Catherine Mengelle
email: mengelle.c@chu-toulouse.fr

Nassim Kamar

Jean Michel Mansuy

Karine Sandres-Sauné

Florence Legrand-Abravanel

Marcel Miédougé

Lionel Rostaing

Jacques Izopet

J-M Mansuy

Miédougé

JC VIRUS DNA IN THE PERIPHERAL BLOOD OF RENAL TRANSPLANT PATIENTS: A ONE-YEAR PROSPECTIVE FOLLOW-UP IN FRANCE

Keywords: Polyomavirus, solid-organ transplantation, immunosuppressive treatment, nephropathy, progressive multifocal leukoencephalopathy Polyomavirus, nephropathy, progressive multifocal leukoencephalopathy, solid-organ 49 transplantation, immunosuppressive treatment 50

HAL is

There is little information on JC virus (JCV) infection in renal transplant patients. A long-term 28 prospective follow-up study was conducted to assess the incidence of JCV DNA in the blood of 29 103 adult renal transplant patients enrolled prospectively between 1 January and 31 December, 30 2006. Patients were monitored until April, 2008. JCV DNA was quantified by a real-time 31 polymerase chain reaction in whole blood samples collected regularly for at least one year post-32 transplant. 33 JCV was detected in seven patients (6.8 %) (31/1487 whole blood samples) at a median time of 34 139 days post-transplant. The median JC virus load of the first positive DNA blood sample was 35 3.4 log 10 copies/mL (1.9 -5.7 log 10 copies/mL). Induction therapy were either anti-CD25 36 monoclonal antibodies (n=5) or antithymocyte globulins (n=2). Post-transplant 37 immunosuppressive treatment included steroids with tacrolimus/mycophenolate mofetil (MMF) 38 (n=2), or ciclosporin/MMF (n=1), or belatacept/MMF (n=4). Two patients were also treated 39 with rituximab. All seven patients infected with JCV had other viral infections(s): BK virus (3), 40

Epstein-Barr virus (2), Cytomegalovirus (1) or both BK virus and Epstein-Barr virus (1). Three 41 patients had BKV-associated nephropathy and decoy cells shedding. JCV infection was not 42 associated with acute rejection episodes or nephropathy, regardless of the virus load. No patient 43 developed progressive multifocal leukoencephalopathy during follow-up. 44

Thus the incidence of JCV infection in renal transplant patients was low and not associated with 45 any specific clinical manifestations. JCV replication must still be diagnosed and differentiated 46 from BK virus infection because of its non-aggressive course. 47 JC virus (JCV) is a member of the Polyomaviridae, which include other human viruses such as BK 52 virus, KI [START_REF] Allander | Identification of a third human polyomavirus[END_REF], WU [START_REF] Gaynor | Identification of a novel polyomavirus from patients with 269 acute respiratory tract infections[END_REF] and SV40. JC and BK virus infections 53 are frequent and occur early in childhood. Seropositivity increases with age; BKV reaches a 54 seroprevalence of 91% in children aged 5-9 years, while seroprevalence of JCV reaches only 50% 55 by the age of 60-69 years [START_REF] Knowles | Population-293 based study of antibody to the human polyomaviruses BKV and JCV and the simian 294 polyomavirus SV40[END_REF]. The transmission routes are not yet clear but are 56 thought to be via the respiratory tract, although oral-fecal transmission is also possible. 57

Both viruses remain latent in the kidney, in B lymphocytes [START_REF] Lafon | JC virus remains latent in peripheral blood B lymphocytes but replicates actively in 300 urine from AIDS patients[END_REF]Monaco et al., 58 1996] and in the central nervous system after primary infection, but they may be reactivated at 59 any time. JCV excretion in the urine can reach 46 % in immunocompetent patients, and the virus 60 usually remains asymptomatic [START_REF] Pagani | Molecular analysis of 309 JC virus genotypes circulating among the Italian healthy population[END_REF][START_REF] Rossi | 321 Presence, quantitation and characterization of JC virus in the urine of Italian 322 immunocompetent subjects[END_REF]. However, reactivation of 61 either virus in immunosuppressed patients can cause several clinical syndromes. JCV can become 62 reactivated in patients infected with HIV, transplant recipients and patients with cancer, and may 63 produce progressive multifocal leukoencephalopathy, a fatal neurological demyelinating disease 64 [START_REF] Hou | Progressive multifocal leukoencephalopathy: JC virus induced 284 demyelination in the immune compromised host[END_REF]. Polyomavirus-associated nephropathy in renal transplant patients is 65 mostly due to BKV, but recent studies have shown that JCV infection may also be involved 66 [START_REF] Drachenberg | Polyomavirus BK versus 258 JC replication and nephropathy in renal transplant recipients: a prospective evaluation[END_REF]. 67

Little is known at present on the incidence of JCV infection and its clinical consequences in these 68 Adult renal transplant recipients under the care of Toulouse University Hospital were enrolled 77 prospectively from 1 January to 31 December, 2006 andwere monitored until April, 2008. 78 Any induction therapy was based on antithymocyte globulins or anti-CD25 monoclonal 79 antibodies. All patients were treated with steroids for post-transplant immunosuppression. They 80 were also given tacrolimus/mycophenolate mofetil (MMF), or cyclosporin A/MMF, 81 belatacept/MMF, or cyclosporin/everolimus. Renal biopsies were taken when acute rejection was 82 suspected. The patients were given three injections of methylprednisolone if acute rejection was 83 confirmed. Antithymocyte globulins were given to cases of steroid-resistant acute rejection. 84

Finally, patients with humoral acute rejection were treated by plasmapheresis and rituximab. 85

Recipients were given prophylaxis for human cytomegalovirus (HCMV). Seropositive recipients 86 who had received a transplant from a seropositive or a seronegative donor, and high-risk patients 87 (a seronegative recipient with a seropositive donor) were given valganciclovir prophylaxis (900 88 mg/d if their creatinine clearance was > 60 ml/min, and 450 mg/d if their creatinine clearance 89 was < 60 ml/min) during the first 3 to 4 months post-transplantation. 90

Virological monitoring 91

All renal transplant recipients were tested prospectively for JCV excretion. Whole blood samples 92 were collected into potassium EDTA tubes from the time of transplantation to at least one year 93 post-transplant. 94 France). The use of specific primers and probes insures no cross reactivity with BKV. Extracted 106 DNA (5 µL) was added to the PCR mixture containing 2 mmol/L MgCl 2 , 0.500 µmol/L of each 107 primer, and 0.100 µmol/L of probe. The PCR conditions were initial denaturation for one cycle 108 of 2 min at 50°C followed by 2 min at 95°C, followed by 45 cycles of 20 s at 95°C and 60 s at 58 109 °C. Cooling was realised 30 s at 40 °C. The reaction, data acquisition, and analyses were all 110 performed on a Light Cycler instrument. The PCR was checked for contamination using a 111 negative sample. JCV DNA load in whole blood is expressed as log 10 genome copy number per 112 mL. Coupled with the MagNA Pure™ extraction, the detection limit was 500 copies genome per 113 mL (i.e. 2.7 log 10 copies/mL). 114

Patients were also tested for other opportunistic viral infections: cytomegalovirus (CMV), 115

Epstein-Barr virus (EBV), and BK virus (BKV) were quantified in whole blood and urine 116 samples (BKV). 117 HCMV DNA, EBV DNA, and BKV DNA were extracted automatically from whole blood with 118 the MagNA Pure® instrument. HCMV DNA was quantified on a Light Cycler 1.0 ™ (Roche), as 119 described previously [START_REF] Mengelle | Automated extraction and quantification of human 303 cytomegalovirus DNA in whole blood by real-time PCR assay[END_REF]. EBV DNA was quantified using a Light Cycler EBV 120 Quantification Kit® (Roche), according to the manufacturer's instructions [START_REF] Gulley | Validation of Roche LightCycler Epstein-Barr virus 278 quantification reagents in a clinical laboratory setting[END_REF]121 Hill et al., 2006;[START_REF] Kozic | Evaluation 296 of a commercial real-time PCR assay for quantitation of Epstein-Barr virus DNA in 297 different groups of patients[END_REF]. BKV DNA was quantified on a Light Cycler 2.0™ (Roche), 122 as described previously [START_REF] Basse | Prospective 250 evaluation of BK virus DNAemia in renal transplant patients and their transplant 251 outcome[END_REF]. 123 The first positive JCV DNA sample was detected at a median time of 139 days post-transplant 156 (43-250). The median JC virus load of the first positive DNA blood sample was 3.4 log 10 157 copies/mL (1.9 -5.7 log 10 copies/mL). JCV DNA was detected in only one blood sample taken 158 from three patients: at 43, 180, 225 days post-transplant and the virus loads were 4.5, 5.7 and 1.9 159 log 10 copies/mL. The patient whose virus load was 5.7 log 10 copies/mL was treated with 160 rituximab. 161 detected five times over 49 days in the one patient in this group who was treated with rituximab, 163 and the virus loads were very low (3.1, 2.5, 2.5, 0.6 and 2.5 log 10 copies/mL). 164

Serological markers 124

All patients had other viral infection(s): BKV (3), EBV (2), CMV (1) and BK+EBV (1). EBV 165 infection occurred soon after transplantation: 27, 29, and 43 days, whereas the other infections 166 occurred later: BK at 51,115,127, transplant (in a HCMV seropositive patient). Fig. 1 summarizes the virological data for those 168 patients in whom JCV was detected. 169

The urine of three patients contained decoy cells. The blood samples of these patients also 170 contained BKV DNA: the first quantified BKV virus loads were 2.8, 4 and 4 log 10 copies/mL, 171 and the urine BKV virus loads were 6.94, 10.83, and 10.89 log 10 copies/mL. First BKV DNA was 172 detected at a median time of 121 days post-transplant (51-223), and was prior to or at the same 173 time as JCV DNAemia. All three patients had a biopsy-confirmed polyomavirus-associated 174 nephropathy and had experienced acute rejection before BKV replication. The acute rejection 175 episodes had been treated with steroid injections alone (1), steroid injections and polyclonal 176 antibodies (1), and steroid injections, muromonab CD3, and rituximab (1). Pathological analysis 177 of a kidney biopsy taken from the fourth patient with BKV replication showed no features of 178 polyomavirus-associated nephropathy. 179

The median CD3 count was 733/mm 3 (178-1717) and the median CD4 count was 470/mm 3 180 (78-1184) at the time of first JCV DNAemia, and the median creatinine was 1.39 mg/dL (1.12-181 2.93). The median microalbuminuria was 94 mg/mL (25-244), the median hematuria was 9 182 cells/mm 3 (0-28), and the median leukocyturia was 3 cells/mm 3 (0-80). 183

  -term follow-up study assessed the incidence of JCV shedding in whole 70 blood samples from renal transplant recipients. The results were correlated with 71 immunosuppressive treatment and the virological parameters of the patients who excreted

  Methods 95Measurement of blood JCV DNA 96 JC virus DNA was extracted automatically from whole blood and detected by quantitative real-97 time PCR. The DNA was extracted from 200 µL samples using a MagNA Pure™ instrument 98 (Roche Diagnostics, Meylan, France) and the MagNA Pure LC DNA Isolation Kit I® according 99 's instructions. JCV DNA was detected using a Light Cycler™ system 100 (Roche). 101The primers were located in the region coding for the agnoprotein: HMJC: 5'-ATA CAg TgC 102 TTT gCC TgA ACC-3' and HMJC6: 5'-CAA CTg AgC AAT AgC ACT ACC-3'. The fluorogenic 103 probe was HSMSJC3: 6FAM-5'ACT ggA gCT CCg ggg gCT gTA 3'TAMRA. Real-time PCR was 104 carried out using Fast Start DNA Master hybridization probes (Roche Diagnostics, Meylan, 105

  were tested using the Ag HBs Architect (Abbott, Abbott Park, Illinois, 125 USA), and anti-HCV antibodies were tested using the anti-HCV Architect (Abbott). HBV DNA 126 and HCV RNA were quantified using the COBAS Ampliprep-COBAS Taqman96™ (CAP-127 CTM) system (Roche). 128 Anti-HCMV IgG and IgM were assayed using the Liaison CMV IgG and Liaison CMV IgM 129 assays according to the manufacturer's instructions (DiaSorin, Saluggie, Italy) Anti-VCA IgG and 130 anti-EBNA antibodies were tested using the Liaison VCA IgG and Liaison EBNA IgG. Anti-131 VCA IgM antibodies were detected with the Liaison EBV IgM assays according to the 132 manufacturer's instructions (DiaSorin). 133 Statistical analyses 134 StatView 5.0 was used for all statistical analyses. The results are shown as means or medians. 135 103 renal transplantations (68 men) were performed at the Toulouse University 137 Hospital between 1 January and 31 December, 2006. The mean age of the patients was 49.8 years 138 (range: 24-77 years). Patients were enrolled for at least one year post-transplant. A total of 1487 139 whole blood samples were collected, and the median frequency of sampling was 12 (2-32) per 140 patient. 141 JCV was detected in 31 whole blood samples from seven of the 103 transplant patients (6.8%), 142 two women and five men. Their mean age was 44 (23-70) years. Six patients had undergone a 143 first transplantation and one patient had undergone a second transplantation. The underlying 144 diagnoses were polycystic kidney disease (n=2), thrombotic microangiopathy (n=1), focal 145 segmental glomerulosclerosis (n=1), interstitial nephropathy (n=1), IgA nephropathy (n=1), and 146 an undetermined cause. 147 Induction therapies were anti-CD25 monoclonal antibodies (n=5) or antithymocyte globulins 148 (n=2). Their post transplant immunosuppressive therapy included steroids with tacrolimus/MMF 149 (n=2), or ciclosporin/MMF (n=1), or belatacept/MMF (n=4). Two patients were given 150 rituximab; one for a posttransplant focal segmental glomerulosclerosis relapse (375 mg/m² 151 twice), and the other for an acute humoral rejection (375 mg/m²/week for 4 weeks). 152 No patient had hepatitis B surface antigens or anti-HCV antibodies. All patients were negative 153 for serum HBV DNA and HCV RNA. Five patients were anti-HCMV seropositive, and all were 154 anti-EBV positive at the time of transplantation. 155

  persistent in the other four (median 66 days, range 21-447). JCV DNA was 162

  the present monocentric study shows that JCV infections are relatively uncommon 235 in renal transplant recipients. They were not associated with acute renal rejection, or with the 236 presence of decoy cells in the JCV DNAemia positive patients, suggesting that the course of JCV 237 infection is lower and less aggressive than of BKV. No association between JCV infection and 238 neurological manifestations was also detected. Overall, these results are comparable to those of 239[START_REF] Razonable | A longitudinal 318 molecular surveillance study of human polyomavirus viremia in heart, kidney, liver, and 319 pancreas transplant patients[END_REF], who found episodes of subclinical JCV DNA in the blood of solid-organ 240 transplant patients. 241 However, JCV infection should be surveyed regularly in transplant recipients as the presence of 242 JCV DNA in the blood may differentiate between JCV and BKV infections. Therefore, detection 243 of JCV indicates over-immunosuppressed patients who may benefit from reduction of

-4 3.9 5.2 5.6 5.4 5.7


Arbusow Viktor

Derfuss Tobias

Held Kathrin

Himmelein Susanne

Strupp Michael

Gurkov Robert

Brandt Thomas

Theil Diethilde
email: diethilde.theil@med.uni-muenchen.de

DVM Diethilde Theil

Latency of Herpes simplex virus type-1 in human geniculate and vestibular ganglia is associated with infiltration of CD8+ T-cells

Keywords: cranial ganglia, qRT-PCR, LAT cranial ganglia, qRT-PCR, LAT, CD8+ T-cells

Herpes simplex virus type-1 latency and CD8+ T-cell occurence were investigated in the trigeminal, geniculate, and vestibular ganglia from seven deceased humans. The HSV-1 ''latency-associated transcript'' was assessed by in situ-hybridization and quantitative RT-PCR. Infiltration of CD8+ T-cell was detected by immunohistochemistry and quantitative RT-PCR.

The data show that HSV-1 latency and CD8+ T-cell infiltration are not solely confined to the trigeminal ganglia but can also occur in other cranial ganglia along the neuroaxis. However, the HSV-1 latency transcripts in the geniculate and vestibular ganglia were expressed at a very low level. The difference in CD8 transcript levels among HSV-1 latently infected trigeminal ganglia, geniculate, and vestibular ganglia was less conspicuous. Colocalisation of latent HSV-1 and CD8+ T-cells in geniculate and vestibular ganglia supports further the hypothesis that HSV-1 reactivation is possible in these ganglia and is the cause of Bell's palsy and vestibular neuritis.

Introduction

Herpes virus simplex type-1 (HSV-1) has been linked to a variety of clinical syndromes of the nervous system. After primary infection of the epithelium (stomatitis aphtosa), HSV-1 enters the axon terminals and is carried by axonal transport to sensory neurons of the human cranial nerve ganglia, where it establishes lifelong latency. HSV-1 latency is characterized by restricted viral expression: an abundant expression of the latency-associated transcript (LAT) [START_REF] Stevens | RNA complementary to a herpesvirus alpha gene mRNA is prominent in latently infected neurons[END_REF] and very low amounts of immediate early (IE) genes [START_REF] Kramer | Quantification of transcripts from the ICP4 and thymidine kinase genes in mouse ganglia latently infected with herpes simplex virus[END_REF][START_REF] Derfuss | Presence of HSV-1 immediate early genes and clonally expanded T-cells with a memory effector phenotype in human trigeminal ganglia[END_REF]. Findings from the HSV-1 mouse model and from humans suggest that a chronic inflammation accompanies HSV-1 latency in the trigeminal ganglia [START_REF] Shimeld | Immune cell infiltration and persistence in the mouse trigeminal ganglion after infection of the cornea with herpes simplex virus type 1[END_REF][START_REF] Theil | Latent herpesvirus infection in human trigeminal ganglia causes chronic immune response[END_REF]. Previous investigations revealed that the highest frequency of neurons, that were positive for the latency-associated transcript, is found in the human trigeminal ganglia. However, latency-associated transcripts were also present, although in lower amounts in the geniculate and vestibular ganglia [START_REF] Theil | Prevalence of HSV-1 LAT in human trigeminal, geniculate, and vestibular ganglia and its implication for cranial nerve syndromes[END_REF]. Decreasing amounts of the latency-associated transcript from the trigeminal ganglia to the geniculate and vestibular ganglia are compatible with the hypothesis that HSV-1 migrates along the lingual nerve to the trigeminal and geniculate ganglia and via the facio-vestibular anastomoses to the vestibular ganglia [START_REF] Arbusow | Distribution of herpes simplex virus type 1 in human geniculate and vestibular ganglia: implications for vestibular neuritis[END_REF]. A large post-mortem study on human trigeminal ganglia demonstrated that latent HSV-1 is accompanied by an abundant CD8+ T-cell infiltration [START_REF] Theil | Latent herpesvirus infection in human trigeminal ganglia causes chronic immune response[END_REF]. According to the HSV-1 mouse model, one would expect that in all ganglia, where HSV-1 becomes latent, there should also be a T-cell infiltration, since this is supposed to maintain the virus in its latent state [START_REF] Khanna | Herpes simplex virus-specific memory CD8+ T cells are selectively activated and retained in latently infected sensory ganglia[END_REF]. However, given that symptomatic HSV-1 reactivations from human trigeminal ganglia occur very frequently in humans (cold sores), the prominent T-cell The aim of the present study was to determine whether immune infiltration occurs in other cranial ganglia (geniculate and vestibular ganglia), which are known to also harbor latent HSV-1. An affirmative answer would indicate viral activity (e.g., symptomatic or asymptomatic reactivations). The presence of latency-associated transcript and T-cells in the geniculate and vestibular ganglia would affirm the hypothesis that HSV-1 reactivations are to be expected at this site and that they cause neurological entities like Bell's palsy and vestibular neuritis.

Material and Methods

The Ethics Committee of the Medical Faculty of the Ludwig-Maximilians University of Munich has approved the use of autopsy samples for the present study. The cause of death of the subjects included in the study was related to trauma. None had lesions suggestive of an active orolabial herpes infection. There were no records that they had suffered from vestibular neuritis or any other neurological disorder during their lifetime.

The trigeminal, geniculate, and vestibular ganglia from seven individuals (ages 5 weeks to 40 years) were collected (Table 1A). Ganglia were embedded in Tissue tek ® compound (Sakura, Zoeterwoude, The Netherlands) and stored at -70ºC until use. Frozen sections were made of 8-µm thickness and mounted on positively charged slides (SuperFrost*/Plus ® , Menzel, Braunschweig, Germany). In parallel a number of tissue sections were collected for RNA extraction. Beforehand, tissue sections from each ganglion were stained with hematoxylin and eosin for morphologic examination and the RNA quality of each ganglion was evaluated.

For immunohistochemistry frozen tissue sections were thawed, dried at 37ºC for 15 min, fixed in acetone for 10 min, and then incubated overnight with mouse anti-CD8 antibody. Afterwards tissue sections were incubated for 30 min in biotinylated rabbit anti-mouse IgG antibody (1:500; DAKO, Hamburg, Germany). The sections were incubated with peroxidase-conjugated streptavidin (DAKO, Hamburg, Germany) for 30 min, followed by a final wash, and then incubated with diaminobenzidine (DAB) (DAKO, Hamburg, Germany) for up to 10 min. For in-situ hybridization the protocol used was described in detail in a previous work [START_REF] Theil | Prevalence of HSV-1 LAT in human trigeminal, geniculate, and vestibular ganglia and its implication for cranial nerve syndromes[END_REF]. The ISH signal was visualized by incubation in nitroblue tetrazolium (NBT) and X-phosphate (BCIP 5-bromo-4-chloro-3-indolyl-phophate) staining solution. RNA extraction and cDNA synthesis were performed using commercially available kits (miRNeasy Mini Kit, Qiagen, Hilden; high capacity cDNA reverse transcription kit, Applied Biosystems, Foster City, CA, USA) according to the manufacturers' protocols. To quantify latency-associated transcripts and CD8 transcripts QuantiTect SYBR  green RT-PCR (Qiagen) was used on a GeneAmp 5700 Sequence Detection System (Applied Biosystems). The results were normalized to the housekeeping gene beta-actin. Primer and PCR conditions were published previously [START_REF] Theil | Latent herpesvirus infection in human trigeminal ganglia causes chronic immune response[END_REF][START_REF] Naoe | Correlation between major histocompatibility complex class I molecules and CD8+ T Page 13 of 15 John Wiley & Sons Journal of Medical Virology lymphocytes in prostate, and quantification of CD8 and interferon-gamma mRNA in prostate tissue specimens[END_REF][START_REF] Derfuss | Presence of HSV-1 immediate early genes and clonally expanded T-cells with a memory effector phenotype in human trigeminal ganglia[END_REF].

The occurrence of latent HSV-1 and T-cell infiltration in the trigeminal ganglia from the subjects included in this study was assessed previously. HSV-1 latency was detected by in-situ hybridization and RT-PCR for the latency-associated transcript.

Cytotoxic T-cells were detected by CD8 immunohistochemistry and quantitative RT-PCR [START_REF] Theil | Latent herpesvirus infection in human trigeminal ganglia causes chronic immune response[END_REF].

Results

Tissue sections and the RNA from all ganglia were checked beforehand for the presence of neurons in the section and for RNA integrity. Due to morphological artifacts and RNA degradation in some ganglia, measurement of CD8 transcripts was not possible in the ganglia from each side of the same individual. Latency-associated transcript and CD8 expression could however be assessed in at least one side of the trigeminal, geniculate, and vestibular ganglia of each individual (Table 1A).

Of the fourteen trigeminal ganglia tested by in situ hybridization or RT-PCR, 6

showed positivity for the latency-associated transcript with either method. None of the 10 geniculate and 11 vestibular ganglia showed a positive signal for the latencyassociated transcript when tested by in situ hybridization, but expression of the latency-associated transcript was detected at low levels by quantitative RT-PCR in the 2 vestibular and 2 geniculate ganglia from one individual (Table 1 (A), subject 5).

The transcripts levels in the control trigeminal ganglia were 1000x higher than in the geniculate and vestibular ganglia (Table 1, B).

Infiltration of CD8+ T-cells was detectable by immunohistochemistry only in the

trigeminal ganglia (data published earlier) but not in the geniculate and vestibular ganglia (data not shown). By means of quantitative RT-PCR, CD8 transcripts were detectable in all tested trigeminal, geniculate, and vestibular ganglia. In the ganglia of three individuals the transcription levels of CD8 were clearly elevated (Table 1, subjects 5, 6 and 7). In these subjects the trigeminal ganglia were also positive for the latency-associated transcript, and in one subject (Table 1 A, subject 5) even the geniculate and vestibular ganglia were positive for the latency-associated transcript.

In the geniculate and vestibular ganglia, that were positive for the latency associated transcript, the CD8 transcript levels were analogous to the CD8 transcript level of the 1B, control 1), although the latency-associated transcripts in the geniculate and vestibular ganglia were very low when compared to the levels in the trigeminal ganglia (Table 1B, control 1).

Discussion

In the present study it was shown that CD8+ T-cells infiltrate human cranial ganglia other than the trigeminal ganglia. The CD8 transcript levels in the geniculate and vestibular ganglia that contained the latency-associated transcripts were comparable with the levels found in the trigeminal ganglia that were positive for the latencyassociated transcripts. These findings emphasize that HSV-1 latency is associated with chronic inflammation not only in the human trigeminal ganglia where frequent HSV-1 reactivation occurs but also in other ganglia linked anatomically (geniculate and vestibular ganglia). This is of clinical relevance, since it supports the view that Bell's palsy and vestibular neuritis are caused by reactivation of HSV-1. Up till now only the surrogate marker for HSV-1 latency, the latency-associated transcript, could be demonstrated in the geniculate and vestibular ganglia [START_REF] Furuta | Latent herpes simplex virus type 1 in human geniculate ganglia[END_REF][START_REF] Furuta | Latent herpes simplex virus type 1 in human vestibular ganglia[END_REF][START_REF] Theil | Prevalence of HSV-1 LAT in human trigeminal, geniculate, and vestibular ganglia and its implication for cranial nerve syndromes[END_REF]. Additional detection of CD8 T-cells in the HSV-1 latently infected geniculate ganglia and vestibular ganglia means that persistently or intermittently low levels of lytic gene expression occur in some neurons, and full reactivation is suppressed by CD8+ T-cells. However, for some unknown reason the protective role of CD8 T-cells can be abrogated and the virus then replicates, leading to a reactivating disease (e.g., Bell's palsy and vestibular neuritis).

Interestingly, the levels of CD8 transcripts in the geniculate and vestibular ganglia, which did not contain the latency-associated transcript but were obtained from subjects who showed positivity for the latency-associated transcript in the trigeminal ganglia, exceeded the values seen in the trigeminal, geniculate, and vestibular ganglia from individuals who had not had any HSV-1 infection. On the one hand, the elevated levels of CD8 in these ganglia could be due to a very low load of latent virus, which is below the sensitivity of the analytical method applied. On the other hand, the CD8+ T-cells might become sequestered at this site after a primary HSV-1 T-cell population persists thereafter in order to maintain the virus in a latent state by constant expression of effector molecules such as granzyme B and IFN-gamma [START_REF] Liu | Gamma interferon can prevent herpes simplex virus type 1 reactivation from latency in sensory neurons[END_REF][START_REF] Bergstrom | Morphology of the vestibular nerve. I. Anatomical studies of the vestibular nerve in man[END_REF], T-cells could persist without depending on viral antigen to provide equipment for a second encounter with the virus. As a matter of fact, a recent study demonstrated that a unique memory T-cell pool remains resident in the skin well after HSV-1 has been cleared [START_REF] Gebhardt | Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus[END_REF]. This CD8+ Tcell population does not seem to depend on ongoing viral gene expression, but shows preferential protection from a subsequent infection with HSV-1.

Correspondingly, entrapment of CD8+ T-cells in the geniculate and vestibular ganglia without latent HSV-1 could stem from a cleared HSV-1 acute infection that encompassed all cranial ganglia or an anterograde infection during the course of HSV-1 reactivation in the trigeminal ganglia [START_REF] Theil | Prevalence of HSV-1 LAT in human trigeminal, geniculate, and vestibular ganglia and its implication for cranial nerve syndromes[END_REF]. These cells probably have the same protective capacity as those found in the skin of HSV-1 infected mice [START_REF] Gebhardt | Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus[END_REF] and humans [START_REF] Zhu | Virus-specific CD8+ T cells accumulate near sensory nerve endings in genital skin during subclinical HSV-2 reactivation[END_REF] and consequently limit viral spread to additional neurological structures.

  a consequence of continuing asymptomatic or symptomatic reactivations that are distinctive of the trigeminal ganglia.

  The trigeminal ganglia from one individual who showed strong positivity for the latency-associated transcript on in-situ hybridization and for T-cells on immunohistochemistry, and the trigeminal ganglia from another individual who was negative for the latency-associated transcript and T-cells were included as controls (references) for quantitation of the latency-associated transcripts and CD8 T-cells (Table1 B).

  cleared. Even though it is generally accepted that immune cells invade the trigeminal ganglia in the course of a primary infection and a certain CD8+

Acknowledgements

This work was supported by a grant from the Deutsche Forschungsgemeinschaft to D.T. and T.D. (TH 894/3-1). We would like to thank Judy Benson and Katie Ogston for carefully copyediting the manuscript.

John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology


Alessandra Zappa

Marta Canuti

Elena Frati

Elena Pariani

Silvana Perin

Lorena Maria Ruzza

Claudio Farina

Alberto Podestà

Alessandro Zanetti

Antonella Amendola

Bocavirus Strains in Young Children with Respiratory Tract Infections in Italy

Keywords: Respiratory infections, Phylogenetic analysis, human Metapneumovirus, human Bocavirus, Recombination events Pediatric respiratory infections, phylogenetic analysis, human Metapneumovirus, 43 human Bocavirus, pediatric hospitalization, viral recombination. 44

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

F o r P e e r R e v i e w

Modified from [START_REF] Huck | Novel human 276 metapneumovirus sublineage[END_REF][START_REF] Peret | Characterization of human metapneumoviruses isolated from patients in North America[END_REF]

II step

MPVF1f F-r CTTTGGACTTAATGACAGATG CCTGTGCTGACTTTGCATG 445 [START_REF] Peret | Characterization of human metapneumoviruses isolated from patients in North America[END_REF] Modified from [START_REF] Huck | Novel human 276 metapneumovirus sublineage[END_REF] Italy. Pharyngeal swabs were collected from 240 children ≤3 years of age (130 males, 110 females; 28 median age, 5.0 months; IQR, 2.0-12.5 months) and tested for respiratory viruses, including hMPV 29 and hBoV, by molecular methods. hMPV-RNA and hBoV-DNA positive samples were 30 characterized molecularly and a phylogenetical analysis was performed. PCR analysis identified 31 131/240 (54.6%) samples positive for at least one virus. The frequency of hMPV and hBoV 32 infections was similar (8.3% and 12.1%, respectively). Both infections were associated with lower 33 respiratory tract infections: hMPV was present as a single infectious agent in 7.2% of children with 34 bronchiolitis, hBoV was associated with 18.5% of pediatric pneumonias and identified frequently as 35 a single etiological agent. Genetically distinct hMPV and hBoV strains were identified in children 36 examined with respiratory tract infections. Phylogenetic analysis showed an increased prevalence of 37 hMPV genotype A (A2b sublineage) compared to genotype B (80% vs. 20%, respectively) and of 38 the hBoV genotype St2 compared to genotype St1 (71.4% vs. 28.6%, respectively). Interestingly, a 39 shift in hMPV infections resulting from A2 strains has been observed in recent years. In addition, 40 the occurrence of recombination events between two hBoV strains with a breakpoint located in the 41 VP1/VP2 region was identified. 42 [Allander et al., 50 2005] identified the etiological causes of several respiratory infections. hMPV is a common cause of 51 respiratory disease in children under five years old and often accounts for severe clinical 52 manifestations, including bronchiolitis or pneumonia [START_REF] Foulongne | Human metapneumovirus infection in 272 young children hospitalized with respiratory tract disease[END_REF]. hMPV is a single-53 stranded negative RNA virus of the Paramyxoviridae family, Pneumovirinae subfamily [van den 54 Hoogen et al., 2001;[START_REF] Broor | Human metapneumovirus: a new respiratory pathogen[END_REF]. Molecular analysis identified two major genotypes (A and 55 B) with genetic lineages A1, A2 and B1, B2, respectively [van den [START_REF] Van Den Hoogen | Antigenic and genetic variability of human Metapneumoviruses[END_REF]. Lineage A2 56 has been recently divided into A2a and A2b sublineages [START_REF] Huck | Novel human 276 metapneumovirus sublineage[END_REF]. 57 hBoV is detected frequently in the respiratory and gastrointestinal tracts, suggesting its 58 pathologic involvement at both sites [START_REF] Lindner | Clinical and 292 epidemiological aspects of human bocavirus infection[END_REF][START_REF] Schildgen | Human 312 Bocavirus: passenger or pathogen in acute respiratory tract infections?[END_REF]. hBoV belongs to 59 the Bocavirus genus of the Parvoviridae family. It is a non-enveloped virus and contains a linear 60 single-stranded DNA molecule. The two major hBoV genotypes correspond to the original St1 61 (Stockholm 1) and St2 (Stockholm 2) isolates [START_REF] Allander | Cloning 241 of a human parvovirus by molecular screening of respiratory tract samples[END_REF]. 62

F o r P e e r R e v i e w

This report describes the molecular epidemiology of hMPV and hBoV identified following 63 surveillance for respiratory viruses in children hospitalized for acute respiratory tract infections in 64 Milan, Italy. A detailed genetic analysis was carried out using the hMPV and hBoV sequences 65 identified. 66 tract infections (i.e., bronchitis, bronchiolitis, pneumonia or asthma) were observed in 66.7% (95% 72 CI: 60.5-72.4) of children. Bronchiolitis and bronchitis were the most frequent outcomes (60.6%, 73 95% CI: 52.9-68.0 and 37.5%, 95% CI: 30.3-45.2, respectively), pneumonia and asthma being less 74 frequent (16.9%, 95% CI: 11.7-23.3 and 9.4%, 95% CI: 5.5-14.7). 75

At the time of hospital admission pharyngeal swabs were collected from each patient for the 76 identification and characterization of respiratory viruses using Plain Swabs and ITM-RT (Copan, 77 Italy). Informed consent was obtained from parents of eligible children and children were divided 78 into four age groups: ≤6 months (60%, 95% CI: 53.7-66.1), 7-12 months (15%, 95% CI: 10.9-79 19.9), 13-24 months (18.8%, 95% CI: 14.2-24.1) and 25-36 months (6.2%, 95% CI: 3.7-9.9). 80

The seasonal distribution of the samples collected is as follows: 35.4% (95% CI: 29.6-41.6) 81 of samples were collected during autumn (October-December), 52.5% (95% CI: 46.2-55.8) during 82 winter (January-March) and 12.1% (95% CI: 8.4-16.7) during spring (April-June). 83 intervals, 95% CI) as appropriate. Comparisons between groups were performed using the Chi-119 squared test or Fisher's exact test. A P-value <0.05 was considered statistically significant (two-120 tailed test). All statistical analyses were performed using the OpenEPI software, version 2.2.1 [Dean 121 et al., 2009] Viral pathogens were associated with 60.6% (95% CI: 52.9-68.0) of lower respiratory-tract 133 infections, particularly in bronchiolitis cases (60.8%, 95% CI: 50.9-70.2). The most frequently 134 detected viruses were RSV (50.8%, 95% CI: 38.2-63.4), hBoV (20.3%, 95% CI: 11.5-32.0), hRV 135 (16.9%, 95% CI: 8.9-28.1), hAdV (16.9%, 95% CI: 8.9-28.1) and hMPV (11.9%, 95% CI: 5.3-136

22.1). 137 138

Epidemiologic and Molecular Features of hMPV and hBoV Infections 139

hMPV or hBoV were detected in 37.4% (95% CI: 29.4-45.9) of respiratory virus-positive 140 samples but only in children ≤24 months old. hMPV prevalence was similar in the first three age 141 groups (range: 8.3-9.0%) while hBoV was identified more frequently in the 13-24 month old group 142 compared to the other groups (24.4% vs. 9.7% and 11.1%, P < 0.05) (Table IIA). 143 hMPV was identified more often as a single pathogen and only 30% of cases were 144 associated with mixed infections (P <0.05). hBoV was detected as a mixed infection in 58.6% 145 (17/29) of cases, mainly in association with RSV (N = 10), hRV (N = 5) and hAdV (N = 4). Among 146 these, two triple-infection (hBoV-RSV-hRV) cases were detected. The frequency of co-infections 147 was similar in all age groups but different viruses were involved. The median age of hBoV co- infected children was 2.0 months (IQR: 1.5-50.0 months), for hBoV-RSV 8.5 months (IQR: 3.5-149 13.0 months) and 14 months for both hBoV-hRV and hBoV-hAdV (IQR: 11.5-17.0 months). No 150 hMPV-hBoV co-infections were identified. 151 hMPV was found as the single source of infection in 7.2% (95% CI: 3.2-13.8) of 152 bronchiolitis cases and part of co-infections in 11.1% of pneumonia (95% CI: 2.9-27.3) and 13.3% 153 of asthma cases (95% CI: 2.3-37.5). hBoV was identified in 12.4% of bronchiolitis cases (95% CI: 154 6.9-20.1), mainly as a hBoV-RSV co-infection. In addition, a high prevalence of hBoV infections 155 (18.5%, 95% CI: 7.1-36.4) were seen in association with pneumonia, most often as a single viral 156 agent. 157 158

Phylogenetic Analysis of hMPV and hBoV Sequences 159

Ten out of 20 hMPV-positive samples were characterized at the molecular level and a 160 phylogenetic tree of the hMPV F gene was generated (Fig. 1A). The identity range between tree 161 sequences was 81.1-100%. Mean evolutionary distances for all sequence pairs are reported in Fig. 162 1B. Phylogenetic analysis showed a prevalence of genotype A strains (8/10) compared to genotype 163 B strains (2/10). In the context of genotype A, six hMPV-F sequences shared similarity with the 164 A2b sublineage, one with A2a and one with the A1 group that presented with a single amino acid 165 change (N358T). Genotype B sequences belonged exclusively to lineage B1. 166

Sixteen out of 29 hBoV-positive samples were analyzed and the hBoV VP1/VP2 region 167 described (Fig. 2). All sequences shared a high degree of similarity with reference strains and other 168 defined sequences (identity range: 97.3-100%). Most sequences (14/16) were genetically similar to 169 genotypes St1 (4/14) and St2 (10/14). A third cluster (bootstrap value: 92), including two Italian 170 sequences (I-MI/04-08 and I-MI/02-07, identity 100%) and the Asian HK7 isolate, were identified 171 between the two main branches. The boot scanning analysis revealed a recombination event with a The prevalence of both hMPV and hBoV infections (8.3% and 12.1%, respectively) were 184 similar to rates reported previously [START_REF] Maggi | Human Metapneumovirus associated 299 with respiratory tract infections in a 3-year study of nasal swabs from infants in Italy[END_REF][START_REF] Bastien | Human Bocavirus infection, Canada[END_REF][START_REF] Foulongne | Human metapneumovirus infection in 272 young children hospitalized with respiratory tract disease[END_REF]185 Allander et al., 2007]. Both hMPV and hBoV infections were associated with lower respiratory tract 186 infections. hMPV was present as a single infectious agent in 7.2% of children aged ≤12 months 187 with bronchiolitis. The involvement of hBoV at the onset of bronchiolitis was difficult to asses due 188 to the frequent association with other viruses, particularly RSV. In this study, hBoV was associated 189 with 18.5% of pediatric pneumonias and frequently identified as a single agent. 190 Twenty percent of hospitalized children had dual or triple co-infections and hBoV was 191 involved in approximately 60% of mixed infections. The extended persistence of hBoV in airways 192 (up to 7 months) increased patient's chances of acquiring a co-infection, explaining the high 193 frequency of co-infections with this virus [START_REF] Brieu | Human bocavirus infection in 256 children with respiratory tract disease[END_REF][START_REF] Lindner | Clinical and 292 epidemiological aspects of human bocavirus infection[END_REF]von Linstow et 194 al., 2008]. The seasonal distribution of hBoV, with a peak incidence between December and 195 January, resembled that observed for RSV, hRV and hAdV. This suggested that the overlapping 196 distribution of these viruses during the seasons could have facilitated hBoV co-infections. The 197 epidemiologic model of hMPV infections was distinct from many other respiratory viruses, with 198 peak incidence rates in late spring, mostly between March and April. This observation may account 199 for the absence of hMPV-hBoV co-infections. 200

Co-circulation of both hMPV genotypes was observed, with predominance of genotype A 201 (particularly the A2b sublineage), supporting previous reports demonstrating an increase in A2 202 strain infections in recent years [START_REF] Boivin | Global genetic diversity of human metapneumovirus fusion gene[END_REF][START_REF] Wang | 337 Co-circulating genetically divergent A2 human metapneumovirus strains among children in 338 southern Taiwan[END_REF]Herbert et al., 2005] identities values between the recombinant sequence and the two reference sequence (MPT8 and 362 HK7) before and after the breakpoint (inset). 363

1*

  Genetic analysis of hMPV. A) Phylogenetic tree of the Italian I-MI hMPV sequences (boldface) demonstrating the phylogenetic relationship of the I-MI sequences based on partial F gene nucleotide sequences. Bootstrap values (in addition to A1, A2a, A2b, B1 and B2 group/sublineages) are indicated. Reference sequences correspond to the following GenBank accession numbers: A1) AF371337, AY145294, DQ453017, AJ867506, AJ867520, EF612444; A2a) AY304360, AY145296, DQ453014, EF612451; A2b) AY530095, DQ453021, AJ867528, AJ867538, EF612457; B1) AY304361, AY145294, DQ453015, AJ867513, EF612440; B2) AY304362, AY145289, DQ452995, AJ867503, EF612441. B) Evolutionary mean distances over all sequence pairs within and between groups. 258x362mm (150 x 150 DPI) Genetic analysis of hBoV sequences. Phylogenetic tree of the Italian I-MI human Bocavirus sequences (boldface) based on partial VP1/VP2 region nucleotide sequences. Bootstrap values (in addition to St1 and St2 genotype clusters) are indicated. Reference sequences correspond to the following GenBank accession numbers: St-1) DQ000497, AM160609, AM160611-AM160613, AM160615, AM689298, AM689299, AM689306, AM689307, EU069434, EU069436, EU069437, EF203921; St-2) DQ000495, AM160610, AM160614, AM689297, AM689301-AM689303, AM689305, EF450717, EF450718, EF450722, EF450723, EF450736, EF450740. 178x302mm (150 x 150 DPI) Recombination recognition of the Italian I-MI/04-08 sequence: bootscan analysis and identities values between the recombinant sequence and the two reference sequence (MPT8 and HK7) before and after the breakpoint (inset). 254x190mm (96 x 96 DPI) Dipartimento di Sanità Pubblica-Microbiologia-Virologia, Università degli Studi di Milano, Via 9 Pascal 36/38, 20133 Milano, Italy; 10 2 Unità di Microbiologia, AO "Ospedale San Carlo Borromeo", Via Pio II 3, 20153 Milano; 11 3 Unità di Pediatria, AO "Ospedale San Carlo Borromeo", Via Pio II 3, 20153 Milano; 12 4 CIRI-IV, Dipartimento di Scienze della Salute, Università degli Studi di Genova, Via Pastore 1Corresponding author: Elisabetta Tanzi, Ph.D. 16 Dipartimento di Sanità Pubblica-Microbiologia-Virologia, Università degli Studi di Milano, human Metapneumovirus (hMPV) and human Bocavirus (hBoV) 24 identified the etiological causes of several cases of acute respiratory tract infections in children. 25 This report describes the molecular epidemiology of hMPV and hBoV infections observed 26 following viral surveillance of children hospitalized for acute respiratory tract infections in Milan, 27

  respiratory tract infections are an important cause of morbidity and mortality worldwide. 46 These infections are ubiquitous and contagious, affecting repeatedly individuals of all ages, 47 particularly children younger than five years of age [World Health Organization, 2009] and many 48 infections have unknown etiologies. The recent discovery of the human Metapneumovirus (hMPV) 49 in 2001 [van den Hoogen et al., 2001] and of the human Bocavirus (hBoV) in 2005

  N=240; 130 males and 110 females; median age, 5.0 months; interquartile 69 range, 2.0-12.5 months) admitted to the San Carlo Borromeo Hospital in Milan between 2004-70 2008 with a diagnosis of acute respiratory infection were enrolled in the study. Lower respiratory 71

  performed by sequence analysis of a 450 bp amplicons of 95 the fusion (F) gene (spanning nucleotides 653-1,102) for hMPV and an 850 bp amplicon of the 96 VP1/VP2 region (spanning nucleotides 1,084-1,933) for hBoV (TableIB). 97Following PCR amplification, amplicons were purified with NucleoSpin ® Extract II 98 (Macherey-Nagel GmbH, Germany). Nucleotide sequences were obtained by automated DNA 99 sequencing on the ABI PRISM 3100 genetic analyzer (Applied Biosystems, Foster City, CA). The 100 hMPV-F gene and hBoV-VP1/VP2 region nucleotide sequences obtained were deposited into as median (interquartile range, IQR) and percentages (95% confidence 118

  more similar to the HK7 strain (identity 97.8% vs. 99%) (Fig.3inset). 175Six amino acid substitutions were found: G415S, N474S, F540Y, N546H, A555T, and 176 G566R. Mutations G415S, F540Y and N546H were identified in the I-MI/04-08 and I-MI/02epidemiologic survey of acute respiratory infections was conducted in children 180 younger than three years old hospitalized between 2004 and 2008 in Milan, Italy. Both 181 hospitalization and positive viral detection were more frequent in children <2 years of age 182 compared to older patients. 183

Fig. 1 .Fig. 3 .

 13 Fig. 1. Genetic analysis of hMPV. A) Phylogenetic tree of the Italian I-MI hMPV sequences 343

Table I .

 I Primers Used in This Study.

	Page 3 of 26			Journal of Medical Virology
	I step	RSV-B	HMPV-M1f RSVB3	GAGTCCTAYCTRGTAGACACC ATCTTCCTAACTCTTGCTRTTAATGCATTG	M	247 611	This study
	A. Primers Used for Respiratory Infection Screening. a HMPV-M1r RSVB4 GATGCGACAGCTCTGTTGATTTACTATG AGTACAGACATDGCWGCACC
	II step hAdV nested-PCR hBoV sequencing nested-PCR	Primer HMPV-M2f HMPV-M2r	Sequence 5' 3' GACCWGCTGTTCAAGTTG YTGTGATGYAGCATACAGAG	Gene Amplicon size (bp) 151	Reference
	I step hPIV1-4 multiplex nested-PCR I step	ADHEX1f VPs1	GCACTTCTGTATCAGATGCCTT	VP1/2	903	[Smuts and Hardie,
	I step		ADHEX1r VPas1	CGTGGTATGTAGGCGTGTAG			2006]
	hPIV-1 II step	PIP1+ VPs2	CYTTAAATTCAGATATGTAT CTTAGAACTGGTGAGAGCACTG	HN	480 850	Modified from
			PIP1-VPas1	GATAAATARTWATTGATACG CGTGGTATGTAGGCGTGTAG			[Echevarría et al.,
	hPIV-2 hPIV-3 hPIV-4 II step hPIV-1 hPIV-2 hPIV-3 hPIV-4 hRV eminested-PCR I step hMPV sequencing nested-PCR I step	F o r P e e r R e v i e w PIP2+ PIP2-AACAATCTGCTGCAGSRTTT ATGTCAGAYAATGGRCAAAT HN PIP3+ PIP3-CTGTAAACTCAGACTTGGTA TTTARGCCYTTGTCAACAAC HN PI4P+ PI4P-CTGAACGGTTGCATTCAGGT TTGCATCAAGAATGAGTCCT P PIS1+ PIS1-CCGGHAATYTCTCATACCTATG CYTTGGAGCGGAGTTGTTAWG HN PIS2+ PIS2-CCATTTACCTAAGTGATGGAAT GCCCTGTTGTATTTGGAAGAGA HN PIS3+ PIS3-ACTCCCAARGTTGATGAAAGAT TAAATCTTGTTGTTGAGATTG HN PI4S+ PI4S-AAAGAATTAGGTGCAACCAGTC GTGTCTGATCCCATAAGCAGC P PR1f CGGACACCCAAAGTAG 5'UTR MPVF1r GTCTTCCTGTGCTAACTTTG F o r P e e r R e v i e w F-f GTYAGCTTCAGTCAATTCAAC F	507 477 441 316 203 102 244 380 532	1998] [Aguilar et al., 2000] Modified from [Echevarría et al., 1998] [Aguilar et al., 2000] [Ireland et al.,
			PR2r	GCACTTCTGTTTCCCC			1993]
	II step	PR1f	CGGACACCCAAAGTAG		202
			PR3r	GGCAGCCACGCAGGCT
	RSV multiplex nested-PCR
	I step
		RSV-A/B	RSVAB1	ATGGAGYTGCYRATCCWCARRRCAARTGCAAT	F	737	[Coiras et al., 2003]
			RSVAB2	AGGTGTWGTTACACCTGCATTRACACTRAATTC
	II step
		RSV-A	RSVA3	TTATACACTCAACAATRCCAAAAAWACC		363
			RSVA4	AAATTCCCTGGTAATCTCTAGTAGTCTGT
				John Wiley & Sons

a Primers used for the molecular surveillance for acute respiratory tract infections (A) and for the molecular characterization of hBoV and hMPV strains (B).

B. Primers Used for the Molecular Characterization of hBoV and hMPV Strains. a

Table II .

 II Molecular detection of respiratory viruses in pharyngeal swabs collected from hospitalized children with acute respiratory tract infections and children with viral respiratory infections.

a p < 0.05, prevalence of hBoV infections vs. that observed in children 0-6 months and 7-12 months of age. b p < 0.05, N (%) viral infections vs. N (%) of children 25-36 months of age.

A.

Number (95% CI) of Infections and Age Distribution Children (N = 240) Hospitalized with Acute Respiratory Tract Infections.

		Total	0-6 months	7-12 months	13-24 months	25-36 months
		240	144 (60; 53.7-66.1)	36 (15; 10.9-19.9)	45 (18.8; 14.2-24.1)	15 (6.3; 3.7-9.9)
	Viral agents
	RSV	46 (19.2; 14.6-24.5)	35 (24.3; 17.8-31.8)	4 (11.1; 3.6-24.7)	7 (15.5; 7.1-28.4)	0
	hAdV	28 (11.7; 8.1-16.2)	13 (9; 5.1-14.6)	7 (19.4; 8.9-34.7)	7 (15.5; 7.1-28.4))	1 (6.7; 0.3-28.7)
	hRV	24 (10; 6.7-14.3)	14 (9.7; 5.6-15.4)	5 (13.9; 5.3-28.1)	4 (8.9; 2.9-20.1)	1 (6.7; 0.3-28.7)
	FLU	11 (4.6; 2.4-7.8)	5 (3.5; 1.3-7.5)	2 (5.5; 0.9-17.2)	4 (8.9; 2.9-20.1)	0
	CoV	7 (2.9; 1.3-5.7)	3 (2.1; 0.5-5.6)	0	3 (6.7; 1.7-17.1)	1 (6.7; 0.3-28.7)
	hPIV	1 (0.4; 0.02-2.0)	0	0	1 (2.2; 0.1-10.5)	0
	hMPV	20 (8.3; 5.3-12.4)	13 (9; 5.1-14.6)	3 (8.3; 2.2-21)	4 (8.9; 2.9-20.1)	0
	hBoV	29 (12; 18.4-16.7)	14 (9.7; 5.6-15.4)	4 (11.1; 3.6-24.7)	11 (24.4;13.6-38.5) a	0
	B.

Number (95% CI ) of Single and Co-infections in Patients (N = 131) in the Context of Age.

	Total	131 (54.6; 48.3-60.8)	76 (52.8; 44.6-60.8) b	22 (61.1; 44.6-75.9) b	30 (66.7; 52-79.2) b	3 (20; 5.4-45.4)
	Single infections	102 (77.9; 70.2-84.4)	58 (76.3; 65.8-84.9)	19 (86.4; 67.2-96.4)	22 (73.3; 55.6-86.8)	3
	Co-infections	29 (22.1; 15.6-29.9)	18 (23.7; 15.2-34.2)	3 (13.6; 3.6-32.8)	8 (26.7; 13.2-44.4)	0

Surveillance in Children with Acute Respiratory Tract Infections 124

  Table IIA). Mixed infections were detected in 22.1% of acute viral respiratory tract 129 infections (Table IIB). Respiratory viruses were detected more frequently in children younger than 130 two years of age (52.8%, 61.1% and 66.7% for children ≤6, 7-12 and 13-24 months of age, 131 respectively) in comparison to children 25-36 months of age (20%, P < 0.05, Table IIB). 132

	123	RESULTS
	125	Viral One hundred and thirty-one out of 240 (54.6%, 95% CI: 48.3-60.8) samples collected from
	126	hospitalized children were PCR-positive for at least one respiratory virus. Forty-six samples
	122 127	. (19.2%) were positive for RSVA/B, 11.7% for hAdV, 10% for hRV, 4.6% for Flu A/B, 2.9% for
	128	hCoV229E/OC43 and 0.4% for hPIV3. hMPV was detected in 8.3% and hBoV in 12.1% of samples
		examined (
		F o r
		P
		e
		e r
		R
		e
		v i e
		w
		8

  World Health Organization. 2009. Acute Respiratory Infections (Update February 2009). Available 340 at: http://www.who.int/vaccine_research/diseases/ari/en/index.html 341

	342	FIGURE LEGENDS
		F o r F o r F o r F o r F o r F o r F o r F o r
		P P P P P P P P
		e e e e e e e e
		e r e r e r e r e r e r e r e r
		R R R R R R R R
		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
		w w w w w w w w
	203	. Guant
		18

John Wiley & SonsJournal of Medical Virology

breakpoint located at nucleotide position 1,302 of the VP1/VP2 region (Fig.3). The 5' region of

these two Italian sequences was more similar to the MPT-8 strain (identity 99% vs. 97.6%) and the

Acknowledgments 229

The Authors thank Dr. D. Colzani for skilful technical assistance. 230

This work was supported by the Italian Ministry of University and Research (MIUR, PRIN-2005, 231 protocol 2005067255_003; MIUR, PRIN-2007, protocol 2007LPAF42_003) 232

Conflict of interest 234

None of the authors have financial or other conflicting interests with regard to the information 235 described in this manuscript. 236


Valeria ; Conte
email: valeria.conte@uniroma2.it

Barbara Floris
email: floris@uniroma2.it

Pierluca Galloni
email: pierluca.galloni@uniroma2.it.

Claudia Bizzarri

SOLVENT EFFECTS OF IONIC LIQUIDS: INVESTIGATION OF FERROCENES AS ELECTROCHEMICAL PROBES

Keywords: Solvent effect, Ionic Liquids, Ferrocenes, Electrochemistry, Kamlet-Taft relationships Solvent effect, Ionic Liquids, Ferrocenes, Electrochemistry, Kamlet-Taft relationships

HAL is

INTRODUCTION

Ionic Liquids (ILs) or, more precisely, room-temperature Ionic Liquids (RTILs) represent a class of compounds more and more investigated, not only as non flammable and non volatile solvents for chemical reactions, but also in a number of electrochemical applications, [1] such as, to name a few, electrochemical mechanical actuator devices, [2] dyes-sensitized photoelectrochemical cells, [3] electrochemical supercapacitors, [4] fuel cells and lithium-ion batteries, [5] and treatment of nuclear waste. [6] Modified ILs have been used as potential electrolytes, [7] as electrodes, [8] and in amperometric flow injection analysis. [9] Other important applications of ILs are analytical ones, [10] with particular attention to electrochemical sensors, [11] protein extraction, [12] or desulfurisation of oils. [13] Recent reviews evidence the importance of RTILs in catalysis [14] and chemical industry. [15] The question "How polar are Ionic Liquids?" was periodically risen and several attempts have been made to describe polarity by macroscopic properties [16] or empirical parameters. [17] Determination of static dielectric constant classified Ils as solvents of moderate polarity, [18] however dielectric constants are not always adequate to correlate solvent polarity effects. [16a] Mixed valence compounds were used as probes to determine the polarity of 1-butyl-3-methylimidazolium based ILs and the conclusion was that they are not "exceptionally" polar. [19] To define the nature of ILs polarity, the molar conductivity ratio was used and compared with solvatochromic polarity scales. [16b] Dielectric continuum calculations used to predict solvation dynamics in dipolar liquids were shown to work poorly for predicting the response in ILs. [20] An important discussion of ILs solvation ability and polarity was recently reported. [21] Physical properties of ILs were investigated both experimentally [22] and by calculations [23] and were discussed by Reichardt, [24] but no clearcut pattern emerged.

Solvent effect of Ils was also qualitatively assayed in a number of reactions, [25] from which similarities and differences with molecular solvents were discussed. Electrochemical reactivity in ambient temperature ILs is also increasingly important: [1,26] advantages and disadvantages of electrochemical studies in room-temperature ILs have been discussed. Among disadvantages, high cost, difficult purification, and high viscosity are very important. From the advantages point of view, we can mention the negligible vapour pressure, the wide electrochemical window, [START_REF] Trulove | Ionic Liquids in Synthesis[END_REF] the high thermal and chemical stability, the solvating ability, and the possibility to work in the absence of supporting electrolyte. [START_REF] Zhang | [END_REF] Moreover, it is possible to observe in ILs transient ionic and radical species that are undetectable in molecular solvents. [29] Therefore, it is not surprising that electrochemical properties are among the physical characteristics determined for newly synthesized ILs. [21c,30] Results of electrochemical reactions appear to depend on the investigated system. For example, electrochemical oxidation of 2,6-di-t-butylphenol gave better results in acetonitrile than in bmim ILs, either pure or in mixture with organic solvents, [31] while, when simple inorganic molecules were used, the electrochemistry in ILs presented similarities and differences with molecular solvents, depending on the nature of the redox couple. [32] In another study, potential utility of 1-ethyl-3-methylimidazolium dicianamide (emimDCA) was evaluated with CuCl. [33] It is worth mentioning a report of an IL that was not only an electrolyte, but also a solvent in poly(vinylferrocene) redox switching processes. [34] Ferrocene-modified ionic liquids in acetonitrile and in ILs showed a reversible electrochemical behaviour, with responses typical of those for unmodified ferrocene. [35] Electrochemical investigation in ILs may be complicated; for example, from studies of the influence of CO 2 in solution on the voltammetric currents of the electroactive species, it was concluded that the voltammetric response in RTILs cannot be assumed to be independent on other solutes. [36] Voltammetric studies in ILs might offer indications about polarities of these media, but an important problem is the choice of a reliable reference system, for which IUPAC dictated requirements. [37] It has been demonstrated that the redox couples ferrocene/ferricenium (FcH/FcH + ) and decamethylferrocene/ decamethylferrocenium (Me 10 Fc/Me 10 Fc + ) meet IUPAC requirements in molecular solvents. [38] After the first extensive investigation on the electrochemical behaviour of FcH/FcH + couple in 1butyl-3-methyl imidazolium salts, that estimated the solvodynamic radii by measuring diffusion coefficients, [START_REF] Lagunas | Ionic Liquids as Green Solvents[END_REF] a number of papers appeared dealing with various aspects of FcH/FcH + electrochemistry in ILs. The ferrocene/ferrocenium couple was tested as reference redox couple for aprotic ionic liquids and it was found suitable for that. [40] The diffusion coefficient of ferrocene was not significantly affected by the concentration, [41] or nitrogen atmosphere; on the other hand in Ar atmosphere an increase of diffusion coefficients with respect to vacuum was detected. [42] The 3 reversible formal potentials and diffusion coefficients of ferrocene were found to be independent of electrode material, concentration, and technique used for the measurement. [43] However, redox potentials of FcH/FcH + do vary with solvent, as a consequence of specific interactions. Taking advantage of this property, the redox potentials of the two couples, FcH/FcH + and Me 10 Fc/Me 10 Fc + , in different ionic liquids were measured in order to investigate whether or not they can be used as probe for ILs polarity. Moreover, such a study was also done with the aim to explore the possibility to use the correlation (if any) to estimate unknown solvent empirical parameters.

Experimental Section

Commercial grade molecular solvents were generally dehydrated and distilled according to literature methods, [START_REF] Perrin | Armarego Purification of Laboratory Chemicals[END_REF] dimethylsulfoxide (DMSO) was partly freezed, discarding the liquid part, and spectroscopic grade anhydrous N,N-dimethylformamide (DMF) was used as such.

HPLC pure Ionic Liquids were prepared by anion exchange of the corresponding bromide, [45] the latter being highly pure after repeated crystallizations. The resulting ILs (Scheme 1) were characterized by 1 H and 13 C NMR, and their identity was confirmed by comparison with literature data. [46] All the ILs have been kept under inert atmosphere and dried before use under vacuum (0.1 mmHg, 60°C) for at least 1 hr.

Ferrocene was purified by sublimation, commercial decamethylferrocene and ferrocenium hexafluorophosphate were used as received.

Voltammetry Experiments

Electrochemical experiments were carried out using an Amel Intrument 7059 at room temperature. A Pt disc (2 mm diameter) and a Pt wire were used as working and counter electrode, respectively, and were polished by standard procedures before use. Saturated calomel electrode (SCE) was used as the reference. Measurements were reproducible even after months with the same reference electrode, the highest difference from the average values being ± 5 mV.

For experiments in molecular solvent, tetrabutylammonium hexafluorophosphate (TBAH) or tetrabutylammonium bromide (TBAB) were used as supporting electrolytes, depending on the solubility. When both salts were soluble, the same results were obtained with either.

In all the runs ferrocene and decamethylferrocene concentration was the same (1 mM), in order to 4 overcome possible concentration effect, as found recently for ferrocene in bmimTf 2 N. [47] Ferrocene and decamethylferrocene solutions were kept under stirring long enough before measurements, to ensure complete solubilization. Due to solubility problems, a ferrocenium salt was used in water.

All the solutions were deoxygenated before use, by bubbling nitrogen. Scan rates were 100 and 200 mV/s, with no significant difference in half-wave potential values, nor in distance between anodic and cathodic peaks. In selected cases, scan rates were varied in the range 50 -500 mV/s, the same results were obtained, within experimental error. All runs were at least duplicated and several cycles were followed each time. Redox potentials, E 1/2 , were calculated from the midpoint of the cathodic and anodic peak potentials observed in the cyclic voltammograms.

The results are reported in the Table 1, as average values of runs at 100 mV/s. Parameters and physical properties used are also reported. Other parameters were also available, [48] however, since values are quite scattered, we tried, as far as possible, to use data sets obtained by the same method.

One example of cyclic voltammetry is shown in Figures 1 (FcH/FcH + in molecular and ionic solvent) and 2 (Me 10 Fc/Me 10 Fc + in molecular and ionic solvent).

insert Figure 1 and Figure 2 here insert Table 1 here

Results and Discussion

The redox potentials of ferrocene and decamethylferrocene were determined in some molecular solvents and in a number of Room Temperature Ionic Liquids (Scheme 1), vs. SCE, at room temperature.

Insert Scheme 1 here

Both compounds satisfy IUPAC standards, as discussed by Lay, [38] who investigated solvent effects of molecular solvents with the same redox systems. However, our results cannot be directly compared with Lay's data, because the latter were determined against a Ag/Ag + reference.

When compared with the data available in the literature for E 1/2(FcH) vs. SCE, [61] our results are, nevertheless, in excellent agreement with those in DMF and bmimBF 4 . The same it is not observed in Since oxidation of ferrocenes leads to charged species, it could be anticipated that solvents should affect redox potentials in that the more polar is the solvent, the easier is the oxidation.

At first, redox potentials were plotted against solvents physical properties. In particular, with dielectric constants ε r , indicative of polarity (Figure 3), a trend -if not a correlation -may be observed in molecular solvents, but not in ILs: where redox potentials are significantly different also when dielectric constants are very similar.

Insert Figure 3 here

To verify whether this behaviour could be ascribed to viscosity, redox potentials of FcH/FcH + and Me 10 Fc/Me 10 Fc + were plotted against this parameter, as shown in Figure 4. From inspection of the graph, it is apparent that viscosity is not a suitable descriptor for the observed differences of redox potentials.

No correlation could be found with macroscopic IL properties, as already found in the literature. [62] Therefore, empirical solvent parameters were tested. A plot of redox potentials vs. solvatochromic parameter E T N (Figure 5) gave scattered points. The same pattern was shown by others empirical solvent parameters, such as π*, α and β (Supporting Material, Figures S1-S3). Therefore a multiparameter approach was tested.

Insert Figure 4 and Figure 5 here

The multi-parameter Kamlet-Taft equation [63] have been relatively little used with RTILs, pure or mixed with other solvents, with results dependent on the reactions investigated. For example, the reactivity of anionic nucleophiles with methyl p-nitrobenzenesulfonate showed no "IL effect", with a correlation indicating hydrogen bond donation by the solvent, [17c,64] although an "IL effect" was evidenced by the change in S N 2 mechanism of the reaction between charged electrophiles and charged nucleophiles. [65] Another S N 2 examined by the Kamlet-Taft equation was quaternization of 1-methylimidazole with benzyl halides, [66] where six solvents, molecular and ionic, were tested. In the substitution reaction of cationic Pt(II) complex with thioacetate, reaction rate increases with increasing hydrogen bond donor ability (α) and dipolarity/polarizability (π*) of ILs, with no special "ionic liquid effect". [67] To the same conclusion led the investigation of the base-catalyzed keto-enol equilibrium with 2-nitrocyclohexanone. [68] The esterification of methoxyacetic acid with benzyl 6 alcohol in ILs was examined using a multiparameter empirical relationship, but the dominant parameter resulted to be the solvent hydrogend bond basicity. [69] The Diels-Alder cycloaddition was one of the most studied reactions. In solvent mixtures bmimBF 4water or methanol, solvophobicity, hydrogen-bond donating, α, and accepting, β, abilities of the mixtures were the more influential factors. [70] When such reaction was investigated in ILs using multiparameter linear solvation energy relationship, the hydrogen-bonding donation ability of the cation resulted important. [56] Later, a theoretical study of solvent effect on the same reaction evidenced that the strong asynchronicity of the Diels-Alder reaction in ILs is due to the polarization effect of the cation. [71] Interestingly, also the influence of ionic liquids on polymerization rate of methyl methacrylate was tested with the Kamlet-Taft equation and found to depend mainly on π*. [72] In its more complete formulation, the multi-parameter Kamlet-Taft equation contains a contribution of solvent dipolarity/polarizability (π*), a term relative to hydrogen-bond donating (α) and accepting (β) abilities, and a contribution of the Hildebrand solubility parameter, δ 2 , that describes the formation of cavities in the solvent to accommodate the solute. However, we preferred, as in the examples above, not to consider this latter parameter, since it did not practically affected the redox potentials (Figure S4 in Supporting Material).

The solvent dependent redox potentials of ferrocene and decamethylferrocene were treated applying the multiparameter linear free energy relationship in the form of eq.1, analogous to that used in the literature. [38] E

(V) = E 0 + sπ* + aα + bβ (1)
In Lay's work, the uncertainty due to liquid junction potentials was taken into consideration by correlating the solvent parameters with ∆E between ferrocene and decamethylferrocene redox potentials. [38] However, we preferred to treat ferrocene and decamethylferrocene data separately, because specific interactions with different solvents are, reasonably, quite different.

The analysis was made with redox potentials measured in all the examined solvents (molecular and ionic) and in molecular solvents only, with the same results. Coefficients of eq.1 are reported in Table 2, together with those obtained by the same equation with literature data, giving the relationships of equations 2 (for ferrocene) and 3 (for decamethylferrocene):

FcH:

E (V) = 0.58 -0.23π* -0.18α + 0.15β (2)
Me 10 Fc:

E (V) = 0.19 -0.36π* -0.24α + 0.20β (3)
Insert Table 2 The correlation improved very slightly, if the δ 2 parameter was introduced, indicating that the improvement is only caused by the higher number of parameters. Therefore we preferred to keep their number as low as possible, to ensure statistical meaning.

If we compare the solvent effect of all the solvents with that of molecular solvents (columns 2 and 3), the same coefficients resulted, within experimental error, especially for ferrocene redox potentials.

This seems to indicate that there is no special "ionic liquid effect". If we compare our data obtained in molecular solvents with those from literature data in the same solvents (columns 3 and 4), they are in good agreement, taking into account the different reference electrodes used for the two measurements sets. This internal coherence makes one comfortable with respect to the consistency of results as well as to the choice of the reference electrode, SCE vs Pt or Au; in fact, with these latter electrodes metal reduction and electrode surface modification have been reported. [1b, 26a,73,74] Thus, it is possible to discuss contribution by individual parameters within the Kamlet-Taft relationship.

The negative values for the π* coefficient is easy to explain: the more polar the solvent, the easiest the oxidation, with ferrocenium species favoured by solvation. It is more difficult to understand the role of the hydrogen bond accepting (β) and donating (α) ability of the solvents. In fact, at a first sight, β should be more effective with the oxidized species, favouring oxidation, whilst α should work better with the neutral substrates, rendering oxidation more difficult. Coefficients obtained by the multiparameter approach say just the opposite.

Let us consider the α contribution first. Hydrogen-bond donation from the solvent to ferrocene might occur toward the non-bonding electron pair on Fe d orbital or toward the ring π system (Figure 6).

insert figure 6 here

Hydrogen-bond with iron atom can be ruled out: in fact, this interaction, that is not possible with ferrocenium cation, is not consistent with the negative value of coefficient a. Moreover, a of ferrocene is very similar to that of decamethylferrocene (Table 2), whereas the interaction with iron should be sterically hindered with the latter. Thus, solvent-to-cyclopentadienyl ring hydrogen-bond bond donation is more likely and the experimental result should be more strictly related to the peculiar nature of ferrocene and ferrocenium species. A tentative explanation may begin with the consideration that, upon oxidation, the 18-electrons closed shell of ferrocene becomes the less stable 17-electrons configuration of ferrocenium cation, with consequent increase of the ring-iron distance (from 1.66 [75] to 1.70 Å [76] in the solid state) and loosening the cyclopentadienyl-iron bond. 2).

We realized that the reasoning is counter-intuitive. Therefore, a calculation of HOMO of ferrocene and ferrocenium ion was performed using Spartan '08 DFT UB3LYP method, with LACVP basis set. [START_REF]Spartan '08[END_REF] The output is shown in figure 7, where it is evident the higher electron density of Cp ring of the oxidized species with respect to the reduced one, thus supporting the above interpretation.

insert figure 7 here

All considered, the multiparameter Kamlet-Taft equation gave a fair correlation with redox potentials of ferrocene (Figure 8), a less satisfactory one with decamethylferrocene.

Insert Figure here

Qualitatively, the equation for decamethylferrocene is the same as that for ferrocene, but the fit is worst; however, the relationship improves significantly if water is not considered (Figure 9).

Insert Figure 9 here

It is important to consider that this LFER has been applied to ionic liquids together with molecular solvents. The relationship indicates that, as far as oxidation of ferrocenes is concerned, specific interaction of reduced and oxidized species with ILs follow the same rules as with molecular solvents. Therefore ferrocene can be considered a suitable probe for solvent polarity in electrochemical measurements, unifying in a single relationship molecular and ionic solvents.

However, it is dangerous to generalize and, with different reactions, solvent effects should be verified each time. The redox potentials of ferrocene and decamethylferrocene were determined in a number of molecular and ionic solvents and used as a probe for polarity of room temperature ionic liquids. No macroscopic solvent property is simply related to redox potentials. Single empirical solvent parameters also are not able to describe the differences in potentials. Instead, Kamlet-Taft multiparameter relationship fits well ferrocenes redox potentials variation, with both molecular solvents and ionic liquids, well describing specific solvent-solute interactions with the two probes.

F

F o r P e e r R e v i e w

SOLVENT EFFECTS OF IONIC LIQUIDS: INVESTIGATION OF FERROCENES AS ELECTROCHEMICAL PROBES

Claudia Bizzarri, Valeria Conte, Barbara Floris, Pierluca Galloni Table 1. Redox potentials of ferrocene (FcH) and decamethylferrocene (Me 10 Fc) in different solvents and empirical solvent parameters used.

Solvent η, mPa s ε Ε Τ Ν π* α β δ 2 J cm -3 E 1/2 (FcH), V E 1/2 (Me 10 Fc), V n. 1
H 2 O 0.890 [START_REF]CRC Handbook of Chemistry and Physics[END_REF] 78.4 [16a ] 1.000 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 1.09 [17a,51] 1.17 [17a, 51] 0.47 [17a,51 ] 2294 [START_REF] Marcus | The Properties of Solvents[END_REF] 0.212 -0.048 2 EtOH 1.074 [START_REF]CRC Handbook of Chemistry and Physics[END_REF] 24.3 [16a ] 0.654 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 0.54 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.86 [17a] 0.75 [17a,50 ] 676 [START_REF] Marcus | The Properties of Solvents[END_REF] 0.407 -0.084 3 THF 0.456 [START_REF]CRC Handbook of Chemistry and Physics[END_REF] 7.47 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.207 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.58 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.00 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.55 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 361 [START_REF] Marcus | The Properties of Solvents[END_REF] 0.580 0.140 4 DMF 0.794 [START_REF]CRC Handbook of Chemistry and Physics[END_REF] 37.06 [START_REF] Abboud | [END_REF] 0.404 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.88 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.00 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.69 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 581 [START_REF] Marcus | The Properties of Solvents[END_REF] 0.476 0.00 5 DMSO 1.987 [START_REF]CRC Handbook of Chemistry and Physics[END_REF] 46.7 [15a ] 0.444 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 1.00 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.00 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.76 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 708 [START_REF] Marcus | The Properties of Solvents[END_REF] 0.459 -0.039 6 MeCN 0.369 [START_REF]CRC Handbook of Chemistry and Physics[END_REF] 36.2 [16a ] 0.460 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.75 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.19 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.31 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 581 [START_REF] Marcus | The Properties of Solvents[END_REF] 0.385 -0.125 7 DCM 0.413 [START_REF]CRC Handbook of Chemistry and Physics[END_REF] 9.05 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 0.309 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.82 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.13 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.1 [17a] 408 [START_REF] Marcus | The Properties of Solvents[END_REF] 0.405 -0.115 8 Me 2 CO 0.306 [START_REF]CRC Handbook of Chemistry and Physics[END_REF] 20.56 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 0.355 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.71 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.08 [START_REF] Reichardt | Solvent and Solvent Effects in Organic Chemistry[END_REF] 0.43 [17a] 488 [START_REF] Marcus | The Properties of Solvents[END_REF] 0.483 0.046 -9 emimTf 2 N 32.0 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 12.3 [16a ] 0.676 [54] 0.97 [17a,55] 0.659 [55] -585 [54] 0.230 -0.31 10 pmim Tf 2 N 6.402 [57] 10.6 [22d] 0.654 [54] ---432,6 [58] 0.258 -0.261 11 bmim Tf 2 N 63.572 [57] 11.6 [16a ] 0.642 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 0.984 [59] 0.617 [59] 0.243 [59] 554 [54] 0.270 -0.25 12 hmim Tf 2 N 68.0 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 7.0 [54] 0.654 [54] 0.958 [55] 0.623 [55] -555,6 [58] 0.250 -0.25 13 bdmim Tf 2 N 88.0 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 11.6 [16a ] 0.546 [54] 1.01 [59] 0.381 [59] 0.239 [59] 625 [54] 0.309 -0.214 14 bupy Tf 2 N 56.8 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 11.5 [16a ] 0.648 [54] 1.01 [55] 0.483 [55] --0.280 -0.25 15 bupy BF 4 103 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 11.7 [16a ] 0.639 [54] ----0.370 -0.03 16 bmim BF 4 92.0 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 12.2 [60] 0.673 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 1.09 [17a] 0.73 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 0.55 [17a] 929 [54] 0.370 -0.08 17 bmim PF 6 173.0 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 11.8 [60] 0.667 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 1.02 [55] 0.77 [START_REF] Wasserscheid | Ionic Liquids in Synthesis[END_REF] 0.44 [17a] 718 [54] 0 [38]a All lit. VOCs [38]

+ N N + N N + (CF 3 SO 2 ) 2 N - (CF 3 SO 2 ) 2 N - X - (CF 3 SO 2 ) 2 N - N N + (CF 3 SO 2 ) 2 N - hmimTf 2 N N X - + + X = (CF 3 SO 2 ) 2 N bmimTf 2 N X = PF 6 bmimPF 6 X = BF 4 bmimBF 5 emimTf 2 N pmimTf 2 N bdmimTf 2 N X = (CF 3 SO 2 ) 2 N bupyTf 2 N X = BF 4 bupyBF 5
Scheme 1. RTILs used as solvents in electrochemical measurements of this paper. 1).

F o r P e e r R e v i e w SOLVENT EFFECTS OF IONIC LIQUIDS: INVESTIGATION OF FERROCENES AS ELECTROCHEMICAL PROBES

Claudia Bizzarri, Valeria Conte, Barbara Floris, Pierluca Galloni

The redox potentials of ferrocene and decamethylferrocene were determined in a number of molecular and ionic solvents and used as a probe for polarity of room temperature ionic liquids. No macroscopic solvent property is simply related to redox potentials. Single empirical solvent parameters also are not able to describe the differences in potentials. Instead, Kamlet-Taft multiparameter relationship fits well ferrocenes redox potentials variation, with both molecular solvents and ionic liquids, well describing specific solvent-solute interactions with both molecules

1 H

 1 -and13 C-NMR spectra were recorded with a Bruker Avance AMX 300 instrument, using D 2 O and CDCl 3 as solvents.

  an hydrophobic IL, whose water content is generally not strictly controlled and can be significantly different.

  rings might be more electron-rich in the oxidized than in the reduced form and the α parameter should ultimately favour oxidation. If this line of reasoning is correct, it should hold also for β parameter: the more electron-rich the cyclopentadienyl ring is, the less acidic it is. Consequently, ferrocene should be more efficiently solvated than ferrocenium as far as solvent hydrogen-bond accepting ability (β) is concerned, in agreement with the positive value of coefficient b (Table

Figure 1 .

 1 Figure 1. CV of FcH/FcH + in acetone (dotted line) and 1-butyl-2,3-dimethylimidazolium bis(trifluoromethanesulfonyl)amide (solid line); sweep rate, 100 mV/s.

Figure 2 .Figure 3 .

 23 Figure 2. CV of Me 10 Fc/Me 10 Fc + in acetone (dotted line) and 1-butyl-2,3-dimethylimidazolium bis(trifluoromethanesulfonyl)amide (solid line); sweep rate, 100 mV/s.

Figure 6 .

 6 Figure 6. Possible hydrogen-bond interactions between ferrocene and donating solvent (α).

Figure 7 .FollowFigure 8 .

 78 Figure 7. DFT calculation of HOMO for ferrocene (left) and ferrocenium ion (right).

  ConclusionsFerrocene and decamethylferrocene redox potentials depend on the solvent, with no difference between molecular and ionic solvents, since the same Kamlet-Taft multiparameter relationship describes well specific solvent-solute interactions with both types. Decamethylferrocene gave a correlation less satisfactory than ferrocene, thus suggesting that each compound may present different problems in interaction with solvents. Ferrocene emerges as the substrate to be used as a probe for solvent properties.

	ABSTRACT
	o r F o r F o r F o r F o r F o r F o r
	P P P P P P P
	e e e e e e e
	e r e r e r e r e r e r e r
	R R R R R R R
	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
	w w w w w w w
	9 15

Table 2 .

 2 Coefficients of the multiparameter Kamlet-Taft correlation obtained with ferrocene (FcH) and decamethylferrocene (Me 10 Fc)

	.270	-0.21

  a

	SOLVENT EFFECTS OF IONIC LIQUIDS: INVESTIGATION OF FERROCENES AS
	ELECTROCHEMICAL PROBES
	Claudia Bizzarri, Valeria Conte, Barbara Floris, Pierluca Galloni
				FcH
	R		0.980	0.970	0.990	0.949
	R 2		0.959	0.942	0.979	0.900
	s		-0.23 ± 0.04	-0.25 ± 0.074	-0.33 ± 0.04	-0.36 ± 0.06
	a	N	-0.18 ± 0.03	-0.18 ± 0.03	-0.21 ± 0.02 N	-0.25 ± 0.02
	b E 0 (V) N	+0.15 ± 0.03 0.583 ± 0.040	+0.13 ± 0.06 0.607 ± 0.060	+0.06 ± 0.04 0.798 ± 0.040 N	+0.02 ± 0.02 0.843 ± 0.040
				Me 10 Fc
	R R 2 s a b		0.959 0.920 -0.36 ± 0.09 -0.24 ± 0.04 +0.20 ± 0.07 F o r 0.959 0.920 -0.56 ± 0.12 -0.35 ± 0.06 +0.22 ± 0.06	0.729 0.532 -0.32 ± 0.30 -0.16 ± 0.10 +0.16 ± 0.10	0.825 0.680 -0.38 ± 0.10 -0.25 ± 0.05 +0.12 ± 0.06
	E 0 (V) a different reference electrode. 0.197 ± 0.090	0.342 ± 0.099 P	0.158 ± 0.100	0.302 ± 0.099
				e
				e r
				R
				e
					v i e
					w

  N, bupyTf 2 N, bmimBF 4 , bmimPF 6 . Redox potentials of ferrocene (FcH) and decamethylferrocene (Me 10 Fc) vs. viscosity values of molecular and ionic solvents. Redox potentials of ferrocene (FcH) and decamethylferrocene (Me 10 Fc) vs. E T N values of molecular solvents and ionic liquids; in the circles: emimTf 2 N, pmimTf 2 N, bmimTf 2 N, hmimTf 2 N, omimTf 2 N.

		0,8 0,8
							FcH, in VOCs FcH
		0,6 0,6	THF	Me 2 CO	DMF DMSO bupyBF 4	EtOH	FcH, in ILs Me 10 Fc, in VOCs Me 10 C, in ILs Me 10 Fc
		0,4 0,4			MeCN		bmimBF 4
	V 1/2 , V 1/2 , E E	0,2 0,2 0,0 0,0	THF	DCM bdmimTf 2 N Me 2 CO DMF DMSO	bmimPF 6 bupyBF 4	H 2 O
	F o r P e e r η, mPa s 20 40 60 80 100 120 P e r e E T N 0,2 0,4 0,6 0,8 Figure 4. F 0 -0,4 -0,2 0,0 -0,4 r o -0,2 bdmimTf 2 N bmimPF 6 MeCN DCM bmimBF 4	140	160 1,0	180 H 2 O	200 1,2
							R R
							e e
							v i e v i e
							w w

EtOH

Figure 5.

Table

  ).

										3
		0,1
	10 Fc 1/2 Me experimental E	-0,3 0,0 -0,2 -0,1	17 11	9	F r o 13	7	2 6	5	8 4 R 2 = 0,920
		-0,3 -0,4		-0,2	r e predicted E 1/2 Me 10 Fc e -0,1 0,0 P	0,1
								R
								e
								v i e
										w

Figure 9. Experimental redox potentials of decamethylferrocene (Me 10 Fc) vs. values calculated with the Kamlet-Taft correlation (numbers refer to solvents, as in Table

  Redox potentials of ferrocene (FcH) and decamethylferrocene (Me 10 Fc) vs. π* values of molecular solvents and ionic liquids; in the circles: emimTf 2 N, bmimTf 2 N, hminTf 2 N. Redox potentials of ferrocene (FcH) and decamethylferrocene (Me 10 Fc) vs. α values of molecular solvents and ionic liquids; in the circles: bmimTf 2 N, hminTf 2 N. Redox potentials of ferrocene (FcH) and decamethylferrocene (Me 10 Fc) vs. β values of molecular solvents and ionic liquids.Figure S4 . Redox potentials of ferrocene (FcH) and decamethylferrocene (Me 10 Fc) vs. δ 2 values of molecular solvents and ionic liquids.

	SOLVENT EFFECTS OF IONIC LIQUIDS: INVESTIGATION OF FERROCENES AS
	ELECTROCHEMICAL PROBES
	0,8 0,8 0,8
	Claudia Bizzarri, Valeria Conte, Barbara Floris, Pierluca Galloni FcH	FcH FcH in VOCs
	0,6 0,6 0,6	Me 10 Fc THF	THF	FcH in ILs Me 10 Fc Me 10 Fc in VOCs
	SUPPORTING INFORMATION Me 2 CO DMF	Me 2 CO	DMF DMSO Me 10 Fc in ILs
	F o r P e e r R e v i e w π* 0,6 0,7 0,8 0,9 1,0 THF Me 2 CO DMF DMSO MeCN DCM bdmimTf 2 N EtOH bmimPF 6 1,1 H 2 O FcH Me 10 Fc bmimBF 4 EtOH THF Me 2 CO MeCN DCM DMF DMSO H 2 O bdmimTf 2 N bupyTf 2 N bupyTf 2 N bmimBF 4 bmimPF 6 R e v i e w R e v i e w R e v i e w Figure S1. F 0,5 E 1/2 , V -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 o r P e e r α 0,0 0,2 0,4 0,6 0,8 1,0 E 1/2 , V -0,4 -0,2 0,0 0,2 0,4 DCM H 2 O 1,2 1,2 DMF DMSO bdmimTf 2 N MeCN EtOH bupyBF 4 bmimBF 4 bmimPF 6 bmimPF 6 H 2 O THF DCM bupyBF 4 bmimBF 4 EtOH emimTf 2 N Figure S2. F o r P e e r β 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 -0,4 -0,2 0,0 bdmimTf 2 N bmimBF 4 bmimPF 6 H 2 O DCM DMF DMSO MeCN EtOH bmimTf 2 N o r P e e r δ 2 , J cm -3 0 500 1000 1500 2000 2500 -0,4 -0,2 H 2 O Figure S3. F 0,0 Me 2 CO Me 2 CO DMSO MeCN bdmimTf 2 N emimTf 2 N E 1/2 , V 0,2 0,4 DCM THF E H 2 O MeCN EtOH bmimBF 4 0,4 bdmimTf 2 N bmimPF 6 bmimTf 2 N 0,2 1/2 , V H 2 O

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Acknowledgments

Experimental contribution by undergraduate student Federico Santilli is thanked.