukocyte cell lines, primary endothelial cells and pancreatic islets.

  system. Infection of endothelial cells may affect viral pathogenesis through several mechanisms: 1) viral progeny production in endothelial cells can provide the virus a route for infecting the parenchymal tissue; 2) viral replication in endothelial cells can increase the local amount of virus, thereby increasing the probability, and possibly altering the outcome, of secondary target tissue infection; 3) the virus-induced activation of endothelial cells can promote chemoattraction and the leukocytes; 4) virally-induced proinflammatory cytokine production can induce inflammation or can be directly harmful for parenchymal cells.

  leading to upregulation of MHC molecules and up-take and presentation of autoantigens by activated antigen-presenting cells, which in turn would promote further β-cell damage by activating autoreactive T-cells(Filippi & Von Herrath, 2005).With regard to the virus-induced development of autoimmune diabetes, it is intriguing that EV-94 is capable both of damaging human pancreatic islet β-cells and of inducing IFN-γ (along with other proinflammatory cytokines and chemokines like IL-1α and CXCL12/SDF-1α) expression in endothelial cells. In addition to antiviral effects, IFN-γ is considered to play a significant role in autoimmune diseases. Among other effects, IFN-γ enhances the expression of MHC-I proteins and increases the repertoire and quantity of peptides displayed to CD8+ T-cells. It also promotes Th1

FIGURESFig. 1 .

 1 FIGURES

Fig. 2 .

 2 Fig. 2. Infectious progeny production of EV-94 (a) and EV-68 (c) in leukocyte cell lines. The cells

Table 1 .

 1 The geometric means of the end-point titres for neutralizing antibodies against EV-68, EV-70 and EV-94 in the sera of Finnish women at the end of the first trimester of pregnancy.

	1983	1993	2002

  [START_REF] Roivainen | Enteroviruses: new findings on the role of enteroviruses in type 1 diabetes[END_REF][START_REF] Blomqvist | Eight years of experience with molecular identification of human enteroviruses[END_REF]. However EV-68 may be underrepresented in clinical samples as a result of mild or subclinical symptoms. In most studies, enteroviruses are sought from stool samples or from waste water; EV-68 may remain undetected in these studies owing to respiratory tract tropism, temperature sensitivity and acid lability. Several EV-68 strains have been detected recently from respiratory tract specimens[START_REF] Savolainen-Kopra | All Known Human Rhinovirus Species Are Present in Sputum Specimens of Military Recruits During Respiratory Infection[END_REF][START_REF] She | Performance of enterovirus genotyping targeting the VP1 and VP2 regions on non-typeable isolates and patient specimens[END_REF]. The mean antibody levels against EV-68 have decreased during the study period, suggesting antigenic drift among circulating EV-68 strains.In conclusion, the results of this study suggest that HEV-D viruses may be more prevalent than has been thought previously, and that they have wide tropism for leukocytes and endothelial cells. It was also shown that EV-94 is able to damage human pancreatic islet β-cells in vitro and to induce proinflammatory and chemoattractive cytokine expression in endothelial cells, and should therefore be considered a potentially diabetogenic enterovirus serotype.

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	P P P P P P P P P P
	e e e e e e e e e e
	e r e r e r e r e r e r e r e r e r e r
	R R R R R R R R R R
	e e e e e e e e e e
	v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e
	w w w w w w w w w w
	Khetsuriani et
	33

John Wiley & SonsJournal of Medical Virology

Journal of Medical Virology

ACKNOWLEDGEMENTS

We thank Olle Korsgren for providing the human pancreatic islets, Hilkka Puttonen for providing the umbilical cords and Carita Savolainen-Kopra and Soile Blomqvist for valuable discussions. This project was funded by the Finnish Cultural Foundation, the European Union (EP7-HEALTH-2007, DIAPREPP N202013), the Academy of Finland and the Juvenile Diabetes Research Foundation (USA).