Puumala Virus in Bank Voles, Lithuania

Little is known about the presence of human pathogenic Puumala virus (PUUV) in Lithuania. We detected this virus in bank voles (Myodes glareolus) in a region of this country in which previously PUUV-seropositive humans were identified. Our results are consistent with heterogeneous distributions of PUUV in other countries in Europe.

Little is known about the presence of human pathogenic Puumala virus (PUUV) in Lithuania. We detected this virus in bank voles (Myodes glareolus) in a region of this country in which previously PUUV-seropositive humans were identified. Our results are consistent with heterogeneous distributions of PUUV in other countries in Europe.

P uumala virus (PUUV) (family Bunyaviridae) is an
enveloped hantavirus that contains a single-stranded trisegmented RNA genome of negative polarity (1). PUUV harbored by the bank vole (Myodes glareolus) is the most prevalent human pathogenic hantavirus in Europe (2). A high population density of bank voles can lead to disease clusters and possible outbreaks of nephropathia epidemica, a mild-to-moderate form of hantavirus disease (3).
In contrast to the Fennoscandian Peninsula and parts of central Europe (4,5), little is known about the epidemiology of PUUV in Poland and the Baltic States. Recent investigations confirmed the presence of PUUV in certain parts of Poland (5,6). A molecular study of bank voles in Latvia identified 2 PUUV lineages (Russian and Latvian) (7). In Estonia, serologic and molecular screening provided evidence of the Russian PUUV lineage (8). For Lithuania, a previous serosurvey indicated the presence of PUUVspecific antibodies in humans from 3 counties (online Technical Appendix Figure 1, http://wwwnc.cdc.gov/EID/ article/23/1/16-1400-Techapp1.pdf). However, molecular evidence of PUUV in humans or in voles is lacking (9).
We report a molecular survey of rodent populations in Lithuania at 5 trapping sites, including 2 sites in counties where PUUV-specific antibodies were previously detected in humans (online Technical Appendix Figure 1). A total of 134 bank voles, 72 striped field mice (Apodemus agrarius), and 59 yellow-necked field mice (A. flavicollis) were captured during 2015. Three trapping sites (Juodkrantė, Elektrėnai, and Lukštas) were located in forests at or near a cormorant colony, and 2 trapping sites (Žalgiriai and Rusnė) were located in a wet forest and flooded meadows. All applicable institutional and national guidelines for the care and use of animals were followed.
For PUUV detection, we extracted RNA from bank vole lung tissue samples by using the Qiazol Protocol (QIAGEN, Hilden, Germany) and conducting screening by using a small segment RNA-specific reverse transcription PCR (RT-PCR) and primers Pu342F and Pu1102R (6). We detected PCR products for 5 (LT15/164, LT15/165, LT15/166, LT15/174, and LT15/201) of 45 bank voles from the Lukštas trapping site. All 9 striped field mice and 2 yellow-necked field mice from Lukštas showed negative results for the PUUV RT-PCR.
We generated phylogenetic trees by using MrBayes 3.2.6 software (http://mrbayes.sourceforge.net/download. php) and MEGA6 software (http://www.megasoftware. net/) for complete (1,302 nt; Figure) and partial (465 nt; online Technical Appendix Figure 2) nucleocapsid protein-encoding sequences. Phylogenetic analysis confirmed results of pairwise nucleotide sequence divergence analysis, which indicated clustering of PUUV sequences from Lithuania with sequences from northern Poland (online Technical Appendix Figure 2) and the Jelgava 1 strain from Latvia (Figure). These sequences of the Latvian clade are well separated from the Russian and all other European PUUV clades.
To evaluate a potential association of PUUV with evolutionary lineages of the bank vole, we determined vole cytochrome b gene sequences, deposited them in GenBank In conclusion, we detected PUUV in bank voles at 1 site (Lukštas) in Lithuania (prevalence of 11.1%). This site is located in a region where PUUV-seropositive persons were identified (9) and near the border with Latvia (online Technical Appendix Figure 1). The absence of PUUV in bank voles at 4 other sites might have been caused by the small number of voles tested. However, our results are consistent with heterogeneous distributions of PUUV in other countries (10).
Detection of this novel PUUV strain by using a specific RT-PCR confirms the reliability of this assay for mo-