2. MATERIALS AND METHODS

Between 1994 and 2000, rodents were trapped alive by using Swedish bridge metal traps (Figure 6) in various areas of Slovakia. The trapping sites were selected on the basis of their proximity to reported human cases of HFRS. A total of 50 or 100 traps were set in the late afternoon, in a standardised, systematic manner on the ground, and collected the next morning. The traps were placed mostly at the edges of fields and forests. After collection and prior to dissection the weight, sex, maturity and the exact trapping sites were recorded for each animal. Blood samples were obtained from the Sinus orbitalis of deeply anaesthetised rodents. The animals were then sacrificed and dissected for lung, liver and spleen tissues. Serum was separated by centrifugation at 240 g for 10 minutes, and frozen at -20ºC. Tissue samples were stored at -70°C until processed further.

	Figure 6: Trapping of small rodents using Swedish bridge metal traps.

The rodent sera were tested by enzyme-linked immunosorbent assay (ELISA) for the presence of hantavirus antibodies. For the detection of hantavirus-specific mouse IgG antibodies, an antiglobulin ELISA was performed. Briefly, microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 100 μl/well of recombinant nucleocapsid antigen. Yeast expressed complete N proteins of HTNV strain Fojnica, PUUV strain Vranica-Hällnäs or DOBV strain Slovenia (A. Razanskiene, unpublished data) were diluted in 0.05 M sodium carbonate buffer, pH 9.0, to final concentration of 0.5 μg/ml. Washing of the plates was undertaken five times between each step. After post-coating with blocking buffer (3% BSA in PBS, 100 μl/well) at room temperature for one hour, rodent serum samples diluted 1:200 in PBS with 1% BSA and 0.05% Tween 20 were incubated for 1 hr at 37°C followed by incubation for 1 hr at 37°C with peroxidase-labelled anti-mouse antibody (DAKO Diagnostica, Hamburg, Germany) diluted 1:1000 in PBS with 1% BSA and 0.05% Tween 20. Staining was performed with ready to use TMB substrate (Seramun, Dolgenbrodt, Germany). The reaction was stopped by addition of 1 M sulphuric acid and optical densities of the reaction products were measured at 450/620 nm. Cut-off values were calculated as the mean optical density value plus 3 standard deviations for values of negative controls.

Lung tissues samples of M. arvalis were screened by immunoblotting for TULV nucleocapsid antigen. Rodent lung tissue samples (2 to 3 mm³) were homogenised by sonification in 500 ml of Laemmli loading buffer; after denaturation, 15 ml of the homogenate was loaded on a sodium dodecyl sulphate (SDS)–12% polyacrylamide gel and separated by electrophoresis. After transfer of the proteins, the membranes were preadsorbed in 4% non-fat dry milk and subsequently incubated with rabbit polyclonal antibodies (raised against TULV/Malacky recombinant N antigen expressed as a His-tagged protein diluted in PBS–0.05% Tween 20). The indicator antibody was a swine anti-rabbit horseradish peroxidase conjugate used at 1:1000 dilution at 37°C for 1 h. Membranes were washed in PBS–0.05% Tween 20, and the bands were stained with o-phenylenediamine dihydrochloride.

The RNA of hantavirus antibody/antigen-positive rodents was extracted from lung or liver tissues using the TRIZOL Reagent (GibcoBRL, Invitrogen, Karlsruhe, Germany). This procedure is based on the acid guanidine isothiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987). Small pieces (2-3 mm³) of tissues were homogenised in 1 ml of TRIZOL using rotor-stator homogeniser. After 10 min of incubation at room temperature, 200 μl of chloroform was added; the mixture was then mixed by pulse-vortexing for 15 sec and incubated at room temperature for 3 min. After centrifugation at 12000 g for 15 min at 4°C, the aqueous phase was transferred to a fresh tube. 500 μl of cold isopropyl alcohol and 20 μl of 4M Lithium Chloride was added and the mixture was incubated at room temperature for 10 min, followed by centrifugation at 12000 g for 10 min at 4°C. The supernatant was removed and the pellet was subsequently washed with 1 ml of 75% and 100% ethanol, using centrifugation at 7500 g for 3 min at 4°C after every step. The pellet was then air-dried and reconstituted in 20 μl of DEPC water.

For special purposes such as amplification of overlapping fragments of the whole M segment, the QIAgen RNeasy Mini kit (Qiagen, Hilden, Germany) was used. The small pieces of tissues were homogenised in 600 μl of RLT Buffer and the standard RNeasy Mini Protocol for isolation of total RNA from animal tissues was followed according to manufacturer’s instructions. RNA was eluted to 30 μl of RNase-free water.

For isolation of hantaviral RNA from cell-culture supernatant, the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) was used. 280 μl of supernatant was added to 1120 μl of Buffer AVL/Carrier RNA and standard QIAamp viral RNA mini spin protocol was performed. RNA was eluted to 60 μl of Buffer AVE.

Total genomic DNA was extracted from small (4 mm³) pieces of liver or lung tissue using DNeasy Tissue Kit (Qiagen). The tissues were digested for 3 hours or overnight at 55°C in a total volume of 200 μl, including 20 μl proteinase K and 180 μl ATL Buffer, and were subjected to standard DNeasy protocol for animal tissues according to manufacturer’s instructions. DNA was eluted to 200 μl of AW2 Buffer from the kit. 5 μl of sample were loaded on gel together with High DNA Mass Ladder to determine approximate yield.

Total RNA was reverse transcribed with a single genus-specific primer complementary to the 3’ and 5’ terminal sequences of all three RNA segments (RTS: 5’ TAGTAGTAGACT 3’) using 200 units of Rnase H- reverse transcriptase Superscript II in RT-buffer (50 mM Tris/HCl pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM DTT; GIBCO, BRL, Gaithersburg, USA) with 500 μM of each dNTP according to manufacturer’s instructions. One fourth (5 μl) was then used for the 1^st PCR reaction.

A standard nested set of generic primers specific for the hantavirus S RNA genome segment was used for the initial screening. Three different nested PCR systems were used for the detection of PUUV, TULV and DOBV. For primer sequences, see the Table 4. The thermal cycling conditions of both 1^st PCRs (40 cycles) and nested PCRs (25 cycles) were: 94°C for 60 sec, 52°C for 60 sec and 72°C for 60 sec, followed by one cycle of final extension for 6 min at 72°C. PCR mixture (50 μl) contained 1.5 mM MgCl₂, 200μM of each dNTP , 15 pmoles of each primer pair and 1.5 U of AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA, USA).

Amplification of the whole S segment was undertaken by use of the Robust RT PCR System (Finnzymes, Espoo, Finland), following the application protocol of the manufacturers. The single genus-specific primer RT-DOB (5´-ttctgcag TAG TAG TAK RCT CCC TAA ARA G) was used. After 2 min of incubation of 20 pmoles of the primer with 5 μl of RNA at 68°C, the Robust RT PCR mixture was added and after 1 h of incubation at 50°C, 35 cycles of 94°C for 30 sec, 58°C for 45 sec and 72°C for 2 min were performed, followed by one cycle of final extension for 7 min at 72°C.

For sequencing of the complete M segment, the nested RT PCR amplifying overlapping M segment fragments was carried out with the Robust RT PCR System (Finnzymes, Espoo, Finland). Six sets of nested primer sequences were designed from published DOBV entire M segment sequences to generate overlapping cDNA fragments for the entire M genome RNA segment (Table 4). Cycling conditions were similar to those for amplification of the whole S segment, only annealing temperature (50-58°C) and elongation time (1-2 min) were always modified according to melting temperature of primers and length of the amplified fragment.

To obtain the partial L segment sequence, single primer pair DOBL89F (5’-TCA YTG ACA GCA GTR GAR TG) and DOBL669R (5’-AAC ATK GCY TCY ARA GCA GC) (Table 4) amplifying 580 nt long L segment fragment (541 nt when excluding the primer sequences) was designed from published DOBV entire L segment sequences.

For amplifying rodent genetic markers 12S rRNA and D-loop, PCR was performed. For primer sequences, see Table 4. PCR mixture (50 μl) contained 1.5 mM MgCl2, 200μM of each dNTP , 50 pmoles of each of primer pair and 1.5 U of AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA, USA). For 12S rRNA, the same cycling conditions as in hantavirus initial screening RT-PCR were used. For D-loop, the annealing temperature of 66°C and 90 sec of elongation were used. 35 cycles were performed.

PCR products were analysed and visualised by electrophoresis in 1.0% agarose gel after 20 min of staining in ethidium bromide (1 μg/ml).

The amplified products were cloned into pCR 2.1 - TOPO vector (TA-Cloning kit, Invitrogen, Leek, Netherlands). At least three recombinant plasmids were sequenced in both directions and the consensus sequence from obtained sequences was determined. Dideoxy sequencing was performed on a LICOR sequencer using the Autoread Kit (Pharmacia-Biotech, Freiburg, Germany) as described by the manufacturer.

Table 4: List of PCR primers used for amplification of hantaviral RNA and rodent DNA

The positions of primer binding sites for hantavirus specific PCR refer to the DOBV/Esl862/Aa/97 strain sequences (S segment: Aj269550, M segment: AY168578), for rodent genetics PCR to the Mus musculus strain C57BL/6J complete mtDNA sequence. Small letters in primer sequences indicate 5’ tails of heterologous sequence integrated for cloning or sequencing purposes.
R=A+G, Y=C+T, M=A+C; S=G+C; K=G+T; W=A+T; I=inozine

The obtained overlapping nucleic acid sequences were combined for analysis and edited with the aid of the SEQMAN program from the Lasergene software package (DNASTAR, Madison, Wis., USA). The sequence data were further analysed using the BioEdit software package (Hall, 1999). Sequences were aligned using CLUSTALW (Thompson et al., 1994) with default parameters. The alignments were then manually checked and corrected where necessary. When aligning the coding sequences, the sequences were first aligned on amino acid level and then reverse-translated to nucleotide sequences using DAMBE software (Xia and Xie, 2001). The reliability of the alignment was checked using DotPlot analysis implemented in BioEdit software package. The alignment was tested for phylogenetic information by Likelihood Mapping analysis (Strimmer and von Haeseler, 1997).

To reconstruct maximum likelihood (ML) phylogenetic trees, we applied quartet puzzling using the TREE-PUZZLE package (Strimmer and von Haeseler, 1997; Schmidt et al ., 2002) . As evolutionary model for the reconstructions we used the Tamura-Nei model, missing parameters were reconstructed from the datasets. The values at the tree branches are the resulting PUZZLE support values, if not otherwise stated. Resulting evolutionary trees were then visualised using TreeView v.1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/ treeview.html)

Bootscan analysis (Salminen et al., 1995) was performed with Stuart Ray’s SimPlot 2.5 (Lole et al., 1999) in combination with the PHYLIP package (Felsenstein, 1993). For the analysis shown we usually used a window size of 200 nucleotides (nt), a step size of 10 nt, and 500 bootstrap samples per window. As underlying phylogeny reconstruction algorithm we used Neighbor-Joining (NEIGHBOR, DNADIST) under the Kimura model with Ts/Tv ratio 2.0 for the distances. For subsequent phylogenetic analysis the alignments were split according to the bootscan diagrams at cross points of a 65% threshold.

For the comparisons, sequence data were obtained from the sequence databases EMBL and Genbank. The list of all hantaviral sequences used in this work together with abbreviated names and accession numbers is shown in Table 5.

Table 5: List of hantavirus strains and their abbreviated names used in sequence analysis

* For hantavirus species abbreviations see Table 2.
n.a., not available
d.s., direct submission

DOBV RT-PCR-positive lung and liver samples from two naturally infected, seropositive A. agrarius trapped in Rozhanovce, Eastern Slovakia, were used for virus isolation attempts. The samples were processed as 10% tissue suspensions in Dulbecco’s medium supplemented with 0.2% bovine serum albumin (BSA). The tissues were triturated in a closed mechanical blender FastPrep Instrument (BIO 101 Systems, Carlsbad, CA, USA). Triturated tissues were briefly centrifuged at low speed to remove larger tissue fragments and inoculated (0.4 ml/flask) onto cultures of confluent Vero E6 cells in 25 cm² flasks (three for each sample). Virus was then allowed to adsorb at 37°C. The cell culture medium (MEM plus 10% fetal calf serum, L-glutamat, penicillin and streptomycin) was changed for the first time after 90 min and then weekly. At 2-3 week intervals, cells, detached by trypsin treatment, were passaged into new culture flask with the addition of the same amount of fresh uninfected cells according to a recently described protocol (Nemirov et al., 1999). While suspended, several slides were prepared and examined for characteristic hantavirus antigen expression following immunofluorescence assay (IFA) techniques.The experiments were performed under biosafety level 3 containment conditions in the Institute of Virology, Charité School of Medicine.

Cells were prepared on Teflon-coated slides with 12 circular areas (“spots”). After detaching with trypsin treatment, the cells were suspended in cell culture medium and washed twice. Washed cells from every flask were resuspended in 5 ml of cell culture medium and 20 μl of the suspension was deposited on each spot of the slides cleaned with 70% ethanol. The slides were put in a moist chamber and incubated in at 37°C and 5% CO₂ overnight. The slides were washed two times in PBS and once in bidistillated water and anhydrous acetone and then fixed in anhydrous acetone at 4°C for 10 minutes, air-dried and used or stored at -20°C till next day.

Slides were first washed in PBS and air-dried. 20 μl of DOBV convalescent antisera pool diluted 1:200 in PBS were deposited on every spot and incubated at room temperature for 30 min in a moist chamber. The slides were washed, with 3 changes of PBS each for 5 min, and air-dried. Fluorescein isothiocyanate (FITC)- conjugated anti-human immunoglobulin, diluted 1:40 in PBS, was added, 20 μl to each spot, and the slides were returned to the moist chamber at room temperature for 30 min. To increase the easy of viewing, Evan’s blue at dilution 1:1500 was added to the conjugate. The slides were again washed, with three changes of PBS each for 5 min, and air-dried. The slides were mounted with the mounting medium (Progen) under cover slips and examined for characteristic cytoplasmic pattern in a fluorescence microscope.

For the antigenic characterisation of the new isolate, a panel of eight monoclonal antibodies (mAbs) directed against N protein of different hantaviruses was used. The reactivity of the mAbs E5/G6, Eco2, C16D11, C24B4, B5D9 (Yoshimatsu et al., 1996; Zöller et al., 1993) and 2E2 (gift of J. Groen) directed against HTNV, R31 against SEOV (Progen Biotechnik, GmbH Heidelberg, Germany) and 5C2/E10 against ANDV (Immunological and Biochemical Testsystems GmbH, Reutlingen, Germany) was analysed in parallel using IFA slides with Vero E6 cells infected with DOBV strains SK/Aa and Slo/Af. After 10 days of incubation the infected cells were mixed with the uninfected cells at a ratio of 1:2. The slides were then stained as described above but FITC-conjugated anti-mouse immunoglobulin was used instead of anti-human conjugate. A positive reaction was stated to be specific if at least one third of the cells showed the fluorescence signal.

The viral stock, prepared from cell-culture supernatant of infected Vero E6 cells, was titrated using the chemiluminiscent focus assay as described by Heider et al. (2001). From the six-well plates with nearly confluent monolayer of Vero E6 cells, the cell-culture growth medium was discarded and the cells were inoculated with 0.2 ml/well of tenfold dilutions of viral stock in Hanks’ balanced salt solution (HBSS; GibcoBRL) supplemented with 2% HEPES (GibcoBRL), 2% fetal calf serum (FCS) and antibiotics mixture PSN (GibcoBRL). After the incubation for 1 h at 37°C in a humidified 5% CO₂ atmosphere, the cells were overlaid with 2.5 ml/well of a pre-warmed (42°C) mixture of one part 1% agarose in water and one part overlay medium (2x Eagle’s basal medium with Earle’s salt and L-glutamine, GibcoBRL) and the plates were then incubated for 10 days under the conditions described above.

Gently injecting 2–3 ml of methanol under the agarose layer and turning the plate upside down then removed the agarose. The cells were fixed with 2 ml/well of methanol for 8 min, allowed to dry and gently washed three times with washing buffer (PBS supplemented with 0.15% Tween 20, Boehringer Ingelheim, Heidelberg). 1 ml/well of anti-DOBV human convalescent serum diluted 1:1000 in washing buffer was added and the plates were incubated for 1 h at 37°C in a humidified 5% CO₂ atmosphere and then five times washed with washing buffer. 1 ml/well of goat anti-human IgG conjugated with horseradish peroxidase and diluted 1:1000 was added and after incubation for 1 h at 37°C in a humidified 5% CO₂ atmosphere, the cells were five times washed. Immediately after adding 0.5 ml/well of the chemiluminescence substrate (Super Signal West Dura Extended Duration Substrate, Pierce, Rockford, USA) diluted 1:5 in water, the plates were evaluated in DIANA Chemiluminescence System (Raytest, Straubenhardt, Germany). On a light screen, the foci of infected cells were enumerated as black-coloured dots.

c-FRNT was performed basically following the protocol described above for the virus titration (Heider et al., 2001). Human convalescent sera were first diluted serially, mixed with an equal volume containing 30–80 focus forming units (FFU) of the respective virus and incubated for 1 h at 37°C prior inoculation the cells. After 10 days of incubation, DOBV-specific rabbit antiserum and goat anti-rabbit IgG (Bio-Rad, Hercules, CA, USA) was used to detect the viral antigen in infected cells. An at least 80% reduction in the number of foci was considered as the criterion for virus neutralisation.