Microevolution of Puumala hantavirus in its host, the bank vole (Myodes glareolus)

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.Puumala-hantaviruksen mikroevoluutio Hantaviruksiin kuuluva Puumala-virus on myyräkuumeen aiheuttaja. Euroopassa todetaan keskimäärin vuosittain yli 10 000 myyräkuumetapausta, ja EU:ssa suurimmat potilasmäärät ovat pohjoisessa - yksin Suomesta tulee yli 70% EU:n tapauksista. Puumala-viruksen isäntä ja levittäjä luonnossa on metsämyyrä, ja myyräkuumetapausten määrä riippuu olennaisesti metsämyyrien runsaudenvaihteluista, jotka meillä Pohjois-Euroopassa esiintyvät 3-4 vuoden jaksoissa. Suomessa on pitkät perinteet jo yli 30 vuoden ajalta myyräkuumeen ja Puumala-viruksen tutkimuksessa. Nyt valmistuneessa väitöskirjassa tutkittiin Puumala-viruksen mikroevoluutiota molekyylibiologisin menetelmin, t.s. maantieteellistä geneettistä vaihtelua ja miten viruksen geneettinen vaihtelu paikallisesti muuttuu isännän voimakkaiden kannanvaihteluiden myötä. Tutkimusaineistot kerättiin Keski- ja Pohjois-Suomesta sekä Latviasta. Viruksen geneettisen vaihtelun pitkäaikaisseuranta tehtiin Konnevedellä vuosina 2005 - 2009, myyrähuipusta myyräromahduksen yli seuraavaan myyrähuippuun ja laskuun. Tutkituista 1369 metsämyyrästä 365 (26.7%) oli viruksen kantajia. Puumala-viruksen genomi muodostuu 3 erillisestä osasta, segmentistä, joiden lyhenteet kookkaimmasta pienimpään ovat L, M ja S. Sekvensoinnin perusteella todettiin, että segmenttien geneettinen diversiteetti oli 10,1% (L), 4,8% (M) and 6,2% (S). L-segmentin diversiteetti oli yllättävän suuri. Kussakin segmentissä mutaatiot olivat kertyneet omana erillisenä kokonaisuutenaan. Suurin osa nukleotidien muutoksista oli hiljaisia , eivätkä ne vaikuttaneet aminohappojärjestykseen. Toisaalta suurin osa tapahtuneista aminohappomuutoksista oli konservatiivisia (aminohappo muuttuu toiseen samantyyppiseen), mikä todistaa voimakkaasta vakauttavasta valinnasta proteiinitasolla. Geneettiset tunnusmerkit auttoivat tunnistamaan kaksi erilaista Puumala-viruskantaa Konneveden myyräpopulaatiossa. Vaikka toisen viruskannan geneettinen diversiteetti oli suurempi, ei mitään syrjäytymistä toisenkaan osalta havaittu. Valtaosa havaituista virustyypeistä (-mutaatioista) oli lyhytaikaisia, ja toisaalta yleisimmät virustyypit löytyivät säännöllisesti tutkimuksen kuluessa kaikista genomin segmenteistä. Vaikka Puumala-virus on RNA-virus, joilla esiintyy runsaasti mutaatiota, voimakas valinta kuitenkin suosi samojen virustyyppien säilymistä. Koska hantavirusten genomi muodostuu 3 segmentistä (osasta), voi rekombinaatiota tapahtua myös virusten lisääntyessä ja segmenttien ryhmittyessä jälkeläistössä uudella tavalla (reassortment). Melkoinen määrä (19,1%) Konneveden metsämyyrien Puumala-viruksista oli syntynyt segmenttien uudelleenryhmittymisen myötä. S ja M olivat selvästi useimmiten vaihtuneet osat. Uudet kombinaatiot eivät kuitenkaan syrjäyttäneet vanhempiaan viruspopulaatiosta, vaan olivat yleensä lyhytaikaisia, vain kerran tutkimusjakson aikana tavattavia. Segmenttien uudelleen ryhmittyminen oli yleisempää syksyllä kuin keväällä, ilmeisesti koska syksyllä on enemmän uusia tuoreita infektioita. Huolimatta sekä pistemutaatioiden ja segmenttien uudeelleenjakaantumisten suurestä määrästä, nämä uudet virusmuodot olivat lyhytaikaisia, ja viruspopulaation rakenne pysyi tutkimuksen aikana melko vakaana, mikä osoittaa voimakkaan valinnan merkitystä uusien virusmuotojen karsijana. Euroopassa esiintyy 7 maantieteellisesti selvästi erilaista Puumala-virus-linjaa. Länsi-Lapissa kohtaavat suomalais-itäinen ja pohjois-skandinaavinen viruslinja. Kohtaamisvyöhykkeellä Pallasjärvellä tutkittiin ensi kertaa kahden Puumala-viruksen maantieteellisen linjan esiintyymistä samassa metsämyyräpopulaatiossa. Tutkimuksella on mielenkiintoa virusevoluution kannalta, koska on pohdittu sitä mahdollisuutta, että tällaiset kontaktivyöhykkeet voivat olla alkuna uusien virusten synnylle, tai että tällaiset hybridivirukset voisivat olla ärhäkkäämpiä kuin emolinjat. Geneettinen vaihtelu emolinjoissa Pallasjärvellä oli pientä (alle 1,7%) ja useimmat mutaatiot olivat hiljaisia. Toisaalta viruslinjojen välinen ero oli suuri (jopa 18,9%). Fylogeneettiset analyysit osoittivat, että nämä kaksi geneettisesti kaukaista viruslinjaa yhdistyvät uusiksi kombinaatioiksi yhtä usein kuin Konneveden paljon vähemmän erilaistuneet linjat. Myös Pallasjärvellä segmenttien uudelleenrymittymisessä oli selvä suuntaus: vain M-segmentti siirtyi viruslinjasta toiseen. Puumala-viruksen maantieteellistä vaihtelua tutkittaessa löydettiin Latviasta aivan uusi Puumala-linja. Geneettiset analyysit osoittivat, että tämän uuden viruslinjan eri segmentit olivat evoluutionsa kuluessa kehittyneet itsenäisesti, toisistaan riippumatta. Myös Puumala-viruksen isännällä, metsämyyrällä, esiintyy erilaisia geneettisiä linjoja Euroopassa, ja on pohdittu, esiintyisivätkö tietyt isäntä- ja viruslinjat yhdessä, eli olisivat kehittyneet yhdessä. Tämän tutkimuksen tulokset eivät tue tätä koevoluutioajatusta metsämyyrä- ja Puumala-viruslinjat eivät ole maantieteellisesti yhteneväisiä. Lisäksi todettiin, että sekä Pallaksella että Latviassa kaksi eri viruslinjaa voi esiintyä yhdessä ja samassa isäntälinjassa. Nämä tulokset korostavat, että metsämyyrän ja Puumala-viruksen nykyisten geneettisten linjojen maantieteelliset levinneisyydet eivät ole vain viimeisen jääkauden ja sen jälkeisen leviämisen seurausta, vaan juontuvat paljon pitemmältä ajalta

Analysis of Puumala hantavirus in a bank vole population in northern Finland: evidence for co-circulation of two genetic lineages and frequent reassortment between strains

Razzauti M, Plyusnina A, Sironen T, Henttonen H, Plyusnin A. Analysis of Puumala hantavirus in a bank vole population in northern Finland: evidence for co-circulation of two genetic lineages and frequent reassortment between strains. J Gen Virol. 2009 Aug;90(Pt 8):1923-31."In this study, for the first time, two distinct genetic lineages of Puumala virus (PUUV) were found within a small sampling area and within a single host genetic lineage (Ural mtDNA) at Pallasjarvi, northern Finland. Lung tissue samples of 171 bank voles (Myodes glareolus) trapped in September 1998 were screened for the presence of PUUV nucleocapsid antigen and 25 were found to be positive. Partial sequences of the PUUV small (S), medium (M) and large (L) genome segments were recovered from these samples using RT-PCR. Phylogenetic analysis revealed two genetic groups of PUUV sequences that belonged to the Finnish and north Scandinavian lineages. This presented a unique opportunity to study inter-lineage reassortment in PUUV; indeed, 32% of the studied bank voles appeared to carry reassortant virus genomes. Thus, the frequency of inter-lineage reassortment in PUUV was comparable to that of intra-lineage reassortment observed previously (Razzauti, M., Plyusnina, A., Henttonen, H. & Plyusnin, A. (2008). J Gen Virol 89, 1649-1660). Of six possible reassortant S/M/L combinations, only two were found at Pallasjarvi and, notably, in all reassortants, both S and L segments originated from the same genetic lineage, suggesting a non-random pattern for the reassortment. These findings are discussed in connection to PUUV evolution in Fermoscandia.""In this study, for the first time, two distinct genetic lineages of Puumala virus (PUUV) were found within a small sampling area and within a single host genetic lineage (Ural mtDNA) at Pallasjarvi, northern Finland. Lung tissue samples of 171 bank voles (Myodes glareolus) trapped in September 1998 were screened for the presence of PUUV nucleocapsid antigen and 25 were found to be positive. Partial sequences of the PUUV small (S), medium (M) and large (L) genome segments were recovered from these samples using RT-PCR. Phylogenetic analysis revealed two genetic groups of PUUV sequences that belonged to the Finnish and north Scandinavian lineages. This presented a unique opportunity to study inter-lineage reassortment in PUUV; indeed, 32% of the studied bank voles appeared to carry reassortant virus genomes. Thus, the frequency of inter-lineage reassortment in PUUV was comparable to that of intra-lineage reassortment observed previously (Razzauti, M., Plyusnina, A., Henttonen, H. & Plyusnin, A. (2008). J Gen Virol 89, 1649-1660). Of six possible reassortant S/M/L combinations, only two were found at Pallasjarvi and, notably, in all reassortants, both S and L segments originated from the same genetic lineage, suggesting a non-random pattern for the reassortment. These findings are discussed in connection to PUUV evolution in Fermoscandia.""In this study, for the first time, two distinct genetic lineages of Puumala virus (PUUV) were found within a small sampling area and within a single host genetic lineage (Ural mtDNA) at Pallasjarvi, northern Finland. Lung tissue samples of 171 bank voles (Myodes glareolus) trapped in September 1998 were screened for the presence of PUUV nucleocapsid antigen and 25 were found to be positive. Partial sequences of the PUUV small (S), medium (M) and large (L) genome segments were recovered from these samples using RT-PCR. Phylogenetic analysis revealed two genetic groups of PUUV sequences that belonged to the Finnish and north Scandinavian lineages. This presented a unique opportunity to study inter-lineage reassortment in PUUV; indeed, 32% of the studied bank voles appeared to carry reassortant virus genomes. Thus, the frequency of inter-lineage reassortment in PUUV was comparable to that of intra-lineage reassortment observed previously (Razzauti, M., Plyusnina, A., Henttonen, H. & Plyusnin, A. (2008). J Gen Virol 89, 1649-1660). Of six possible reassortant S/M/L combinations, only two were found at Pallasjarvi and, notably, in all reassortants, both S and L segments originated from the same genetic lineage, suggesting a non-random pattern for the reassortment. These findings are discussed in connection to PUUV evolution in Fermoscandia."Peer reviewe

Microevolution of Puumala hantavirus during a Complete Population Cycle of Its Host, the Bank Vole (Myodes glareolus)

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Phylogeography of Puumala orthohantavirus in Europe

Puumala virus is an RNA virus hosted by the bank vole (Myodes glareolus) and is today present in most European countries. Whilst it is generally accepted that hantaviruses have been tightly co-evolving with their hosts, Puumala virus (PUUV) evolutionary history is still controversial and so far has not been studied at the whole European level. This study attempts to reconstruct the phylogeographical spread of modern PUUV throughout Europe during the last postglacial period in the light of an upgraded dataset of complete PUUV small (S) segment sequences and by using most recent computational approaches. Taking advantage of the knowledge on the past migrations of its host, we identified at least three potential independent dispersal routes of PUUV during postglacial recolonization of Europe by the bank vole. From the Alpe-Adrian region (Balkan, Austria, and Hungary) to Western European countries (Germany, France, Belgium, and Netherland), and South Scandinavia. From the vicinity of Carpathian Mountains to the Baltic countries and to Poland, Russia, and Finland. The dissemination towards Denmark and North Scandinavia is more hypothetical and probably involved several independent streams from south and north Fennoscandia

Phylogeography of Puumala orthohantavirus in Europe

Puumala virus is an RNA virus hosted by the bank vole (Myodes glareolus) and is today present in most European countries. Whilst it is generally accepted that hantaviruses have been tightly co-evolving with their hosts, Puumala virus (PUUV) evolutionary history is still controversial and so far has not been studied at the whole European level. This study attempts to reconstruct the phylogeographical spread of modern PUUV throughout Europe during the last postglacial period in the light of an upgraded dataset of complete PUUV small (S) segment sequences and by using most recent computational approaches. Taking advantage of the knowledge on the past migrations of its host, we identified at least three potential independent dispersal routes of PUUV during postglacial recolonization of Europe by the bank vole. From the Alpe-Adrian region (Balkan, Austria, and Hungary) to Western European countries (Germany, France, Belgium, and Netherland), and South Scandinavia. From the vicinity of Carpathian Mountains to the Baltic countries and to Poland, Russia, and Finland. The dissemination towards Denmark and North Scandinavia is more hypothetical and probably involved several independent streams from south and north Fennoscandia

Detection of Orientia sp. DNA in rodents from Asia, West Africa and Europe

Article Open AccessInternational audienceOrientia bacterium is the agent of the scrub typhus, a seriously neglected life-threatening disease in Asia. Here, we report the detection of DNA of Orientia in rodents from Europe and Africa. These findings have important implications for public health. Surveillance outside Asia, where the disease is not expected by sanitary services, needs to be improved

Comparison between Transcriptome Sequencing and 16S Metagenomics for Detection of Bacterial Pathogens in Wildlife

Background Rodents are major reservoirs of pathogens responsible for numerous zoonotic diseases in humans and livestock. Assessing their microbial diversity at both the individual and population level is crucial for monitoring endemic infections and revealing microbial association patterns within reservoirs. Recently, NGS approaches have been employed to characterize microbial communities of different ecosystems. Yet, their relative efficacy has not been assessed. Here, we compared two NGS approaches, RNA-Sequencing (RNA-Seq) and 16S-metagenomics, assessing their ability to survey neglected zoonotic bacteria in rodent populations.Methodology/Principal Findings : We first extracted nucleic acids from the spleens of 190 voles collected in France. RNA extracts were pooled, randomly retro-transcribed, then RNA-Seq was performed using HiSeq. Assembled bacterial sequences were assigned to the closest taxon registered in GenBank. DNA extracts were analyzed via a 16S-metagenomics approach using two sequencers: the 454 GS-FLX and the MiSeq. The V4 region of the gene coding for 16S rRNA was amplified for each sample using barcoded universal primers. Amplicons were multiplexed and processed on the distinct sequencers. The resulting datasets were de-multiplexed, and each read was processed through a pipeline to be taxonomically classified using the Ribosomal Database Project. Altogether, 45 pathogenic bacterial genera were detected. The bacteria identified by RNA-Seq were comparable to those detected by 16S-metagenomics approach processed with MiSeq (16S-MiSeq). In contrast, 21 of these pathogens went unnoticed when the 16S-metagenomics approach was processed via 454-pyrosequencing (16S-454). In addition, the 16S-metagenomics approaches revealed a high level of coinfection in bank voles. Conclusions/Significance :We concluded that RNA-Seq and 16S-MiSeq are equally sensitive in detecting bacteria. Although only the 16S-MiSeq method enabled identification of bacteria in each individual reservoir, with subsequent derivation of bacterial prevalence in host populations, and generation of intra-reservoir patterns of bacterial interactions. Lastly, the number of bacterial reads obtained with the 16S-MiSeq could be a good proxy for bacterial prevalence

Microevolution of bank voles (Myodes glareolus) at neutral and immune-related genes during multiannual dynamic cycles : Consequences for Puumala hantavirus epidemiology

Understanding howhost dynamics, including variations of population size and dispersal, may affect the epidemiology of infectious diseases through ecological and evolutionary processes is an active research area. Here we focus on a bank vole (Myodes glareolus) metapopulation surveyed in Finland between 2005 and 2009. Bank vole is the reservoir of Puumala hantavirus (PUUV), the agent of nephropathia epidemica (NE, a mild form of hemorrhagic fever with renal symptom) in humans. M. glareolus populations experience multiannual density fluctuations that may influence the level of genetic diversity maintained in bank voles, PUUV prevalence and NE occurrence. We examine bank vole metapopulation genetics at presumably neutral markers and immunerelated genes involved in susceptibility to PUUV (Tnf-promoter, Tlr4, Tlr7 and Mx2 gene) to investigate the links between population dynamics, microevolutionary processes and PUUV epidemiology. We show that genetic drift slightly and transiently affects neutral and adaptive genetic variability within the metapopulation. Gene flow seems to counterbalance its effects during the multiannual density fluctuations. The low abundance phase may therefore be too short to impact genetic variation in the host, and consequently viral genetic diversity. Environmental heterogeneity does not seem to affect vole gene flow, which might explain the absence of spatial structure previously detected in PUUV in this area. Besides, our results suggest the role of vole dispersal on PUUV circulation through sex-specific and density-dependent movements. We find little evidence of selection acting on immune-related genes within this metapopulation. Footprint of positive selection is detected at Tlr-4 gene in 2008 only. We observe marginally significant associations between Mx2 genotype and PUUV genogroups. These results show that neutral processes seem to be the main factors affecting the evolution of these immune-related genes at a contemporary scale, although the relative effects of neutral and adaptive forces could vary temporally with density fluctuations. Immune related gene polymorphism may in turn partly influence PUUV epidemiology in this metapopulation. (C) 2016 Published by Elsevier B.V.Peer reviewe