Krisztian ; Banyai

Baranya

J J Hull

Freeman Mm

Frace Ma

Gentsch Jr
email: jgentsch@cdc.gov

Sequencing and phylogenetic analysis of the coding region of six common rotavirus strains: evidence for intragenogroup reassortment among co-circulating G1P[8] and G2P[4] strains from the United States

Keywords: evolution, genome sequencing, Wa-like rotaviruses, DS1-like rotaviruses, United States of America evolution, genome sequencing, Wa-like rotaviruses, DS1-like rotaviruses, United States of America

 for future genomics analyses. The full or nearly complete coding region of all 11 genes for 3 G1P[8] (LB2719, LB2758 and LB2771) and 3 G2P[4] (LB2744, LB2764 and LB2772) strains isolated from children hospitalized with severe diarrhea in Long Beach, California, where these strains were circulating at comparable rates during 2005-2006 are described in this study. Based on the full-genome classification system, all G1P[8] strains had a conserved genomic constellation: G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-E1-H1 and were mostly identical to the few Wa-like strains whose genome sequences have already been determined. Similarly, the genome sequences of the 3 G2P[4] strains were highly conserved: G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-E2-H2 and displayed an overall lesser genetic divergence with reference DS-1-like strains.

While intergenogroup reassortment was not seen between the G1P[8] and G2P[4] strains studied here, evidence for intragenogroup reassortment events was identified. Similar studies in the post rotavirus genomic era will help uncover whether intergenogroup reassortment affecting the backbone genes could play a significant role in any potential vaccine breakthrough events by evading immunity of vaccinated children.

Introduction

Rotavirus A is the most important species within the Rotavirus genus (Sedoreovirinae, Reoviridae) and the main cause of acute dehydrating diarrhea in infants and young children worldwide [START_REF] Estes | Rotaviruses[END_REF]. The traditional classification scheme to designate individual strains based upon serotype specificities of the neutralizing antigens (G and P types) has been recently extended to the entire genome to allow specifying the genotype of all 11 genome segments. Use of this new classification system during sequencing studies confirmed the existence of the two main human genogroups (Wa and DS1) and permits more detailed studies of inter-strain variability and evolutionary history [Matthijnssens et al., 2008a, b].

The epidemiology of rotaviruses is complex. Typically, each year multiple strain variants co-circulate in a given area and strain prevalence changes yearto-year [Matthijnssens et al., 2009;[START_REF] Martella | Zoonotic aspects of rotaviruses[END_REF]. The existing genetic and antigenic diversity within many serotypes enables intratypic shifts to preserve the dominance of a particular antigenic type even in those epidemic years when serotype specificity of dominating strains remains the same [Bányai et al., 2009]. During the 1980s four serotypes (G1P[8], G2P [START_REF] Pietsch | Molecular characteristics of German G8P[rotavirus strain GER1H-09 suggest that a genotyping and subclassification update is required for G8[END_REF], G3P[8], and G4P[8]) were predominant worldwide, mostly in temperate climates and an additional serotype, G9P[8], emerged to become epidemiologically important during the 1990s. While these 5 antigenic types remained important during the 2000s, additional globally emerging strains (G12P[6], G12P[8]) have been introduced and clonally expanded in many areas [Matthijnssens et al., 2009].

The first synchronized rotavirus strain surveillance network in the United States was launched in 1995-1996 by the Centers for Disease Control and Prevention (CDC; Atlanta, GA) with a group of participating laboratories located across the United States volunteering to provide rotavirus positive stool samples during each rotavirus season [START_REF] Ramachandran | Detection and characterization of novel rotavirus strains in the United States[END_REF][START_REF] Griffin | Surveillance of rotavirus strains in the United States: identification of unusual strains. The National Rotavirus Strain Surveillance System collaborating laboratories[END_REF]. As summarized recently, during the 9-year pre rotavirus vaccine surveillance period (1996-2005) G1P[8] was extremely predominant in the United States followed by G2P [START_REF] Pietsch | Molecular characteristics of German G8P[rotavirus strain GER1H-09 suggest that a genotyping and subclassification update is required for G8[END_REF], and then by G9P[8], G9P[6], G3P[8], G4P [8], and several rare strains, even though temporal and geographical variation in strain distribution was marked [Gentsch et al., 2009].

Historic observations suggest that rotavirus strains belonging to different genogroups do not exchange readily their genome segments except for the outer capsid coding genes although a few exceptions have been noted [START_REF] Nakagomi | Identification of rotavirus genogroups by RNA-RNA hybridization[END_REF]Nakagomi and Nakagomi, 2002;[START_REF] Medici | Characterization of inter-genogroup reassortant rotavirus strains detected in hospitalized children in Italy[END_REF]. For example, [START_REF] Kaga | Recurrent circulation of single nonstructural gene substitution reassortants among human rotaviruses with a short RNA pattern[END_REF] described an outbreak in Japan where a single segment reassortant between Wa and DS1 genogroup strains were identified. Also, [START_REF] Palombo | Multiple-gene rotavirus reassortants responsible for an outbreak of gastroenteritis in central and northern Australia[END_REF] have described a multi-segment reassortant outbreak between Wa and DS1 genogroup strains in Australia. These findings contrast with the original perception on the restricted capability of strains belonging to different genogroups to reassort and thus it remains to be determined whether such reassortment events of non-serotype genes are truly rare or might be more common than expected based on the few studies published in this subject.

Ironically, while the number of genome sequences of epidemiologically minor strains increases continuously [Bányai et al., 2009b[Bányai et al., , 2009c[Bányai et al., , 2009d;;[START_REF] De Grazia | Unusual assortment of segments in 2 rare human rotavirus genomes[END_REF]Esona et al., 2009aEsona et al., , 2009b;;Matthijnssens et al., 2006aMatthijnssens et al., , 2006bMatthijnssens et al., , 2008aMatthijnssens et al., , 2008bMatthijnssens et al., , 2008cMatthijnssens et al., , 2009b[START_REF] Matthijnssens | Reassortment of human rotavirus gene segments into G11 rotavirus strains[END_REF][START_REF] Mukherjee | Full genomic analysis of a human group A rotavirus G9P[6] strain from Eastern India provides evidence for porcine-to-human interspecies transmission[END_REF][START_REF] Pietsch | Molecular characteristics of German G8P[rotavirus strain GER1H-09 suggest that a genotyping and subclassification update is required for G8[END_REF][START_REF] Tsugawa | Whole genome sequence and phylogenetic analyses reveal human rotavirus G3P[3] strains Ro1845 and HCR3A are examples of direct virion transmission of canine/feline rotaviruses to humans[END_REF][START_REF] Wang | Full genomic analysis of a porcine-bovine[END_REF], very few genome sequences are available for representatives of the epidemiologically major Wa-like G1P [8] and DS1-like G2P [START_REF] Pietsch | Molecular characteristics of German G8P[rotavirus strain GER1H-09 suggest that a genotyping and subclassification update is required for G8[END_REF] strains, an important shortcoming that does not allow evaluating comprehensively the significance of intergenogroup reassortment in their epidemiologic fitness and evolution. Because it seemed reasonable to assume that high frequency of co-circulating Wa-like G1P[8] and DS1-like G2P [START_REF] Pietsch | Molecular characteristics of German G8P[rotavirus strain GER1H-09 suggest that a genotyping and subclassification update is required for G8[END_REF] strains could favor the generation of rotavirus strains with mixed gene configuration a pilot study was undertaken at CDC to analyze epidemiologically important strains at the genomic level. A single US surveillance area and season was sought in the records where high co-circulation of G1P[8] and G2P [START_REF] Pietsch | Molecular characteristics of German G8P[rotavirus strain GER1H-09 suggest that a genotyping and subclassification update is required for G8[END_REF] strains was observed.

Materials and Methods

Study design

In this study the 2005-2006 season in Long Beach, California was chosen, where G1P[8] and G2P [START_REF] Pietsch | Molecular characteristics of German G8P[rotavirus strain GER1H-09 suggest that a genotyping and subclassification update is required for G8[END_REF] strains co-circulated at comparable rates, 38% to 32%, respectively. A total of six strains (G1P[8], LB2719, LB2758, and LB2771; G2P [START_REF] Pietsch | Molecular characteristics of German G8P[rotavirus strain GER1H-09 suggest that a genotyping and subclassification update is required for G8[END_REF], LB2744, LB2764, and LB2772) were selected for genetic analysis; to increase the chance to find any reassortants, these 6 strains were selected on the basis of distinctness in their RNA profiles in a polyacrylamide gel (not shown). Stool specimens were utilized in order to avoid any potential nucleotide changes that may be associated with cell culture adaptation.

Genome sequencing

The strategy to obtain the nearly full length genomes of the selected strains included the application of type common primer sets designed to the ends of each genome segment in individual RT-PCR assays followed by the primer walking strategy. Briefly, genomic RNA was extracted from 10% stool suspension by TRIzol as described elsewhere [START_REF] Jakab | Oneyear survey of astrovirus infection in children with gastroenteritis in a large hospital in Hungary: occurrence and genetic analysis of astroviruses[END_REF]. Then, 6 µl RNA was mixed with the appropriate combination of gene specific primer pairs (2 µl of 20 µM of each primer) in a total volume of 10 µl and then denatured at 97°C for 5 min. Then the RT-PCR mixture (One-step RT-PCR kit, QIAGEN) was added in a 30 µl final reaction volume. The thermal profile included incubation at 42°C for 30 min for reverse transcription, followed by activation of the Taq DNA polymerase at 95°C for 15 min, and 40 cycles of amplification (94°C, 30 sec; 42°C, 30 sec; 72°C, 3 min) followed by final extension at 72°C for 7 min. Amplicons were run in low melting point agarose gels and stained with ethidium bromide, followed by excision and purification using the Qiaquick gel extraction kit (QIAGEN) according to the manufacturer's instructions. To avoid contamination of samples the genes of each strain were amplified and purified on separate days. Sequence contigs were generated by Sequencher 4.8 (Gene Codes Corporation, Inc., Ann Arbor, MI). The phylogenetic analysis was done by the neighbor-joining method in MEGA4.1 software [START_REF] Tamura | MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0[END_REF]. The significance of tree topologies was estimated by bootstrap analysis with 1,000 pseudoreplicates.

Computer analyses

GenBank accession numbers

Sequences were deposited in GenBank under the following accession numbers: HM467916-HM467981 .

Results

In general, the complete or nearly complete ORFs of all 11 genes for 6 strains were determined. Short stretches remained undetermined for the VP1, VP2, VP4, and NSP1 genes, where PCR primers overlapped with few nucleotides at the 5' ends of the ORF. This resulted in the loss of 7 amino acids in the predicted coding region (~0.1% of the full length polypeptide sequence of Wa; Table 1).

The missing sequences are highly conservative regions of 5' ends of the respective genes. As expected, the ORFs of Wa-like and DS1-like strains were similar in terms of sequence lengths; exceptions include the VP2 gene, where notable variance was seen in length at the N-terminal end of the protein. For example, the 3 Wa-like US G1P[8] strains had 4, 8 and 10 aa long inserts, respectively, when compared to the reference strain Wa, while DS1-like US G2P [START_REF] Pietsch | Molecular characteristics of German G8P[rotavirus strain GER1H-09 suggest that a genotyping and subclassification update is required for G8[END_REF] strains did not display variation in this region. Concerning the segment lengths, only the NSP5/NSP6 encoding genome segments were remarkably different between Wa-like and DS1-like strains, due to a known 143 bp long insertion at the 3' end, downstream of the coding region [START_REF] Matsui | Sequence analysis of gene 11 equivalents from "short" and "super short" strains of rotavirus[END_REF].

Phylogenetic analysis (Fig. 1) of the nt sequences of all 11 genes of 3 G1P [8] strains revealed a conserved genomic constellation (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1) with, but moderate sequence similarity to strains, Wa, KU, Dhaka 16-03, and D, the few G1P[8] strains whose genome sequences have already been determined [Matthijnssens, et al., 2008b]. Evidence for reassortment among these 3 Californian strains was revealed by demonstrating an inverse phylogenetic relationship among the 3 strains in various genes. Thus, in the NSP1, NSP2, NSP3, NSP5, and VP3 gene phylogenies the strains LB2758 and LB2771 clustered together while the strain LB2719 was more divergent from these strains, whereas in the NSP4, VP1, VP2, VP4, and VP6 gene phylogenies the strains LB2719 and LB2771 clustered together and the strain LB2758 was more divergent from these 2 strains. Of interest, the VP7 gene of all 3 G1P [8] strains belonged to the same genetic cluster, designated lineage I (nt similarity, >96.4%; Table 2) .

Phylogenetic analysis (Fig. 1) of the 3 G2P [START_REF] Pietsch | Molecular characteristics of German G8P[rotavirus strain GER1H-09 suggest that a genotyping and subclassification update is required for G8[END_REF] strains identified a conserved genomic constellation (G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2) with reference G2P [START_REF] Pietsch | Molecular characteristics of German G8P[rotavirus strain GER1H-09 suggest that a genotyping and subclassification update is required for G8[END_REF] strains, DS1 and TB-Chen, and also displayed a lesser extent of genetic divergence to these strains and within the 3 selected strains in most gene phylogenies. In these comparisons, strain LB2744 and LB2772 always clustered together, although no absolute sequence identity was seen among them (range of sequence similarity, 98.7% for VP1 to 99.8% for NSP2 and NSP5), while strain LB2764 was slightly divergent from these strains (range of sequence similarity, 95.3% for VP7 to 99.6% for NSP2). Despite the moderate sequence identity within the VP7 gene, all 3 Californian strains belonged to the same genetic cluster, designated lineage II, with up to 99.6% sequence identity to reference strains (Table 3). In the full genomic constellation, only NSP2 could have represented a shared gene among the 3 G2P [START_REF] Pietsch | Molecular characteristics of German G8P[rotavirus strain GER1H-09 suggest that a genotyping and subclassification update is required for G8[END_REF] strains where the mean genetic similarity (99.6%, range, 99.4%-99.8%) was identical to the genetic similarity seen for the G1P[8] strains, LB2758 and LB2771, for which the common origin of NSP2 was anticipated. Thus, it is possible, that the strain LB2764 acquired its NSP2 gene through reassortment with a co-circulating LB2744-like strain.

Altogether, intragenogroup reassortment events within 3 co-circulating G1P [8] strains and likely among G2P [START_REF] Pietsch | Molecular characteristics of German G8P[rotavirus strain GER1H-09 suggest that a genotyping and subclassification update is required for G8[END_REF] strains were identified, but no intergenogroup reassortment events were detected by the whole genome sequencing approach.

The overall low detection rate of mixed infections (~5%) with involvement of strains belonging to different genogroups in hospitalized Californian children might provide one explanation for this failure. It is planned that in the future additional strains from the CDC's strain collection will be analyzed in similar studies to better understand if intergenogroup reassortment of the non serotype genes is key in evolution and epidemiologic success of various strains detected in either US children treated in hospital due to rotavirus gastroenteritis or in other countries and settings, where mixed infection are more abundant.

Discussion

Although these six strains represent the most important antigen combinations in the US and in many regions of the world [START_REF] Gentsch | Serotype diversity and reassortment between human and animal rotavirus strains: implications for rotavirus vaccine programs[END_REF][Gentsch et al., , 2009;;[START_REF] Santos | Global distribution of rotavirus serotypes/genotypes and its implication for the development and implementation of an effective rotavirus vaccine[END_REF]Matthijnssens et al., 2009a], their representation in terms of whole genome sequence data is limited [Matthijnssens et al., 2008a[Matthijnssens et al., , 2008b]].

Thus, in addition to the primary objective to identify intergenogroup reassortment events among co-circulating strains, the present study was also conducted to provide additional reference genome sequences of G1P[8] and G2P [START_REF] Pietsch | Molecular characteristics of German G8P[rotavirus strain GER1H-09 suggest that a genotyping and subclassification update is required for G8[END_REF] rotaviruses for future genomics and epidemiologic studies conducted on these medically important strains. As nucleotide sequencing becomes routine, it seems likely that surveillance studies in the post rotavirus vaccine era will utilize whole genome sequencing as part of the standard strain characterization algorithm. Such studies are needed to determine whether intergenogroup reassortment affecting the backbone genes could play a significant role in any potential vaccine breakthrough events by evading immunity of vaccinated children. [START_REF] Phan | Detection and genetic characterization of group A rotavirus strains circulating among children with acute gastroenteritis in Japan[END_REF] Note: A 720 bp long fragment was utilized for comparison due to sequence length differences of various reference strains. Note: A 994 bp long fragment was utilized for comparison due to sequence length differences of various reference strains.

  rotavirus strain R479 isolated from an infant in China. J Med Virol 82:1094-1102. Ward RL, Nakagomi O, Knowlton DR, McNeal MM, Nakagomi T, Clemens JD, Sack DA, Schiff GM. 1990. Evidence for natural reassortants of human rotaviruses belonging to different genogroups. J Virol 64:3219-3225.

Fig. 1 .

 1 Figure legend.Fig. 1. Phylogenetic analysis of nucleotide sequences of 3 G1P[8] and 3 G2P[4] Long Beach strains (indicated by arrowheads) along with sequences obtained from GenBank. The trees were constructed using neighbor-joining method of the MEGA4 package with bootstrap of 1000 replicates and rooted with sequences of human rotavirus G3P[9] strain Au-1. Bootstrap values (≥50%) are indicated at the branch nodes. Abbreviations used are Po (Porcine), Bo (Bovine), Hu (Human) and Eq (Equine).

Table 1 .

 1 Overview of sequence completeness of 6 selected US strains. Strain specific insertions in the VP2 gene of Wa-like strains and genogroup specific insertions in the NSP5 gene of DS1-like strains are not considered in the calculations of the percentile values.

	Strain	Length of the sequenced coding region based on the deduced amino acid sequences (%) in comparison to strain Wa *
		VP1,	VP2,	VP3,	VP4,	VP6,	VP7,	NSP1,	NSP2,	NSP3,	NSP4,	NSP5,
	1088 aa	890 aa **	835 aa	775 aa	397 aa	327 aa	486 aa	317 aa	310 aa	175 aa	197 aa ***
	LB2719	99.9	99.8	100	99.5	100	100	100	100	100	100	100
	LB2758	99.9	99.8	100	99.5	100	100	100	100	100	100	100
	LB2771	99.9	99.8	100	99.5	100	100	99.9	100	100	100	100
	LB2744	99.9	99.8	100	99.5	100	100	100	100	100	100	100
	LB2764	99.9	99.8	100	99.5	100	100	100	100	100	100	100
	LB2772	99.9	99.8	100	99.5	100	100	100	100	100	100	100
	* Missing data: VP1, missing 1 aa at N-terminal in all 6 US strains; VP2, missing 2 aa at N terminal in all 6 US strains;
	VP4, missing 4 aa at N terminal in all 6 US strains; NSP1, missing 1 aa at N terminal in strain LB2771
	** Predicted length of the deduced amino acid sequences of the VP2 of contemporary US G1P[8] and G2P[4] strains
	differed from the length of VP2 of Wa: LB2719, 894 aa; LB2758, 898 aa; LB2771, 900; all 3 DS1-like US strains, 877 aa.
	*** 200 aa in the NSP5 of DS1-like strains
	** and ***

Table 2 .

 2 VP7 gene nucleotide identities of 3 Californian G1P[8] strains to 69 reference G1 strains.

	Range of nt similarities (%)
	to reference strains

* According to classification scheme proposed by

Table 3 .

 3 VP7 gene nucleotide identities of 3 Californian G2P[START_REF] Pietsch | Molecular characteristics of German G8P[rotavirus strain GER1H-09 suggest that a genotyping and subclassification update is required for G8[END_REF] strains to 59 reference G2 strains

	Range of nt similarities (%)
	to reference strains

* According to classification scheme proposed by

[START_REF] Trinh | Sequence analysis of the VP7 gene of human rotaviruses G2 and G4 isolated in Japan, China, Thailand, and Vietnam during 2001-2003[END_REF]

Journal of Medical Virology

Acknowledgements

At the time the study was launched, K.B. was an international fellow of the Emerging Infectious Diseases Fellowship Program awarded by the Association of Public Health Laboratories.

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


Dr Philippe Halfon
email: philippe.halfon@alphabio.fr

Pierre Berger

Hacène Khiri

Agnès Martineau

Guillaume ; Pénaranda

Michèle Merlin

Catherine Faucher

Hacène Khiri

Alphabio Laboratoire

Hacene Khiri

Algorithm based on CMV kinetics DNA viral load for preemptive therapy initiation after hematopoietic cell transplantation

Keywords: Cytomegalovirus, plasma real-time PCR, Preemptive therapy

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The aim of this study was to use the viral dynamics of CMV DNA viral to start 5 preemptive therapy, and to prospectively compare the CMV viral load kinetics to 6 pp65 antigenemia. 7 263 blood samples were collected prospectively from 93 patients. All clinical 8 decisions regarding use of preemptive therapy were based on CMV antigenemia. 9

Based on the positivity of the antigen assay and clinical CMV outcome in 10 allotransplant patients, an optimal threshold of 3.05 log10 (1130 copies/ml) was 11 found to discriminate patients who required preemptive therapy and those who did 12 not (sensitivity, 71%; specificity, 65%). A DNAemia level increase of 2.24 log10 (174 13 copies/ml) per day was the optimal threshold to discriminate between patients who 14 required preemptive therapy and those who did not (sensitivity, 93%; specificity, 15 43%). Sensitivity of PCR assay was 92.4% compared with 39% for the antigen assay 16 (p<.001). 17 CMV remains one of the most important complications after allogeneic hematopoietic 25 stem cell transplantation (HCT) [START_REF] Boeckh | Late cytomegalovirus disease and 259 mortality in recipients of allogeneic hematopoietic stem cell transplants: importance of 260 viral load and T-cell immunity[END_REF][START_REF] Krause | Screening for CMV-specific T cell 313 proliferation to identify patients at risk of developing late onset CMV disease[END_REF]Razonable 26 and Emery, 2004]. Both DNA and CMV pp65 antigen detection methods have largely 27 replaced viral tube cultures and shell vial culture in CMV surveillance because of 28 their improved sensitivity, predictive value, and rapidity [START_REF] Boeckh | 251 Cytomegalovirus pp65 antigenemia-guided early treatment with ganciclovir versus 252 ganciclovir at engraftment after allogeneic marrow transplantation: a randomized 253 double-blind study[END_REF]Boeckh 29 et al., 2004;[START_REF] Boivin | 264 Quantitative analysis of cytomegalovirus (CMV) viremia using the pp65 antigenemia 265 assay and the COBAS AMPLICOR CMV MONITOR PCR test after blood and 266 marrow allogeneic transplantation[END_REF][START_REF] Caliendo | Evaluation of real-time PCR laboratory-developed tests using 269 analyte-specific reagents for cytomegalovirus quantification[END_REF][START_REF] Caliendo | Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV 273 antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test 274 in transplant recipients[END_REF]Einsele 30 et al., 1995]. The CMV load detected in blood and in tissues is an important factor in 31 predicting subsequent or ongoing CMV disease in transplant patients and individuals 32 infected with human immunodeficiency virus (HIV) [START_REF] Ghaffari | Monitoring of cytomegalovirus reactivation in bone marrow transplant 295 recipients by real-time PCR[END_REF]Humar et al., 33 2009]. Antigenemia assay and culture assays are imprecise, have poor sensitivity, or 34 require long time processing. These drawbacks may be improved by real time PCR 35

with automated DNA extraction [START_REF] Abbate | Evaluation of an automated extraction system in 246 combination with Affigene CMV Trender for CMV DNA quantitative determination: 247 comparison with nested PCR and pp65 antigen test[END_REF][START_REF] Gimeno | Quantification of DNA in plasma by an 298 automated real-time PCR assay (cytomegalovirus PCR kit) for surveillance of active 299 cytomegalovirus infection and guidance of preemptive therapy for allogeneic 300 hematopoietic stem cell transplant recipients[END_REF]Gouarin et 36 al., 2007;[START_REF] Ksouri | Analysis of cytomegalovirus 317 (CMV) viremia using the pp65 antigenemia assay, the amplicor CMV test, and a semi-318 quantitative polymerase chain reaction test after allogeneic marrow transplantation[END_REF][START_REF] Tang | Cytomegalovirus DNA 358 measurement in blood and plasma using Roche LightCycler CMV quantification 359 reagents[END_REF]. Earlier studies suggested that 37 plasma-based PCR assays result in delayed detection of CMV DNA because the 38 sensitivity of these assays is lower than that of cell-based and whole blood assays 39 [START_REF] Razonable | The clinical use of various blood compartments for cytomegalovirus (CMV) 348 DNA quantitation in transplant recipients with CMV disease[END_REF]. Plasma does not require lengthy cell separation and offers a 40 much better opportunity to detect CMV viremia during periods of severe cytopenia 41 when cell-based assays perform poorly. Plasma-based assays were validated 42 clinically by direct comparison with CMV detection [START_REF] Kalpoe | Validation of clinical application of cytomegalovirus plasma DNA load 310 measurement and definition of treatment criteria by analysis of correlation to antigen 311 detection[END_REF]Lengerke et 43 al., 2006]. Quantitation of virus load may also be useful in preemptive treatment 44 strategies; a certain threshold or increasing levels of viral load could be used to start 45 antiviral therapy [START_REF] Boeckh | How we treat cytomegalovirus in hematopoietic cell transplant 262 recipients[END_REF][START_REF] Lilleri | Clinically-based determination of safe DNAemia cutoff levels for preemptive 326 therapy or human cytomegalovirus infections in solid organ and hematopoietic stem 327 cell transplant recipients[END_REF]Machida et al., 46 2000;[START_REF] Mori | Dose-adjusted 334 preemptive therapy for cytomegalovirus disease based on real-time polymerase chain 335 reaction after allogeneic hematopoietic stem cell transplantation[END_REF]. Recent guidelines confirmed that initial viral load is probably 47 the best indicator of viral dynamics because it indicates the slope of CMV replication 48 without intervention [START_REF] Boeckh | How we treat cytomegalovirus in hematopoietic cell transplant 262 recipients[END_REF][START_REF] Verkruyse | Once daily ganciclovir as 361 initial pre-emptive therapy delayed until threshold CMV load > or =10000 copies/ml: 362 a safe and effective strategy for allogeneic stem cell transplant patients[END_REF]. These 49 guidelines reported that an initial viral load of 1000 copies/ml should be considered 50 as the parameter to start preemptive therapy. It was demonstrated that the doubling 51 time of CMV is only 1 to 2 days on average and that the degree of 52 immunosuppression determines in vivo replication dynamics [START_REF] Emery | The dynamics of human 284 cytomegalovirus replication in vivo[END_REF]. were subjected the same day to a pp65 antigenemia assay and a quantitative real-64 time PCR assay of plasma DNA (Table 1). 65 66

Monitoring and management of active CMV infection. The surveillance for active 67

CMV infection was performed routinely with the pp65 antigenemia assay and the 68 real-time PCR assay. An episode of active CMV infection was defined either by a 69 the Artus CMV LC PCR kit (Qiagen GmbH, Hilden, Germany) was performed using 87 the ABI PRISM 7000 system (Applied Biosystems Inc., Foster City, CA), or the 88 m2000RT (Abbott Molecular) according to the manufacturer's instructions. The limit of 89 detection of the assay was 64.2 copies/ml (linear range: [200;10 6 copies/ml]. DNA 90 extractions were performed using the Abbott sample preparation system DNA kit on 91 the m2000 instrument (Abbott Molecular) according to the manufacturer instructions 92 [START_REF] Gimeno | Quantification of DNA in plasma by an 298 automated real-time PCR assay (cytomegalovirus PCR kit) for surveillance of active 299 cytomegalovirus infection and guidance of preemptive therapy for allogeneic 300 hematopoietic stem cell transplant recipients[END_REF]. 93 Data analysis. Non parametric Cuzick test for trend was used to compare multiple 94 distributions [START_REF] Cuzick | A Wilcoxon-type test for trend[END_REF] 2). Among the 59 samples with detectable viral load by PCR, 29 (49%) were 110 not detected by the antigenemia assay. Two samples were positive by the 111 antigenemia assay but non detectable by the PCR assay. These samples were 112 reanalyzed by PCR using different aliquots and found to be undetectable (the internal 113 control was amplified, ruling out the possibility of PCR inhibition or inefficient DNA 114 extraction). The concordance between the two assays was 90% (kappa=.65) and 115 PCR assay was more sensitive than antigenemia assay (92.4% versus 39.0% 116 p<.001) for detection of CMV in blood. 117

In samples detectable by PCR, CMV DNA increased significantly with antigenemia 118 cell number (Cuzick test for trend p=0.02) (data not shown). 119

Based on the follow-up of the positive detection by PCR and/or the CMV clinical 120 outcome, 14 episodes of active CMV infection were diagnosed (Table 3) Based on the positivity of the antigenemia assay in allotransplant patients, an optimal 141 threshold of 3.05 log10 (1130 copies/ml) was found to discriminate patients who 142 required preemptive therapy and those who did not (sensitivity, 71%; specificity, 143 65%). The same analysis was performed on the entire cohort: 3.17 log10 (1494 144 copies/ml) was the optimal threshold for ongoing preemptive therapy (sensitivity, 145 72%; specificity, 61%). 146

-Kinetics viral load threshold 147

In allotransplant patients, as long as CMV DNAemia is below a cut-off value of 3.05 148 log10 (1130 copies/ml), any slope of viral load change ≥ 2.24 log10 per day should 149 set off an alarm for preemptive therapy. This threshold slope corresponds with a viral 150 load increase of at least 174 copies/ml/day. The analysis performed on the entire 151 cohort showed that the same threshold (2.24 log10) was optimal. 152 153 Discussion 154 155

Using CMV viral load in PCR tests for CMV DNA detection in place of pp65 antigen 156 test in order to identify CMV infections earlier constitutes an important medical 157 challenge for many reasons. Firstly, the positive predictive value of plasma PCR for 158 the development of CMV disease has been evaluated in different settings with 159 somewhat contradictory results [START_REF] Boeckh | Optimization of quantitative detection of cytomegalovirus DNA in plasma by 256 real-time PCR[END_REF][START_REF] Boeckh | How we treat cytomegalovirus in hematopoietic cell transplant 262 recipients[END_REF]; 160 et al., 2007;[START_REF] Caliendo | Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV 273 antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test 274 in transplant recipients[END_REF][START_REF] Gerna | 286 Validation of a DNAemia cutoff for preemptive therapy of cytomegalovirus infection 287 in adult hematopoietic stem cell transplant recipients[END_REF][START_REF] Gerna | Human 290 cytomegalovirus (HCMV) leukodnaemia correlates more closely with clinical 291 symptoms than antigenemia and viremia in heart and heart-lung transplant recipients 292 with primary HCMV infection[END_REF]. 161

The consistently positive CMV DNA results during CMV infection, whereas pp65 tests 162

shows false negatives, lead to a more accurate description of CMV 163 dynamics/kinetics. Secondly, the quantitative nature of the assay may increase its 164 specificity by using a certain viral load threshold or increases over time, thereby 165 avoiding unnecessary treatment of patients who are at low risk of progression to 166 disease. CMV DNA has a much higher stability in blood/plasma than pp65 antigen in 167 polynuclear cell lymphocyte and is therefore less sensitive to transport and does not 168 require immediate processing of samples [START_REF] Boeckh | How we treat cytomegalovirus in hematopoietic cell transplant 262 recipients[END_REF]Boivin et al., 169 2000;[START_REF] Ksouri | Analysis of cytomegalovirus 317 (CMV) viremia using the pp65 antigenemia assay, the amplicor CMV test, and a semi-318 quantitative polymerase chain reaction test after allogeneic marrow transplantation[END_REF][START_REF] Weinberg | Molecular methods for cytomegalovirus surveillance 370 in bone marrow transplant recipients[END_REF][START_REF] Yakushiji | Monitoring of cytomegalovirus 374 reactivation after allogeneic stem cell transplantation: comparison of an antigenemia 375 assay and quantitative real-time polymerase chain reaction[END_REF]. 170

In the present study, based on the positivity of the antigenemia assay and clinical 171 CMV outcome, it has been demonstrated that an optimal initial viral load threshold of 172 3.17 log10 (1494 copies/ml) discriminated patients who required preemptive therapy 173 (sensitivity, 72%; specificity, 61%). A threshold of 3.00 log10 was applied as to fit 174 with a recent study by [START_REF] Boeckh | How we treat cytomegalovirus in hematopoietic cell transplant 262 recipients[END_REF]: Results 175 were similar with the ones obtained with 3.17 log10 threshold: sensitivity of 75%, 176 specificity of 57%, negative predictive value of 62%, and positive predictive value of 177

71%. 178

In the present study, the PCR assay was more sensitive than the antigenemia assay 179 for detecting CMV in blood (sensitivity, 92.4% versus 39.0% respectively, p<.001). In 180 fact, 14 episodes were defined solely on the basis of positive results with the PCR 181 assay, whereas only two non detectable PCR/antigenemia-positive episodes were 182 observed. If the decision to initiate preemptive therapy in our cohort had been based 183 upon either the first detection of CMV DNA in plasma or the occurrence of two 184 consecutive detectable PCR results, the first detection at 3.05 log10 (1130 copies/ml) 185 would have allowed preemptive therapy to be started 8 days before the first positivity 186 of antigenemia. On the other hand, as long as CMV DNAemia is below a cut-off 187 value of 3.05 log10 (1130 copies/ml), any slope of viral load change ≥ 2.24 log10 per 188 value, even on the following day, were at least 174 copies/ml higher, one should 216 consider to initiate preemptive therapy. 217

One limitation of this study might be the lack of standardization of CMV DNA 218 monitoring. Such a standardization could be used to calibrate all the different PCR 219 methods, but it represents a major obstacle in obtaining universally acceptable and 220 validated thresholds for preemptive therapy [START_REF] Deback | Monitoring of human cytomegalovirus infection in immunosuppressed patients 278 using real-time PCR on whole blood[END_REF]. This limitation can 221 be improved by the development of an international standard using International 222 Units. Another point to be discussed is the use of whole blood (which is known to get 223 a higher viral load than plasma) for assessing CMV DNA with a possible earlier 224 detection of CMV DNA in whole blood, due to the higher sensitivity of cell-based and 225 whole blood assays. 226

227

In conclusion, data presented in this study indicate that the Qiagen Artus CMV LC 228 PCR kit, a standardized assay, is more suitable than the antigenemia assay for 229 detecting CMV in blood. It allowed earlier diagnosis of active CMV infection and 230 monitoring of the response to anti-CMV treatment. This study confirmed the threshold 231 plasma viral load of 1000 copies/ml as a cut-off for initiating preemptive therapy 232 [START_REF] Boeckh | How we treat cytomegalovirus in hematopoietic cell transplant 262 recipients[END_REF]

  hematopoietic cell transplantation is initiated by a diagnostic 3 technique at first detection of cytomegalovirus (CMV). 4

A

  standardized real-time PCR assay is more appropriate than the antigen assay for 18 detecting CMV. It allowed earlier diagnosis of active CMV infection and monitoring of 19 the response to anti-CMV treatment. 20 21 Keywords: Cytomegalovirus, plasma real-time PCR, Preemptive therapy 22 23 Background 24

53 54

 53 The primary aim of the present study was to use the viral dynamics of CMV viral load 55 as a parameter to start preemptive therapy. The secondary objective was to compare 56 prospectively the CMV viral load kinetics to pp65 antigenemia. recruited at the hematology unit of the Institute Paoli Calmettes in 61 Marseille, France between September 2006 and October 2006. A total of 263 blood 62 samples were collected prospectively from 93 patients (59 men, 34 women) and 63

  single positive PCR, by antigenemia, or by both. Two consecutive negative results on 70 both tests defined the end of a given episode. Preemptive therapy with oral 71 valganciclovir (900 mg/12 h) and intravenous ganciclovir (5 mg/kg of body weight/12 72 h) was initiated upon a positive antigenemia result ( 1 pp65-positive cells/200,000 73 cells) as indicated previously. 74 75 pp65 antigenemia assay 76 77 The CMV pp65 antigenemia assay is an indirect immunofluorescence test used for 78 the detection of internal matrix phosphoprotein (pp65) in peripheral blood leukocytes. It was carried out using the CINAkit® (Argene, France) according to the 80 manufacturer's recommendations. The presence of one or more pp65-positive cells 81 was considered a positive result. 82 83 Real-Time PCR assay. Blood samples were obtained in EDTA-treated tubes and 84 were processed within 2 hours. Polymorphonuclear leukocytes (PBMCs) and plasma 85 were separated by the standard dextran sedimentation method. Real-time PCR with 86

--Figure 2

 2 Figure2shows the kinetics of six cases that were CMV DNA PCR detectable and 135

Figure

  Figure 1: Description of the 263 blood samples included

Page

  Asymptomatic (N=11) ; CMV Pneumonia (N=3) ; Cytolysis* (N=2) ; Fever (N=2); Cutaneous Rash (N=1); Other (N=1); Not Defined (N=1) Not Defined (N=30) Asymptomatic (N=6) ; Fever (N=3) ; CMV Pneumonia (N=2) ; Not Defined (N=22) *Cytolysis is considered when >2 x upper normal limit 391

of PCR and pp65 antigenemia assays for detection CMV. 103

  . Chi-square test was used to compare proportions. 95 PCR, and 2 among the 204 non detectable by PCR. Nineteen samples could not be 107 analyzed by the antigenemia assay because of low neutrophil counts, among which 108 six were detectable by PCR. CMV was detected by both assays in 30 samples (11%) 109

	106	antigenemia assay, 32 samples (12%) were positive, 30 among the 59 detectable by
		(
	96 97 98 99 100 101 102	Statistical significance was considered if p<0.05. Receiver operating curve (ROC) was used and Youden index was used to determine determine the most appropriate DNAemia threshold. All calculations were assessed using SAS 9.1.3 (SAS Institute Inc. Cary, NC). Results o r P e e r R e v i e Performance F w
		6

Among the 263 samples from the 93 patients, 59 (22%) had a detectable viral load 104 by PCR and 204 (78%) had non detectable viral load (Figure

1

). With the pp65 105

Table

  . In addition, in case of initial viral load detection of 233 CMV DNA below this range, the kinetics of CMV DNA should be monitored every 234

		F o r F o r F o r F o r
		P P P P
		e e e e
		e r e r e r e r
		R R R R
		e e e e
		v i e v i e v i e v i e
	235	three days and preemptive therapy initiated when DNAemia level increases by 2.24 w w w w
	236	log10 (174 copies/ml) per day.
	237
	238
	239	Conflict of interest disclosure: All authors declare that they have no conflict of
	240	interest.
	241
	242
		14

Table 1 : Clinical characteristics of the 263 study samples

 1

Table 2 : Comparison of PCR assay and the antigenemia assay for detection of CMV in the 263 392

 2

	393	blood samples
	394
		Result of antigenemia	Viral Load by PCR assay
		assay	Positive (59)	Negative (204)
		Positive (32)	30	2
		Negative (212)	23	189
		Uninterpretable (19)	6	13
	395

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John Wiley & SonsJournal of Medical Virology

Page 12 of 20 John Wiley & Sons Journal of Medical Virology

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


M.Sc Kirsi ; Nurminen
email: kirsi.nurminen@uta.fi

Ph.D Vesna Blazevic

M.Sc Leena Huhti

M.D Sirpa Räsänen

M.Sc Tiia Koho

Ph.D Vesa P Hytönen

M.D Timo Vesikari

Prevalence of norovirus GII-4 antibodies in Finnish children

Keywords: NoV, antibody responses, ELISA, avidity, blocking antibodies NoV, antibody responses, ELISA, antibody avidity, blocking antibodies

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION 84 85

Noroviruses (NoVs) are common causative agents of acute non-bacterial gastroenteritis in children 86 and adults worldwide [START_REF] Green | Caliciviridae: the noroviruses[END_REF]. After rotavirus, NoVs are the major cause of paediatric 87 gastroenteritis, often requiring hospitalization [START_REF] Pang | Human calicivirus-associated sporadic gastroenteritis in 510 Finnish children less than two years of age followed prospectively during a rotavirus vaccine trial[END_REF][START_REF] Moreno-Espinosa | Human caliciviruses and pediatric gastroenteritis. 491[END_REF]. 88

NoVs belong to a genetically diverse family of Caliciviridae and there are currently at least 25 NoV 89 genotypes infectious to humans, of which 8 belong to genogroup GI and 17 to genogroup GII 90 [START_REF] Zheng | Norovirus classification 545 and proposed strain nomenclature[END_REF]]. The prevalence of genotypes fluctuates annually [START_REF] Gallimore | Inter-seasonal diversity of 428 norovirus genotypes: emergence and selection of virus variants[END_REF] but 91 during the last decade variants of the GII-4 genotype have become the dominant genotypes in NoV 92 epidemics worldwide [START_REF] Siebenga | Norovirus 530 illness is a global problem: emergence and spread of norovirus GII.4 variants, 2001-2007[END_REF]. 93 94

The inability to grow NoVs in in vitro cell cultures [START_REF] Duizer | Laboratory efforts to 417 cultivate noroviruses[END_REF]] has impeded the 95 development of serotype-specific antibody assays. Thus, NoV capsid antigens used in serological 96 diagnostic assays have to be produced by recombinant protein production techniques including 97 baculovirus-insect cell expression system [START_REF] Jiang | Expression, self-assembly, and antigenicity of the 454 Norwalk virus capsid protein[END_REF]. NoV capsid genes are cloned into the 98 baculovirus genome and expressed in insect cells to yield capsid proteins which self-assemble to 99 form VLPs, mimicking the structural, antigenic and immunogenic properties of the native virus 100 [START_REF] Jiang | Expression, self-assembly, and antigenicity of the 454 Norwalk virus capsid protein[END_REF]. 101 102 NoV seroprevalence studies using NoV VLPs have been conducted in several countries worldwide 103 [START_REF] Gray | Prevalence of antibodies to 436 Norwalk virus in England: detection by enzyme-linked immunosorbent assay using baculovirus-437 expressed Norwalk virus capsid antigen[END_REF], Lew et al. 1994, Nakata et al. 1998, Jing et al. 2000, Talal et al. 2000, Kobayashi 104 et al. 2009]. The prevalence of NoV specific antibodies has been observed to follow an age-related 105 pattern [START_REF] Gray | Prevalence of antibodies to 436 Norwalk virus in England: detection by enzyme-linked immunosorbent assay using baculovirus-437 expressed Norwalk virus capsid antigen[END_REF], Jing et al. 2000, Talal et al. 2000]; children acquire antibodies against 106

NoV at an early age and almost 100% of adults have NoV-specific antibodies [START_REF] Jing | Seroprevalence against Norwalk-like human 461 caliciviruses in beijing, China[END_REF]. 107 To distinguish primary from recurrent infections, the avidity of the immunoglobulin G (IgG) 109 antibodies may be determined [START_REF] Gutierrez | Are IgG antibody avidity assays useful in the diagnosis of infectious 443 diseases? A review[END_REF]Maroto. 1996, Kanno and[START_REF] Kanno | Immunoglobulin G antibody avidity assay for serodiagnosis of 464 hepatitis C virus infection[END_REF]. The 110 significance of antibody level or avidity as a correlate for protection from NoV infection is not 111 clear but some studies have shown that high preexisting IgG levels may be protective in children 112 but not in adults [START_REF] Ryder | Evidence of 524 immunity induced by naturally acquired rotavirus and Norwalk virus infection on two remote 525 Panamanian islands[END_REF], Gray et al. 1994, Lew et al. 1994]. In the absence of a cell 113 culture model, a surrogate NoV neutralization antibody assay is used to demonstrate the ability of 114 antibodies to block the binding of NoV VLPs to ABH histo-blood group antigens (HBGAs) [Rockx 115 et al. 2005[START_REF] Cannon | Herd immunity to 414 GII.4 noroviruses is supported by outbreak patient sera[END_REF]. HBGAs likely play an important role in virus entry to the gut 116 mucosal cells being the docking site or receptor for NoVs [START_REF] Harrington | Binding of Norwalk virus-like 446 particles to ABH histo-blood group antigens is blocked by antisera from infected human volunteers 447 or experimentally vaccinated mice[END_REF]. High blocking 117 ability of the antibodies has been suggested to correlate with protection against infection 118 [START_REF] Harrington | Binding of Norwalk virus-like 446 particles to ABH histo-blood group antigens is blocked by antisera from infected human volunteers 447 or experimentally vaccinated mice[END_REF][START_REF] Lindesmith | Human susceptibility and resistance to Norwalk virus infection[END_REF], LoBue et al. 2006 linked immunosorbent assays (ELISA). The VLPs were produced using baculovirus-insect cell 153 expression system as described previously [START_REF] Jiang | Expression, self-assembly, and antigenicity of the 454 Norwalk virus capsid protein[END_REF]]. VLP production and purification are 154 described in details elsewhere [T. Koho et al., manuscript Sera were tested for anti-GII-4 and anti-GII-12 immunoglobulin G (IgG) and immunoglobulin A 161 (IgA) by ELISA as previously described [START_REF] Iritani | Humoral immune responses against norovirus infections of children[END_REF]] with modifications. VLPs were used to 162 coat (4°C overnight) 96-well microtiter plates (Nunc Immuno Maxisorp,Thermo Fisher Scientific 163 Inc., Waltham, MA, USA) in 0.01 M carbonate-bicarbonate buffer (pH 9.6) at a concentration 0.5 164 µg/ml for IgG and 2 µg/ml for IgA detection (100 µl/well). The optimal antigen concentration for 165 coating was determined in preliminary experiments by testing serial dilutions of the antigens with 166 positive and negative sera and plotting saturation curves. After washing three times with 10 mM 167 phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBS-T) the plates were blocked at 168 room temperature (RT) for 1 h with PBS containing 5% skimmed milk (Sigma-Aldrich, St. Louis, 169 MO, USA). The wells were then washed three times with PBS-T and incubated 1 h at 37°C with 170 100 µl of serum diluted 1:100 or two-fold dilution series in PBS-T containing 1% skimmed milk. 171 All serum samples were tested in duplicate wells. After washing six times, horseradish peroxidase 172 (HRP) conjugated anti-human IgG or IgA (Zymed, Invitrogen, Carlsbad, CA, USA) diluted 1:4000 173 in 1% milk in PBS-T was added to the wells. After incubation (1 h, 37°C) the plates were washed 174 and o-phenylenediamine dihydrochloride (SIGMAFAST OPD, Sigma-Aldrich) substrate was added 175 at a concentration of 0.4 mg/ml. The plates were incubated at RT in the dark for 30 minutes and the 176 reaction was stopped with 2 M sulphuric acid (H 2 SO 4 ). Absorbance (optical density, OD) at a 177 wavelength of 490 nm was measured in a microplate reader (Victor 2 1420, Perkin Elmer, Waltham, 178 MA, USA). One known positive and one known negative serum sample was added to all plates as 179 controls. Background signal from the blank wells (wells without serum) was subtracted from all of the 180 OD readings at a plate. A sample was considered positive if the net absorbance value was above the 181 set cut-off value, calculated as follows: > 2x mean OD readings from the negative control serum 182 wells at the same dilution and at least 0.100 OD. Negative antigen control wells included Sf-9 mock 183 antibodies [START_REF] Kanno | Immunoglobulin G antibody avidity assay for serodiagnosis of 464 hepatitis C virus infection[END_REF]. After incubation of sera on VLP coated plates the sera 188 were aspirated from the plate and 8 M urea in PBS-T was added. After 5 minutes of incubation the 189 treatment was repeated. Plates were washed 4 times prior to the addition of HRP-conjugated anti-190 human IgG as described above. The avidity index was calculated as [OD with urea/OD without 191 urea] x 100% and the index value >50% was considered as high avidity. 192 193

Carbohydrate blocking assay 194 195

The assay for blocking the binding of HBGAs to NoV VLPs with human serum was performed as 196 described previously [START_REF] Harrington | Binding of Norwalk virus-like 446 particles to ABH histo-blood group antigens is blocked by antisera from infected human volunteers 447 or experimentally vaccinated mice[END_REF]. Briefly, microtiter plates were coated with VLPs in PBS (pH 7.2) at a concentration of 2 µg/ml and incubated 4 h at RT. After washing, the 198 plates were incubated with 5% milk in PBS overnight at 4˚C for blocking. Sera diluted 1:100 were 199 added to the wells and the plates were incubated 1 h at 37°C and then aspirated from the plate. 100 200 µl of biotinylated H-type-3 or Lewis B (for negative control) (Lectinity Holdings,Inc.,Moscow,201 Russia) were added at a concentration of 40 µg/ml in 1% milk in PBS-T. After 4 h at 37°C the wells 202 were washed and a 1:2000 dilution of streptavidin-conjugated HRP (Thermo Fisher Scientific Inc.) 203 was added and incubated 1 h at 37°C. The development of the colour reaction and the measurement 204 of absorbance at a wavelength 490 nm were conducted as described above. OD reading from the 205 wells incubated without serum was considered as a maximum signal for the binding of H-type 3 to 206 GII-4 VLP. No binding of VLPs to the negative control HBGA (Lewis B) was detected. 207 antibodies was detected in the primary infections whereas the highest frequency was observed in the 259 recurrent infections (P<0.001). All the adult control sera (n=6) contained high avidity antibodies 260 (data not shown). 261 262

Cross reactivity of NoV antibodies 263 264

To determine the immunological cross-reactivity of the sera the seroprevalence was assayed against 265 GII-12 which so far has not been reported in Finland [Maunula and Von Bonsdorff. 2005, S. 266 Räsänen et al., manuscript in preparation]. Of the GII-4 positive sera 70.8% were also IgG positive 267 against GII-12 VLPs. A subset of GII-4 IgA positive samples (96 out of 197) was tested and found 268 that 47.6% of these were IgA cross-reactive for GII-12. When the GII-12 antibody responses were 269 compared to the responses obtained with GII-4 VLPs a positive correlation was detected overall 270 (r=0.64) although the mean ODs were generally lower for GII-12-specific responses (data not 271 shown). Sera of the adult individuals were all positive for GII-12 (data not shown). Cross-reactivity 272 between the two genotypes was further confirmed using paired sera of three patients infected with 273 GII-4 NoV (Fig. 3). All the paired sera showed IgG seroconversion not only to GII-4 (Fig. 3A) but 274 also to GII-12 antigen, although with lower magnitude of the response (Fig. 3B). 275 (mean OD 0.66 ± 0.06) as well as antibody prevalence (47.3%, Fig. 1) were detected in the age 286 group 7-23 months (Fig. 4). In the age group 2-4 years mean OD level increased (1.11 ± 0.07) and 287 by the age of 14 years children had high levels of preexisting antibodies to NoV (mean OD 1.62 ± 288 0.10) in the majority of individuals (91.2%, Fig. 1) and the least infections (14.7%) (Fig. 4). 289

Therefore, a trend to a negative association between the incidence of infection and the level of pre-290 existing antibodies was observed. 291 292

Antibody blocking 293 294

Acute sera obtained from children infected during a waterborne outbreak of AGE in the town of 295 Nokia, Finland [START_REF] Räsänen | Mixed 517 viral infections causing acute gastroenteritis in children in a waterborne outbreak[END_REF] with NoV GII-4 or other agent causing acute gastroenteritis 296 were tested for blocking of the GII-4 VLPs binding to H-type-3 HBGA. Higher blocking activity 297 was seen in the sera of children not infected with NoV than in the sera of children with NoV 298 infection (P<0.05) (Fig 5A). In addition, blocking of the VLPs binding to H-type-3 antigen was 299 performed with 1:100 dilutions of paired sera from eight children with NoV gastroenteritis (Fig. 300 5B). The blocking activity increased after a recent NoV infection. The acute sera could block on 301 average 24.8% of the maximum GII-4 binding to H-type 3 whereas 61.6% of the binding was 302 blocked by the convalescent sera (P<0.05). 303 VLPs was stronger. Other reports also found that seroconversion can be observed in convalescent 336 sera using VLPs from genetically distinct virus from the one causing the primary infection [Belliot 337 et al. 2001]. Despite the cross-reactivity, greater seroresponses are detected against homologous 338 strain, and strains belonging to the same genogroup react more strongly than the strains across the 339 genogroups [START_REF] Treanor | Subclass-specific serum antibody responses to 538 recombinant Norwalk virus capsid antigen (rNV) in adults infected with Norwalk, Snow Mountain, 539 or Hawaii virus[END_REF], Farkas et al. 2003[START_REF] Lindesmith | Heterotypic humoral and cellular immune responses following Norwalk virus infection[END_REF]]. Because of the cross 340 reactive immunity and the presence of the pre-existing antibodies detected, ELISA method as the 341 one described in this study and routinely used by others [START_REF] Rockx | Characterization of the homo-and 521 heterotypic immune responses after natural norovirus infection[END_REF][START_REF] Iritani | Humoral immune responses against norovirus infections of children[END_REF] does 342 not give precise serotype-specific determination of the immune response. 343

344

The avidity and the level of NoV specific IgG antibodies are low after primary infections in young 345 children. The avidity increases with time and particulary as a result of repeated infections when 346 molecular evolution specifies the antigen binding domains of the antibodies to recognize the target 347 epitopes more precisely [START_REF] Murphy | The adaptive immune response[END_REF]]. Indeed, high avidity antibodies were detected in older 348 children and adults who are likely to have experienced NoV infections repeatedly throughout life. 349

The high avidity antibodies detected in infants less than 6 months of age presumably represent 350 transplancentally acquired maternal IgG antibodies [START_REF] Gray | Prevalence of antibodies to 436 Norwalk virus in England: detection by enzyme-linked immunosorbent assay using baculovirus-437 expressed Norwalk virus capsid antigen[END_REF], Numata et al. 1994]. The 351 results show that 20% of infants under 6 months of age already had NoV infection as judged by the 352 presence of NoV IgA antibodies. Such incidence of infection suggests that transplacentally acquired 353 IgG antibodies do not offer full protection. 354

355

The present study showed that children who were exposed to NoV in a waterborne outbreak of 356 AGE [START_REF] Räsänen | Mixed 517 viral infections causing acute gastroenteritis in children in a waterborne outbreak[END_REF]] but were not infected by NoV, had higher blocking activity of GII-4 357 NoV infection has been hypothesised to be associated with a short-term immunity to NoV infection 361 [START_REF] Rockx | Characterization of the homo-and 521 heterotypic immune responses after natural norovirus infection[END_REF]]. The results shown in the current study also suggest that high blocking activity 362 of VLPs binding to HBGA in the acute serum may confer protection against reinfection. Blocking 363 activity of the antibodies may be a relevant surrogate marker of NoV protection when considering 364 different vaccine approaches. 365 366 In this study, the majority of sporadic NoV cases treated in hospital occurred in children 7-23 367 months of age. This age group also had the lowest pre-existing antibody levels. Therefore, a trend to 368 a negative correlation between the susceptibility to NoV infection and pre-existing IgG antibodies 369 was observed. A previous study conducted in Finland [START_REF] Lew | Detection of 470 Norwalk virus or Norwalk-like virus infections in Finnish infants and young children[END_REF]] suggested that children with 370 low NoV-specific IgG titers (<1:50) are more susceptible to NoV infection in the next 10 months 371 than children with higher titers (>1:50). In the current study it was shown that older children with 372 higher NoV specific antibody levels and avidity had also fewer infections than children less than 2 373 years old. It may be speculated that NoV infections before the age of two years induce protection 374 for later ages as the children develop protective immune response to NoV. This is in support of the 375 concept of NoV vaccination in young infants in order to decrease the burden NoV of infection as 376 well as the hospitalization cases due to NoV gastroenteritis. 377

  this study was to determine the seroprevalence of NoV GII-4 IgG and IgA 121 antibodies in Finnish children, and to study if the magnitude and the avidity of the antibodies 122 correlate with susceptibility to infection. In addition, cross-reactivity of NoV antibodies between a 123 common (GII-4) and a rare (GII-12) genotype was investigated. 492 acute stage sera were collected from 0 to 14 year-old children hospitalized for AGE 138 in Tampere University Hopital and Kuopio University Hospital from August 2006 to August 2008 139 [S. Räsänen et al., manuscript in preparation]. The study protocol and consent forms had been 140 approved by the ethics committee in the Pirkanmaa hospital district in 2006 and the patients or the 141 legal guardians volunteered for the study after having given an informed consent. NoV infection 142 was detected in the stool samples of 97 children by reverse transcription (RT)-PCR which is 143 described in details elsewhere [S. Räsänen et al., manuscript in preparation]. Paired serum samples 144 were obtained from 14 of the children with NoV infection. Acute sera were collected within five 145 days and convalescent sera 2-6 weeks after onset of the disease. In addition, acute sera from six 146 adults were collected as a control. The sera were kept at -20° C before testing. in the format of VLPs of the two NoV genotypes, GII-4 [004/95M-151 14/1995/AU] and GII-12 [HU/NLV/Wortley/90/UK] were used as coating antigens in enzyme-152

  cell lysate and were routinely <0.100 OD. A seroconversion was 184 considered as a ≥4-fold rise in the titer between acute and convalescent sera. 185 186 To determine the IgG avidity of NoV antibodies a urea elution was used to remove the low avidity 187

  the blank wells (wells lacking HBGA) was subtracted from all of the OD 208 values of tested samples. chi-square test was performed to examine the differences in antibody prevalence between 213 age groups. Student's t-test was used to assess the intergroup differences in antibody magnitude and 214 avidity. All hypothesis testing was two-tailed. Pearson's correlation coefficient was used to 215 determine the correlation between GII-4 and GII-12 specific antibody levels. Mann-Whitney U-test 216 was used to compare the intergroup differences in the blocking ability of the sera. Statistical 217 significance was defined as P<0.05. serum specimens 63.4% and 38.9% sera were positive for GII-4 specific IgG and IgA, 237 respectively. The age-related seroprevalence is shown in Figure1. The prevalence of IgG antibodies 238 to GII-4 was 66.7% in the age group 0-6 months and 47.3% in the age group 7-23 months. After the 239 age of two years the prevalence increased to 73.1% and continued to increase up to 91.2% in 240 children 5-14 years of age. The NoV IgG antibody prevalence in the age group 7-23 months was 241 significantly lower compared to the other age groups (P<0.001 to 0.05). The corresponding 242 seroprevalence for GII-4 specific IgA antibodies was low (20.8%) in infants 0-6 months of age and 243 rose with increasing age. Overall the prevalence for IgG was significantly higher in each age group 244 compared to IgA seroprevalence (P<0.001 to 0.05). Samples positive for both IgG and IgA were 245 detected in 16.8% of 0-6 months old infants, 20.5% of 7-23 months old, 51.3% of 2-4 years and 246 79.4% of 5-14 years old children (data not shown). IgG and IgA GII-4 specific antibodies were 247 detected in 100% of the adult sera (Fig.1of NoV IgG antibodies in the three age groups representing newborns (0-6 252 months of age), presumable primary/recent infections (7-12 months of age) and recurrent infections 253 (9-14 years of age) are shown on Figure2. High avidity IgG antibodies (mean avidity index 61.5 ± 254 5.1%) were detected in infants <6 months of age. The mean avidity index was significantly lower in 255 children aged 7-12 months (40.4 ± 3.9%) than in children aged 0-6 months (P<0.05). The highest of 256 all mean avidity indexes (85.6 ± 4.5%) were detected in older children, who had most probably 257 infections during their lifetime. The lowest frequency of the high avidity 258

  children encountered in hospital because of AGE, 97 (19.7%) were diagnosed with NoV 279 infection by RT-PCR [S.Räsänen et al., manuscript in preparation]. The incidence of NoV 280 gastroenteritis was highest in children aged 7-23 months (53 out of 97 cases; 54.6%). Of these cases 281 60.4% were primary infections as judged by the absence of NoV antibodies at the acute stage while 282 had pre-existing antibodies to NoV. To determine the possible protective effect of the pre-283 existing NoV-specific antibodies against a subsequent NoV infection the OD values in the different 284 age groups obtained in ELISA IgG assay were compared. The lowest pre-existing antibody level 285

  a NoV infection. The increase in the blocking ability of the serum after the 360

Fig. 5 .Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .

 52345 FIGURE CAPTIONS 556

7 Serum antibody ELISA and avidity assay 159 160

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	155	in preparation]. The morphology and the
	156	antigenicity of the VLPs produced were verified with electron microscopy and western blot (data
	157	not shown).

preparation]. In addition, these results imply the existence of highly conserved antibody epitopes

between the genotypes within a genogroup. We also showed that a seroconversion caused by

infection could be detected with a heterotypic antigen (GII-12) although the response to homotypic

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John Wiley & SonsJournal of Medical Virology

ACKNOWLEDGEMENTS 383 384

We thank Eeva Jokela for her technical assistance during the study. We are grateful for Marjo


Lianet Monzote

Osmany Cuesta-Rubio

An Matheeussen

Tim Van Assche

Louis Maes

Cos Paul
email: paul.cos@ua.ac.be

P Cos

Antimicrobial evaluation of the polyisoprenylated benzophenones nemorosone and guttiferone A

Keywords: nemorosone, guttiferone A, antibacterial, antiprotozoal nemorosone, guttiferone A, antibacterial, antiprotozoal

published or not. The documents may come

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INTRODUCTION

Infectious diseases are still a major threat to public health despite the tremendous progress in human medicine and their control remains a huge challenge since vaccines are only available against a limited number of pathogens. Most of the current antiinfectives suffer from considerable limitations in terms of antimicrobial spectrum and side-effects, and their widespread overuse has led to drug resistance [START_REF] Fauci | Infectious diseases: considerations for the 21st century[END_REF].

Research on new anti-infectives must therefore be continued and all possible strategies should be explored.

In the area of infectious diseases, 75% of new drugs originated from natural sources between 1981[START_REF] Cuesta-Rubio | Polyisoprenylated benzophenones in Cuban propolis; biological activity of nemorosone[END_REF](Newman et al., 2003)). As less than 10% of the world's biodiversity has yet been tested for biological activity, many more natural lead compounds are awaiting discovery [START_REF] Bedoya | Anti-infectious activity in the Cistaceae family in the Iberian Peninsula[END_REF]. The plant kingdom has been used as medicines for thousands of years and is undoubtedly a valid source of drug leads. They offer structural diversity that is not rivaled by the creativity or synthetic ingenuity of medicinal chemists. Well-known examples are taxol (paclitaxel) and camptothecin derivatives (topotecan and irinotecan) as antitumor agents and artemisinin derivatives (artemether and artesunate) as antimalarial agents [START_REF] Koehn | The evolving role of natural products in drug discovery[END_REF]. (Cuesta-Rubio et al., 2005). For several polyisoprenylated benzophenones, cytotoxic, antimicrobial, antioxidant and anti-inflammatory activities have been reported [START_REF] Acuña | Polyisoprenylated benzophenones from clusiaceae: potential drugs and lead compounds[END_REF].

The aim of this study was to investigate the polyisoprenylated benzophenones nemorosone (Fig. 1A) and guttiferone A (Fig. 1B) for their antimicrobial effect against a panel of bacteria, fungi and protozoan parasites.

MATERIALS AND METHODS

Extraction of products.

Flowers of Clusia rosea were collected in Havana (Cuba) in February 2010 and a voucher specimen (No.705) was deposited in the Herbarium of the Instituto de Investigaciones Fundamentales de la Agricultura, Havana, Cuba. Floral resins were extracted with 90% EtOH . Following filtration, the extract was dried under reduced pressure to yield a yellowish gum. The residue was subjected to RP-HPLC (90% MeOH) and nemorosone was crystallized using a mixture of EtOH-H 2 O (70%-30%; v-v). HPLC separations were performed on a Waters 590 series pumping system equipped with a Waters R401 refractive index detector and a 300 x 7.8 mm i.d. 10 µm µ-Bondapak C-18 column (Waters). The structure of nemorosone was confirmed by 1 H NMR, 13 C NMR and ESI-MS spectra, and its purity was verified by HPLC-DAD (>99%). All spectroscopic data were consistent with those previously reported (Piccinelli et al., 2009).

Guttiferone A was obtained from Garcinia aristata fresh fruits by the same procedure employed to obtain aristofenone [START_REF] Cuesta-Rubio | A New tautomeric pair of polyisoprenylated benzophenones from Garcinia aristata[END_REF]: fresh fruits were extracted with n-hexane; the yellow solid obtained after concentration under reduced pressure was purified by HPLC; successive crystallizations from hexane gave crystalline guttiferone A. 1D-and 2-NMR experiments were in agreement with NMR data previously reported [START_REF] Gustafson | The guttiferones, HIV-inhibitory benzophenones from Symphonia globulifera, Garcinia livigstonei and Clusia rosea[END_REF]. The purity of the compounds was analyzed by HPLC-DAD and HPLC-MS and was found to be 98 %.

Microorganisms and cells.

The following microorganisms were used in this study: Reference drugs. Erythromycin and chloramphenicol were purchased from Sigma-Aldrich (Bornem, Belgium), while miconazole and flucytosine were kindly supplied by Janssen Pharmaceuticals (Beerse, Belgium). Benznidazol, chloroquine, miltefosine and suramine were kindly provided by WHO-TDR. Amphotericin B was purchased from Imefa (Havana, Cuba).

Escherichia coli ATCC8739,

Test plate production.

The experiments were performed in 96-well plates (Greiner, Germany) at four-fold dilutions in a dose-titration range of 64 µg/mL to 0.25 µg/mL. Dilutions were carried out by a programmable precision robotic station (BIOMEK 2000, Beckman, USA). Each plate also contained medium-controls (blanks: 0% growth), infected untreated controls (negative control: 100 % growth) and reference controls (positive control). All tests were run in duplicate. Biological screening tests. The integrated panel of microbial screens for the present study and the standard screening methodologies were adopted as have been described

by [START_REF] Cos | Anti-infective potential natural products: How to develop a stronger in vitro "proof-of-concept[END_REF] Antibacterial activity: S. aureus and E. coli were cultured at 37 °C in MHB (Mueller Hinton broth) medium. Assays were performed by adding 5x10 3 CFU /well. After 17 hours incubation, bacterial viability was assessed fluorimetrically after adding resazurin [START_REF] Räz | The Alamar Blue assay to determine drug sensitivity of African trypanosomes (T.b. rhodesiense and T.b. gambiense) in vitro[END_REF] for 30 min at 37°C. Fluorescence was measured using a GENios Tecan fluorimeter (excitation 530 nm, emission 590 nm).

Antifungal activity: T. rubrum and C. albicans were cultured in RPMI medium at 27 °C and 37 °C respectively. Assays were performed by adding 5x10 3 CFU /well. After 7 days (for T. rubrum) or 24 h (for C. albicans) incubation, fungal viability was assessed fluorimetrically after adding resazurin for 24 h at 27 °C for T. rubrum and for 4 h at 37 °C for C. albicans.

Antiplasmodial activity: Parasites were cultured in human A + erythrocytes at 37 °C under a low oxygen atmosphere (3% O 2 , 4% CO 2 , and 93% N 2 ) [START_REF] Trager | Human malaria parasites in continuous culture[END_REF] in RPMI-1640 culture medium, supplemented with 0.5 (g/v) % Albumax TM .

Infected human red blood cell suspension (1% parasitemia, 2% hematocrit) were added to each well with test compounds and incubated for 72 h. After incubation, test plates were frozen at -20 °C. Parasite multiplication was measured by the Malstat method [START_REF] Makler | Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity[END_REF]. One hundred microliters of Malstat TM reagent were transferred in a new plate and mixed with 20 µL of the hemolysed parasite suspension for 15 min at room temperature. After addition of 20 µL NBT (2 mg/mL)/PES (0.1 mg/mL) solution and 2 h incubation at room temperature in the dark, absorbance was read at 655 nm Antitrypanosomal activity: trypomastigotes of T. brucei were cultured at 37 °C and 5% CO 2 in Hirumi-9 medium [START_REF] Hirumi | Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers[END_REF], supplemented with 10% inactivated fetal calf serum (FCSi). Assays were performed by adding 1.5 x 10 4 trypomastigotes/well. After 72 h incubation, parasite growth was assessed fluorimetrically by adding resazurin for 24 h at 37 °C. Amastigotes of T. cruzi were maintained on MRC-5 cells in minimal essential medium (MEM) supplemented with 20 mM L-glutamine, 16.5 mM sodium hydrogen carbonate and FCSi (5%) at 37 °C and 5% CO 2 . To determine in vitro anti-trypanosomal activity, 4 x 10 3 MRC-5 cells and 4 x 10 4 parasites were added to each well of test plate with compound. After incubation at 37 °C for 7 days, parasite growth was assessed by adding the β-galactosidase substrate chlorophenol red β-D-galactopyranoside [START_REF] Buckner | Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase[END_REF] for 4 h at 37 °C. The colour reaction was read at 540 nm and absorbance values were expressed as a percentage of the blank controls.

Antileishmanial activity: L. infantum amastigotes were collected from an infected donor hamster and used to infect primary peritoneal mouse macrophages (PMM). To determine in vitro antileishmanial activity, 3 x 10 4 macrophages were seeded in each well of a 96-well plate. After 48 h incubation at 37 °C and 5% CO 2 , the cells were washed and infected with L. infantum amastigotes at a multiplicity of 15 parasites per cell. Two hours after infection, pre-diluted compounds were added and the plates were further incubated for 120 h at 37 °C and 5% CO 2 . Then, the cells were fixed with methanol and stained with 10 % Giemsa for microscopic reading. The total parasite burden (= average number of amastigotes per cell) in treated wells was compared with control wells. For L. amazonensis, PMM were harvested and plated at 10 6 /mL in a 24- well Lab-Tek (Costar  , USA) and incubated at 37 ºC and 5% CO 2 for 2 h. Non-adherent cells were removed and stationary-phase promastigotes were added at a 4:1 parasite/macrophage ratio for 4 h. The cells were washed to remove free parasites, prediluted compounds were added and the plates were further incubated for 48 h [START_REF] Caio | Selective effect of 2',6'-dihydroxy-4'-methoxychalcone isolated from Piper aduncum on Leishmania amazonensis[END_REF]. The total parasite burden was determined as described above.

Cytotoxicity assay: MRC-5 cells were cultivated in MEM, supplemented with Lglutamine (20 mM), 16.5 mM sodium hydrogen carbonate and 5 % FCSi at 37 °C and 5% CO 2 . For the assay, 10 4 MRC-5 cells/well were seeded onto the test plates containing the pre-diluted compounds and incubated at 37 °C and 5% CO 2 for 72 h. Cell viability was assessed fluorimetrically 72 h after addition of resazurin. For the highest antimicrobial activities, selectivity indices (SI) were calculated by the ratio of the CC 50 for MRC-5 cells and the IC 50 for microorganism.

Statistical Analysis.

In each experiment, the 50 % of microbial growth (IC 50 ) and human cell growth (CC 50 ) inhibition value was determined from the lineal concentration-response curves and the results were expressed as the mean ± standard deviation of two independent experiments.

RESULTS

Nemorosone and guttiferone A were profiled in vitro for their antimicrobial activities.

The benzophenones showed a moderate activity against the Gram-positive bacterium S. aureus, while no activity was demonstrated against E. coli and the fungi T. rubrum and C. albicans (Table 1). More interesting results were obtained against protozoan parasites (Table 2). The highest activity was found for P. falciparum with IC 50 values lower than 1 µM. The cytotoxicity on MRC-5 cells revealed CC 50 values of 15.5 ± 2.9 µM and 12.0 ± 1.0 µM for respectively nemorosone and guttiferone A. In general, a low selectivity was found, except for P. falciparum with an SI value of 39 for nemorosone and 24 for guttiferone A.

DISCUSSION

Plants are an important source in the search of new and selective drugs for the treatment of infectious diseases. The antimicrobial activity of plant-derived agents has been attributed to compounds belonging to diverse chemical groups, such as alkaloids, saponins and terpenes [START_REF] Alviano | Plant extracts: search for new alternatives to treat microbial diseases[END_REF]. Benzophenones represent another interesting class of bioactive compounds with anti-inflammatory, antioxidant and antitumoral effects [START_REF] Acuña | Polyisoprenylated benzophenones from clusiaceae: potential drugs and lead compounds[END_REF]. In addition, several benzophenones have been shown to possess antibacterial activities, like peroxysampsone A [START_REF] Xiao | Prenylated benzophenone peroxide derivatives from Hypericum sampsonii[END_REF], garcinol [START_REF] Rukachaisirikul | An antibacterial biphenyl derivative from Garcinia bancana MIQ[END_REF] and microsphaerin (Yoganathan et al., 2008) against S. aureus. Antiplasmodial effects have also been demonstrated, for example for coccinone, isogarcinol and symphonone [START_REF] Marti | Antiplasmodial benzophenone derivatives from the root barks of Symphonia globulifera (Clusiaceae)[END_REF].

In the current study, the natural benzophenones nemorosone and guttiferone A were evaluated against a wide panel of microorganisms, including a Gram-negative (E. coli) and a Gram-positive (S. aureus) bacterium, a dermatophyte (T. rubrum), a yeast (C. albicans) and protozoa that cause relevant parasitic diseases, namely malaria (P. falciparum), Chagas disease (T. cruzi), sleeping disease (T. brucei), and visceral (L. infantum) and cutaneous (L. amazonensis) leishmaniasis.

Nemorosone has been isolated from plants, like Tovomitopsis saldanhae [START_REF] Bittrich | Floral resin of Tovomitopsis saldanhae (Guttiferae) and 7-epi-nemorosone: structural revision[END_REF] and Clusia grandiflora (Lokvam et al., 2000), as well as from propolis [START_REF] Cuesta-Rubio | Polyisoprenylated benzophenones in Cuban propolis; biological activity of nemorosone[END_REF][START_REF] Tomás-Barberán | Phytochemical evidence for the botanical origin of tropical propolis from Venezuela[END_REF]. Antitumoral, antioxidant [START_REF] Cuesta-Rubio | Polyisoprenylated benzophenones in Cuban propolis; biological activity of nemorosone[END_REF] and antibacterial activity against Salmonella typhi, Proteus [START_REF] Rojas | Actividad antimicrobiana de nemorosona aislada de Clusia rosea[END_REF] have been documented. In addition, activity against honeybee pathogens Paenibacillus larvae and Paenibacillus alvei was also reported (Lokvam et al., 2000).

Benzoylphloroglucinol derivatives isolated from different plants have been named guttiferones A-F, H and O. Guttiferone A was first isolated from Symphonia globulifera as an anti-HIV constituent [START_REF] Gustafson | The guttiferones, HIV-inhibitory benzophenones from Symphonia globulifera, Garcinia livigstonei and Clusia rosea[END_REF]. Other studies have described their isolation from Garcinia brasiliensis [START_REF] Pereira | Leishmanicidal activity of benzophenones and extracts from Garcinia brasiliensis Mart. fruits[END_REF] and Allanblackia monticola [START_REF] Lenta | Leishmanicidal and cholinesterase inhibiting activities of phenolic compounds from Allanblackia monticola and Symphonia globulifera. Molecules 12: 1548-1557. benzophenones from the trunk latex of Clusia grandiflora (Clusiaceae)[END_REF] and antiproliferative effect on T. cruzi [START_REF] Abe | Trypanocidal constituents in plants 3. Leaves of Garcinia intermedia and heartwood of Calophyllum brasiliense[END_REF], L. amazonensis [START_REF] Pereira | Leishmanicidal activity of benzophenones and extracts from Garcinia brasiliensis Mart. fruits[END_REF] and P. falciparum [START_REF] Ngouela | Anti-plasmodial and antioxidant activities of constituents of the seed shells of Symphonia globulifera Linn f[END_REF]. In parallel, a potent free radical scavenging activity compared to the well-known antioxidant caffeic acid was observed [START_REF] Ngouela | Anti-plasmodial and antioxidant activities of constituents of the seed shells of Symphonia globulifera Linn f[END_REF].

Except for P. falciparum, an unspecific activity was observed as demonstrated by the low SI. Both compounds were inactive against E. coli and C. albicans, which correspond with previous reports [START_REF] Naldoni | Antimicrobial activity of benzophenones and extracts from the fruits of Garcinia brasiliensisis[END_REF]. The highest activity for the two benzophenones was demonstrated against P. falciparum, with IC 50 values below 1 µM and with SI values of 39 for nemorosone and 24 for guttiferone A. The mechanism of action of benzophenones has not been studied in detail. Nevertheless, guttiferone A caused a potent in vitro inhibition of cysteine protease cathepsins B and papain and serine peptidases cathepsins G and trypsin [START_REF] Martins | Natural polyprenylated benzophenones inhibiting cysteine and serine proteases[END_REF], which are present in Plasmodium parasites and play an important role in their survival and development in host cells [START_REF] Rosenthal | Cysteine proteases of malaria parasites[END_REF]. Further experiments in animal models of malaria should be performed with the aim to validate these compounds as potential antimalarial drug leads. Benzophenones could be used as a pharmacophore group to design new prenylated benzophenone derivatives. This was shown in previous structure-activity studies where the biological activity increased proportionally with the number of prenyl groups and consequently with their lipophilicity [START_REF] Pereira | Leishmanicidal activity of benzophenones and extracts from Garcinia brasiliensis Mart. fruits[END_REF][START_REF] Urzúa | Antibacterial properties of 3-H-Spiro[1-benzofuran-2,1-cyclohexane] derivatives from Heliotropium filifolium[END_REF]. Previous studies have demonstrated that the more lipophilic benzophenones showed the highest activity against Leishmania parasites. This could be correlated with an increased capacity to cross the macrophage and to reach the amastigote form [START_REF] Pereira | Leishmanicidal activity of benzophenones and extracts from Garcinia brasiliensis Mart. fruits[END_REF]. Other benzophenones displayed toxicity and non-specific action such as symphonone H [START_REF] Marti | Antiplasmodial benzophenone derivatives from the root barks of Symphonia globulifera (Clusiaceae)[END_REF], 2,5-diaminobenzophenone [START_REF] Altenkämper | Antimalarial and antitrypanosomal activity of a series of amide[END_REF], guttiferone O and guttiferone E [START_REF] Lannang | Cytotoxicity of natural compounds isolated from the seeds of Garcinia afzelii[END_REF].

Although some studies are available on the antimicrobial activity of guttiferone A, to our knowledge, this is the first study on nemorosone against a wide panel of microbial pathogens. A selective activity was demonstrated against P. falciparum for nemorosone and guttiferone A, requiring for chemical derivatisation and further exploration in animal models.

  Polyisoprenylated benzophenones have been isolated from plants, particularly in the Clusiaceae family, and their biological properties have received considerable attention from a pharmacological point of view in recent years. These compounds are thought to be of mixed shikimate and acetate biosynthetic origin in which the acetate-derived benzene ring is modified by intervention of prenyl groups. Structural modifications of this ring results in the formation of complex molecules as bicyclo-[3.3.1]-nonane derivatives. Most of them show keto-enol equilibrium in solution and this hydroxyl group seems to play an important role in the biological effects of many

  Staphylococcus aureus ATCC6538, Trichophyton rubrum B68183, Candida albicans B59630, chloroquine-resistant P. falciparum K1, suraminsensitive T. b. brucei Squib-427, nifurtimox-sensitive T. cruzi Tulahuen CL2, L. infantum MHOM/MA(BE)/67 and L. amazonensis MHOM/77BR/LTB0016. Cytotoxicity was tested with human simian virus 40-immortalized lung fibroblasts (MRC-5 SV 2 cells; European Collection of Cell Cultures, United Kingdom).

(

  Biorad 3550-UV microplate reader). The percentage growth inhibition was calculated compared to the blanks.

  sonnei and Streptococcus faecalis

Figure 1 .

 1 Figure 1. Chemical structures of polyisoprenylated benzophenones used in this study.

Table 1 .

 1 Activity of nemorosone and guttiferone A against bacteria and fungi.

	Products		IC 50 a ± SD b (µM)
		S. aureus	E. coli	T. rubrum	C. albicans
	Nemorosone	16.1 ± 0.1	> 64.00	> 64.00	> 64.00
	Guttiferone A	7.5 ± 0.8	> 64.00	19.6 ± 17.5	> 64.00
	Erythromycin	6.6 ± 4.3	-	-	-
	Chloramphenicol	-	6.9 ± 3.4	-	-
	Miconazole	-	-	0.3 ± 0.2	-
	Flucytosine	-	-	-	0.7 ± 0.1

a : IC 50 : Concentration of compound that caused 50 % of inhibition growth b : SD: Standard deviation

Table 2 .

 2 Activity of nemorosone and guttiferone A against protozoa.

	Products			IC 50 a ± SD b (µM)
		P. falciparum	T. cruzi	T. brucei	L. infantum L. amazonensis
	Nemorosone	0.4 ± 0.2	12.5 ± 1.7	17.5 ± 1.9	32.9 ± 5.4	11.2 ± 0.6
	Guttiferone A	0.5 ± 0.2	11.8 ± 1.1	13.5 ± 0.1	13.5 ± 0	15.6 ± 0.1
	Chloroquine	0.3 ± 0.1	-	-		-
	Benznidazol	-	2.2 ± 0.5	-		-
	Suramine	-	-	0.05 ± 0.05		-
	Miltefosine	-	-		7.7 ± 4.5	-
	Amphotericin B	-	-			0.03 ± 0.006

a : IC 50 : Concentration of compounds that caused 50 % of inhibition growth b : SD: Standard deviation 254x142mm (96 x 96 DPI)

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