Life-long shedding of Puumala hantavirus in wild bank voles (Myodes glareolus).

The knowledge of viral shedding patterns and viraemia in the reservoir host species is a key factor in assessing the human risk of zoonotic viruses. The shedding of hantaviruses (family Bunyaviridae) by their host rodents has widely been studied experimentally, but rarely in natural settings. Here we present the dynamics of Puumala hantavirus (PUUV) shedding and viraemia in naturally infected wild bank voles (Myodes glareolus). In a monthly capture-mark-recapture study, we analysed 18 bank voles for the presence and relative quantity of PUUV RNA in the excreta and blood from 2 months before up to 8 months after seroconversion. The proportion of animals shedding PUUV RNA in saliva, urine and faeces peaked during the first month after seroconversion, but continued throughout the study period with only a slight decline. The quantity of shed PUUV in reverse transcription quantitative PCR (RT-qPCR) positive excreta was constant over time. In blood, PUUV RNA was present for up to 7 months but both the probability of viraemia and the virus load declined with time. Our findings contradict the current view of a decline in virus shedding after the acute phase and a short viraemic period in hantavirus infection - an assumption widely adopted in current epidemiological models. We suggest the life-long shedding as a means of hantaviruses to survive over host population bottlenecks, and to disperse in fragmented habitats where local host and/or virus populations face temporary extinctions. Our results indicate that the kinetics of pathogens in wild hosts may differ considerably from those observed in laboratory settings.

Analysis of complete Puumala virus genome, Finland.

Puumala virus causes nephropathia epidemica, a rodent-borne zoonosis that is endemic to Europe. We sequenced the complete Puumala virus genome that was directly recovered from a person who died and compared it with those of viruses from local bank voles. The virus strain involved was neither a unique nor rare genetic variant.

Co-circulation of two Puumala hantavirus lineages in Latvia: a Russian lineage described previously and a novel Latvian lineage.

Puumala hantavirus (PUUV) causes a mild form of haemorrhagic fever with renal syndrome in Europe. Seven genetic lineages of PUUV have thus far been recorded, which exhibit geographic structure within the distribution of its natural host, the bank vole (Myodes glareolus). This study presents evidence for two distinct PUUV lineages co-circulating in Latvia: one previously described from Russia and a novel one that appears to be endemic. The Latvian lineage (LAT) is considerably divergent and several amino acid markers make it easily distinguishable. Phylogenetic analysis suggested a possibility of different evolutionary histories for the PUUV genome segments of LAT.

Impact of Landscape on Host-Parasite Genetic Diversity and Distribution Using the Puumala orthohantavirus-Bank Vole System.

In nature, host specificity has a strong impact on the parasite's distribution, prevalence, and genetic diversity. The host's population dynamics is expected to shape the distribution of host-specific parasites. In turn, the parasite's genetic structure is predicted to mirror that of the host. Here, we study the tandem Puumala orthohantavirus (PUUV)-bank vole system. The genetic diversity of 310 bank voles and 33 PUUV isolates from 10 characterized localities of Northeast France was assessed. Our findings show that the genetic diversity of both PUUV and voles, was positively correlated with forest coverage and contiguity of habitats. While the genetic diversity of voles was weakly structured in space, that of PUUV was found to be strongly structured, suggesting that the dispersion of voles was not sufficient to ensure a broad PUUV dissemination. Genetic diversity of PUUV was mainly shaped by purifying selection. Genetic drift and extinction events were better reflected than local adaptation of PUUV. These contrasting patterns of microevolution have important consequences for the understanding of PUUV distribution and epidemiology.

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.

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

Understanding how host 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 immune-related 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.

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 musculus domesticus, 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. IMPORTANCE Several recent public health crises have shown that the surveillance of zoonotic agents in wildlife is important to prevent pandemic risks. High-throughput sequencing (HTS) technologies are potentially useful for this surveillance, but rigorous experimental processes are required for the use of these effective tools in such epidemiological contexts. In particular, HTS introduces biases into the raw data set that might lead to incorrect interpretations. We describe here a procedure for cleaning data before estimating reliable biological parameters, such as positivity, prevalence, and coinfection, using 16S rRNA amplicon sequencing on an Illumina MiSeq platform. This procedure, applied to 711 rodents collected in West Africa, detected several zoonotic bacterial species, including some at high prevalence, despite their never before having been reported for West Africa. In the future, this approach could be adapted for the monitoring of other microbes such as protists, fungi, and even viruses.

A 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.

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

Orientia 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.

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.

Microevolution of Puumala hantavirus during a complete population cycle of its host, the bank vole (Myodes glareolus).

Microevolution of Puumala hantavirus (PUUV) was studied throughout a population cycle of its host, the bank vole (Myodes glareolus). We monitored PUUV variants circulating in the host population in Central Finland over a five-year period that included two peak-phases and two population declines. Of 1369 bank voles examined, 360 (26.3%) were found infected with PUUV. Partial sequences of each of the three genome segments were recovered (approx. 12% of PUUV genome) from 356 bank voles. Analyses of these sequences disclosed the following features of PUUV evolution: 1) nucleotide substitutions are mostly silent and deduced amino acid changes are mainly conservative, suggesting stabilizing selection at the protein level; 2) the three genome segments accumulate mutations at a different rate; 3) some of the circulating PUUV variants are frequently observed while others are transient; 4) frequently occurring PUUV variants are composed of the most abundant segment genotypes (copious) and new transient variants are continually generated; 5) reassortment of PUUV genome segments occurs regularly and follows a specific pattern of segments association; 6) prevalence of reassortant variants oscillates with season and is higher in the autumn than in the spring; and 7) reassortants are transient, i.e., they are not competitively superior to their parental variants. Collectively, these observations support a quasi-neutral mode of PUUV microevolution with a steady generation of transient variants, including reassortants, and preservation of a few preferred genotypes.

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.

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 Pallasjärvi, 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 Pallasjärvi 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 Fennoscandia.

Accumulation of point mutations and reassortment of genomic RNA segments are involved in the microevolution of Puumala hantavirus in a bank vole (Myodes glareolus) population.

The genetic diversity of Puumala hantavirus (PUUV) was studied in a local population of its natural host, the bank vole (Myodes glareolus). The trapping area (2.5 x 2.5 km) at Konnevesi, Central Finland, included 14 trapping sites, at least 500 m apart; altogether, 147 voles were captured during May and October 2005. Partial sequences of the S, M and L viral genome segments were recovered from 40 animals. Seven, 12 and 17 variants were detected for the S, M and L sequences, respectively; these represent new wild-type PUUV strains that belong to the Finnish genetic lineage. The genetic diversity of PUUV strains from Konnevesi was 0.2-4.9 % for the S segment, 0.2-4.8 % for the M segment and 0.2-9.7 % for the L segment. Most nucleotide substitutions were synonymous and most deduced amino acid substitutions were conservative, probably due to strong stabilizing selection operating at the protein level. Based on both sequence markers and phylogenetic clustering, the S, M and L sequences could be assigned to two groups, 'A' and 'B'. Notably, not all bank voles carried S, M and L sequences belonging to the same group, i.e. S(A)M(A)L(A) or S(B)M(B)L(B). A substantial proportion (8/40, 20 %) of the newly characterized PUUV strains possessed reassortant genomes such as S(B)M(A)L(A), S(A)M(B)L(B) or S(B)M(A)L(B). These results suggest that at least some of the PUUV reassortants are viable and can survive in the presence of their parental strains.