Association between the DQA MHC class II gene and Puumala virus infection in Myodes glareolus, the bank vole

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Abstract

Puumala virus (PUUV) is a hantavirus specifically harboured by the bank vole, Myodes (earlier Clethrionomys) glareolus. It causes a mild form of hemorrhagic fever with renal syndrome (HFRS) in humans, called Nephropathia epidemica (NE). The clinical severity of NE is variable among patients and depends on their major histocompatibility complex (MHC) genetic background. In this study we investigated the potential role of class II MHC gene polymorphism in the susceptibility/resistance to PUUV in the wild reservoir M. glareolus. We performed an association study between the exon 2 of the DQA gene and PUUV antibodies considering a natural population of bank voles. Because immune gene polymorphism is likely to be driven by multiple parasites in the wild, we also screened bank voles for other potential viral and parasitic infections. We used multivariate analyses to explore DQA polymorphism/PUUV associations while considering the potential antagonist and/or synergistic effects of the whole parasite community. Our study suggests links between class II MHC characteristics and viral infections including PUUV and Cowpox virus. Several alleles are likely to be involved in the susceptibility or in the resistance of bank voles to these infections. Alternatively, heterozygosity does not seem to be associated with PUUV or any other parasite infections. This result thus provides no evidence in favour of the hypothesis of selection through overdominance. Finally this multivariate approach reveals a strong antagonism between ectoparasitic mites and PUUV, suggesting direct or indirect immunogenetic links between infections by these parasites. Other datasets are now required to confirm these results and to test whether the associations vary in space and/or time.

Introduction

Polymorphism at immune genes may be involved in specific recognition of pathogens as variation allows the recognition of spectra of epitopes (Doherty and Zinkernagel, 1975). In this context, one of the leading goals of immunogenetics, i.e. the analysis of genetic polymorphisms in specific recognition and immune regulation, has been to understand the genetic basis of susceptibility to complex diseases (Geraghty, 2002). The profound influence of the host genetics on resistance to infections has been established in numerous studies, which mainly concerned human infections such as malaria, HIV and hepatitis (review in Cooke and Hill, 2001, Hill, 2001). In wild animal populations, immunogenetics provides key insight into the relative influence of genetic variation and environmental factors on host-pathogen interactions. A number of zoonoses, i.e. infections transmitted from animals to humans (Taylor et al., 2001), has (re-)emerged during the past 15 years (Kallio-Kokko et al., 2005). Therefore, an important application of immunogenetics concerns the assessment of emergent or re-emergent disease risks in natural populations. (i) The study of immune genes may explain why hosts differ in their susceptibility to different parasites. The reasons why hosts differ in their susceptibility/resistance to different parasites could rely on the degree of matching between immune genes and parasite antigens. Immunogenetics may thus contribute to the identification of unknown zoonotic agents, which is essential for understanding zoonoses epidemiology (Mills and Childs, 1998). (ii) Natural population studies of immune gene polymorphism may provide key insight into the factors determining the appearance, spread and distribution of resistance/immuno-modulating alleles within populations and across geographical landscapes. Such information is essential to study the spatial and temporal variations in disease risk or incidence.

Hantaviruses are one of the three main emerging infectious agents in Europe (Kallio-Kokko et al., 2005). They are specific rodent-borne viruses belonging to the Bunyaviridae family, and are transmitted to humans primarily from aerosols of rodent excreta. Puumala virus (PUUV) is a hantavirus, which causes a mild form of haemorrhagic fever with renal syndrome (HFRS) in humans, called Nephropathia epidemica (NE). The mortality rate ranges between 0.1 and 0.4% (Vapalahti et al., 2003). Infections are frequent in Northern Europe, European Russia and parts of Central Western Europe (Vapalahti et al., 2003), with about 6000 cases reported per year. In France, it has been classified as an important emerging disease by the Institut de Veille Sanitaire (INVS, Capek, 2006).

The specific reservoir host of PUUV is the bank vole Myodes (earlier Clethrionomys) glareolus. In this rodent, PUUV produces a chronic, long-lasting infection. Host and virus have geographical ranges that do not completely overlap. Many parts of Europe, such as the Mediterranean peninsulas and Britain remain blank of PUUV infections although M. glareolus is present. Two recent studies provided arguments in favour of selection acting on bank voles and mediated by PUUV. First, Kallio et al., 2006a, Kallio et al., 2006b observed that PUUV infection could affect bank vole fecundity (earlier breeding). Second, experimental infections of M. glareolus laboratory colonies with PUUV revealed that some bank voles could not be successfully infected. Neither antibodies, nor PUUV RNA could be detected in these individuals reared in PUUV-contaminated beddings, providing evidence of variability in vole susceptibility to PUUV. This variability was not significantly explained by age, sex, maturation status or individual weight (Kallio et al., 2006a, Kallio et al., 2006b). This suggests that immunogenetic factors could be involved not only in the disease progression (Klein et al., 2004a) but also in controlling infection itself. The absence of HFRS in some parts of M. glareolus geographic distribution could therefore be partly explained by selected vole immunogenetic factors associated with resistance to PUUV infections.

Association studies have provided evidence for the role of immunogenetics in the course of hantavirus infections in rodents. Using the hantavirus Seoul and the rat Rattus norvegicus, Klein et al., 2004a, Klein et al., 2004b characterized immunologic pathways that differ between males and females in response to infection. They showed that most of the 192 identified immune-related genes that encode for these immune responses were differentially expressed in infected females compared with infected males. Several MHC genes were associated with these sex differences in gene expression. Alternatively, the considerable variability in the clinical severity of NE among humans depends on MHC genes (Mustonen et al., 1996, Mustonen et al., 1998, Mustonen et al., 2004, Plyusnin et al., 1997). The HLA-B227 haplotype is associated with a mild course of NE, whereas the extended haplotypes HLA-B8-DR3 and HLA-DRB1-0301 are associated with severe clinical courses. The complete MHC class II region has been sequenced in rats (Günther and Walter, 2001, Hurt et al., 2004) and mice (Blake et al., 2003). It has a similar regional organization as the human MHC, and orthologous relationships exist between class II regions in all mammals (Takahata and Nei, 1990). Moreover, in laboratory mouse and rats, the DQA gene (called RT1.B in rats, H2-A in mice) is tightly linked to DRB genes (Blake et al., 2003, Hurt et al., 2004). As DRB is at least quadriduplicated in bank voles (Axtner and Sommer, 2007), what makes genotyping difficult to perform, DQA was a relevant candidate to look for associations between MHC class II genes and PUUV infection.

In this study we investigate the role of the class II MHC DQA gene polymorphism on susceptibility/resistance to PUUV by the mean of an association study conducted in a natural population of Myodes glareolus, the specific reservoir of this hantavirus. Vole sampling was performed within the second most important French endemic area of HFRS (Jura, Franche Comté), which has had an unusually high number of human cases in 2005 (230 in France, 38 in the Jura, INVS).

Section snippets

Trapping design

The main study area is located around Mignovillard (46°8′N, 6°13′E and elevation 850 m) in Jura, France. It consists of a 4-km2 site, half of which is composed of wooded meadows and the other half of forests (man-made spruce or semi-natural forests). Bank voles were sampled in September 2004, July and September 2005 using French Agricultural Research Institute (INRA) live traps, which were fitted out with dormitory boxes, baited with hay and a piece of apple. Twenty 100-m trap-lines composed of

Results

The 98 bank voles, 53 males and 45 females, trapped between September 2004 and 2005 in Mignovillard were included in the analyses.

Discussion

In humans, the clinical severity of NE depends on the MHC genetic background of the patients, with haplotypes associated to a benign clinical course and other with a severe one (Mustonen et al., 1996, Mustonen et al., 1998, Mustonen et al., 2004, Plyusnin et al., 1997). Investigating the existence of such associations in reservoirs of hantaviruses is thus particularly important to better understand the risks of HFRS emergence related to wild rodents populations.

Conclusions

Our study provides evidence for the role of immunogenetics in viral infections. Several alleles are suggested for either susceptibility or resistance to these infections. However, these results have to be taken cautiously as they concern few seropositive individuals. Moreover, as demonstrated for passerines and malaria parasites, links between host immunogenetics and resistance can result from local adaptation processes (Bonneaud et al., 2006). The associations detected might thus involve

Acknowledgements

We thank Patrice Cadet for help in the statistical analyses, Audrey Scala and Florian Holon for help in field work and Stanislas Kalùz for mite identification. This research was partially funded by EU grant GOCE-2003-010284 EDEN and the paper is catalogued by the EDEN Steering Committee as EDEN 044 (http://www.eden-fp6project.net/).

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