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Genetic variability of the S segment of Toscana virus
Virus Research 200 (2015) 35–44
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Genetic variability of the S segment of Toscana virus Melissa Baggieri, Antonella Marchi, Paola Bucci, Loredana Nicoletti, Fabio Magurano ∗ Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
a r t i c l e
i n f o
Article history: Received 10 December 2014 Received in revised form 12 January 2015 Accepted 12 January 2015 Available online 20 January 2015 Keywords: Toscana virus Phlebovirus Neurological disease Phylogenetic analysis Negative strand
a b s t r a c t Toscana virus (TOSV) was originally isolated in 1971 from a pool of Phlebotomus perniciosus sandflies collected in Grosseto province (Central Italy). Since its first isolation, several studies have been conducted in Italy and other Mediterranean countries in order to identify its possible animal reservoirs, spread of infection and genetic variability. Phylogenetic analysis conducted on TOSV genome demonstrated the co-circulation of two major lineages in the Mediterranean areas, TOSV A and TOSV B. This study reports the results of the genetic analysis of 32 viral strains isolated in Italy in the last 30 years from patients hospitalized with neurological disease, from sandflies and from the brain of a bat. The genetic diversity of TOSV was investigated by determining the sequences of the whole S segment. Phylogenetic analysis showed that TOSV A lineage represents the lineage circulating in Italy. Moreover, the current variability of lineage A is similar to that of lineage B. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Toscana virus (TOSV) is an arthropod-borne virus (Bunyaviridae family, genus Phlebovirus) transmitted to humans by bites of phlebotominae sandflies. TOSV was first isolated from a pool of Phlebotomus perniciosus sandflies collected in Grosseto province (Central Italy) in 1971. After its first isolation, it has been also isolated from Phlebotomus perfiliewi and Sergentomyia minuta sandflies (Verani et al., 1982; Charrel et al., 2006). Since 1983, TOSV has been associated to neurological diseases such as meningitis and meningoencephalitis in humans (Ehrnst et al., 1985; Nicoletti et al., 1991; Braito et al., 1998). However, most of TOSV infections are asymptomatic or cause mild symptoms, such as fever and headaches that resolve spontaneously (Braito et al., 1997). The diffusion of TOSV infections is strictly linked to the distribution of the insect vectors, with a peak of occurrence in August during the maximum activity of the insects (Tesh, 1988; Braito et al., 1998; Valassina et al., 2000). According to results from viral isolation and serologic surveys cases of patients infected with TOSV have been documented in Italy, France, Spain, Portugal, Greece, Turkey and other countries of the
Abbreviations: TOSV, Toscana virus; ORFs, open reading frames; CSF, cerebrospinal fluid; RT-PCR, reverse transcriptase polymerase chain reaction; MEGA, Molecular Evolutionary Genetics Analyses; BIC, Bayesian information criterion. ∗ Corresponding author. Tel.: +39 06 49902448. E-mail addresses: [email protected] (M. Baggieri), [email protected] (A. Marchi), [email protected] (P. Bucci), [email protected] (L. Nicoletti), [email protected] (F. Magurano). http://dx.doi.org/10.1016/j.virusres.2015.01.013 0168-1702/© 2015 Elsevier B.V. All rights reserved.
Mediterranean basin (Ergünay et al., 2011; Alkan et al., 2013). In Italy, TOSV cases have frequently been reported from rural areas of Centre and South. Recently, the circulation in urban areas of Emilia Romagna (Northern Italy) was also shown (Vocale et al., 2012). TOSV has a tripartite genome with three negative ssRNA segments coding for the nucleoprotein N and non-structural protein NSs (segment S), the glycoproteins Gn and Gc and the nonstructural protein NSm (segment M), and the viral polymerase L (segment L), respectively (Giorgi et al., 1991; Accardi et al., 1993; Di Bonito et al., 1997). TOSV utilizes an ambisense coding strategy for S segment: NSs protein is codified in the genomic sense and N protein in antigenomic sense. The N and NSs open reading frames (ORFs) represent two potential distinct targets for genetic analysis of the S segment. Several studies have been conducted on the molecular variability of TOSV RNA segments. Studies based on the L and M segments demonstrated a co-circulation of two major viral lineages in the Mediterranean areas, proposed as TOSV A and TOSV B (SanchezSeco et al., 2003; Sanbonmatsu-Gamez et al., 2005; Venturi et al., 2007; Collao et al., 2009). TOSV A is found in Italy and France, and TOSV B in the Iberian Peninsula and France (SanbonmatsuGamez et al., 2005; Sanchez-Seco et al., 2003). Recently, findings of an autochthon Croatian TOSV suggested the presence of a new geographical lineage inside the TOSV serotype (Punda-Polic´ et al., 2012). Our work aimed to investigate the genetic variability of the TOSV isolates in Italy overtime, by performing a genetic analysis on the coding sequences of the S segment obtained from Italian TOSV strains collected in Central Italy during the years 1980–2013.
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2. Methods 2.1. Clinical and environmental TOSV strains A total of 32 TOSV strains were analyzed. Twenty-five specimens were collected between 1980 and 1995, and had already been identified as TOSV by neutralization test and complement-fixation test (Verani et al., 1988). They were collected from cerebrospinal fluid (CSF) of patients with neurological disease (N = 18), from pools of sandflies (N = 6) and from the brain of a Pipistrellus kuhli (N = 1). Seven more strains came from CSF samples collected during 2009 and 2013 from patients hospitalized with neurological disease. All samples were from Italy (regions of Tuscany, Umbria, Marche and Lombardy) except one collected from a patient from Portugal (“Portugal 1983”). Relevant features are listed in Table 1. 2.2. Amplification and sequencing Viral RNA was extracted using QIAmp Viral RNA Mini Kits (Qiagen) according by manufacturer’s protocol. Two rounds of amplification, a reverse transcriptase polymerase chain reaction (RT-PCR) followed by hemi-nested PCR, were performed to amplify NSs and N ORFs of S segment by using primers designed over the original sequence TOSV ISS Phl3 (GenBank, X53794). NSs: Primer pair Tos1 (5 -ACAAAGACCTCCCGTATTGC-3 , bases 2–20)/Tos2R (5 -GGGGTTAGGGGAATTAGGAT-3 , bases 1053–1035) was used for RT-PCR on NSs; pairs Tos1/Tos1R (5 -GAATCTCCACTATCTGCTCC-3 , bases 547–566) and Tos2 (5 GAGCTCCTGTTCTTGTCAGA-3 , bases 452–471)/Tos2R were used for hemi-nested PCR. Thermal cycling conditions are available on request.
N: Primers TN1 (5 -CCGTGTATTAAACAAAAGCT-3 , bases 1837– 1856)/F4 (5 -AATCCCCATCCCAATCCTAA-3 , bases 1028–1042) were used for RT-PCR on the N region; pairs TN1/TV2 (Valassina et al., 1996) and F3 (5 -GCATTTGTTCCGTGGACTGT-3 , bases 1469–1488)/F4 were used for the hemi-nested round. Degenerate primers were used for N amplification of a patient from Portugal (Sanbonmatsu-Gamez et al., 2005). PCR products were analyzed by electrophoresis using 1.5% agarose gel and bands visualized by gel-red staining. Amplicons were purified using QIAquick PCR Purification Kit (Qiagen), and sequencing reactions performed by Macrogen DNA Sequencing Service (dna.macrogen.com). 2.3. Sequence alignment and phylogenetic analysis From each sample, a final 1060 bp long sequence was obtained for the NSs region and a 800 bp long sequence was obtained for the N one. Sequence data were first analyzed by Chromas software (version 2. 1.1, Technelysium Pty Ltd.). Forward and reverse sequences were aligned using BioEdit software (version 7.2.3, Hall, 1999). The two overlapping NSs and N sequences were assembled to obtain the whole genomic S segment, including non-coding 3 and 5 ends. Representative sequences of the whole S segment were deposited in the GenBank database under Accession numbers KM275763-KM275787 and KM275237, sequences of NSs under Accession numbers KM275788-KM275793. Different alignments were constructed for S nucleotide, and for both N and NSs nucleotide and amino acid sequences. All sequences were aligned and compared with the reference strain ISS Phl.3 (GenBank, X53794) and with isolates from Italy (GenBank, EU327772; JF330274; JF330275), Tunisie (GenBank, JX867536), Spain (GenBank, EF120631; FJ153285), Portugal
Table 1 TOSV strains analyzed, source features and clinical profile.
Clinical samplesb
Environmental samples
a b
Name
Clinical manifestationa and isolation source
Origin
Year
Accession No.
Tuscany 1983/1 Tuscany 1983/2 Portugal 1983 Tuscany 1984/1 Tuscany 1985 Marche 1990/1 Marche 1990/2 Tuscany 1991 Tuscany 1992 Marche 1992 Tuscany 1993/1 Marche 1993 Tuscany 1993/2 Tuscany 1994 Tuscany 1995/1 Tuscany 1995/2 Tuscany 1995/3 Tuscany 1995/4 Tuscany 2009/1 Tuscany 2009/2 Tuscany 2009/3 Tuscany 2009/4 Tuscany 2009/5 Abruzzo 2010 Lombardy 2013 Tuscany 1980 Tuscany 1981 Tuscany 1982/1 Tuscany 1982/2 Tuscany 1982/3 Marche 1990/3 Tuscany 1984/2
Meningitis Meningitis Meningitis Meningitis Meningitis Meningoencephalitis Meningitis Meningitis Meningitis Meningitis Meningitis Meningitis Meningitis Headache Meningitis Meningitis Meningoencephalitis Meningitis Meningitis Meningitis Meningitis Meningitis Meningitis Meningitis Encephalitis P. perniciosus P. perniciosus P. perfiliewi P. perfiliewi P. perfiliewi P. perfiliewi Pip. Kuhli
Florence (Tuscany) Florence (Tuscany) Portugal Florence (Tuscany) Florence (Tuscany) Macerata (Marche) Macerata (Marche) Siena (Tuscany) Siena (Tuscany) Pesaro (Marche) Siena (Tuscany) Macerata (Marche) Siena (Tuscany) Siena (Tuscany) Florence (Tuscany) Siena (Tuscany) Florence (Tuscany) Florence (Tuscany) Sesto (Tuscany) Sesto (Tuscany) Florence (Tuscany) Florence (Tuscany) Lastra a Signa (Tuscany) Chieti (Abruzzo) Mantua (Lombardy) Sesto (Tuscany) Sesto (Tuscany) Ville di Corsano (Tuscany) Ville di Corsano (Tuscany) Le Ripi (Tuscany) Fermo (Marche) Montalcino (Tuscany)
1983 1983 1983 1984 1985 1990 1990 1991 1992 1992 1993 1993 1993 1994 1995 1995 1995 1995 2009 2009 2009 2009 2009 2010 2013 1980 1981 1982 1982 1982 1990 1984
KM275764 KM275765 KM275763 KM275766 KM275767 KM275768 KM275769 KM275770 KM275771 KM275772 KM275773 KM275774 KM275775 KM275776 KM275777 KM275778 KM275792 KM275779 KM275790 KM275780 KM275781 KM275791 KM275782 KM275793 KM275783 KM275784 KM275785 KM275789 KM275786 KM275787 KM275788 KM275237
Clinical manifestations refer only to human. Environmental samples were isolated from different hosts, and sequences were obtained from the isolate. All clinical samples came from cerebrospinal fluid.
M. Baggieri et al. / Virus Research 200 (2015) 35–44
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Table 2 Mean nucleotide and amino acid p-distance ± standard deviation (SD). To estimate the evolutionary divergence over sequence pairs between groups, the average number of nucleotide differences per site over all sequence pairs between groups was calculated. For each pairwise sequence comparison, all positions containing alignment gaps and missing data were eliminated. S, small genome segment; NSs nonstructural protein; N, nucleoprotein. Nucleotide
TOSVA TOSVB TOSV(A + B)
Amino acid
S
N
NSs
N
NSs
0.023 ± 0.013 (0–0.044) 0.023 ± 0.010 (0–0.033) 0.061 ± 0.057 (0–0.151)
0.020 ± 0.012 (0–0.042) 0.014 ± 0.006 (0–0.024) 0.052 ± 0.049 (0–0.131)
0.025 ± 0.015 (0–0.055) 0.029 ± 0.013 (0–0.044) 0.063 ± 0.063 (0–0.172)
0 0 0
0.018 ± 0.013 (0–0.044) 0.016 ± 0.007 (0–0.025) 0.042 ± 0.041 (0–0.114)
Tuscany 1991
90
Marche 1990/1 Tuscany 1995/2 Tuscany 1993/2 Tuscany 1993/1 Tuscany 1994 Tuscany 2009/5 EU327772 Tuscany 2007 JF330274 Tuscany 2009 JF330275 Tuscany2009 Tuscany 1984/1 Tuscany 1980 Tuscany 1982/3 Tuscany 1981 85
Tuscany 1995/4
TOSV A
Tuscany 2009/3 Tuscany 2009/2 Tuscany 1995/1 Marche 1992
82 93 95
Tuscany 1985 Marche 1990/2 Marche 1993 Tuscany 1983/2
Tuscany 1984/2 Tuscany 1983/1 Lombardy 2013 98
100
Tuscany 1992
Tuscany 1982/2 X53794 Tuscany 1971 JX867536 Tunisie 2013 100
EF120631 Spain 2008 FJ153285 Spain 2008
100
100
Portugal 1983 EF201833 Portugal 2006
93 97
TOSV B
FJ153286 France 2008 KC776214 France 2010 EF656361 France 2007 HM566170 SFNV
0.05
Fig. 1. Molecular phylogenetic analysis of the S nucleotide sequences by Maximum Likelihood method based on the Kimura 2-parameter model. A discrete Gamma distribution was used to model evolutionary rate differences among sites. Significant bootstrap values (>80%) are indicated. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Strain HM566170 SFNV was used as outgroup strain.
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Table 3 Comparison of amino acid substitutions among TOSV based on NSs protein sequences. Strain
Amino acid positions
X53794 ISS Phl3 Tuscany 1983/1 Tuscany 1983/2 Tuscany 1984/1 Tuscany1985 Marche 1990/1 Marche 1990/2 Tuscany 1991 Tuscany 1992 Marche 1992 Tuscany1993/1 Marche 1993 Tuscany 1993/2 Tuscany 1994 Tuscany 1995/1 Tuscany 1995/2 Tuscany 1995/3 Tuscany 1995/4 Abruzzo 2010 Tuscany 2009/1 Tuscany 2009/2 Tuscany 2009/3 Tuscany 2009/4 Tuscany 2009/5 Lombardy 2013 Tuscany 1980 Tuscany 1981 Tuscany 1982/1 Tuscany 1982/2 Tuscany 1982/3 Marche 1990/3 Tuscany 1984/2 JX867536 Tunisie 2013 JF330274 Tuscany 2009 JF330275 Tuscany 2009 EU327772 Tuscany 2007 Portugal 1983 EF201833 Portugal 2006 EF120631 Spain 2008 FJ153285 Spain 2008 EF656361 France 2007 KC776214 France 2010 FJ153286 France 2008 Strain
X53794 ISS Phl3 Tuscany 1983/1 Tuscany 1983/2 Tuscany 1984/1 Tuscany1985 Marche 1990/1 Marche 1990/2 Tuscany 1991 Tuscany 1992 Marche 1992 Tuscany1993/1 Marche 1993 Tuscany 1993/2 Tuscany 1994 Tuscany 1995/1 Tuscany 1995/2 Tuscany 1995/3 Tuscany 1995/4 Abruzzo 2010 Tuscany 2009/1 Tuscany 2009/2 Tuscany 2009/3 Tuscany 2009/4 Tuscany 2009/5 Lombardy 2013 Tuscany 1980 Tuscany 1981
10
11
15
37
44
45
50
53
59
67
68
69
79
92
106
123
138
160
Y H H H H H H H – H H H H H H H – H – H H
R – – – – – – – – – – – – – – – – – – – – – – – K – – – – – – – – – – – K K K K K K K
G – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – S S S S S S S
D G G G G G G G – G G G G G G G – G – G G G G G – G G – – G – G G G G G – – – – – – –
E – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – G G – – – – –
F – – – – – – – – – – – – – – – L – – – – – – – – – – – – – – – – – – – – – – – – – –
N – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – K K K K K K K
K E E E E E E E – E E E E E E E – E – E E E E E E E E – – E – E E E E E E E E E E E E
K – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – R R R R R R R
I – M – M – M – – M – M – – M – – – – – – – – – – – – – – – – – – – – – M M – – – – –
E – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – N N N N N N N
K – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – G G G G G G G
M – – – – – – – – – – – – – – – – – – – – – – – – – – V – – – – – – – – – – – – – – –
M – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – I
S N N N N N N N – N N N N N – N – N – N N N N N – N N – – N – N N N N N – – N N – – –
T – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – S S S S S S S
S – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – P – – – – – – –
N – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – S S S S C C C
H H – H H – – H – H H H H H H H H H H H H
Amino acid positions 163
176
185
196
207
212
214
215
216
220
221
222
225
236
240
242
245
249
N I I I I I I I I I I I I I I I – I I I I I I I I I I
I – M – M – M – – M – M – – – – – – – – – – – – – – –
A – – – – – – – – – – – – – – – – – T – – – – – – – –
D – – – – – – – V – – – – – – – – – – – – – – – – – –
R – – – – – – – – – – – – – – – – – – – – – – – – – –
T – – – – – – – – – – – – – – – – – – – – – – – – – –
S – – – – – – – – – – – – – – – – – – – – – – – – – –
K – – – – – – – – – – – – – – – – – – – – – – – – – –
M – – – – – – – – – – – – – – – – – – – – – – – – – –
T – – – – – – – – – – – – – – – – – – – – – – – – – –
H – – – – – – – – – – – – – – – – – – – – – – – – – –
L – – – – – – – – – – – – – – – – – P – – – – – – – –
Y – – – – – – – – – – – – – – – – – – – – – – – – – –
S N N – – N – N – – N – N N N N – N – N N N N N – N N
K – – – – – – – – – – – – – – – – – – – – – – – – – –
G E E – – E – E – E E E E E E E – E – E E E E E – E E
R – – – – – – – – – – – – – – – – – – – – – – – – – –
K – – – – – – – – – – R – – – – – – – – – – – – – – –
M. Baggieri et al. / Virus Research 200 (2015) 35–44
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Table 3 (Continued ) Strain
Amino acid positions 163
176
185
196
207
212
214
215
216
220
221
222
225
236
240
242
245
249
Tuscany 1982/1 Tuscany 1982/2 Tuscany 1982/3 Marche 1990/3 Tuscany 1984/2 JX867536 Tunisie 2013 JF330274 Tuscany 2009 JF330275 Tuscany 2009 EU327772 Tuscany 2007 Portugal 1983 EF201833 Portugal 2006 EF120631 Spain 2008 FJ153285 Spain 2008 EF656361 France 2007 KC776214 France 2010 FJ153286 France 2008
– – I – I I I I I I I I I I I I
– – – – – – – – – L L L L L L L
– – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – –
– – – – – – – – – K K K K K K K
– – – – – I – – – A A A A A A A
– – – – – – – – – – – – – F – –
– – – – – – – – – R R R R R R R
– – – – – – – – – – – – T – – –
– – – – – – – – – S S S S S S S
– – – – – – – – – Q Q Q Q Q Q Q
– – – – – – – – – – – – – – – –
– – – – C – – – – – – – – – – –
– – N – N – N N N – – – – – – –
– – – – – – – – – R R R R R R R
– – E – E E E E E D D D D D D D
– – – – – – – – – K K K K K K K
– – – – – – – – – – – – – – – –
Strain
X53794 ISS Phl3 Tuscany 1983/1 Tuscany 1983/2 Tuscany 1984/1 Tuscany1985 Marche 1990/1 Marche 1990/2 Tuscany 1991 Tuscany 1992 Marche 1992 Tuscany1993/1 Marche 1993 Tuscany 1993/2 Tuscany 1994 Tuscany 1995/1 Tuscany 1995/2 Tuscany 1995/3 Tuscany 1995/4 Abruzzo 2010 Tuscany 2009/1 Tuscany 2009/2 Tuscany 2009/3 Tuscany 2009/4 Tuscany 2009/5 Lombardy 2013 Tuscany 1980 Tuscany 1981 Tuscany 1982/1 Tuscany 1982/2 Tuscany 1982/3 Marche 1990/3 Tuscany 1984/2 JX867536 Tunisie 2013 JF330274 Tuscany 2009 JF330275 Tuscany 2009 EU327772 Tuscany 2007 Portugal 1983 EF201833 Portugal 2006 EF120631 Spain 2008 FJ153285 Spain 2008 EF656361 France 2007 KC776214 France 2010 FJ153286 France 2008
Amino acid positions 252
255
258
265
266
269
286
287
289
290
293
294
296
297
299
301
309
M – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – L L L L L L L
S C C C C C C C – C C C C C C C – C – C C C C C – C C – – C – – – C – C – – – – – – –
R – – – K – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – K K K K K K K
R – – – – – – – –
T – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – K – – – –
G – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – R R R R R R R
P L – L – – – L – – L – L L – L – L – L L L L L – L L – – L – T – L L L I I I I T I I
Q – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – H H H H H H H
T – I – I – I – I I – I – – I – – – – – – – – – – – – – – – – – – – – – S S S S S S S
I – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – V V M M V V V
D – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – N N V V N N N
K E – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – R R R R R R R
C – W – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
L – – – P – – – – P – P – – – – – – – – – – – – – – – – – – – – – – – – P P P P P P P
E – – – K – K – – K – K – – K – – – – – – – – – – – – – – – – – – – – – – – – – – – –
M – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – K K K K K K K
– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – G – – – – – – – – –
– – – – – – – – – – – – – – – – – – – – – – – – – – K K – – K K K
(GenBank, EF201833) and France (GenBank, EF656361; KC776214; FJ153286). Sandfly Naples virus (SFNV, Genbank EF201832) was chosen as outgroup strain in the analysis of S segment. P-distance values (proportions of nucleotide sites at which two sequences being compared are different) were calculated using Molecular Evolutionary Genetics Analyses (MEGA) software version 6 (Tamura et al., 2013).
To examine selective pressure among TOSV NSs sequences, synonymous and nonsynonymous substitution rates were calculated for the nucleotide data set using the Jukes–Cantor correction method in the DnaSP software (version 4.10.7) (Rozas et al., 2003). Bayesian information criterion (BIC) was used to determine the model of nucleotide substitution that best fit the data using
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Tuscany 2009/3
72
Tuscany 2009/2 Tuscany 1981 Tuscany 1995/4 Tuscany 1995/1 Tuscany 1980 Tuscany 1982/3 Tuscany 1984/1 Tuscany 1993/1 73
I
Tuscany 1993/2 JF330274 Tuscany 2009 JF330275 Tuscany 2009 Tuscany 2009/5 Tuscany 1994 Tuscany 1995/2 EU327772 Tuscany 2007 Tuscany 1991
Marche 1990/1 Tuscany 1984/2 Tuscany 1982/2 70
X53794 Tuscany 1971
84
Tuscany 1983/1 72
II
Tuscany 1992
75
Lombardy 2013
Marche 1992 Tuscany 1985 Marche 1993 Marche 1990/2 Tuscany 1983/2 Portugal 1983 0.05
Fig. 2. Molecular phylogenetic analysis of the N nucleotide sequences by Maximum Likelihood method based on the Kimura 2-parameter model. A discrete Gamma distribution was used to model evolutionary rate differences among sites. Significant bootstrap values (>70%) are indicated. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Strain Portugal 1983 was used as outgroup strain.
the selection tool available in MEGA software. Models with the lowest BIC scores were considered to describe the substitution pattern the best. For each model, AICc value (Akaike Information Criterion, corrected), Maximum Likelihood value (lnL). The model that best fit the data was the Kimura 2 +G model for the nucleotide analysis (Kimura, 1980), and JTT +I matrix-based method for the amino acid analysis (Jones et al., 1992). Phylogenetic trees were constructed using the Maximum Likelihood method. Then, the reliability of each tree was estimated with a bootstrap analysis by 1000 replicates. 3. Results and discussion A total of 32 strains isolated from Italian samples collected over a period of more than 30 years (1980–2013) from hospitalized patients with neurological disease, from sandflies and from the brain of a bat, were included in this study. Sequences of whole S genomic segment from each isolated TOSV strain, containing the N and NSs ORFs plus the non-coding region, were aligned and compared with the Italian reference
strain ISS Phl3 (GenBank, X53794) and with all other sequences of the S segment available in GenBank database. The same analysis was conducted for N and NSs ORFs sequences, singularly. Virtual translation of N and NSs nucleotide sequences was also performed, and respective amino acid sequences were compared. Nucleotide and amino acid p-distance values were calculated (Table 2). For the whole segment S, N and NSs ORFs, p-distance was calculated within three different groups. Group TOSV A included 26 sequences of the whole S segment, 26 sequences of the N ORF and 32 of the NSs ORF, plus four sequences taken from GenBank. Group TOSV B included, for all the three analyses, 7 sequences, one from our lab (“Portugal 1983”) and 6 taken from GenBank. Group TOSV A + B included both TOSV A and TOSV B groups. Data showed that the diversity within the entire population of sequences (group TOSV A + B) is small for the S segment, with a mean p-distance of 0.061 (range, 0–0.151). As expected, p-distance values calculated within a single group – either TOSV A or TOSV B – were each one lower than whose calculated for TOSV A + B group
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Tuscany 1995/2 EU327772 Tuscany 2007 Tuscany 1993/1 Tuscany 1993/2 Tuscany 1991
96
Marche 1990/1 Tuscany 1994 JF330274 Tuscany 2009 JF330275 Tuscany 2009 Tuscany 2009/5 Tuscany 2009/4
I
Tuscany 1984/1 Tuscany 1983/1 Tuscany 1980
83
Tuscany 1995/4 Tuscany 2009/3 77
Tuscany 2009/2 Tuscany 2009/1 Tuscany 1982/3 Tuscany 1981 Tuscany 1984/2 Tuscany 1995/1 Marche 1990/2
88
Tuscany 1983/2 91
Marche 1992
II
Tuscany 1985 Marche 1993 Lombardy 2013 Tuscany 1992 86
Abruzzo 2010 Tuscany 1995/3 X53794 Tuscany 1971
78
III
Tuscany 1982/2 Marche 1990/3 Tuscany 1982/1 Portugal 1983
0.05
Fig. 3. Molecular phylogenetic analysis of the NSs nucleotide sequences by Maximum Likelihood method based on the Kimura 2-parameter model. A discrete Gamma distribution was used to model evolutionary rate differences among sites. Significant bootstrap values (>70%) are indicated. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Strain Portugal 1983 was used as outgroup strain.
and similar to each other (mean 0.023, range 0–0.044 and 0–0.033, respectively). The sequence variations calculated for N and NSs ORFs showed similar trends. No differences in p-distance values were found by adding sequences derived from GenBank (data not shown). At amino acid level, the analysis on N and NSs showed no sequence variation for the N protein, and p-distance values of 0.018 (range 0–0.044) and 0.016 (range 0–0.025) for the NSs protein within TOSV A and TOSV B, respectively. On the entire alignment of NSs protein amino acid mutations were randomly distributed
along the primary sequence and there was no evidence of particularly variable regions. The amino acid substitutions are shown in Table 3. The distribution of synonymous and nonsynonymous substitution rates along the NSs ORF was also analyzed. The mean nonsynonymous/synonymous rate ratio (ω = dN/dS) was 0.091 within TOSV A and 0.074 within TOSV B lineages, indicating that the NSs ORF is not under a positive pressure. KinasePhos 2.0 analysis (Wong et al., 2007) based on kinase-specific phosphorylation site prediction was also performed on the NSs amino acid sequences. Two putative tyrosine phosphorylation sites were
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M. Baggieri et al. / Virus Research 200 (2015) 35–44
Tuscany 1982/3 JF330274 Tuscany 2009 Tuscany 1993/1 Tuscany 2009/1 Tuscany 2009/3 Tuscany 2009/2 Tuscany 1993/2 Tuscany 2009/4 Tuscany 1995/2 Tuscany 2009/5 Tuscany 1984/1 Tuscany 1995/4 EU327772 Tuscany 2007 Tuscany 1980
I
Tuscany 1991 Tuscany 1994 Tuscany 1981 Marche 1990/1 72
Tuscany 1983/1 JF330275 Tuscany 2009 Tuscany 1984/2 Tuscany 1995/1 Marche 1990/2
57
Tuscany 1983/2
II
Tuscany 1985 Marche 1992 Marche 1993 Lombardy 2013 Tuscany 1992
78 79
Abruzzo 2010 X53794 Tuscany 1971 Marche 1990/3
III
Tuscany 1982/2 Tuscany 1982/1 Tuscany 1995/3 Portugal 1983 0.01
Fig. 4. Molecular phylogenetic analysis of the NSs amino acidic sequences by Maximum Likelihood method based on the Kimura 2-parameter model. A discrete Gamma distribution was used to model evolutionary rate differences among sites. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Strain Portugal 1983 was used as outgroup strain. The evolutionary distances were computed using the JTT matrix-based method. Significant bootstrap values (>55%) are indicated.
identified (aa 24, 48) located in a highly conserved region. TOSV NSs has been shown to possess two distinct functions: (i) to limit IFN- upregulation (Gori Savellini et al., 2001) and (ii) to promote RNA-dependent protein kinase degradation (Kalveram and Ikegami, 2013). Further studies will be necessary to understand if and how these phosphorylation sites are involved in these functions. Phylogenetic analysis based on the S segment showed that all but one strains clustered within TOSV A lineage (Fig. 1). The only one strain clustering in lineage TOSV B, was from a case imported from Portugal in 1983 (“Portugal 1983”) that showed similarities with sequences from Portugal (GenBank, EF201833), Spain (GenBank, EF120631; FJ153285) and France (GenBank, EF656361; KC776214; FJ153286). This suggested that TOSV A represents the
main or only lineage that has circulated in Italy since TOSV first isolation in 1971. Phylogenetic trees constructed on N and NSs ORFs showed different patterns (Figs. 2 and 3). In N analysis TOSV A strains clustered into two groups, whereas NSs analysis showed three main clusters. Phylogenetic trees constructed on the deduced NSs amino acid sequences showed the same pattern as NSs ORF with three main clusters (Fig. 4), and the high-support bootstrap values indicated that the analyses were reliable. Italian strains in each cluster appeared to have no geographical nor temporal/host correlation within the groups. In addition, they seemed to be co-evolved and randomly widespread in the area under study.
M. Baggieri et al. / Virus Research 200 (2015) 35–44
These findings reflect previous results achieved from analysis of the M segment which demonstrated a co-circulation of four TOSV clusters in Central Italy, with no correlation between viral strains and area/year of isolation (Venturi et al., 2007). Furthermore, TOSV A lineage showed a low sequence variability over the years since the reference strain ISS Phl3 (GenBank, X53794) clustered with Italian strains circulating until 2013 in all the three trees. A low sequence variability was also showed within lineage TOSV B, despite the small number of TOSV B strains involved in the analysis. Studies based on sequence comparison showed that RNA arboviruses are relatively stable in nature, suggesting that a cycle with host alternation (between vertebrate and invertebrate hosts) can interfere on the viral evolution by a strong conservative sequence selection. This genome stability may result from the requirements for viral replication in two hosts that present competitive niches for replication and adaptation (Strauss and Strauss, 1994). This was supported by experimental data on the variability within phleboviruses (Sall et al., 1997; Simons et al., 1990). As reported above, nucleotide sequences variability of NSs gene was higher than that of N gene, implying that the N gene is more stable, in agreement with recent studies (Magurano et al., 2014). In addition, the N protein seems to be well conserved with almost no amino acid changes. The N protein interacts with genome segments to form the ribonucleocapsids (Raymond et al., 2012), it also represents the major antigen responsible for both IgM and IgG responses (Magurano and Nicoletti, 1999). It is probable that changes in the N amino acid sequence that make this protein less efficient in its interaction with viral nucleic acid are lethal for the virus and have no chance to be selected. 4. Conclusions The oligonucleotide set used for this analysis appeared to be a good choice for the amplification of the NSS specific region of both A and B TOSV lineages, and contribute to define the basis for rapid diagnostic tests for TOSV based on RT-PCR. This study was conducted with the aim to determine the fulllength sequences of S segments of different Italian strains in order to better understand their evolutionary and phylogenetic relationships. Although the TOSV is an important pathogen, little genomic information is available and more studies should be conducted on the ecology and the epidemiology of this phlebovirus. This study on phylogenesis of S segment of TOSV reveals a low genetic variability. This finding confirms data from previous study (Magurano et al., 2014), providing further indications that RNA Arboviruses are relatively stable in nature. Obtaining genome sequence information for viruses with poor representation in public databases is an important task, in particular for programs of virus surveillance and studies on emerging pathogens. Acknowledgements The authors thank Dr. Stefano Buttò for critical reading the manuscript and for fruitful discussion; Dr. Raffaella Bucciardini for statistical support and Dr. Robert Peter Parker for linguistic revision. References Accardi, L., Grò, M.C., Di Bonito, P., Giorgi, C., 1993. Toscana L segment: molecular cloning, coding strategy and amino acid sequence in comparison with other negative strand RNA viruses. Virus Res. 27, 119–131. Alkan, C., Bichaud, L., de Lamballerie, X., Alten, B., Gould, E.A., Charrel, R.N., 2013. Sandfly-borne phleboviruses of Eurasia and Africa: epidemiology, genetic diversity, geographic range, control measures. Antiviral Res. 100 (1), 54–74. Braito, A., Corbisiero, R., Corradini, S., Marchi, A., Sancasciani, N., Fiorentini, C., Ciufolini, M.G., 1997. Evidence of Toscana virus infections without central nervous system involvement: a serological study. Eur. J. Epidemiol. 13, 761–764.
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Virus Research journal homepage: www.elsevier.com/locate/virusres
Genetic variability of the S segment of Toscana virus Melissa Baggieri, Antonella Marchi, Paola Bucci, Loredana Nicoletti, Fabio Magurano ∗ Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
a r t i c l e
i n f o
Article history: Received 10 December 2014 Received in revised form 12 January 2015 Accepted 12 January 2015 Available online 20 January 2015 Keywords: Toscana virus Phlebovirus Neurological disease Phylogenetic analysis Negative strand
a b s t r a c t Toscana virus (TOSV) was originally isolated in 1971 from a pool of Phlebotomus perniciosus sandflies collected in Grosseto province (Central Italy). Since its first isolation, several studies have been conducted in Italy and other Mediterranean countries in order to identify its possible animal reservoirs, spread of infection and genetic variability. Phylogenetic analysis conducted on TOSV genome demonstrated the co-circulation of two major lineages in the Mediterranean areas, TOSV A and TOSV B. This study reports the results of the genetic analysis of 32 viral strains isolated in Italy in the last 30 years from patients hospitalized with neurological disease, from sandflies and from the brain of a bat. The genetic diversity of TOSV was investigated by determining the sequences of the whole S segment. Phylogenetic analysis showed that TOSV A lineage represents the lineage circulating in Italy. Moreover, the current variability of lineage A is similar to that of lineage B. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Toscana virus (TOSV) is an arthropod-borne virus (Bunyaviridae family, genus Phlebovirus) transmitted to humans by bites of phlebotominae sandflies. TOSV was first isolated from a pool of Phlebotomus perniciosus sandflies collected in Grosseto province (Central Italy) in 1971. After its first isolation, it has been also isolated from Phlebotomus perfiliewi and Sergentomyia minuta sandflies (Verani et al., 1982; Charrel et al., 2006). Since 1983, TOSV has been associated to neurological diseases such as meningitis and meningoencephalitis in humans (Ehrnst et al., 1985; Nicoletti et al., 1991; Braito et al., 1998). However, most of TOSV infections are asymptomatic or cause mild symptoms, such as fever and headaches that resolve spontaneously (Braito et al., 1997). The diffusion of TOSV infections is strictly linked to the distribution of the insect vectors, with a peak of occurrence in August during the maximum activity of the insects (Tesh, 1988; Braito et al., 1998; Valassina et al., 2000). According to results from viral isolation and serologic surveys cases of patients infected with TOSV have been documented in Italy, France, Spain, Portugal, Greece, Turkey and other countries of the
Abbreviations: TOSV, Toscana virus; ORFs, open reading frames; CSF, cerebrospinal fluid; RT-PCR, reverse transcriptase polymerase chain reaction; MEGA, Molecular Evolutionary Genetics Analyses; BIC, Bayesian information criterion. ∗ Corresponding author. Tel.: +39 06 49902448. E-mail addresses: [email protected] (M. Baggieri), [email protected] (A. Marchi), [email protected] (P. Bucci), [email protected] (L. Nicoletti), [email protected] (F. Magurano). http://dx.doi.org/10.1016/j.virusres.2015.01.013 0168-1702/© 2015 Elsevier B.V. All rights reserved.
Mediterranean basin (Ergünay et al., 2011; Alkan et al., 2013). In Italy, TOSV cases have frequently been reported from rural areas of Centre and South. Recently, the circulation in urban areas of Emilia Romagna (Northern Italy) was also shown (Vocale et al., 2012). TOSV has a tripartite genome with three negative ssRNA segments coding for the nucleoprotein N and non-structural protein NSs (segment S), the glycoproteins Gn and Gc and the nonstructural protein NSm (segment M), and the viral polymerase L (segment L), respectively (Giorgi et al., 1991; Accardi et al., 1993; Di Bonito et al., 1997). TOSV utilizes an ambisense coding strategy for S segment: NSs protein is codified in the genomic sense and N protein in antigenomic sense. The N and NSs open reading frames (ORFs) represent two potential distinct targets for genetic analysis of the S segment. Several studies have been conducted on the molecular variability of TOSV RNA segments. Studies based on the L and M segments demonstrated a co-circulation of two major viral lineages in the Mediterranean areas, proposed as TOSV A and TOSV B (SanchezSeco et al., 2003; Sanbonmatsu-Gamez et al., 2005; Venturi et al., 2007; Collao et al., 2009). TOSV A is found in Italy and France, and TOSV B in the Iberian Peninsula and France (SanbonmatsuGamez et al., 2005; Sanchez-Seco et al., 2003). Recently, findings of an autochthon Croatian TOSV suggested the presence of a new geographical lineage inside the TOSV serotype (Punda-Polic´ et al., 2012). Our work aimed to investigate the genetic variability of the TOSV isolates in Italy overtime, by performing a genetic analysis on the coding sequences of the S segment obtained from Italian TOSV strains collected in Central Italy during the years 1980–2013.
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2. Methods 2.1. Clinical and environmental TOSV strains A total of 32 TOSV strains were analyzed. Twenty-five specimens were collected between 1980 and 1995, and had already been identified as TOSV by neutralization test and complement-fixation test (Verani et al., 1988). They were collected from cerebrospinal fluid (CSF) of patients with neurological disease (N = 18), from pools of sandflies (N = 6) and from the brain of a Pipistrellus kuhli (N = 1). Seven more strains came from CSF samples collected during 2009 and 2013 from patients hospitalized with neurological disease. All samples were from Italy (regions of Tuscany, Umbria, Marche and Lombardy) except one collected from a patient from Portugal (“Portugal 1983”). Relevant features are listed in Table 1. 2.2. Amplification and sequencing Viral RNA was extracted using QIAmp Viral RNA Mini Kits (Qiagen) according by manufacturer’s protocol. Two rounds of amplification, a reverse transcriptase polymerase chain reaction (RT-PCR) followed by hemi-nested PCR, were performed to amplify NSs and N ORFs of S segment by using primers designed over the original sequence TOSV ISS Phl3 (GenBank, X53794). NSs: Primer pair Tos1 (5 -ACAAAGACCTCCCGTATTGC-3 , bases 2–20)/Tos2R (5 -GGGGTTAGGGGAATTAGGAT-3 , bases 1053–1035) was used for RT-PCR on NSs; pairs Tos1/Tos1R (5 -GAATCTCCACTATCTGCTCC-3 , bases 547–566) and Tos2 (5 GAGCTCCTGTTCTTGTCAGA-3 , bases 452–471)/Tos2R were used for hemi-nested PCR. Thermal cycling conditions are available on request.
N: Primers TN1 (5 -CCGTGTATTAAACAAAAGCT-3 , bases 1837– 1856)/F4 (5 -AATCCCCATCCCAATCCTAA-3 , bases 1028–1042) were used for RT-PCR on the N region; pairs TN1/TV2 (Valassina et al., 1996) and F3 (5 -GCATTTGTTCCGTGGACTGT-3 , bases 1469–1488)/F4 were used for the hemi-nested round. Degenerate primers were used for N amplification of a patient from Portugal (Sanbonmatsu-Gamez et al., 2005). PCR products were analyzed by electrophoresis using 1.5% agarose gel and bands visualized by gel-red staining. Amplicons were purified using QIAquick PCR Purification Kit (Qiagen), and sequencing reactions performed by Macrogen DNA Sequencing Service (dna.macrogen.com). 2.3. Sequence alignment and phylogenetic analysis From each sample, a final 1060 bp long sequence was obtained for the NSs region and a 800 bp long sequence was obtained for the N one. Sequence data were first analyzed by Chromas software (version 2. 1.1, Technelysium Pty Ltd.). Forward and reverse sequences were aligned using BioEdit software (version 7.2.3, Hall, 1999). The two overlapping NSs and N sequences were assembled to obtain the whole genomic S segment, including non-coding 3 and 5 ends. Representative sequences of the whole S segment were deposited in the GenBank database under Accession numbers KM275763-KM275787 and KM275237, sequences of NSs under Accession numbers KM275788-KM275793. Different alignments were constructed for S nucleotide, and for both N and NSs nucleotide and amino acid sequences. All sequences were aligned and compared with the reference strain ISS Phl.3 (GenBank, X53794) and with isolates from Italy (GenBank, EU327772; JF330274; JF330275), Tunisie (GenBank, JX867536), Spain (GenBank, EF120631; FJ153285), Portugal
Table 1 TOSV strains analyzed, source features and clinical profile.
Clinical samplesb
Environmental samples
a b
Name
Clinical manifestationa and isolation source
Origin
Year
Accession No.
Tuscany 1983/1 Tuscany 1983/2 Portugal 1983 Tuscany 1984/1 Tuscany 1985 Marche 1990/1 Marche 1990/2 Tuscany 1991 Tuscany 1992 Marche 1992 Tuscany 1993/1 Marche 1993 Tuscany 1993/2 Tuscany 1994 Tuscany 1995/1 Tuscany 1995/2 Tuscany 1995/3 Tuscany 1995/4 Tuscany 2009/1 Tuscany 2009/2 Tuscany 2009/3 Tuscany 2009/4 Tuscany 2009/5 Abruzzo 2010 Lombardy 2013 Tuscany 1980 Tuscany 1981 Tuscany 1982/1 Tuscany 1982/2 Tuscany 1982/3 Marche 1990/3 Tuscany 1984/2
Meningitis Meningitis Meningitis Meningitis Meningitis Meningoencephalitis Meningitis Meningitis Meningitis Meningitis Meningitis Meningitis Meningitis Headache Meningitis Meningitis Meningoencephalitis Meningitis Meningitis Meningitis Meningitis Meningitis Meningitis Meningitis Encephalitis P. perniciosus P. perniciosus P. perfiliewi P. perfiliewi P. perfiliewi P. perfiliewi Pip. Kuhli
Florence (Tuscany) Florence (Tuscany) Portugal Florence (Tuscany) Florence (Tuscany) Macerata (Marche) Macerata (Marche) Siena (Tuscany) Siena (Tuscany) Pesaro (Marche) Siena (Tuscany) Macerata (Marche) Siena (Tuscany) Siena (Tuscany) Florence (Tuscany) Siena (Tuscany) Florence (Tuscany) Florence (Tuscany) Sesto (Tuscany) Sesto (Tuscany) Florence (Tuscany) Florence (Tuscany) Lastra a Signa (Tuscany) Chieti (Abruzzo) Mantua (Lombardy) Sesto (Tuscany) Sesto (Tuscany) Ville di Corsano (Tuscany) Ville di Corsano (Tuscany) Le Ripi (Tuscany) Fermo (Marche) Montalcino (Tuscany)
1983 1983 1983 1984 1985 1990 1990 1991 1992 1992 1993 1993 1993 1994 1995 1995 1995 1995 2009 2009 2009 2009 2009 2010 2013 1980 1981 1982 1982 1982 1990 1984
KM275764 KM275765 KM275763 KM275766 KM275767 KM275768 KM275769 KM275770 KM275771 KM275772 KM275773 KM275774 KM275775 KM275776 KM275777 KM275778 KM275792 KM275779 KM275790 KM275780 KM275781 KM275791 KM275782 KM275793 KM275783 KM275784 KM275785 KM275789 KM275786 KM275787 KM275788 KM275237
Clinical manifestations refer only to human. Environmental samples were isolated from different hosts, and sequences were obtained from the isolate. All clinical samples came from cerebrospinal fluid.
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Table 2 Mean nucleotide and amino acid p-distance ± standard deviation (SD). To estimate the evolutionary divergence over sequence pairs between groups, the average number of nucleotide differences per site over all sequence pairs between groups was calculated. For each pairwise sequence comparison, all positions containing alignment gaps and missing data were eliminated. S, small genome segment; NSs nonstructural protein; N, nucleoprotein. Nucleotide
TOSVA TOSVB TOSV(A + B)
Amino acid
S
N
NSs
N
NSs
0.023 ± 0.013 (0–0.044) 0.023 ± 0.010 (0–0.033) 0.061 ± 0.057 (0–0.151)
0.020 ± 0.012 (0–0.042) 0.014 ± 0.006 (0–0.024) 0.052 ± 0.049 (0–0.131)
0.025 ± 0.015 (0–0.055) 0.029 ± 0.013 (0–0.044) 0.063 ± 0.063 (0–0.172)
0 0 0
0.018 ± 0.013 (0–0.044) 0.016 ± 0.007 (0–0.025) 0.042 ± 0.041 (0–0.114)
Tuscany 1991
90
Marche 1990/1 Tuscany 1995/2 Tuscany 1993/2 Tuscany 1993/1 Tuscany 1994 Tuscany 2009/5 EU327772 Tuscany 2007 JF330274 Tuscany 2009 JF330275 Tuscany2009 Tuscany 1984/1 Tuscany 1980 Tuscany 1982/3 Tuscany 1981 85
Tuscany 1995/4
TOSV A
Tuscany 2009/3 Tuscany 2009/2 Tuscany 1995/1 Marche 1992
82 93 95
Tuscany 1985 Marche 1990/2 Marche 1993 Tuscany 1983/2
Tuscany 1984/2 Tuscany 1983/1 Lombardy 2013 98
100
Tuscany 1992
Tuscany 1982/2 X53794 Tuscany 1971 JX867536 Tunisie 2013 100
EF120631 Spain 2008 FJ153285 Spain 2008
100
100
Portugal 1983 EF201833 Portugal 2006
93 97
TOSV B
FJ153286 France 2008 KC776214 France 2010 EF656361 France 2007 HM566170 SFNV
0.05
Fig. 1. Molecular phylogenetic analysis of the S nucleotide sequences by Maximum Likelihood method based on the Kimura 2-parameter model. A discrete Gamma distribution was used to model evolutionary rate differences among sites. Significant bootstrap values (>80%) are indicated. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Strain HM566170 SFNV was used as outgroup strain.
38
M. Baggieri et al. / Virus Research 200 (2015) 35–44
Table 3 Comparison of amino acid substitutions among TOSV based on NSs protein sequences. Strain
Amino acid positions
X53794 ISS Phl3 Tuscany 1983/1 Tuscany 1983/2 Tuscany 1984/1 Tuscany1985 Marche 1990/1 Marche 1990/2 Tuscany 1991 Tuscany 1992 Marche 1992 Tuscany1993/1 Marche 1993 Tuscany 1993/2 Tuscany 1994 Tuscany 1995/1 Tuscany 1995/2 Tuscany 1995/3 Tuscany 1995/4 Abruzzo 2010 Tuscany 2009/1 Tuscany 2009/2 Tuscany 2009/3 Tuscany 2009/4 Tuscany 2009/5 Lombardy 2013 Tuscany 1980 Tuscany 1981 Tuscany 1982/1 Tuscany 1982/2 Tuscany 1982/3 Marche 1990/3 Tuscany 1984/2 JX867536 Tunisie 2013 JF330274 Tuscany 2009 JF330275 Tuscany 2009 EU327772 Tuscany 2007 Portugal 1983 EF201833 Portugal 2006 EF120631 Spain 2008 FJ153285 Spain 2008 EF656361 France 2007 KC776214 France 2010 FJ153286 France 2008 Strain
X53794 ISS Phl3 Tuscany 1983/1 Tuscany 1983/2 Tuscany 1984/1 Tuscany1985 Marche 1990/1 Marche 1990/2 Tuscany 1991 Tuscany 1992 Marche 1992 Tuscany1993/1 Marche 1993 Tuscany 1993/2 Tuscany 1994 Tuscany 1995/1 Tuscany 1995/2 Tuscany 1995/3 Tuscany 1995/4 Abruzzo 2010 Tuscany 2009/1 Tuscany 2009/2 Tuscany 2009/3 Tuscany 2009/4 Tuscany 2009/5 Lombardy 2013 Tuscany 1980 Tuscany 1981
10
11
15
37
44
45
50
53
59
67
68
69
79
92
106
123
138
160
Y H H H H H H H – H H H H H H H – H – H H
R – – – – – – – – – – – – – – – – – – – – – – – K – – – – – – – – – – – K K K K K K K
G – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – S S S S S S S
D G G G G G G G – G G G G G G G – G – G G G G G – G G – – G – G G G G G – – – – – – –
E – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – G G – – – – –
F – – – – – – – – – – – – – – – L – – – – – – – – – – – – – – – – – – – – – – – – – –
N – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – K K K K K K K
K E E E E E E E – E E E E E E E – E – E E E E E E E E – – E – E E E E E E E E E E E E
K – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – R R R R R R R
I – M – M – M – – M – M – – M – – – – – – – – – – – – – – – – – – – – – M M – – – – –
E – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – N N N N N N N
K – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – G G G G G G G
M – – – – – – – – – – – – – – – – – – – – – – – – – – V – – – – – – – – – – – – – – –
M – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – I
S N N N N N N N – N N N N N – N – N – N N N N N – N N – – N – N N N N N – – N N – – –
T – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – S S S S S S S
S – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – P – – – – – – –
N – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – S S S S C C C
H H – H H – – H – H H H H H H H H H H H H
Amino acid positions 163
176
185
196
207
212
214
215
216
220
221
222
225
236
240
242
245
249
N I I I I I I I I I I I I I I I – I I I I I I I I I I
I – M – M – M – – M – M – – – – – – – – – – – – – – –
A – – – – – – – – – – – – – – – – – T – – – – – – – –
D – – – – – – – V – – – – – – – – – – – – – – – – – –
R – – – – – – – – – – – – – – – – – – – – – – – – – –
T – – – – – – – – – – – – – – – – – – – – – – – – – –
S – – – – – – – – – – – – – – – – – – – – – – – – – –
K – – – – – – – – – – – – – – – – – – – – – – – – – –
M – – – – – – – – – – – – – – – – – – – – – – – – – –
T – – – – – – – – – – – – – – – – – – – – – – – – – –
H – – – – – – – – – – – – – – – – – – – – – – – – – –
L – – – – – – – – – – – – – – – – – P – – – – – – – –
Y – – – – – – – – – – – – – – – – – – – – – – – – – –
S N N – – N – N – – N – N N N N – N – N N N N N – N N
K – – – – – – – – – – – – – – – – – – – – – – – – – –
G E E – – E – E – E E E E E E E – E – E E E E E – E E
R – – – – – – – – – – – – – – – – – – – – – – – – – –
K – – – – – – – – – – R – – – – – – – – – – – – – – –
M. Baggieri et al. / Virus Research 200 (2015) 35–44
39
Table 3 (Continued ) Strain
Amino acid positions 163
176
185
196
207
212
214
215
216
220
221
222
225
236
240
242
245
249
Tuscany 1982/1 Tuscany 1982/2 Tuscany 1982/3 Marche 1990/3 Tuscany 1984/2 JX867536 Tunisie 2013 JF330274 Tuscany 2009 JF330275 Tuscany 2009 EU327772 Tuscany 2007 Portugal 1983 EF201833 Portugal 2006 EF120631 Spain 2008 FJ153285 Spain 2008 EF656361 France 2007 KC776214 France 2010 FJ153286 France 2008
– – I – I I I I I I I I I I I I
– – – – – – – – – L L L L L L L
– – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – –
– – – – – – – – – K K K K K K K
– – – – – I – – – A A A A A A A
– – – – – – – – – – – – – F – –
– – – – – – – – – R R R R R R R
– – – – – – – – – – – – T – – –
– – – – – – – – – S S S S S S S
– – – – – – – – – Q Q Q Q Q Q Q
– – – – – – – – – – – – – – – –
– – – – C – – – – – – – – – – –
– – N – N – N N N – – – – – – –
– – – – – – – – – R R R R R R R
– – E – E E E E E D D D D D D D
– – – – – – – – – K K K K K K K
– – – – – – – – – – – – – – – –
Strain
X53794 ISS Phl3 Tuscany 1983/1 Tuscany 1983/2 Tuscany 1984/1 Tuscany1985 Marche 1990/1 Marche 1990/2 Tuscany 1991 Tuscany 1992 Marche 1992 Tuscany1993/1 Marche 1993 Tuscany 1993/2 Tuscany 1994 Tuscany 1995/1 Tuscany 1995/2 Tuscany 1995/3 Tuscany 1995/4 Abruzzo 2010 Tuscany 2009/1 Tuscany 2009/2 Tuscany 2009/3 Tuscany 2009/4 Tuscany 2009/5 Lombardy 2013 Tuscany 1980 Tuscany 1981 Tuscany 1982/1 Tuscany 1982/2 Tuscany 1982/3 Marche 1990/3 Tuscany 1984/2 JX867536 Tunisie 2013 JF330274 Tuscany 2009 JF330275 Tuscany 2009 EU327772 Tuscany 2007 Portugal 1983 EF201833 Portugal 2006 EF120631 Spain 2008 FJ153285 Spain 2008 EF656361 France 2007 KC776214 France 2010 FJ153286 France 2008
Amino acid positions 252
255
258
265
266
269
286
287
289
290
293
294
296
297
299
301
309
M – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – L L L L L L L
S C C C C C C C – C C C C C C C – C – C C C C C – C C – – C – – – C – C – – – – – – –
R – – – K – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – K K K K K K K
R – – – – – – – –
T – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – K – – – –
G – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – R R R R R R R
P L – L – – – L – – L – L L – L – L – L L L L L – L L – – L – T – L L L I I I I T I I
Q – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – H H H H H H H
T – I – I – I – I I – I – – I – – – – – – – – – – – – – – – – – – – – – S S S S S S S
I – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – V V M M V V V
D – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – N N V V N N N
K E – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – R R R R R R R
C – W – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
L – – – P – – – – P – P – – – – – – – – – – – – – – – – – – – – – – – – P P P P P P P
E – – – K – K – – K – K – – K – – – – – – – – – – – – – – – – – – – – – – – – – – – –
M – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – K K K K K K K
– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – G – – – – – – – – –
– – – – – – – – – – – – – – – – – – – – – – – – – – K K – – K K K
(GenBank, EF201833) and France (GenBank, EF656361; KC776214; FJ153286). Sandfly Naples virus (SFNV, Genbank EF201832) was chosen as outgroup strain in the analysis of S segment. P-distance values (proportions of nucleotide sites at which two sequences being compared are different) were calculated using Molecular Evolutionary Genetics Analyses (MEGA) software version 6 (Tamura et al., 2013).
To examine selective pressure among TOSV NSs sequences, synonymous and nonsynonymous substitution rates were calculated for the nucleotide data set using the Jukes–Cantor correction method in the DnaSP software (version 4.10.7) (Rozas et al., 2003). Bayesian information criterion (BIC) was used to determine the model of nucleotide substitution that best fit the data using
40
M. Baggieri et al. / Virus Research 200 (2015) 35–44
Tuscany 2009/3
72
Tuscany 2009/2 Tuscany 1981 Tuscany 1995/4 Tuscany 1995/1 Tuscany 1980 Tuscany 1982/3 Tuscany 1984/1 Tuscany 1993/1 73
I
Tuscany 1993/2 JF330274 Tuscany 2009 JF330275 Tuscany 2009 Tuscany 2009/5 Tuscany 1994 Tuscany 1995/2 EU327772 Tuscany 2007 Tuscany 1991
Marche 1990/1 Tuscany 1984/2 Tuscany 1982/2 70
X53794 Tuscany 1971
84
Tuscany 1983/1 72
II
Tuscany 1992
75
Lombardy 2013
Marche 1992 Tuscany 1985 Marche 1993 Marche 1990/2 Tuscany 1983/2 Portugal 1983 0.05
Fig. 2. Molecular phylogenetic analysis of the N nucleotide sequences by Maximum Likelihood method based on the Kimura 2-parameter model. A discrete Gamma distribution was used to model evolutionary rate differences among sites. Significant bootstrap values (>70%) are indicated. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Strain Portugal 1983 was used as outgroup strain.
the selection tool available in MEGA software. Models with the lowest BIC scores were considered to describe the substitution pattern the best. For each model, AICc value (Akaike Information Criterion, corrected), Maximum Likelihood value (lnL). The model that best fit the data was the Kimura 2 +G model for the nucleotide analysis (Kimura, 1980), and JTT +I matrix-based method for the amino acid analysis (Jones et al., 1992). Phylogenetic trees were constructed using the Maximum Likelihood method. Then, the reliability of each tree was estimated with a bootstrap analysis by 1000 replicates. 3. Results and discussion A total of 32 strains isolated from Italian samples collected over a period of more than 30 years (1980–2013) from hospitalized patients with neurological disease, from sandflies and from the brain of a bat, were included in this study. Sequences of whole S genomic segment from each isolated TOSV strain, containing the N and NSs ORFs plus the non-coding region, were aligned and compared with the Italian reference
strain ISS Phl3 (GenBank, X53794) and with all other sequences of the S segment available in GenBank database. The same analysis was conducted for N and NSs ORFs sequences, singularly. Virtual translation of N and NSs nucleotide sequences was also performed, and respective amino acid sequences were compared. Nucleotide and amino acid p-distance values were calculated (Table 2). For the whole segment S, N and NSs ORFs, p-distance was calculated within three different groups. Group TOSV A included 26 sequences of the whole S segment, 26 sequences of the N ORF and 32 of the NSs ORF, plus four sequences taken from GenBank. Group TOSV B included, for all the three analyses, 7 sequences, one from our lab (“Portugal 1983”) and 6 taken from GenBank. Group TOSV A + B included both TOSV A and TOSV B groups. Data showed that the diversity within the entire population of sequences (group TOSV A + B) is small for the S segment, with a mean p-distance of 0.061 (range, 0–0.151). As expected, p-distance values calculated within a single group – either TOSV A or TOSV B – were each one lower than whose calculated for TOSV A + B group
M. Baggieri et al. / Virus Research 200 (2015) 35–44
41
Tuscany 1995/2 EU327772 Tuscany 2007 Tuscany 1993/1 Tuscany 1993/2 Tuscany 1991
96
Marche 1990/1 Tuscany 1994 JF330274 Tuscany 2009 JF330275 Tuscany 2009 Tuscany 2009/5 Tuscany 2009/4
I
Tuscany 1984/1 Tuscany 1983/1 Tuscany 1980
83
Tuscany 1995/4 Tuscany 2009/3 77
Tuscany 2009/2 Tuscany 2009/1 Tuscany 1982/3 Tuscany 1981 Tuscany 1984/2 Tuscany 1995/1 Marche 1990/2
88
Tuscany 1983/2 91
Marche 1992
II
Tuscany 1985 Marche 1993 Lombardy 2013 Tuscany 1992 86
Abruzzo 2010 Tuscany 1995/3 X53794 Tuscany 1971
78
III
Tuscany 1982/2 Marche 1990/3 Tuscany 1982/1 Portugal 1983
0.05
Fig. 3. Molecular phylogenetic analysis of the NSs nucleotide sequences by Maximum Likelihood method based on the Kimura 2-parameter model. A discrete Gamma distribution was used to model evolutionary rate differences among sites. Significant bootstrap values (>70%) are indicated. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Strain Portugal 1983 was used as outgroup strain.
and similar to each other (mean 0.023, range 0–0.044 and 0–0.033, respectively). The sequence variations calculated for N and NSs ORFs showed similar trends. No differences in p-distance values were found by adding sequences derived from GenBank (data not shown). At amino acid level, the analysis on N and NSs showed no sequence variation for the N protein, and p-distance values of 0.018 (range 0–0.044) and 0.016 (range 0–0.025) for the NSs protein within TOSV A and TOSV B, respectively. On the entire alignment of NSs protein amino acid mutations were randomly distributed
along the primary sequence and there was no evidence of particularly variable regions. The amino acid substitutions are shown in Table 3. The distribution of synonymous and nonsynonymous substitution rates along the NSs ORF was also analyzed. The mean nonsynonymous/synonymous rate ratio (ω = dN/dS) was 0.091 within TOSV A and 0.074 within TOSV B lineages, indicating that the NSs ORF is not under a positive pressure. KinasePhos 2.0 analysis (Wong et al., 2007) based on kinase-specific phosphorylation site prediction was also performed on the NSs amino acid sequences. Two putative tyrosine phosphorylation sites were
42
M. Baggieri et al. / Virus Research 200 (2015) 35–44
Tuscany 1982/3 JF330274 Tuscany 2009 Tuscany 1993/1 Tuscany 2009/1 Tuscany 2009/3 Tuscany 2009/2 Tuscany 1993/2 Tuscany 2009/4 Tuscany 1995/2 Tuscany 2009/5 Tuscany 1984/1 Tuscany 1995/4 EU327772 Tuscany 2007 Tuscany 1980
I
Tuscany 1991 Tuscany 1994 Tuscany 1981 Marche 1990/1 72
Tuscany 1983/1 JF330275 Tuscany 2009 Tuscany 1984/2 Tuscany 1995/1 Marche 1990/2
57
Tuscany 1983/2
II
Tuscany 1985 Marche 1992 Marche 1993 Lombardy 2013 Tuscany 1992
78 79
Abruzzo 2010 X53794 Tuscany 1971 Marche 1990/3
III
Tuscany 1982/2 Tuscany 1982/1 Tuscany 1995/3 Portugal 1983 0.01
Fig. 4. Molecular phylogenetic analysis of the NSs amino acidic sequences by Maximum Likelihood method based on the Kimura 2-parameter model. A discrete Gamma distribution was used to model evolutionary rate differences among sites. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Strain Portugal 1983 was used as outgroup strain. The evolutionary distances were computed using the JTT matrix-based method. Significant bootstrap values (>55%) are indicated.
identified (aa 24, 48) located in a highly conserved region. TOSV NSs has been shown to possess two distinct functions: (i) to limit IFN- upregulation (Gori Savellini et al., 2001) and (ii) to promote RNA-dependent protein kinase degradation (Kalveram and Ikegami, 2013). Further studies will be necessary to understand if and how these phosphorylation sites are involved in these functions. Phylogenetic analysis based on the S segment showed that all but one strains clustered within TOSV A lineage (Fig. 1). The only one strain clustering in lineage TOSV B, was from a case imported from Portugal in 1983 (“Portugal 1983”) that showed similarities with sequences from Portugal (GenBank, EF201833), Spain (GenBank, EF120631; FJ153285) and France (GenBank, EF656361; KC776214; FJ153286). This suggested that TOSV A represents the
main or only lineage that has circulated in Italy since TOSV first isolation in 1971. Phylogenetic trees constructed on N and NSs ORFs showed different patterns (Figs. 2 and 3). In N analysis TOSV A strains clustered into two groups, whereas NSs analysis showed three main clusters. Phylogenetic trees constructed on the deduced NSs amino acid sequences showed the same pattern as NSs ORF with three main clusters (Fig. 4), and the high-support bootstrap values indicated that the analyses were reliable. Italian strains in each cluster appeared to have no geographical nor temporal/host correlation within the groups. In addition, they seemed to be co-evolved and randomly widespread in the area under study.
M. Baggieri et al. / Virus Research 200 (2015) 35–44
These findings reflect previous results achieved from analysis of the M segment which demonstrated a co-circulation of four TOSV clusters in Central Italy, with no correlation between viral strains and area/year of isolation (Venturi et al., 2007). Furthermore, TOSV A lineage showed a low sequence variability over the years since the reference strain ISS Phl3 (GenBank, X53794) clustered with Italian strains circulating until 2013 in all the three trees. A low sequence variability was also showed within lineage TOSV B, despite the small number of TOSV B strains involved in the analysis. Studies based on sequence comparison showed that RNA arboviruses are relatively stable in nature, suggesting that a cycle with host alternation (between vertebrate and invertebrate hosts) can interfere on the viral evolution by a strong conservative sequence selection. This genome stability may result from the requirements for viral replication in two hosts that present competitive niches for replication and adaptation (Strauss and Strauss, 1994). This was supported by experimental data on the variability within phleboviruses (Sall et al., 1997; Simons et al., 1990). As reported above, nucleotide sequences variability of NSs gene was higher than that of N gene, implying that the N gene is more stable, in agreement with recent studies (Magurano et al., 2014). In addition, the N protein seems to be well conserved with almost no amino acid changes. The N protein interacts with genome segments to form the ribonucleocapsids (Raymond et al., 2012), it also represents the major antigen responsible for both IgM and IgG responses (Magurano and Nicoletti, 1999). It is probable that changes in the N amino acid sequence that make this protein less efficient in its interaction with viral nucleic acid are lethal for the virus and have no chance to be selected. 4. Conclusions The oligonucleotide set used for this analysis appeared to be a good choice for the amplification of the NSS specific region of both A and B TOSV lineages, and contribute to define the basis for rapid diagnostic tests for TOSV based on RT-PCR. This study was conducted with the aim to determine the fulllength sequences of S segments of different Italian strains in order to better understand their evolutionary and phylogenetic relationships. Although the TOSV is an important pathogen, little genomic information is available and more studies should be conducted on the ecology and the epidemiology of this phlebovirus. This study on phylogenesis of S segment of TOSV reveals a low genetic variability. This finding confirms data from previous study (Magurano et al., 2014), providing further indications that RNA Arboviruses are relatively stable in nature. Obtaining genome sequence information for viruses with poor representation in public databases is an important task, in particular for programs of virus surveillance and studies on emerging pathogens. Acknowledgements The authors thank Dr. Stefano Buttò for critical reading the manuscript and for fruitful discussion; Dr. Raffaella Bucciardini for statistical support and Dr. Robert Peter Parker for linguistic revision. References Accardi, L., Grò, M.C., Di Bonito, P., Giorgi, C., 1993. Toscana L segment: molecular cloning, coding strategy and amino acid sequence in comparison with other negative strand RNA viruses. Virus Res. 27, 119–131. Alkan, C., Bichaud, L., de Lamballerie, X., Alten, B., Gould, E.A., Charrel, R.N., 2013. Sandfly-borne phleboviruses of Eurasia and Africa: epidemiology, genetic diversity, geographic range, control measures. Antiviral Res. 100 (1), 54–74. Braito, A., Corbisiero, R., Corradini, S., Marchi, A., Sancasciani, N., Fiorentini, C., Ciufolini, M.G., 1997. Evidence of Toscana virus infections without central nervous system involvement: a serological study. Eur. J. Epidemiol. 13, 761–764.
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Braito, A., Ciufolini, M.G., Pippi, L., Corbisiero, R., Fiorentini, C., Gistri, A., Toscano, L., 1998. Phlebotomus-transmitted Toscana virus infections of the central nervous system: a seven-year experience in Tuscany. Scand. J. Infect. Dis. 30, 505–508. Charrel, R.N., Izri, A., Temmam, S., de Lamballerie, X., Parola, P., 2006. Toscana virus RNA in Sergentomyia minuta files. Emerg. Infect. Dis. 12 (8), 1299–1300. Collao, X., Palacios, G., Sanbonmatsu-Gámez, S., Pérez-Ruiz, M., Negredo, A.I., Navarro-Marí, J.M., Grandadam, M., Aransay, A.M., Ian Lipkin, W., Tenorio, A., Sánchez-Seco, M.P., 2009. Genetic diversity of Toscana virus. Emerg. Infect. Dis. 15 (4), 574–577. Di Bonito, P., Mochi, S., Grò, M.C., Fortini, D., Giorgi, C., 1997. Organization of the M genomic segment of Toscana phlebovirus. J. Gen. Virol. 78, 77–81. Ehrnst, A., Peters, C.J., Niklasson, B., Svedmayr, A., Holmgren, B., 1985. Neurovirulent Toscana virus (a sandfly fever virus) in Swedish man after visit to Portugal. 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