1 Department of Pathology and Center for Tropical Diseases, University of Texas Medical Branch, Galveston, Texas 77555-0588, USA
2 Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-0588, USA
3 Institute of Virology, Medical School of Wuhan University, Wuhan, Hubei Province, PR of China
Correspondence
Shu-Yuan Xiao
syxiao{at}utmb.edu
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ABSTRACT |
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The sequences obtained in this study have been deposited in GenBank under the accession numbers AY129732AY129752.
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Introduction |
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The Phlebovirus genus currently consists of 68 distinct virus serotypes, most of which have not been genetically characterized. These viruses are antigenically unrelated to members of other genera within the family Bunyaviridae, but they show various degrees of cross-reactivity among themselves in serological tests, such as haemagglutinin-inhibition (HI) tests and complement-fixation (CF) tests (Tesh et al., 1976, 1982
; Travassos da Rosa et al., 1983
). The 68 known phleboviruses are divided into two major antigenic groups: the phlebotomus fever (or sandfly fever) group with 55 members and the Uukuniemi group with 13 members. The phlebotomous fever group is further divided into 13 serocomplexes and 12 members not assigned to any serocomplex. These include the eight serocomplexes listed in the Seventh Report of the International Committee on Taxonomy of Viruses (Elliott et al., 2000
) and five additional serocomplexes recently defined in our laboratory, namely Aguacate, Chargres, Arboledas, Arumowot and Tapara (A. P. A. Travassos da Rosa, unpublished data). The phlebotomus fever viruses are transmitted by sandflies, mosquitoes or ceratopogonids of the genus Culicoides, whereas the Uukuniemi group viruses are transmitted by ticks (Elliott et al., 2000
).
Because of the paucity of genetic information about the phleboviruses, viruses within this genus are presently defined by their serological relationships. Antigenic relationships between the viruses as determined by HI tests are used for genus placement, relationships by CF tests for serogroup and complex assignment, and plaque reduction neutralization tests (PRNTs) for serotype and subtype differentiation (Tesh et al., 1982; Travassos da Rosa et al., 1983
). The current antigenic (serological) classification of the phleboviruses is unsatisfactory for the following reasons: (i) to date, a total of 68 phlebovirus species have been discovered and undoubtedly many more exist in nature; (ii) based on their genetic structure, abundance and complex patterns of antigenic cross-reactivity, it is probable that natural reassortment occurs among some of these viruses, confounding their antigenic classification and identification; (iii) some of the phleboviruses do not produce readable plaques under agarose and do not readily infect or kill common laboratory animals, making reagent production and antigenic comparisons difficult; and (iv) relatively few research laboratories are still capable of performing the classical serological tests necessary for characterization of phleboviruses. Therefore, other techniques are needed to study and characterize fully this large and diverse genus.
In the present study, we have searched for relatively homologous regions in the M segment sequence of selected viruses of the phlebovirus genus and designed primers to amplify cDNA products using RT-PCR. The products were sequenced and phylogenetic analyses were carried out based on the nucleotide sequences of the RT-PCR products. The cluster pattern of the viruses studied matched the grouping by serological methods; thus, we believe that the technique can serve as a framework for taxonomic placement of other uncharacterized or yet to be discovered phleboviruses.
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Methods |
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DNase I treatment and reverse transcription.
RNA was treated with Amplification Grade RNase-free DNase I (Invitrogen) before reverse transcription to eliminate DNA contamination. The reaction included 1 µg total RNA, 1 unit Amplification Grade RNase-free DNase I, 20 mM Tris/HCl, pH 8·3, 50 mM KCl and 2 mM MgCl2, in a total volume of 10 µl. After incubation at room temperature for 15 min, the reaction was terminated by adding 1 µl 25 mM EDTA, heated for 10 min at 65 °C and then chilled on ice. The reaction mixture was used directly for reverse transcription. dNTPs (0·01 µmol; Sigma) and 0·1 µg random hexamers (Promega) were added into the 11 µl terminated DNase I reaction mixture, followed by heating at 65°C for 5 min and quick chilling on ice. Four µl 5x times; buffer (250 mM Tris/HCl, pH 8·3, 375 mM KCl, 15 mM MgCl2), 0·2 µmol DTT, 40 units RNaseOUT recombined ribonuclease inhibitor (Invitrogen) and 50 units SuperScript II RNase H- Reverse Transcriptase (Invitrogen)] were added; the final volume was 20 µl. The reaction was incubated at 25 °C for 10 min followed by 42 °C for 60 min and terminated at 70 °C for 15 min.
Primer design.
The amino acid sequences deduced from the available nucleic acid sequences of the M segment of Punta Toro (PT) virus, Rift Valley fever (RVF) virus, Toscana (TOS) virus and sandfly fever Sicilian (SFS) virus (see Table 1 for GenBank accession numbers) were aligned with the CLUSTAL W program (Fig. 1
). Four conserved regions (designated 14) were selected for primer location. Initially, degenerate primers were synthesized according to the most conserved bases from these regions, but these primers failed to amplify most of the viruses. Subsequently, a different approach was used. Individual oligonucleotides specific for each of the sequences were synthesized from these same regions. These specific primers from one region were pooled as a cocktail primer for RT-PCR. The sequences and position of primers are listed in Table 2
. As shown, the forward primer Ph-M-2FM cocktail consisted of Ph-M-2F-PT, Ph-M-2F-RVF, Ph-M-2F-SFS and Ph-M-2F-TOS, which corresponded to sequences of PT, RVF, SFS and TOS at the conserved region 2, respectively. The reverse primer Ph-M-3RM mix was initially composed of Ph-M-3R-PT, Ph-M-3R-RVF, Ph-M-3R-SFS and Ph-M-3R-TOS, which corresponded to sequences of PT, RVF, SFS and TOS viruses at the conserved region 3, respectively. Later, as more sequences were obtained during the study, two additional oligonucleotides were made and added to this cocktail as new components, namely, Ph-M-3R-ELB and Ph-M-3R-27. Another reverse primer, Ph-M-4R2I, corresponding to the conserved region 4, was synthesized using inosine to reduce the degeneracy of the primer. In addition, another forward primer mix, Ph-M-1FM, was designed for conserved region 1. It consisted of Ph-M-1TF and Ph-M-1SF, specific for Toscana and Sicilian viruses, respectively. This primer mix (Ph-M-1FM) was paired with Ph-M-3RM to amplify the Naples prototype (Sabin) virus.
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Sequence and phylogenetic analyses.
The sequences were examined with BLASTX in the MacVector 7.0 program (Accelrys) to search for the homologous sequences in GenBank. All nucleotide sequences of the amplified viruses and available sequences from GenBank were aligned with CLUSTAL W in the MacVector 7.0 program.
Phylogenetic analyses were carried out with PAUP 4.0 Beta version (Swofford, 2002). A bootstrap consensus tree was generated by the neighbour-joining method. The number of bootstrap replicates was 1000. The M segment sequences of Uukuniemi virus and RFV virus were used as outgroups (see Table 1
for GenBank accession numbers).
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Results |
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To ensure the fidelity of the PCR products, we designed a specific primer pair for each of the Naples and the Sicilian complex viruses, based on the sequences obtained for each virus; these specific primer pairs (Table 3) were used to amplify the individual viruses again. All of these reactions generated products with sequences that matched those obtained with the cocktail primers. Therefore, the cocktail approach of designing primers to amplify these phleboviruses appears to be reliable.
Sequence analysis
All sequences obtained from the PCR-amplified products were deposited in GenBank. A BLASTX search confirmed that these sequences were homologous to M segment sequences of phleboviruses previously available from GenBank.
Phylogenetic analyses based on the partial M segment nucleotide sequences
Since all of the PCR products encompassed a common 600 bp region of the M segment, the sequences of this region (between primers Ph-M-2FM and Ph-M-3RM) from these viruses were utilized for alignment, using the CLUSTAL W program of the MacVector software. Phylogenetic analysis was carried out by the neighbour-joining procedure, using the PAUP 4.0 program (Beta version 8). The resultant phylogenetic tree is shown in Fig. 2. Phylogenic analysis of these viruses resulted in three distinct genotypic lineages, corresponding to the Sicilian, Naples and Punta Toro complexes, which are shown as lineages I, II and III, respectively. The percentage differences of amino acid and nucleic acid sequences among the different groups of phleboviruses are listed in Table 4
.
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Discussion |
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In order to characterize genetically the many phleboviruses in our reference collection, we wanted to develop a method to amplify any phlebovirus by RT-PCR. We first attempted to find a universal primer pair, as was done in the original phylogenetic study for hantaviruses (Xiao et al., 1992, 1994
). However, this approach did not work, due to the high sequence divergence among the phleboviruses as later discovered in this study (Table 4
). Nevertheless, the second approach, which used cocktail primers (Table 2
), allowed us to amplify successfully the vast majority of the viruses examined.
As shown in Fig. 2, the phylogenetic clustering of individual viruses studied corresponded well with their serological groupings. Lineage I consisted of the Sicilian-like viruses and Corfou virus. Viruses isolated from the same localities and in the same year were closely related, but not identical. By CF test, Corfou cross-reacted with sandfly fever Sicilian virus (unpublished data). It was compatible with the position of Corfou in this lineage.
Lineage II consisted of viruses in the sandfly fever Naples serocomplex, which were distributed into three branches: Toscana prototype (ISS PHL3) and Toscana (ELB); Tehran and Yu 8-76; and Naples prototype (Sabin), Poona 7101795, R-3 and NAMRU-840055. No geographical clustering could be inferred from this group.
Lineage III consisted of Punta Toro serocomplex viruses. All members of this group were isolated from South America. In one branch, Punta Toro virus strains Adames, Pa Ar 2381, GML 902878 and Balliet, all from Panama, were grouped together. The two viruses, Pa Ar 2381 and GML 902878, isolated from Bayano, Panama, in 1975 and 1976, respectively, were very similar; the difference between their nucleotide sequences was 0·2 %, while their amino acid sequences were identical. Balliet virus, an isolate from western Panama, is less virulent in hamsters than Adames, an isolate from eastern Panama (Anderson et al., 1990); the percentage differences between their partial M segment nucleotide and amino acid sequences were 11 % and 6 %, respectively. Another PT branch contained three viruses from Colombia and two from Panama.
As shown in Fig. 2 and Table 4
, the sequence diversities within a serogroup and among the different serogroups were variable and larger than those of many other viruses. For example, the sequence difference between Corfou and the other members in the Sicilian serogroup was up to 42 %, yet by both CF test and phylogenetic analysis, they fall into the same serogroup or lineage. Similar diversities were seen in all the other serogroups.
In summary, phleboviruses from both the Old and New Worlds group into different lineages. The positions of phleboviruses in the phylogenetic tree are not necessarily related to their geographical distribution. Furthermore, the geographical distributions of some phleboviruses overlap (Tesh et al., 1976). Consequently, viruses present in the same area could theoretically co-infect a single host and produced reassortants.
The approach of using a mixture or cocktail of specific primers, representing different groups of viruses, appears to be an effective alternative to the previously used consensus' primer; the method is particularly useful for virus genera that have high genetic diversity, such as the phleboviruses. We are currently using the same approach for amplification of the S and L segment sequence of these viruses.
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ACKNOWLEDGEMENTS |
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Received 9 August 2002;
accepted 15 October 2002.