Institute of Microbiology and Immunology, Medical Faculty of Ljubljana, Zaloska 4, 1000 Ljubljana, Slovenia1
Slovenian Museum of Natural History, Ljubljana, Slovenia2
Haartman Institute, University of Helsinki, Helsinki, Finland3
Author for correspondence: Tatjana Avsic-Zupanc. Fax +386 1 543 7401. e-mail tatjana.avsic{at}mf.uni-lj.si
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Abstract |
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Introduction |
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Two pathogenic hantaviruses that cause HFRS have so far been proven to circulate in Europe: PUU and DOB. DOB was isolated originally from the lungs of a yellow-necked mouse (Apodemus flavicollis) trapped in the Dolenjska region of Slovenia, where a number of severe HFRS cases had occurred (Avsic-Zupanc et al., 1999 ). Extensive antigenic and genetic characterization identified DOB as a unique hantavirus type (Avsic-Zupanc et al., 1995
). It was shown that DOB, transmitted to humans by A. flavicollis, is the aetiological agent of a severe form of HFRS that occurs in the Balkans (Antoniadis et al., 1996
; Lundkvist et al., 1997b
; Papa et al., 1998
; Avsic-Zupanc et al., 1999
). DOB has also been found in striped field mice (Apodemus agrarius) in Estonia (Plyusnin et al., 1997
; Nemirov et al., 1999
), Russia (Plyusnin et al., 1999a
) and Slovakia (Sibold et al., 1999
). These findings suggest that striped field mice, which are known to harbour HTN in Asia, also carry DOB in Central and Eastern Europe. Human DOB infections have been detected in Russia (Lundkvist et al., 1997a
), Estonia (Lundkvist et al., 1998
) and Germany (Meisel et al., 1998
). Notably, no casualties were associated with any of the DOB-HFRS cases in these countries, where the virus is probably carried by A. agrarius. This is in sharp contrast to the Balkans, where 912% fatality rates have been reported for hospitalized DOB-HFRS cases (Papa et al., 1998
; Avsic-Zupanc et al., 1999
).
In Slovenia, hantavirus infection has been demonstrated in multiple rodent species and other mammalian orders by detection of viral antigen and antibodies (Avsic-Zupanc, 1999 ). Earlier epidemiological surveys indicated that A. flavicollis and bank voles (Clethrionomys glareolus), which are common throughout Central Europe, were most frequently infected with hantaviruses. The purpose of the present study was to estimate the rate of DOB infection in rodents and to evaluate the genetic variability of DOB in Slovenia.
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Methods |
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RTPCR.
RNA was extracted from kidney tissue of seropositive rodents. Approximately 100 mg kidney tissue was ground in 1 ml TRIzol Reagent (Gibco BRL) and mixed with 0·2 ml chloroform. After incubation at room temperature for 23 min, the mixture was centrifuged at 12000 g for 15 min at 4 °C. After centrifugation, the RNA was precipitated from the aqueous phase by mixing with 0·5 ml ice-cold 2-propanol and subsequent centrifugation at 12000 g for 10 min at 4 °C. The RNA pellet was washed with 75% ethanol by vortexing and centrifuged at 8000 g for 5 min at 4 °C. The RNA was briefly air-dried and dissolved in 50 µl nuclease-free water.
RTPCR was performed initially don all seropositive rodent samples by using the cross-reactive outer primers MOF103 and MOR204, described previously (Chu et al., 1995 ), which amplify a 490 bp region from the M segment (encoding G1) of a number of different hantaviruses (nt 11901680). RT was carried out with the SuperScript pre-amplification system for first-strand cDNA synthesis (Gibco BRL) according to the manufacturers instructions. Ten µl first-strand cDNA was then mixed with 4 µl 10x PCR buffer, 2 µl 25 mM MgCl2, 0·5 µl (100 pmol) each of primers MOF103 and MOR204, 32·7 µl distilled water and 0·3 µl (2·5 U) Taq DNA polymerase. The reaction mixture was then subjected to 35 cycles, each consisting of 30 s at 94 °C, 30 s at 50 °C and 2 min at 72 °C. A second-round PCR (for nt 13091599) was carried out with DOB-specific primers DOB G1F (5' ATGCCAGCGAGTCGACCAA 3') and DOB G1R (5' GAGCTATTATGTAAGATTGC 3'), which reside within the amplified region of the RTPCR primers in a nested fashion. For nested PCR, the annealing temperature was increased to 55 °C. The amplified products were analysed by electrophoresis in 2% agarose gels in Trisacetate buffer. After staining with ethidium bromide, the PCR products were visualized by UV trans-illumination.
In addition, four partial S segment sequences were recovered. Previously described nested primers (Papa et al., 1998 ), designed to detect all known hantaviruses associated with rodents of the subfamily Murinae (HTN, DOB, SEO), were used in nested RTPCR to obtain partial S segment sequences (nt 364963) from two samples, Slo-1 and Slo-3 (from Dobrava and Kocevje). Two other sequences (Slo-9 and Slo-10, originating from Prekmurje) were recovered by using a different protocol. Briefly, RT and first-round PCR were performed with the DOB-specific primers DOBS1 (5' CAATTGGTGATAGCCAGGCAGAAGG 3') and DOBS2 [5' GCCATGCCTGCAAT(A/G)AACAGGCAGG 3'], which should yield a 929 bp product (nt 841012). Primers DOBS3 [5' CTTGACATTGATGAACC(A/T)ACAG 3'] and DOBS4 [5' G(C/T)CAGATA(A/G)TAGCTTGCA(A/C)GG 3'] were used for the second-round PCR in order to obtain a product that corresponds to nt 354782 of the S segment. Amplification products of the correct length were separated in agarose gels and purified with the QIAquick kit (Qiagen).
For comparison, the partial M segment sequence (nt 13091599) was recovered for the wild-type (wt) DOB strain Kurkino/Aa44/97 (Plyusnin et al., 1999a ) from Russia by using primers DOB G1F and DOB G1R.
Sequencing.
The PCR products were purified from gel slices by using the Geneclean kit (BIO 101) according to the manufacturers instructions. ABI PRISM Dye Terminator cycle sequencing ready reaction kits with AmpliTaq DNA polymerase FS (PE Applied Biosystems) and 4 pM of each of the nested primers were used in the sequencing reactions. The products were purified by using Centri-sep spin columns (Princeton Separations) and sequenced on an ABI 310 Genetic Analyser (PE Applied Biosystems). The chromatograms were analysed and assembled by using the Staden software (MRC Laboratory of Molecular Biology, Cambridge, UK) run on a Linux operating system.
All sequence alignments were done with the SeqApp program, version 1.9a169. The DISTANCES program from the GCG package was used for calculations of distances between nucleotide sequences and amino acid sequences.
Phylogenetic analysis.
The PHYLIP program (Felsenstein, 1993 ) was used to make 500 bootstrap replicates of the sequence data (SEQBOOT). Distance matrices were calculated by using Kimuras two-parameter model (DNADIST) and analysed by the FitchMargoliash tree-fitting algorithm (FITCH). Alternatively, the DNApars program was used to find the trees with maximum parsimony. The bootstrap support percentages of particular branching points were calculated from these trees (CONSENSE).
For comparisons, existing sequence data were obtained from sequence databases. The S segment sequences included: HTN strain 76-118 (GenBank accession number M14627); SEO strain SR-11 (M34881); PUU strains Sotkamo (X61035), CG13891 (U22423), Vindeln/L-20 (Z48586), Udmurtia/894Cg/91 (Z21497) and Cg1820 (M32750); Tula virus (TUL) strains Tula/Moravia/5302v/95 (Z69991) and Tula/76Ma/87 (Z30941); Prospect Hill virus (PH) strain PH-1 (Z49098); Isla Vista virus (ILV) strain MC-SB-1 (U31534); SN strain H10 (L25784); NY strain RI-1 (U09488); El Moro Canyon virus (ELMC) strain RM-97 (U11427); BAY strain Louisiana (L36929); BCC (L39949); LN strain 510B (AF005727); Rio Segundo virus (RIOS) strain RMx-Costa-1 (U18100); Khabarovsk virus (KBR) strain MF-43 (U35255); Topografov virus (TOP) strain Topografov/Ls136V (AJ011646); and DOB prototype strain (L41916) and strains DOB-HA (AF060021), DOB-GA (AF060019), DOB-SZ (AF060022), DOB-EA (AF060020), DOB-PR (AF060018), DOB-SI (AF060017), DOB-CG (AF060016), DOB-TI (AF060015), DOB-NF (AF060014), DOB-PA (AF060024), DOB-TD (AF060023), Saaremaa/160v (AJ009775), Saaremaa/90Aa/97 (AJ009776), Kurkino/44Aa/98 (AJ131672), Kurkino/53Aa/98 (AJ131673), Ano Poroija/9Af/99 (AJ276305) and Ano Poroija/13Af/99 (AJ276306). Partial sequences of two DOB strains from Slovakia, DOB/Slovakia/Apa862/97 and DOB/Slovakia/Apa872/97, were kindly provided by Detlev Krüger and Claus Sibold (Humboldt University, Berlin, Germany).
The M segment sequences included HTN strain 76-118 (M14627); SEO strain SR-11 (M34882); Thailand virus (THAI) strain 749 (L08756); PUU strains Sotkamo (X61034), CG13891 (U22418), Cg1820 (M29979) and L-20 (U14136); TUL strains Tula/Moravia/5302v/95 (TULv) (Z69993) and Tula/Moravia/5286Ma/94 (TUL86) (Z66538); PH strain PH-1 (Z55129); SN strain H10 (L25783); NY strain RI-1 (U36801); ELMC strain RM-97 (U26828); BAY strain Louisiana (L36930); LN strain 510B (AF005728); BCC (L39950); Blue River virus strain Indiana (BR-IN) (AF030551); KBR strain MF-43 (AJ011648); TOP strain Topografov/Ls136V (AJ011647); and DOB prototype strain (L33685) and strain Saaremaa/160Aa/96 (AJ009774).
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Results |
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Comparative analysis of the DOB M segment sequences (Table 2) included nine partial sequences recovered from Slovenian A. flavicollis [Dob (prototype), Slo-1 to Slo-8 and Slo-10] and three sequences recovered from A. agrarius: one Slovenian (Slo-9, from Prekmurje), one Russian (Rus-53) and one Estonian (Est-160). All sequences derived from A. flavicollis were closely related to each other (nucleotide diversity 05·8%) and showed clear geographical clustering: nucleotide diversity between strains from the same locality did not exceed 0·4%. In contrast, the sequence derived from A. agrarius (Slo-9) was remarkably different from the other Slovenian strains, including the strain Slo-10 from the same locality (nucleotide diversity of 12·613·7%).
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It should be noted that the values of sequence divergence presented in Tables 2 and 3
are calculated for the partial sequences of the M and S segments. When two isolates, Dobrava (prototype) and Saaremaa/160v, are compared, the values calculated for the partial S/N sequences (11·2 and 2·3% nucleotide and amino acid sequence divergence, respectively) are close to the values determined for the complete sequences (12·2 and 3·0%; Nemirov et al., 1999
); the same can be seen for the nucleotide sequences of the M segment (20·6% for the partial sequences vs 18·8% for the complete sequences). However, the amino acid sequence divergence of the deduced G1 sequences shown in Table 2
are almost half those determined for the complete ORF of the M segment (3·3% for the partial sequences vs 6·2% for the complete sequences), suggesting that the divergences presented in Table 2
for the G1 amino acid sequences might be underestimates.
Phylogenetic analysis
The phylogenetic analysis based on partial sequences of the S segment revealed that sequences originating from A. flavicollis, together with those recovered from HFRS cases, constitute a well-supported group (Fig. 2a). It is divided further into three lineages that include (i) human sequences from north-western Greece, (ii) sequences from A. flavicollis trapped in Slovenia (Dobrava and Kocevje) and Bosnia and (iii) sequences from north-eastern Greece recovered either from A. flavicollis (Gre-9 and Gre-13) or from HFRS patients (Gre-PR and Gre-TD). The sequence originating from A. flavicollis from Prekmurje (Slo-10) is placed within the third lineage, but with low bootstrap support (48%).
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Similar to what was observed for the S segment sequences, all DOB strains originating from A. flavicollis grouped together on the phylogenetic tree based on partial M segment sequences (nt 13141590) (Fig. 2b). Also, a geographical clustering can be seen for the strains from different locations in Slovenia: sequences from Dobrava (Dob), Kocevje (Slo-3 and Slo-4) and Gorjanci (Slo-7 and Slo-8), all situated in the south-eastern part of Slovenia, are clustered together, as are two sequences from the Tenetise area (Slo-5 and Slo-6), which is in the central part of the country. At the same time, like on the S-derived tree, the sequence originating from A. agrarius from Slovenia (Slo-9) is placed apart from all sequences recovered from A. flavicollis, as are two other sequences originating from A. agrarius, from Estonia and Russia. These three are grouped together, albeit with low bootstrap support (4244%).
Thus, on both the S- and M-derived phylogenetic trees, the A. agrarius-derived Slo-9 strain does not group together with strains originating from A. flavicollis of the same geographical origin, but instead groups with other A. agrarius-derived strains from Russia, Slovakia and Estonia; i.e. it shows host-dependent rather than geography-dependent clustering.
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Discussion |
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All but three serologically positive animals (94%) were found to be positive by a DOB-specific nested RTPCR assay for the partial M segment and sequences were recovered from the two Apodemus species.While A. flavicollis mice were abundant in all areas studied, A. agrarius mice were trapped in only two areas (Gorjanci and Prekmurje). That is in concordance with the findings of the mammalogists from the Slovenian Museum of Natural History (Krystufek, 1991 ). According to their studies of rodent populations in Slovenia, A. flavicollis is distributed widely all over the country, while A. agrarius inhabits only the north-eastern area along the border with Hungary and Croatia and the sub-Mediterranean lowland.
It should be mentioned that DOB-positive A. flavicollis were found in all five trapping sites, while positive A. agrarius were trapped in a single site only (Prekmurje). This probably reflects the limited distribution of the latter species in Slovenia.
Has a host-switch resulted in two subtypes of DOB?
Genetic and phylogenetic analyses provide strong evidence that DOB in Slovenia is represented by two distinct genetic lineages. The first lineage is harboured by A. flavicollis and, at the moment, is represented by eight strains from five different locations distributed across the country. The genetic distances calculated for these strains show that they are closely related to each other, with a direct correlation of the similarity to their geographical distribution (in general, the genetic distances between the strains increase with increasing geographical separation). The data on genetic comparisons of the DOB sequences originating from A. flavicollis are in good agreement with their phylogeny: on both the S- and M-derived trees they group together and, within this group, they form small separate clusters according to their geographical origin. The reason(s) why such a clustering is seen better on the S-derived tree than on the M-derived tree remains unclear. One factor might be that the region of the M segment selected for the analyses is sub-optimal.
The second DOB lineage in Slovenia is so far represented by a single A. agrarius-derived strain from Prekmurje. This variant seems to occur rather rarely in Slovenia, where its rodent host species is not widely distributed. It is genetically and phylogenetically distinct from all DOB strains harboured by A. flavicollis (including those from Slovenia, Greece and Bosnia) and instead shows closer relatedness to DOB strains harboured by A. agrarius from Slovakia, Russia and Estonia. Most notably, both DOB lineages were found in co-circulation within the same location, suggesting that they are not mutually exclusive, i.e. they are sympatric.
These observations, taken together with other currently available data, raise several important questions. While the finding of distinct DOB genetic lineages in A. flavicollis and A. agrarius supports the hypothesis of hantavirushost co-speciation and co-evolution (Plyusnin et al., 1996 ; Nichol, 1999
; Plyusnin & Morzunov, 2000
), the large genetic distance between HTN and the DOB variant found in A. agrarius is inconsistent with this hypothesis. In fact, the phylogenetic relationships of HTN and the DOB lineage in A. agrarius seem not to mirror those of their rodent hosts. Even taking into consideration the fact that A. agrarius from Europe (the host for one of the DOB variants) and from the Far East (the host for HTN) belong to two distinct subspecies, A. agrarius agrarius and A. agrarius mantchuricus (Chernukha et al., 1986
), one would expect the two hantaviruses to be monophyletic, but the analysis shows them not to be (Fig. 2
). Such a discrepancy might be explained by a host-switch event occurring during evolution of these viruses, similar to that reported recently for the hantaviruses TOP and KBR (Vapalahti et al., 1999
). If that was the case for the DOB variants harboured by A. flavicollis and A. agrarius, the first could have acted as the donor and the second as the recipient for a host-switching hantavirus. To date, several other examples of host-switch events in hantaviruses are known (for reviews see Nichol, 1999
; Plyusnin & Morzunov, 2000
) and all of them are considered exceptions from the general flow of virushost co-speciation and co-evolution.
The question of whether the two DOB variants represent distinct subtypes or even distinct hantavirus types (species) remains to be answered. Nevertheless, separation of the classic DOB, with the Slovenian A. flavicollis-derived Dobrava isolate (Avsic-Zupanc et al., 1995 ) as the prototype, from Saaremaa virus (SAA), with an Estonian A. agrarius-derived isolate (Nemirov et al., 1999
) as the prototype, does not seem totally illogical. Indeed, DOB and SAA, besides occupying distinct ecological niches (i.e. primary rodent reservoirs), show at least 4-fold differences in titres in neutralization tests (Nemirov et al., 1999
;
. Lundkvist, personal communication) and up to 6·2% diversity of amino acid sequences for the complete glycoprotein precursor; i.e. they fulfil two of the criteria currently accepted to define distinct hantavirus species (Elliott et al., 2000
) and are very close to fulfilling the third (7% diversity), at least for the G1/G2 sequences. To clarify the issue, comparative serology of the DOB and SAA isolates should be studied in greater detail. Also, phylogenetic studies of the natural hosts of DOB and SAA from different areas of Europe will be needed, similar to those performed for Peromyscus mice, the hosts for SN and related hantaviruses in North America (Morzunov et al., 1998
).
Finally, it is worth mentioning that DOB and SAA seem to possess different pathogenicity for humans. Although there is no direct evidence to date for this conclusion, none of the existing data contradict such a statement. The most severe HFRS cases (fatality rate among hospitalized patients of 912%) have been reported from the Balkans, where the classic DOB is dominant (Antoniadis et al., 1996 ; Papa et al., 1998
; Avsic-Zupanc et al., 1999
). In contrast, in other parts of Europe, where one might expect SAA to dominate, together with its host rodent species, no fatality associated with DOB or DOB-like virus types has been registered (Lundkvist et al., 1997a
, b
, 1998
; Meisel et al., 1998
). The most prominent example here is the large DOB-associated outbreak in central Russia in 19911992, when 130 HFRS patients were hospitalized and no fatal cases occurred (Lundkvist et al., 1997a
).
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Acknowledgments |
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Footnotes |
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Received 18 January 2000;
accepted 24 March 2000.