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1 Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome 00161, Italy
2 Division of Infectious and Tropical Diseases, University of Florence, Italy
3 Division of Infectious Diseases, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Italy
Correspondence
Maria Grazia Ciufolini
mariagrazia.ciufolini{at}iss.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences of the TosV M segment are DQ479890–DQ479916.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). TosV is endemic in Mediterranean countries, where the insect vectors (P. perniciosus and Phlebotomus perfiliewi) are present (Nicoletti et al., 1996). It is an enveloped, negative-stranded RNA virus, with a genome consisting of three segments: small (S), medium (M) and large (L), encoding the nucleoprotein (N) and a non-structural protein (NSs), the envelope glycoproteins (GN and GC) and a non-structural protein (NSm), and the large protein (L, the viral RNA-dependent RNA polymerase), respectively (Accardi et al., 1993; Di Bonito et al., 1997; Giorgi et al., 1991; Gro et al., 1997).
Among phleboviruses, TosV is the only sandfly-transmitted virus that demonstrates neurotropic activity (Nicoletti et al., 1991). TosV is responsible for acute neurological disease in humans, particularly during the summer with a peak in August, correlating with the life cycle of the phlebotomus vectors (Braito et al., 1998a; Tesh, 1988; Valassina et al., 2000). Most disease cases have been reported in residents or travellers in central Italy or Spain and sporadically from other Mediterranean countries, such as Portugal, Cyprus, southern France and Greece (Charrel et al., 2005).
TosV infection has been reported to be associated with aseptic meningitis or, less frequently, meningoencephalitis or encephalitis without meningitis (Braito et al., 1998a, b; Dionisio et al., 2001; Kuhn et al., 2005). Asymptomatic infection and infection without central nervous system involvement have also been described (Braito et al., 1997). An unusual presentation has been documented for two brothers with diagnosis of TosV infection, both presenting with severe meningoencephalitis, deep coma, disseminated intravascular coagulation and hydrocephalus; one of them had also orchitis (Baldelli et al., 2004).
The molecular variability of TosV strains and its eventual effect on pathogenicity and virulence has been poorly investigated. The co-circulation of four variants of TosV in Tuscany has been suggested, based on the detection of few silent mutations found by sequencing a short amplicon from the N gene in cases of acute meningitis (Valassina et al., 1998). Sequence analysis of the TosV L segment indicated the circulation of at least two different lineages of TosV in the Mediterranean basin, one in Italy and one in Spain (Sanbonmatsu-Gamez et al., 2005; Sanchez-Seco et al., 2003), and the Spanish strains could not be detected by a PCR test used widely as a diagnostic tool in Italy (Sanchez-Seco et al., 2003).
In this study, sequencing of an M gene portion of viral strains detected in the cerebrospinal fluid (CSF) of infected patients, in a bat, the only wild mammal from which TosV has been isolated, and in insect vectors, collected in different years and from different regions, was performed in order to assess TosV genetic variability and evolution.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was available for 10 patients. Most patients originated from different provinces of three regions of central Italy (Florence and Siena provinces of Tuscany region, Perugia province of Umbria region and Macerata province of Marche region); one viral isolate was from a Swedish tourist (Pt 3) who probably acquired TosV infection in Portugal (Ehrnst et al., 1985). Year of isolation, area of origin and clinical symptoms are shown in Table 1(a).
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), were analysed. Most viral strains originated from the Florence and Siena provinces of the Tuscany region (central Italy) and one from Palermo province in Sicily (southern Italy). Host species, number of individuals in the pool and their gender, year of isolation and area of capture are shown in Table 1(b)
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Amplification and sequencing from viral RNA.
RNA was extracted by using an RNeasy Mini kit (Qiagen) directly from CSF samples of infected patients or from TosV strains isolated in mouse brain following several passages in mice or in Vero cell culture.
Two overlapping amplicons of 968 and 1351 bp, comprising the portion of the M gene encoding the GN glycoprotein, were obtained by RT-PCR followed by nested PCR. RT-PCR was performed by using the primer pairs M3 (5'-GGAAACCATTTTGGACAGAG-3', bases 400–419)/M10 (5'-GTTTGAAGGTGATGATTCCTCT-3', bases 2096–2075) and M7 (5'-CCTCATAAAGAAGAAATGTGGA-3', bases 1211–1232)/M16 (5'-TGGCCATGGGATCTATCATGTT-3', bases 3375–3354), followed by a nested PCR with the primer pairs M5 (5'-GATGCTCAAGAAGAAGGAGC-3', bases 773–792)/M8 (5'-TCAACATTTGCAGTTTCACATG-3', bases 1741–1720) and M9 (5'-AACATCATCAGCCTCTAAGTGC-3', bases 1667–1688)/M14 (5'-ATGCCACACATCTAAAGATCTT-3', bases 3018–2997), respectively.
PCR products were purified by centrifugation with Centricon 100 filters (Millipore) and used as templates for sequencing reactions, performed by using an ABI Prism BigDye Terminator v. 1.1 cycle sequencing kit (Applied Biosystems). Oligonucleotides M5 and M7, and M9, M14 and M12 (5'-GAGCAACTATGCACATTACC-3', bases 2530–2511), were used for sequencing reactions with the M5–M8 amplicons and the M9–M14 amplicons, respectively. Further sequencing reactions with oligonucleotides M8, M10 and M11 (5'-GTACCTACCGTACCCAGGAA-3', bases 2015–2034) were performed when necessary. Sequencing reactions were purified by precipitation and finally run on an ABI Prism 310 Genetic Analyzer (Applied Biosystems).
Phylogenetic analysis.
Multiple sequence alignments of 2013 nt of the M segment were carried out by using the CLUSTAL_X program (Thompson et al., 1997) and edited manually with the Bioedit program (Hall, 1999). The HKY+I+
evolutionary model was chosen as the best-fitting nucleotide-substitution model, according to the hierarchical likelihood-ratio test (LRT) implemented in the MODELTEST v. 3.0 software (Posada & Crandall, 1998). Phylogenetic analysis was performed by using parsimony in the PAUP package version 4b.10 (Swofford, 2003). Genetic distances (p-distances) of the deduced amino acid sequences were estimates according to Nei (1978), using the MEGA version 3.0 program (Kumar et al., 2004). Clustering was performed by the unweighted pair-group method of analysis with arithmetic mean (UPGMA). The statistical robustness and reliability of the branching order within each phylogenetic tree were confirmed with a bootstrap analysis using 1000 replicates. The strain isolated from Portugal (Pt 3-1983-P) was chosen as outgroup due to its separated geographical location. Analysis of p-distances was conducted by using MEGA v. 3.0 (Kumar et al., 2004). All of the sequences were compared with the only TosV M segment complete sequence available in GenBank (ISS Phleb.3; accession no. X89628
[GenBank]
). The 577 bp sequence of the Portuguese ELB TosV strain described by Liu et al. (2003) (GenBank accession no. AY129737
[GenBank]
) was aligned with the corresponding portion of our M segment sequences and p-distance values were calculated.
Evolutionary analysis.
Probabilistic models of codon substitution that allow for variable non-synonymous/synonymous substitution-rate ratios (dN/dS or 
) to identify positively selected sites were applied (Nielsen & Yang, 1998). A maximum-likelihood tree was used as the input tree. The nucleotide-sequence alignment was fitted to six models with different hypotheses regarding the distribution of estimated values of (Yang et al., 2000). All of the models implemented in the CODEML program of the PAML package (Yang, 1997) are nested with different degrees of freedom. The LRT was used to determine whether allowing for sites with a >1 improved the fit of the model to the data significantly. When the likelihood of the positive-selection model is significantly higher than that of the nested neutral-hypothesis model, the Bayes empirical Bayes (BEB) procedure is used to predict which codons are under positive selection and to calculate their probabilities (Yang et al., 2000). Although PAML LRTs are conservative for short sequences (e.g. positive selection could be underestimated), BEB prediction for positively selected sites is unaffected by sequence length (Anisimova et al., 2001, 2002).
The nucleotide sequences of the TosV M segment were deposited in GenBank under accession numbers DQ479890 [GenBank] –DQ479916 [GenBank] .
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Phylogenetic analysis
A phylogenetic analysis of 27 M segment nucleotide sequences was performed using the HKY+I+ model. The value of the 
parameter of the distribution, which measures the variability of the nucleotide-substitution rates among sites, was 0.281. TosV sequences clustered in four main lineages (G1, G2, G3 and G4) with bootstrap values above 80 % (Fig. 1a). The same clustering pattern was obtained with the phylogenetic analysis of the deduced amino acid sequences (Fig. 1b).
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The mean p-distance for both nucleotide and amino acid sequences did not show high genetic variability, with a nucleic acid sequence variation ranging from 0 to 5.7 % (mean, 3.5 %) and amino acid sequence variation from 0 to 3.4 % (mean, 2.2 %). Mean nucleotide and amino acid p-distance values were calculated between TosV strains within each cluster and between clusters, and between the outgroup sequence of the Portuguese strain Pt 3-1983-P and each of the four clusters; these are shown in Table 2.
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and the corresponding portion of our M sequences was 0.181±0.003 (range, 0.177–0.186) at the nucleotide level and 0.090±0.007 (range, 0.078–0.104) at the amino acid level.
Analysis of selective pressure
The mean non-synonymous/synonymous substitution rate in the M protein ranged from 0.1760 to 0.1826 among all models compared, suggesting that non-synonymous mutation had only about 18 % as much chance as synonymous mutation of being fixed in the population (=0.18). Parameters estimated from the models suggest the presence of positively selected sites for only 6 % of total sites. Only one site (aa 338) with a posterior probability >99 % was identified; other sites were hotspots, with a posterior probability ranging from 53 to 95 %.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Changes in the amino acid sequence could easily make this protein less efficient in its interaction with the viral nucleic acid. A phylogenetic analysis of the L segment showed that Spanish TosV isolates sequences were related closely to one another and related distantly to the Italian reference strain ISS Phleb.3 (Sanbonmatsu-Gamez et al., 2005; Sanchez-Seco et al., 2003). This finding suggests the presence of at least two geographically distinct populations of TosV. Analysis of the NSs coding region of Rift Valley fever virus (RVFV) isolates collected over a period of 38 years and isolated from diverse localities in Africa and from various hosts (human, animal and arthropod) showed divergence ranging from 0 to 9.6 % at the nucleotide level and from 0 to 9.6 % at the amino acid level (Sall et al., 1997). The genetic variation of RVFV, estimated by sequencing a portion of the M segment RNA of 22 isolates from a variety of host species collected over 34 years in six African countries, ranged from 0 to 4.5 % at the nucleotide level and from 0 to 2.4 % at the amino acid level (Battles & Dalrymple, 1988). Liu et al. (2003) analysed the sequences of a portion of the M segment of different phleboviruses, showing that the sequence divergence was 27.6 % (nucleotide) and 25.7 % (amino acid) within the Sicilian serocomplex, 33.7 % (nucleotide) and 34.4 % (amino acid) within the Naples serocomplex and 35.6 % (nucleotide) and 37.5 % (amino acid) within the Punta Toro serocomplex.
The phlebovirus envelope glycoproteins GN and GC, encoded by the M genomic segment, are important for viral infection, pathogenesis and immunity; they serve as neutralizing and haemagglutinin-inhibiting antibody targets (Battles & Dalrymple, 1988; Besselaar & Blackburn, 1991; Keegan & Collett, 1986; Pifat et al., 1988). These virion surface proteins, being exposed to the selective pressure of the host, can be expected to vary noticeably from one strain to another, particularly for those epitopes that are important for reaction with antibodies.
In our study, we have analysed an M gene portion, comprising the GN coding region and the first 100 aa of the GC coding region, of 27 TosV strains collected over a period of 23 years in different areas of central Italy (majority of cases) and also in Sicily and Portugal (one case each), and isolated from different host species (arthropods, humans and a bat).
Our analyses of this very heterogeneous group of TosV strains revealed a low degree of genetic variability, with a mean nucleic acid sequence variation of 3.5 % and a mean amino acid sequence variation of 2.2 %.
Phylogenetic analysis revealed four different lineages (Fig. 1). Analysis of this small number of TosV sequences did not reveal a close correspondence between viral strains and area or year of isolation, or with host species. Our result of sequences both from patients and from environmental sources, obtained from the same area and time period, clustering in different groups suggests the co-circulation of different viral variants. Finally, no correlation between phylogenetic clusters and clinical presentation of the disease could be observed in this small population.
Differences in the methods of sequence analysis, in the number and origin of the viral isolates studied, in the genome segment and also in the length of the sequences obtained and analysed make it difficult to compare different studies. In our investigation, a lower degree of both nucleotide and amino acid sequence divergence for M sequences between the Portuguese strain Pt 3-1983-P, the most geographically distant, and the Italian strains has been observed, with respect to the distances calculated by comparing a 577 bp M sequence of the Portuguese ELB TosV strain reported by Liu et al. (2003) with the corresponding portion of our M sequences. The mean nucleotide p-distance value between M sequences of the Portuguese ELB TosV strain and our Italian strains is similar to the genetic distances reported previously between the Spanish TosV strains and the Italian reference strain ISS Phleb.3 for L and S nucleotide sequences [mean p-distance values of 0.197 and 0.122 for L and S sequences, respectively, calculated from data of Sanbonmatsu-Gamez et al. (2005)]. Indeed, by comparing mean amino acid p-distance values calculated by Sanbonmatsu-Gamez et al. (2005) for the L and S segments (mean p-distances values of 0.018 and 0.000, respectively) between Spanish and Italian strains with those observed for the M segment between the Portuguese ELB TosV strain and our Italian strains, a higher degree of variability for the M segment can be hypothesized, whilst the N gene seems to be more conserved. In our study, phylogenetic analysis of M amino acid sequences revealed the same clusters as did analysis of the nucleotide sequences, whilst discrimination of the two genotypes (Spanish and Italian) based on both partial L genomic segment and the N gene could only be obtained by analysing nucleotide sequences (Sanbonmatsu-Gamez et al., 2005). It must be noted that the relationship of the lineages G3 and G4 is different for the nucleotide and the amino acid trees, probably due a very low genetic variability and consequently a very low phylogenetic signal for the amino acid sequences. In our opinion, further studies are required to investigate whether the M sequence actually reflects a greater potential for variability.
The observed mean ratio of non-synonymous to synonymous substitution in the M segment was very low (=0.18) and did not surpass the threshold of >1, i.e. was not indicative of positive selection; moreover, all of the sequences showed a low nucleotide substitution-rate heterogeneity across sites (=0.281), suggesting that whilst most of the sites along the gene may be invariable because they are under purifying and/or neutral selection, only a few mutational hotspots are present, which may accumulate mutations at a faster rate. The only residue under positive selection (aa 338) was located at the N terminus of the GN glycoprotein.
Our preliminary data would suggest a strong selective constraint on the evolution of the GN glycoprotein. The life cycle of TosV is not understood fully. The data available suggest that the reservoir of this virus is probably the vector. The role of vertebrates in the maintenance of the transmission cycle remains unclear (Ciufolini et al., 1989; Verani et al., 1988). A strong selective pressure from the immune system of a vertebrate host reservoir seems to be lacking. However, genetic heterogeneity of TosV is currently based on the analysis of a very limited number of strains that may not represent a clear picture of the biological reality.
In this study, we chose oligonucleotides based on the only TosV M sequence available in GenBank. Not all of the TosV strains assayed could be amplified (data not shown). We particularly failed to amplify the M segment of most of the viral strains available from the Marche region, which were detectable both from patients and from insect vectors with RT-PCR followed by a nested PCR with oligonucleotides specific for the S segment. Several attempts have also been made to amplify the M segment with degenerate primers; further studies are in progress to characterize these TosV strains.
In conclusion, our study indicates that TosV strains belonging to at least four different clusters co-circulate in central Italy. Results of the phylogenetic analysis show a low evolutionary rate of this virus under natural ecological conditions. Further studies are needed to characterize viral strains that we failed to amplify in the current analysis and to evaluate whether they differ substantially from identified strains.
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ACKNOWLEDGEMENTS |
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Received 26 June 2006;
accepted 8 December 2006.
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