Prevalence of Hantaviruses Harbored by Murid Rodents in Northwestern Ukraine and Discovery of a Novel Puumala Virus Strain

Abstract

In Europe, two species of hantaviruses, Puumala orthohantavirus (PUUV) and Dobrava orthohantavirus (DOBV), cause hemorrhagic fever with renal syndrome in humans. The rodent reservoirs for these viruses are common throughout Ukraine, and hence, the goal of this study was to identify the species and strains of hantaviruses circulating in this region. We conducted surveillance of small rodent populations in a rural region in northwestern Ukraine approximately 30 km from Poland. From the 424 small mammals captured, we identified nine species, of which the most abundant were Myodes glareolus, the bank vole (45%); Apodemus flavicollis, the yellow-necked mouse (29%); and Apodemus agrarius, the striped field mouse (14.6%) Using an indirect immunofluorescence assay, 15.7%, 20.5%, and 33.9% of the sera from M. glareolus, A. glareolus, and A. flavicollis were positive for hantaviral antibodies, respectively. Additionally, we detected antibodies to the hantaviral antigen in one Microtus arvalis, one Mus musculus, and one Sorex minutus. We screened the lung tissue for hantaviral RNA using next-generation sequencing and identified PUUV sequences in 25 small mammals, including 23 M. glareolus, 1 M. musculus, and 1 A. flavicollis, but we were unable to detect DOBV sequences in any of our A. agrarius specimens. The percent identity matrix and Bayesian phylogenetic analyses of the S-segment of PUUV from 14 M. glareolus lungs suggest the highest similarity (92-95% nucleotide or 99-100% amino acid) with the Latvian lineage. This new genetic information will contribute to future molecular surveillance of human cases in Ukraine.

Keywords: Puumala orthohantavirus; Ukraine; field survey; next generation sequencing.

Figure 1

Survey location in northwestern Ukraine. The survey was conducted in northwestern Ukraine (top) at two distinct locations (lower left panel), which were ten km from each other: site 1, lines A–D and site 2, lines E–I (lower right panel).

Figure 2

Weight range of rodents and percent hantavirus for A. agrarius, A. flavicollis, and M. glareolus.

Figure 3

Percent identity matrix of PUUV S-segment cRNA of Ukrainian isolates and representative sequences of the eight PUUV lineages. Nucleotide similarity is reported above the black diagonal, and amino acid similarity is reported below the black diagonal. Light grey highlight represents the Ukraine isolates’ amino acid similarity; medium grey highlight represents the Ukraine isolates’ nucleotide similarity; and dark grey highlight represents the LAT lineage similarity with the Ukraine isolates and the DAN sequence, which had 100% amino acid similarity but 86% nucleotide similarity. Each virus isolate is listed by the GenBank accession number, the country the isolate was collected from, the host species (Myodes glareolus (M.g)), the year collected, and its associated PUUV lineage. Ukraine isolates are listed according to the host species, specimen number, and capture line.

Figure 4

Phylogenetic tree of the partial PUUV S-segment. The tree was generated from an alignment to a 765 nt sequence at position 1–765 nt/1–241 aa (genome position is noted from reference sequence NC_005224). Branch support values for each major node are displayed. The Latvian (LAT) lineage is highlighted within the grey box, and the line from which each Ukraine isolate was collected is shown in parenthesis. Each virus isolate is listed by GenBank accession number, the country the isolate was collected from, the host species (Myodes glareolus (M.g)), and the year collected. The eight PUUV lineages are listed to the right of the tree.

Figure 5

Phylogenetic tree of a partial PUUV M-segment. The tree was generated from an alignment of a 633nt sequence at position 2190–2823 nt/717–928 aa (genome position is noted from reference sequence NC_005223). Branch support values for each major node are displayed. Ukraine isolates are highlighted within the grey box, and the line from which each Ukraine isolate was collected is shown in parenthesis. Each virus isolate is listed according to the GenBank accession number, the country the isolate was collected from, the host species (Myodes glareolus (M.g.), Homo sapiens (H.s.), and Clethrionomys rufocanus (C.r.)), the year collected, and whether the isolate was sequenced directly from the host or amplified in another host (cell lines or Syrian hamster).

Figure 6

Phylogenetic tree of a partial PUUV L-segment. The tree was generated from an alignment of a 542 nt sequence at position 2278–2820 nt/748–927 aa (genome position is noted from reference sequence NC_005225). Branch support values for each major node are displayed. Ukraine isolates are highlighted within the grey box, and the line from which each Ukraine isolate was collected is shown in parenthesis. Each virus isolate is listed according to GenBank accession number, the country the isolate was collected from, the host species (Myodes glareolus (M.g)), the year collected, and whether the isolate was sequenced directly from the host or amplified in another host (cell lines or Syrian hamster).