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 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

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

Changes in Diversification Patterns and Signatures of Selection during the Evolution of Murinae-Associated Hantaviruses

In the last 50 years, hantaviruses have significantly affected public health worldwide, but the exact extent of the distribution of hantavirus diseases, species and lineages and the risk of their emergence into new geographic areas are still poorly known. In particular, the determinants of molecular evolution of hantaviruses circulating in different geographical areas or different host species are poorly documented. Yet, this understanding is essential for the establishment of more accurate scenarios of hantavirus emergence under different climatic and environmental constraints. In this study, we focused on Murinae-associated hantaviruses (mainly Seoul Dobrava and Hantaan virus) using sequences available in GenBank and conducted several complementary phylogenetic inferences. We sought for signatures of selection and changes in patterns and rates of diversification in order to characterize hantaviruses' molecular evolution at different geographical scales (global and local). We then investigated whether these events were localized in particular geographic areas. Our phylogenetic analyses supported the assumption that RNA virus molecular variations were under strong evolutionary constraints and revealed changes in patterns of diversification during the evolutionary history of hantaviruses. These analyses provide new knowledge on the molecular evolution of hantaviruses at different scales of time and space

16S rRNA amplicon sequencing for epidemiological surveys of bacteria in wildlife

The human impact on natural habitats is increasing the complexity of human-wildlife interactions and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential for recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of the large numbers of samples needed to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to mitigate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a comprehensive trimming strategy for managing these biases. We demonstrated the application of this strategy using 711 commensal rodents, including 208 Mus musculusdomesticus, 189 Rattus rattus, 93 Mastomys natalensis, and 221 Mastomys erythroleucus, collected from 24 villages in Senegal. Seven major genera of pathogenic bacteria were detected in their spleens: Borrelia, Bartonella, Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia. Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90% of individuals per site, depending on the bacterial taxon, rodent species, and site considered, and 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of bacterial pathogens without requiring assumptions about their presence in the samples. This approach is therefore particularly suitable to continuous pathogen surveillance in the context of disease-monitoring programs

Utilisation de la métagénomique 16S pour la surveillance de l’émergence de zoonoses bactériennes dans les populations animales

International audienc

Utilisation de la métagénomique 16S pour la surveillance de l’émergence de zoonoses bactériennes dans les populations animales

International audienc