Puumala hantavirus (PUUV) is a zoonotic virus that in humans causes nephropathia epidemica (NE) in humans, a mild form of haemorrhagic fever with renal syndrome. An average of 10 000 cases are reported annually in Europe, many of which occur in Fennoscandia. The incidence of NE is connected to the distribution and population density of the the bank vole (Myodes glareolus), the main virus host. In Fennoscandia, high incidences of NE occur at 3-4 year intervals due to the characteristic population cycles of this woodland rodent. This study aimed to gain a better understanding of PUUV microevolution by examining genetic features of the virus in several bank vole populations of Finland and Latvia. Genetic variation in PUUV circulating in a bank vole population at Konnevesi in Central Finland was examined and monitored over five-years throughout a complete bank vole cycle, including two peak-phases in 2005 and 2008 and two population declines in 2006 and 2009 (i.e., viral bottlenecks). Altogether, 1369 bank voles were captured and 26.3% were detected PUUV-infected. Partial sequences of the three viral genome segments (Small, Medium and Large) were inspected from 365 PUUV genomes. Genetic diversity was 6.2% for the S segment, 4.8% for the M segment, and a surprisingly high 10.1% for the L segment. Each genome segment had accumulated mutations as a separate gene pool. The majority of nucleotide substitutions were synonymous and most of the deduced amino acid substitutions were conservative, suggesting a strong stabilizing selection operating at the protein level. Genetic markers found along the genome segments allowed for the recognition of two genogroups of PUUV co-circulating in the host population. Even though, one of the genogroups presented higher genetic diversity, no signs of completion were observed between them. Nearly 80% the variants exhibited a transient existence, and frequently occurring variants were integrated by most abundant segment genotypes suggesting a viral mutational robustness. A substantial portion (19.1%) of genomes appeared to be reassorted, with S and M typically being exchanged. Reassorted variants did not outcompete parental variants and were commonly transient. Reassortment was seasonal, occurring more frequently in autumn when recent infection risk increases. An imperceptible intra-genogroup reassortment could contribute to the steady state of the viral population, counteracting the effects of Muller s ratchet. Co-circulation and interaction of two distinct PUUV lineages (Finnish and North-Scandinavian) was monitored in a bank vole population at Pallasjärvi in Northern Finland. To date, seven genetic lineages have been detected, all of which exhibit geographic structure within the host distribution. Here, we present new evidence of two lineages circulating in the same bank vole phylogroup (Ural clade). Genetic diversity within each PUUV lineage was modest (up to 1.7%) and most substitutions were synonymous. However, genetic differences between the two lineages were as high as 18.9%. Phylogenetic analyses revealed that these distinct lineages naturally reassort with a frequency comparable to that genogroups circulating at Konnevesi, i.e., 32%. In contrast to Konnevesi, only M segment was exchanged between PUUV lineages at Pallasjärvi.

Two distinct PUUV lineages were also found to co-circulate in Latvia. One (Russian) has been previously described and the other awaits formal description. The novel Latvian lineage is considerably divergent from other PUUV lineages and several amino acid markers made it easily distinguishable. Phylogenetic analysis suggested an independent evolutionary history for the segments of Latvian lineage. Similar to Pallasjärvi, both Russian and Latvian lineages were found in a single bank vole phylogroup (Carpathian clade), confirming earlier observations that PUUV lineages are not limited to a single host phylogroup.