Puumala Virus Variants Circulating in Forests of Ardennes, France: Ten Years of Genetic Evolution.

In Europe, Puumala virus (PUUV) transmitted by the bank vole (Myodes glareolus) is the causative agent of nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome. In France, very little is known about the spatial and temporal variability of the virus circulating within bank vole populations. The present study involved monitoring of bank vole population dynamics and PUUV microdiversity over a ten-year period (2000-2009) in two forests of the Ardennes region: Elan and Croix-Scaille. Ardennes region is characterised by different environmental conditions associated with different NE epidemiology. Bank vole density and population parameters were estimated using the capture/marking/recapture method, and blood samples were collected to monitor the overall seroprevalence of PUUV in rodent populations. Phylogenetic analyses of fifty-five sequences were performed to illustrate the genetic diversity of PUUV variants between forests. The pattern of the two forests differed clearly. In the Elan forest, the rodent survival was higher, and this limited turn-over resulted in a lower seroprevalence and diversity of PUUV sequences than in the Croix-Scaille forest. Uncovering the links between host dynamics and virus microevolution is improving our understanding of PUUV distribution in rodents and the NE risk.

Revisiting the genetic diversity of emerging hantaviruses circulating in Europe using a pan-viral resequencing microarray.

Hantaviruses are zoonotic agents transmitted from small mammals, mainly rodents, to humans, where they provoke diseases such as Hemorrhagic fever with Renal Syndrome (HFRS) and its mild form, Nephropathia Epidemica (NE), or Hantavirus Cardio-Pulmonary Syndrome (HCPS). Hantaviruses are spread worldwide and monitoring animal reservoirs is of primary importance to control the zoonotic risk. Here, we describe the development of a pan-viral resequencing microarray (PathogenID v3.0) able to explore the genetic diversity of rodent-borne hantaviruses endemic in Europe. Among about 800 sequences tiled on the microarray, 52 correspond to a tight molecular sieve of hantavirus probes covering a large genetic landscape. RNAs from infected animal tissues or from laboratory strains have been reverse transcribed, amplified, then hybridized to the microarray. A classical BLASTN analysis applied to the sequence delivered through the microarray allows to identify the hantavirus species up to the exact geographical variant present in the tested samples. Geographical variants of the most common European hantaviruses from France, Germany, Slovenia and Finland, such as Puumala virus, Dobrava virus and Tula virus, were genetically discriminated. Furthermore, we precisely characterized geographical variants still unknown when the chip was conceived, such as Seoul virus isolates, recently emerged in France and the United Kingdom.

Complete Genome and Phylogeny of Puumala Hantavirus Isolates Circulating in France.

Puumala virus (PUUV) is the agent of nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome (HFRS) in Europe. NE incidence presents a high spatial variation throughout France, while the geographical distribution of the wild reservoir of PUUV, the bank vole, is rather continuous. A missing piece of the puzzle is the current distribution and the genetic variation of PUUV in France, which has been overlooked until now and remains poorly understood. During a population survey, from 2008 to 2011, bank voles were trapped in eight different forests of France located in areas known to be endemic for NE or in area from where no NE case has been reported until now. Bank voles were tested for immunoglobulin (Ig)G ELISA serology and two seropositive animals for each of three different areas (Ardennes, Jura and Orleans) were then subjected to laboratory analyses in order to sequence the whole S, M and L segments of PUUV. Phylogenetic analyses revealed that French PUUV isolates globally belong to the central European (CE) lineage although isolates from Ardennes are clearly distinct from those in Jura and Orleans, suggesting a different evolutionary history and origin of PUUV introduction in France. Sequence analyses revealed specific amino acid signatures along the N protein, including in PUUV from the Orleans region from where NE in humans has never been reported. The relevance of these mutations in term of pathophysiology is discussed.

Unknown age in health disorders: A method to account for its cumulative effect and an application to feline viruses interactions.

Parasite interactions have been widely evidenced experimentally but field studies remain rare. Such studies are essential to detect interactions of interest and access (co)infection probabilities but face methodological obstacles. Confounding factors can create statistical associations, i.e. false parasite interactions. Among them, host age is a crucial covariate. It influences host exposition and susceptibility to many infections, and has a mechanical effect, older individuals being more at risk because of a longer exposure time. However, age is difficult to estimate in natural populations. Hence, one should be able to deal at least with its cumulative effect. Using a SI type dynamic model, we showed that the cumulative effect of age can generate false interactions theoretically (deterministic modeling) and with a real dataset of feline viruses (stochastic modeling). The risk to wrongly conclude to an association was maximal when parasites induced long-lasting antibodies and had similar forces of infection. We then proposed a method to correct for this effect (and for other potentially confounding shared risk factors) and made it available in a new R package, Interatrix. We also applied the correction to the feline viruses. It offers a way to account for an often neglected confounding factor and should help identifying parasite interactions in the field, a necessary step towards a better understanding of their mechanisms and consequences.

True versus false parasite interactions: a robust method to take risk factors into account and its application to feline viruses.

BACKGROUND: Multiple infections are common in natural host populations and interspecific parasite interactions are therefore likely within a host individual. As they may seriously impact the circulation of certain parasites and the emergence and management of infectious diseases, their study is essential. In the field, detecting parasite interactions is rendered difficult by the fact that a large number of co-infected individuals may also be observed when two parasites share common risk factors. To correct for these "false interactions", methods accounting for parasite risk factors must be used. METHODOLOGY/PRINCIPAL FINDINGS: In the present paper we propose such a method for presence-absence data (i.e., serology). Our method enables the calculation of the expected frequencies of single and double infected individuals under the independence hypothesis, before comparing them to the observed ones using the chi-square statistic. The method is termed "the corrected chi-square." Its robustness was compared to a pre-existing method based on logistic regression and the corrected chi-square proved to be much more robust for small sample sizes. Since the logistic regression approach is easier to implement, we propose as a rule of thumb to use the latter when the ratio between the sample size and the number of parameters is above ten. Applied to serological data for four viruses infecting cats, the approach revealed pairwise interactions between the Feline Herpesvirus, Parvovirus and Calicivirus, whereas the infection by FIV, the feline equivalent of HIV, did not modify the risk of infection by any of these viruses. CONCLUSIONS/SIGNIFICANCE: This work therefore points out possible interactions that can be further investigated in experimental conditions and, by providing a user-friendly R program and a tutorial example, offers new opportunities for animal and human epidemiologists to detect interactions of interest in the field, a crucial step in the challenge of multiple infections.

Pathocenosis: a holistic approach to disease ecology.

The history of medicine describes the emergence and recognition of infectious diseases, and human attempts to stem them. It also throws light on the role of changing environmental conditions on disease emergence/re-emergence, establishment and, sometimes, disappearance. However, the dynamics of infectious diseases is also influenced by the relationships between the community of interacting infectious agents present at a given time in a given territory, a concept that Mirko Grmek, an historian of medicine, conceptualized with the word "pathocenosis". The spatial and temporal evolution of diseases, when observed at the appropriate scales, illustrates how a change in the pathocenosis, whether of "natural" or anthropic origin, can lead to the emergence and spread of diseases.

Using dynamic stochastic modelling to estimate population risk factors in infectious disease: the example of FIV in 15 cat populations.

BACKGROUND: In natural cat populations, Feline Immunodeficiency Virus (FIV) is transmitted through bites between individuals. Factors such as the density of cats within the population or the sex-ratio can have potentially strong effects on the frequency of fight between individuals and hence appear as important population risk factors for FIV. METHODOLOGY/PRINCIPAL FINDINGS: To study such population risk factors, we present data on FIV prevalence in 15 cat populations in northeastern France. We investigate five key social factors of cat populations; the density of cats, the sex-ratio, the number of males and the mean age of males and females within the population. We overcome the problem of dependence in the infective status data using sexually-structured dynamic stochastic models. Only the age of males and females had an effect (p = 0.043 and p = 0.02, respectively) on the male-to-female transmission rate. Due to multiple tests, it is even likely that these effects are, in reality, not significant. Finally we show that, in our study area, the data can be explained by a very simple model that does not invoke any risk factor. CONCLUSION: Our conclusion is that, in host-parasite systems in general, fluctuations due to stochasticity in the transmission process are naturally very large and may alone explain a larger part of the variability in observed disease prevalence between populations than previously expected. Finally, we determined confidence intervals for the simple model parameters that can be used to further aid in management of the disease.

Emergence of infectious diseases: when hidden pathogens break out.

Our understanding of disease emergence is largely limited by the assumption that disease emergence is the result of increased exposure to pathogenic agents. Pathogen exposure is thought to arise through an increase in the number of interactions between humans and their natural environment, changes in demography and mobility, or through genetic variation in the infectious agents which may alter virulence or ability to infect new host species. The study of new diseases (which are often revealed by unusually severe symptoms or atypical epidemiological patterns) applies the most effort to the research of new pathogens. Here, using examples, we discuss alternative but non-exclusive mechanisms that may either reveal the presence of long-term circulating pathogens or explain changes in their nosologic properties in relation to their pattern of circulation and infection conditions. A better understanding of the ecology of pathogenic organisms and their host populations should help to define more efficient health management strategies.

When domestic cat (Felis silvestris catus) population structures interact with their viruses.

Many theoretical studies have proposed different causal mechanisms by which the structure of a host population could have important implications for life history traits of pathogens. However, little information is available from real systems to test these hypotheses. The domestic cat, Felis silvestris catus, whose populations exhibit a great variability in social and spatial structure, represent an ideal case study to assess this question. In the present article, we show how cat population structure may have influenced the evolution of feline viruses and, in return, how these viruses may have modified the genetic structure of cat populations.

The canine distemper epidemic in Serengeti: are lions victims of a new highly virulent canine distemper virus strain, or is pathogen circulation stochasticity to blame?

In the year 1994, the Serengeti lion population was decimated by a canine distemper disease outbreak. Retrospective investigations showed that this host population had already been in contact with the pathogen in 1981 without any detected sign of disease. As an alternative to the virus mutation hypothesis to explain this difference in virulences observed in 1981 and 1994, we propose a novel mechanism of disease emergence based on variation in population immunity. We use a stochastic model to show that stochastic fluctuations in pathogen circulation, owing to a low probability of virus transmission from its reservoir to the target host and thereby resulting in variations in the global immunity level of the target host population, can explain the observations made in Serengeti. This mechanism may also be involved in other infectious disease emergences or re-emergences.

Antibodies to Aleutian mink disease parvovirus in free-ranging European mink (Mustela lutreola) and other small carnivores from southwestern France.

Owing to the rapid decline of the European mink (Mustela lutreola) in France, a national conservation action plan has been initiated, in which scientific research to improve understanding of the causes of the decline is one of the primary objectives. In order to investigate the possible role of Aleutian disease parvovirus (ADV) in decline of the species, a serologic survey was conducted from March 1996 to March 2002 in 420 free-ranging individuals of six species of small carnivores distributed in eight departments of southwestern France. Antibodies to ADV were detected in 17 of 75 American mink (Mustela vison), 12 of 99 European mink, 16 of 145 polecats (Mustela putorius), four of 17 stone martens (Martes foina), one of 16 pine martens (Martes martes), and three of 68 common genets (Genetta genetta). Seroprevalence was significantly higher in American mink than in other species. Seropositive individuals with gamma globulin levels >20% were observed in four European mink, four American mink, two stone martens, and one pine marten. Geographic distribution of positive animals indicates the virus has spread to all areas where European mink are found. Furthermore, a trend of increasing prevalence seems to appear in Mustela sp. sympatric with American mink. Although further investigations are necessary to evaluate the role of ADV in decline of European mink, evidence of the virus in the wild at the levels found in our study has implications for conservation of this species.