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Department of Pathology and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX 77555-0609, USA
Correspondence
Shu-Yuan Xiao
syxiao{at}utmb.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are EF201815–EF201844.
A table showing primers used for RT-PCR amplification of the whole S segment of phleboviruses is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) in two main groups: the sandfly fever (SF) group with 40 members and the Uukuniemi (UUK) group with 13 members. Some of the SF viruses, but not the UUK group viruses, are associated with human diseases, including several clinical syndromes ranging from classical phlebovirus fever to meningoencephalitis to haemorrhagic fever (Bartelloni & Tesh, 1976; Braito et al., 1998, Meegan & Bailey, 1989) with varying severity, depending in part on the infecting virus serotype. Among these phleboviruses, Rift Valley fever (RVFV), Toscana (TOSV), sandfly fever Sicilian (SFSV), sandfly fever Naples (SFNV) and Punta Toro (PTV) viruses are of great public health importance (Tesh, 1988). The SF group viruses are usually transmitted to humans by the bite of infected arthropods (sandflies, mosquitoes or ceratopogonid midges of the genus Culicoides); however, in the case of RVFV, aerosol transmission can also occur (Tesh, 1988). The UUK group viruses are transmitted by ticks (Nichol et al., 2005). Available evidence suggests that many of the phleboviruses are maintained in their arthropod vectors by vertical (transovarial) transmission and that vertebrate hosts play little or no role in the basic maintenance cycle of these agents (Tesh, 1988). This maintenance mechanism has important ecological implications for the phleboviruses, as it allows them to persist during periods when adult vectors are absent or when susceptible vertebrate hosts are not available.
Without biochemical and genetic information, classification of the phleboviruses and differentiation of virus types has been dependent on serology. Briefly, viruses differentiated by plaque reduction neutralization test (PRNT) from other members of the genus are considered to be distinct serotypes. Using more broadly reacting serological techniques, such as haemagglutination inhibition (HI) and complement fixation (CF) tests, the phleboviruses show varying degrees of cross-reactivity (Tesh et al., 1976, 1982; Travassos da Rosa et al., 1983). However, this classification may not be straightforward in some cases, as we found recently in another study of the Rift Valley fever group viruses (Xu et al., 2007). As the bunyaviruses are segmented RNA viruses, they can partially exchange genetic material between isolates from different geographical regions (Flick & Bouloy, 2005; Sall et al., 1999; Nunes et al., 2005).
We previously analysed the phylogenetic relationships among 26 phleboviruses based on partial M segment sequences (Liu et al., 2003). The results showed a high divergence in the M segment sequences among these viruses. Studies by others (Giorgi et al., 1991) on the small (S) segment showed that the deduced amino acid sequences of the non-structural NSs protein for five different phleboviruses shared similarities of only 17–30 %. The S segment of the phlebovirus genome contains two open reading frames separated by a C-rich intergenic region, one encoding the nucleoprotein (N) in the 5' half of the viral complementary-sense RNA and the other encoding the NSs protein in the 3' half (Giorgi et al., 1991). Among several strains of RVFV, comparison of NSs gene sequences showed that certain areas were highly conserved (Sall et al., 1997). A more recent report on sequence analysis of 33 RVFV strains showed the NSs gene to be slightly more variable than the N gene, reflecting less constraint on the evolution of the NSs protein (Bird et al., 2007). These data suggest that there may be strong evolutionary pressure to maintain distinct regions of the NSs protein for individual viruses. Therefore, phylogenetic analysis of the full S segment of different phleboviruses may reveal how they have evolved and their relationships during this evolution. The present study was conducted to determine and compare the sequences for the N and NSs proteins of 33 selected members of the genus Phlebovirus (Table 1). The resulting data will contribute towards a better understanding of phlebovirus phylogeny; in addition, they provide evidence of whether or not reassortment occurs among naturally occurring phleboviruses.
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METHODS |
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along with their host source, geographical origin and year of isolation. All of these viruses were obtained from the collection at the World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch (UTMB) in Galveston, USA. Viruses were propagated once in Vero E6 cells before RNA extraction. Other phleboviruses used for comparison are also listed in Table 1; their sequences were obtained from GenBank: TOSV (X53794), RVFV (X53771) and UUK virus (UUKV; NC_005221).
Preparation of RNA, RT-PCR and sequencing.
Total RNA was extracted from infected cell monolayers with Trizol reagent (Invitrogen) as described previously (Liu et al., 2003). To design primers for RT-PCR, sequences of phleboviruses available from GenBank were aligned by the CLUSTAL W program of MEGALIGN implemented in the DNASTAR package (DNASTAR Inc.). Primer designs were based on conserved regions using PRIMER SELECT in the DNASTAR package. The sequences, polarities and positions of primers are listed in Supplementary Table S1, available in JGV Online. Forward primer PH-END corresponds to the 5' consensus region of S segment cDNA. Reverse primer PH-S-DR is located in the N or NS protein-coding region. The CF and Ph-S-DF primers were paired with Ph-S-ENDR1 and/or Ph-S-ENDR2, respectively, to amplify the remaining regions. RT-PCR was performed as described previously (Liu et al., 2003). The PCR products were purified from the agarose gel using a QIAquick gel extraction kit (Qiagen) and subjected to sequencing at the Protein Chemistry Core facility at the UTMB. Raw sequence data were assembled and finalized using the SEQMAN module within the DNASTAR software package. To ensure the authenticity of the sequences, new specific primers were designed according to the individual newly obtained sequences and used to perform new rounds of RT-PCR from the RNA samples, which were resequenced. The amino acid sequences of the N and NSs proteins of the S segment were deduced using the EDITSEQ module implemented in the DNASTAR software package.
Phylogenetic analyses.
The nucleotide sequences of the N and NSs genes in the S segments of the 30 phleboviruses and their deduced amino acid sequences were aligned with representative sequences of other known phleboviruses from GenBank, including RVFV, TOSV and UUKV, using CLUSTAL_W version 1.4 implemented in MACVECTOR version 7.1.1 (Accelrys). Phylogenetic trees were constructed using the neighbour-joining (NJ) (Saitou & Nei, 1987), maximum-parsimony (MP) and maximum-likelihood (ML) methods implemented in the PAUP version 4.0 software package (Kumar & Gadagkar, 2000; Swofford, 2002). For NJ analysis, a distance matrix was calculated from the aligned sequences using the Hasegawa, Kishino and Yano (HKY85) formula by allowing transitions and transversions to occur at different rates and also allowing base frequencies to vary. The distance from the aligned deduced amino acid was measured by the mean character difference. Parsimony analyses were used by selecting the tree or trees that minimized the number of evolutionary steps, including homoplasies to explain the data. For the likelihood analyses, a general time-reversible model was used and nucleotide frequencies were estimated empirically. The data were sampled by 1000 bootstrap replicates to determine the confidence indices within the phylogenetic tree and the statistical confidence of the topologies (Felsenstein, 1988). Bayesian analysis was conducted with MRBAYES version 3.0 using two replicates of 1 million generations. Bayesian posterior probabilities were calculated from the consensus of 19 602 trees after excluding the first 400 trees as burn-in (Huelsenbeck & Ronquist, 2001). The resulting trees were plotted using TREEVIEW version 1.6.6 (Page, 1996).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and 3. As shown based on the nucleotide and deduced amino acid sequences of the N protein (Table 2) of viruses in the Sicilian serocomplex, the identities ranged from 72.5 to 99.6 % and from 83.4 to 99.2 %, respectively. Among the Naples serocomplex, the nucleotide and deduced amino acid sequence identities ranged from 74.7 to 92.8 %, and from 83.1 to 99.6 %, respectively. Among the Punta Toro serocomplex, the nucleotide and deduced amino acid sequence identities ranged from 68.3 to 99.6 % and from 70.9 to 99.6 %, respectively. Within the Icoaraci serocomplex, nucleotide and deduced amino acid sequence identities ranged from 79.9 to 98.1 % and from 91.9 to 99.6 %, respectively. Within the Frijoles serocomplex, the identities were 78.5 and 90.2 %, respectively. For genes of the NSs protein (Table 3), the mean nucleotide and amino acid identities were lower than those for the N protein. In contrast, the identity among the different phlebovirus serocomplexes (species) was much lower than that within the same serocomplex.
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Analysis of the N gene revealed the presence of five major phylogenetic groups (Figs 1a, 2a) in addition to the RVF group (only the prototype was included in this study). The prototype Balliet strain of PTV was related to PAN 483391 and PAN 479603, together with PA AR 2381, GML 902878 and PTV Adames. Buenaventura virus (BUEV) clustered with Co Ar 170255, VP-334K and VP-366G. Co Ar 171616 formed another clade. These three clades constituted the Punta Toro serocomplex. The Sicilian serocomplex was composed of four clades: R-18 with RM-09; the SFSV (Sabin) prototype with I-701735; 91045I with 91025 B; and Corfou. The Naples serocomplex was subdivided into four clades: TOSV with ELB; YU 8-76 clustered with NAMRU 840055 and Naples (Sabin); R-3; and P-7101795. The Icoaraci serocomplex consisted of Salobo virus (SLBOV) 1983, SLBOV 1997, Belterra virus (BELTV) and Icoaraci virus (ICOV); and the Frijoles serocomplex included Joa virus (JOAV) and Frijoles virus (FRIV). When constructed using amino acid sequences, the phylogenetic relationship was similar (Fig. 2a).
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), which corresponded to the serocomplexes: Punta Toro, Sicilian, Naples, Icoaraci and Frijoles. RVFV was on its own. When constructed using amino acid sequences, the phylogenetic tree was similar (Fig. 2b).
Notably, although Corfou virus clustered with the Sicilian serocomplex most of the time (Figs 1a, 2a, b), it did not cluster with any group in the tree based on the nucleotide sequence of the NSs gene (Fig. 1b). Nevertheless, this separation from the original group into a new branch had a rather low bootstrap value of 60 %.
The reliability of these trees was examined by bootstrap analysis. For the N gene, according to the nucleotide sequences, the clustering was strongly supported by the bootstrap value of 100 % for the five serocomplexes (Fig. 1a). Within the Punta Toro serocomplex, the clustering of different viruses was strongly supported by a bootstrap value of 100 % for PTV Balliet, PAN 483391, PAN 479603, PA AR 2381, GML 902878 and PTV Adames; and 98 % for Buenaventura, Co Ar 170255, VP-334K and VP-366G. The clustering of the other groups was also supported by high bootstrap values, as shown in Fig. 1(a).
According to amino acid sequences, the clustering of different serocomplexes was also strongly supported by bootstrap values of 100 % for all of the five serocomplexes (Fig. 2a).
As for the nucleotide sequences of the NSs gene, the clustering of different serocomplexes was strongly supported by a bootstrap value of 89 % for the Naples serocomplex, 98 % for the Punta Toro serocomplex, 62 % for the Sicilian serocomplex, 75 % for the Icoaraci serocomplex and 82 % for Frijoles serocomplex (Fig. 1b). For amino acid sequences, the clustering of different serocomplexes was supported by bootstrap values of 100 % (Fig. 2b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The complex antigenic relationships among the phleboviruses may be caused by potential genomic reassortment occurring in nature, either in distant evolutionary time or current time. The CF test generally detects N protein antibodies, a marker for the S segment. Some of these viruses are more closely related by HI or PRNT (M gene products responsible for surface glycoprotein targets) than by CF test (Tesh et al., 1982). This suggests the possibility of S and M (and potentially L) segment interchange. Natural genetic reassortment has been demonstrated in a number of bunyaviruses (Borucki et al., 1999; Bowen et al., 2001; Nunes et al., 2005; Saeed et al., 2001). Homologous reassortment also has been shown in laboratory studies using RVFV in Vero cells to analyse the contribution of each of the RNA segments of the vaccine to its attenuated phenotype (Rossi & Turell, 1988; Sall et al., 1999).
The L, M and S genome segments of phleboviruses each contribute differently to viral pathogenesis. The S RNA exhibits an ambisense coding strategy; it is transcribed by the virion RNA polymerase as a subgenomic virus complementary-sense mRNA that encodes the N protein and, from a full-length antigenomic S RNA, as a subgenomic virus-sense mRNA that encodes the non-structural NSs protein (Nichol et al., 2005). The N and NSs proteins have been found to be the most important determinants in the pathogenesis of phleboviruses (Billecocq et al., 2004; Bouloy et al., 2001; Ikegami et al., 2006; Sall et al., 1997; Vialat et al., 2000). In order to understand better their evolutionary and phylogenetic relationships, we conducted this study to determine the full-length sequences of S segments of different phleboviruses.
As shown in Figs 1 and 2, the phylogenetic clustering of the individual viruses studied corresponded well with their serological groupings (Tesh et al., 1982; Travassos da Rosa et al., 1983). The phylogenetic trees constructed using their complete N and NSs nucleotide sequences and deduced amino acid sequences presented similar patterns. They also corresponded well to our earlier M segment phylogenetic analysis (Liu et al., 2003) of the same serocomplexes. The high-support bootstrap values indicated that they were confident and reliable phylogenetic analyses.
For the Punta Toro serocomplex, all members were isolated from Panama or Colombia during the period 1964–2000. PTV Balliet, PTV Adames, PAN 483391, PAN 479603, Pa Ar 2381 and GML 902878 were all from Panama and grouped together. The other two serotypes, VP-334K and VP-336G, also from Panama, clustered together with Co Ar 170255 and Buenaventura (Co Ar 3319), which were from Colombia. The mean nucleotide identity for the N and NSs genes within the Punta Toro serocomplex was 78.8 and 68.8 %, whilst their mean amino acid identity was 82.4 and 66.8 %. The position of this group in the phylogenetic tree correlated closely with their geographical distribution.
For the Sicilian serocomplex, the locations and years of isolation for these viruses were closely related, but not identical. High identity was observed from the phylogenetic tree among the Sicilian-like viruses. The mean nucleotide identity for the N and NSs genes within this group was 82.9 and 74.9 %, and the mean amino acid identity was 92.6 and 77.2 %, respectively. In the nucleotide tree based on the NS gene (Fig. 1b), one of the members, Corfou virus, did not show clustering with Sicilian serocomplex viruses. However, serologically by CF test it was indistinguishable from these viruses and the amino acid tree of the same gene, and trees based on the N gene all confirmed its placement within this group. The sandfly fever Naples serocomplex was distributed between two branches: TOS (X53794) and TOS (ELB); and Naples-like POONA 7101795, Naples prototype (Sabin), Naples-like NAMRU 840055 and Naples-like R-3. All of these viruses were isolated between 1944 and 1985, but no geographical clustering could be inferred from this group. The mean nucleotide identity for the N and NSs genes within this group was 80.6 and 68.7 %, respectively, whilst their mean amino acid identity was 90.7 and 63.7 %.
The Icoaraci serocomplexes contained four viruses, isolated from Brazil. The mean nucleotide identities for the N and NSs genes within this group were 86.1 and 83.0 %, whilst their mean amino acid identities were 95.4 and 90.4 %. FRIV from Panama was closely related to JOAV from Brazil, which formed the Frijoles serocomplex. Their mean identities were 78.5 and 65.5 % for the N and NSs genes, respectively, and 90.2 and 65.7 % for the deduced amino acid sequences. These data are consistent with another phylogenetic study based on partial S, M and L segment sequences, showing that BELTV, SLBOV, ICOV, FRIV and JOAV are distinct from RVFV (Xu et al., 2007).
Our results also showed that the identities of nucleotide and amino acid sequences for the N gene were higher than that of the NSs gene. This implies that the N gene is more stable, which has also been observed by others (Sall et al. 1997; Simons et al., 1990). It also indicates that the NSs protein is more specific to each group, and it may play a more important role in pathogenesis and replication of these viruses and serve as a main virus virulence factor (Ikegami et al., 2006; Vialat et al., 2000). Furthermore, as shown in Table 3, at the amino acid level, the sequence divergence of the NSs protein among different groups was disproportionately higher than at the nucleotide level. The underlying mechanism for this phenomenon is unknown, but may be related to specific environmental pressure unique to each group within their geographical regions. In addition, compared with our previous findings based on the M genome segment (Liu et al., 2003), it seems that no reassortment has occurred among the viruses included in this study. Finally, to our knowledge, this study is by far the most comprehensive phylogenetic analysis, based on N and NSs genomic sequences of 30 natural isolated phleboviruses. It provides information for more definitive genotypic classification of these important viruses.
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ACKNOWLEDGEMENTS |
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Received 18 January 2007;
accepted 3 April 2007.
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