During 2000–2006, blood samples from ≈600 patients around Sochi who had acute febrile illness with suspected hantavirus infection were tested for hantavirus antibody by an indirect immunofluorescence assay (IFA). Of these, 26 patients were found to be hantavirus antibody–positive. During the HFRS outbreak in the Lipetsk region in the winter of 2001–02, hantavirus infection of >100 patients was serologically confirmed by IFA.

Small mammals were trapped in the Sochi region during the 3 summer and autumn seasons of 2000–2002 and in the Lipetsk region during the winter of 2001–02. Lung tissues of the mammals were screened for the presence of hantavirus antigens by an antigen-capture ELISA as described (22).

Suspensions (10%) of ELISA-antigen–positive lungs were added to Vero E6 cells as described (23). Then, with serum from HFRS patients, the cells were checked for hantavirus antigen by IFA. On the 32nd day of passage, positive cells were detected in a flask containing cells originating from an A. agrarius mouse from the Lipetsk region (Aa1854/Lipetsk-02 strain [Aa/Lipetsk]) and on the 70th day in another flask with cells from an A. ponticus mouse from Sochi (Ap1584/Sochi-01 [Ap/Sochi]).

HFRS patient serum was screened for the presence of hantavirus antibody by IFA as described (24); slides with combined antigens from Vero E6 cells infected with PUUV, DOBV, HTNV, and SEOV were used as substrates. Slides with monovalent antigens of these viruses were used for serotyping hantavirus antibodies.

For confirmation and serotyping, all IFA-positive serum samples were tested by focus-reduction neutralization test (FRNT). The viral stocks, prepared from cell-culture supernatants of infected Vero E6 cells, were titrated with the chemiluminescence focus assay (25) or a protein A–peroxidase conjugate/DAB-NiCl₂ (26). For FRNT, human convalescent serum samples were diluted serially in 2-fold steps, mixed with an equal volume of the respective virus containing 30–100 focus-forming units of this virus, incubated for 1 h at 37°C or overnight at 4°C–6°C, and then used to inoculate the cells. After 6–10 days of incubation, DOBV-N–specific rabbit antiserum or convalescent-phase human serum was used to detect the viral antigen as described above. A reduction in the number of foci of at least 80% was considered as the criterion for virus neutralization.

Hantavirus RNA was extracted from cell-culture supernatant by using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). The standard QIAamp viral RNA mini spin protocol was performed. Amplification and sequencing of the entire S and M segments and partial L segment sequences were performed as described for the DOBV SK/Aa isolate (15).

Sequences were aligned by using ClustalW (27). The reliability of the alignment was checked by using DotPlot (28). The alignment was tested for phylogenetic information by likelihood-mapping analysis (29).

We calculated maximum-likelihood and neighbor-joining phylogenetic trees by using TREE-PUZZLE 5.2 (30) and PAUP* 4.0 Beta 10 software packages (31), respectively. The Tamura-Nei and Hasegawa-Kishino-Yano evolutionary models with and without gamma distribution of rate heterogeneity were used for the tree reconstructions. We used bootstrap analysis with 10,000 replicates to evaluate the statistical significance of the topology for the neighbor-joining trees. Similarity plots and bootscanning (32) were performed by using Stuart Ray’s SimPlot 3.2 with default parameters (33).

Altogether we characterized 126 patients with DOBV-associated HFRS (Table 1) from 2 geographically distant areas of European Russia. Of these, 108 were from the Lipetsk region (during 2001–02) and 18 were from the Sochi region (sporadic cases during 2000–2006).

In terms of clinical markers, a significantly higher proportion of the 18 patients from Sochi than from Lipetsk had abdominal pain, vision disturbance, nausea and vomiting, diarrhea, hyperemia of the face, hemorrhagic sclerae, liver enlargement, oliguria, and anuria; 1 patient died. On the other hand, hypertension developed in a significantly higher proportion of the 108 patients from Lipetsk; 1 patient died (Table 2).

The clinical course of the disease was classified as mild, moderate, or severe, following the standard criteria used in the Russian Federation (34). According to these criteria, 55% of the cases in the Sochi region were classified as severe, 39% as moderate, and 6% as mild. In contrast, only 27% of the cases in the Lipetsk region were classified as severe, 54% as moderate, and 19% as mild (Table 3).

Association of all investigated HFRS cases with DOBV infection was established by IFA serotyping. Antibody titers against DOBV, HTNV, SEOV, and PUUV were determined. In all cases, antibody titers against PUUV were substantially lower. However, for most HFRS cases, IFA could not differentiate between DOBV, HTNV and SEOV; antibody specificities were finally characterized by FRNT.

During 2000–2002, an epizootiologic study was performed to identify and isolate the etiologic agents of the above-described HFRS cases (21,26). A total of ≈600 small animals (8 species) were trapped during 2000–2002 in the Sochi region and ≈300 animals (10 species) in the winter of 2001–2002 in the Lipetsk region. A. ponticus in the Sochi and A. agrarius in the Lipetsk region were the species that most frequently carried hantavirus antigen; 19.6% and 57.6% of animals, respectively, were positive according to ELISA.

To ensure correct classification of the reservoir hosts, tissue samples of the 2 animals that served as the sources of virus isolation were subjected to DNA extraction and sequence analysis. Nucleotide sequence of the mitochondrial DNA fragment containing the control region, D-loop, was determined for both animals and compared with Apodemus spp. D-loop sequences from GenBank. Neighbor-joining phylogenetic analysis demonstrated that the Aa1854/Lipetsk animal was identified correctly as A. agrarius. However, no A. ponticus D-loop nucleotide sequence was available in GenBank for comparison. Nevertheless, phylogenetic analysis of Ap1584/Sochi showed that the obtained D-loop sequence was distinct from all other analyzed sequences (Figure 1). This finding at least confirms that Ap1584/Sochi was not a misidentified member of A. sylvaticus, A. flavicollis, or another morphologically similar Apodemus species.

Complete S- and M-segment and partial L-segment nucleotide sequences of both isolates, Sochi/Ap and Lipetsk/Aa, were determined. The complete S segment of Sochi/Ap was found to be 1,649 nt long. It contained a single open reading frame (ORF; nt 36–1325) that encoded a putative nucleocapsid protein (N) of 429 amino acids. The complete S segment of Lipetsk/Aa was 24 nt longer (1,673 nt) due to a longer 3′ noncoding region. The Sochi/Ap M segment consisted of 3,616 nt that encoded a single ORF (nt 47–3448) of putative 1,133-aa glycoprotein precursor. The first putative start codon at positions 41–43, present in all other DOBV as well as HTNV M segment sequences, was missing, but the next one was located just 6 nt downstream in the same frame (as observed also in SEOV M-segment sequences). The M-segment sequence of Lipetsk/Aa was 3,643 nt long (ORF positions 41–3448; 1,135 aa); the difference in length is again the result of insertions/deletions in the 3′ noncoding region. In addition, a partial L-segment sequence of 541 nt (nt positions 109–649, according to the complete L-segment sequence of DOBV AP/Af; AJ410617) was determined for the Sochi/Ap and Lipetsk/Aa strains.

The sequence similarities between the 2 Russian DOBV isolates were rather low (Table 4). From the existing DOBV cell culture isolates, the Sochi/Ap strain shared the highest similarity with AP/Af19 isolate from Greece. Lipetsk/Aa virus was most similar to the SK/Aa strain. Between other available DOBV sequences, the Sochi/Ap virus S-segment sequence was highly similar to a partial sequence found in an HFRS patient from Krasnodar (P-s1223/Krasnodar-2000) as well as to the sequence Ap-1/Goryachiy Klyuch-2000 amplified from A. ponticus. (Krasnodar and Goryachiy Klyuch are places not far from Sochi.) As expected, the Lipetsk/Aa strain was most similar to Kurkino, another A. agrarius–associated strain from Russia (Table 4).

Sequences of all 3 segments were analyzed by using maximum-likelihood and neighbor-joining phylogenetic methods with various evolutionary models. If not otherwise stated, all the trees for the particular dataset showed similar tree topology and statistical support, but only maximum-likelihood trees with Tamura-Nei evolutionary model are shown (Figure 2).

In the S-segment analysis, the Sochi/Ap sequence clustered with high statistical support with the patient-associated sequence from Krasnodar, Russia (AF442623), and the A. ponticus–associated sequence from the same region (AF442622) and formed a distinct lineage, which we named DOBV-Ap (Figure 2, panel A). Whereas DOBV-Ap and DOBV-Af share a common ancestor in the S-segment phylogenetic tree, in M and L segment analysis Sochi/Ap formed an outgroup from all other DOBV sequences and did not directly cluster with DOBV-Af sequences (Figure 2, panels B, C). Besides putative genetic reassortment processes, incomplete and unequal sequence datasets (fewer sequences for M- and L-segment datasets are available) could be the reason for these conflicting results. More sequence data are necessary to confirm these findings.

Lipetsk/Aa sequences unambiguously clustered within the DOBV-Aa lineage in the analyses of all 3 segments (Figure 2, panels A–C). As expected, it formed a well-supported monophyletic group with DOBV-Aa strains from Kurkino, Russia. In M and L segment analysis, for which the number of available sequences is rather limited, DOBV-Aa strains from Slovakia were most closely related, although the statistical support for this clustering was below the cutoff value of 70%.

We observed slight differences between maximum-likelihood and neighbor-joining trees in S segment analysis in which Saaremaa strains clustered with DOBV-Af and DOBV-Ap lineages in maximum-likelihood analyses but with DOBV-Aa in neighbor-joining analyses. However, in both instances, the statistical support was below the cutoff limit.

Moreover, we performed recombination analyses by using similarity plots and bootscanning. However, reliable results confirming homologous recombination events that affected Sochi/Ap or Lipetsk/Aa sequences could not be obtained (data not shown).

The availability of the 2 novel DOBV strains as cell culture isolates enabled us to use them in chemiluminescence FRNT (c-FRNT) and characterize serum from selected patients also by neutralizing antibody titers. The DOBV prototype strain (Slo/Af; ref.1) and both novel isolates were used in c-FRNT to serotype all 18 patients from the Sochi region (27 serum samples; consecutive samples available for 8 patients). For 15 of these 18 patients, at least 1 serum sample showed the highest neutralizing antibody titer against the local Sochi virus. However, >4-fold differences in titers (considered to be significant) were found in serum samples of only 10 patients. For 8 patients, such significant difference was not found (5 patients with 2-fold difference, 3 with no difference). Table 5 shows examples of convalescent-phase serum samples representing these different groups.

From patients in the Lipetsk region, 6 serum samples were characterized by c-FRNT (Table 5). To verify whether some differences can also be found between 2 DOBV-Aa isolates, DOBV strain SK/Aa isolated in Slovakia, Central Europe (15), was included in the analysis. Four samples exhibited the highest titer against Lipetsk/Aa when compared with Slo/Af and Sochi/Ap, although in only 2 was the difference 4-fold. One sample reacted equally with Lipetsk/Aa and Slo/Af, and 1 showed even higher reactivity against the Slo/Af strain. When we directly compared Lipetsk/Aa and SK/Aa, equal proportions of serum showed equal titers and 2-fold or 4-fold higher titers against Lipetsk/Aa than against SK/Aa.

In addition, 2 DOBV convalescent-phase serum samples from Slovakia, previously serotyped as anti–DOBV-Af reactive (15), were analyzed to determine whether Sochi/Ap, Lipetsk/Aa, and Slo/Af could be distinguished by these samples. In both instances, Slo/Af virus was neutralized best, although in only 1 case was the difference in neutralizing antibody titer 4-fold (Table 5).