A five-year perspective on the situation of haemorrhagic fever with renal syndrome and status of the hantavirus reservoirs in Europe, 2005–2010

P Heyman (paul.heyman@mil.be)1, C S Ceianu2, I Christova3, N Tordo4, M Beersma5, M João Alves6, A Lundkvist7, M Hukic8, A Papa9, A Tenorio10, H Zelená11, S Eßbauer12, I Visontai13, I Golovljova14, J Connell15, L Nicoletti16, M Van Esbroeck17, S Gjeruldsen Dudman18, S W Aberle19, T Avšić-Županc20, G Korukluoglu21, A Nowakowska22, B Klempa23, R G Ulrich24, S Bino25, O Engler26, M Opp27, A Vaheri28 1. Research Laboratory for Vector-borne Diseases and National Reference Laboratory for Hantavirus Infections, Brussels, Belgium 2. Cantacuzino Institute, Vector-Borne Diseases Laboratory, Bucharest, Romania 3. National Center for Infectious and Parasitic Diseases, Sofia, Bulgaria 4. Unit of the Biology of Emerging Viral Infections (UBIVE), Institut Pasteur, Lyon, France 5. Department of Virology, Erasmus University Hospital, Rotterdam, the Netherlands 6. Centre for Vectors and Infectious Diseases Research (CEVDI), National Institute of Applied Sciences (INSA), National Institute of Health Dr. Ricardo Jorge, Águas de Moura, Portugal 7. Swedish Institute for Communicable Disease Control (SMI), Karolinska Institute, Stockholm, Sweden 8. Clinical Centre, University of Sarajevo, Institute of Clinical Microbiology, Sarajevo, Bosnia and Herzegovina 9. Aristotle University of Thessaloniki, School of Medicine, Thessaloniki, Greece 10. Arbovirus and Imported Viral Disease Unit, National Centre for Microbiology, Institute for Health Carlos III, Majadahonda, Spain 11. Institute of Public Health, Ostrava, Czech Republic 12. Department of Virology and Rickettsiology, Bundeswehr Institute for Microbiology, Munich, Germany 13. National Centre for Epidemiology, Budapest, Hungary 14. Department of Virology, National Institute for Health Development, Tallinn, Estonia 15. Department of Medical Microbiology, University College Dublin, Dublin, Ireland 16. Department of Infectious, Parasitic and Immunomediate Diseases, Istituto Superiore di Sanitá, Laboratory of Virology, Rome, Italy 17. Institute of Tropical Medicine, Antwerp, Belgium 18. Norwegian Institute of Public Health, Division of Infectious Disease Control, Oslo, Norway 19. Department of Virology, Medical University of Vienna, Vienna, Austria 20. University of Ljubljana, Medical Faculty, Institute of Microbiology and Immunology, Ljubljana, Slovenia 21. Refik Saydam National Public Health Agency, Sihhiye, Ankara, Turkey 22. Medical Diagnostic Laboratory, Voivodeship Sanitary-Epidemiological Station, Rzeszów, Poland 23. Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovakia 24. Friedrich-Loeffler Institute, Federal Research Institute for Animal Health, World Organisation for Animal Health Collaborating Centre for Zoonoses in Europe, Institute for Novel and Emerging Infectious Diseases, Greifswald Insel Riems, Germany 25. Institute of Public Health, Control of Infectious Diseases, Tirana, Albania 26. Spiez Laboratory, Biology Virology Group, Spiez, Switzerland 27. Laboratoire National de Santé, Virologie, Luxembourg 28. Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland


Introduction
Hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped RNA viruses that have rodents and insectivores as hosts and are transmitted by aerosols of host excreta or by direct contact to humans. At least five hantaviruses, Puumala (PUUV), Dobrava (DOBV), Saaremaa (SAAV), Tula (TULV) and Seoul virus (SEOV), circulate in Europe. The most prominent and most widely occurring hantavirus in Europe is PUUV, transmitted by the bank vole (Myodes glareolus). PUUV causes a mild form of haemorrhagic fever with renal syndrome (HFRS), called nephropathia epidemica (NE). DOBV is transmitted by the yellow-necked field mouse (Apodemus flavicollis) and is known to cause more severe HFRS [1,2]. SAAV, which is closely related to DOBV, is carried by the striped field mouse (A. agrarius). It should be noted that the hantavirus strains associated with A. agrarius in central Europe and Russia have been shown to be phylogenetically distinct from the north-eastern European SAAV strains as well as from strains associated with A. flavicollis (DOBV-Af lineage) or the strains associated with the Black Sea field mouse (A. ponticus) (DOBV-Ap lineage). It is from an epidemiological point of view currently impossible to distinguish between the lineages by routine diagnostics when the viral RNA sequence is not available [3,4]. TULV is transmitted by the common vole (Microtus arvalis), the field vole (M. agrestis) and the southern vole (M. levis, also known as M. rossiaemeridionalis). This virus has not definitely been linked to human disease. SEOV, transmitted by the brown and black rat (Rattus norvegicus and R. rattus), causes mild HFRS in Asia and in many harbour cities worldwide. In Europe, it has so far only been identified once as a human pathogen, in an unpublished case in France that was confirmed by focus reduction neutralisation test [1]. During the past decade several hantaviruses have been discovered that have insectivores as carriers. In Europe these are Laihia, Asikkala and Seewis virus, transmitted, respectively, by the Eurasian water shrew (Neomys fodiens), the Eurasian pygmy shrew (Sorex minutus) and the common shrew (Sorex araneus) ( Table 1).
In the past decade (2000-2009) oscillations in the number of hantavirus infections have been reported [5]. The unusually high number of hantavirus infections in Germany in 2010, with 327 cases between January and April in Baden-Württemberg [6], prompted the European Centre for Disease Prevention and Control (ECDC) to request an update on the hantavirus situation in Europe from the European Network for diagnostics of Imported Viral Diseases (ENIVD) and its Collaborative Laboratory Response Network (CLRN). The present article summarises the current knowledge on the occurrence of hantaviruses based on a survey in 30 European countries. In general HFRS is characterised by high fever for up to four days and unspecific symptoms at the onset of the disease such as headache, thrombocytopenia and influenza-like symptoms, followed by nausea, abdominal pain and vomiting. After four to 10 days renal manifestations characterised by oliguria and transient renal failure and later polyuria may occur [1,2].

Methods
The ENIVD hantavirus working group sent a questionnaire to all ENIVD members (N=30, see Table 1 when the number of cases matched the average case numbers, plus or minus 10% recorded for the respective country during the decade ending in the respective year. Case numbers 10-50% higher than the 10-year average were considered moderate activity, numbers 50-100% higher were considered slightly elevated and numbers at least 100% higher than the average number were considered increased activity.

Results
The annual number of cases diagnosed per country in the years 2006 to 2009 and 2010 up to end of August is summarised in  Figure 3). This is in accordance with already recognised epidemic years in different European countries (Table 2).
Further information we obtained on the carrier species present in the participating countries and the viruses detected in those rodents is summarised in Table 1. It confirmed earlier observations regarding the prominent role of PUUV and DOBV in Europe. Hantaviruses transmitted by insectivores were only found in Finland and Austria in this survey (see Table 1). No link to human disease has been shown so far for these viruses. Given the established role of the rodent-borne viruses PUUV, DOBV and possibly SAAV as human HFRS pathogens in Europe, it seems unlikely that insectivore-borne hantaviruses play a major role as pathogens.
Fatal cases due to hantavirus infection are rare in Europe and mostly linked to DOBV infection. Although some fatal cases have been linked to PUUV infection, the mortality rate due to this virus remains lower than 0.1%.

Discussion
Data on human hantavirus infections have been registered in 30 European countries since 2005. Our knowledge of the disease, virus geno-and serotypes, hosts and diagnostic capacities has increased over the past decade. However, there seem to be large regional differences in the case numbers. The update on endemic regions in the participating countries confirmed the focal aspect of hantavirus infections (see Table 1). In the majority of countries, the endemic regions are forested areas that provide sufficient shelter and food for rodent populations.  1990 1991 1992 1993 1994 1995 1996 1997 1998 1999    Epidemic peaks may be linked to times of favourable climatic conditions when an abundance of available food triggers a peak in the rodent population [8,10]. A relation between climate, high density of the rodent population and increased virus prevalence in rodent populations was also observed [4]. This puts humans at increased risk of contact with infected rodents and their excreta. According to preliminary findings, it was such a scenario that led to the spectacular increase in cases in Germany in 2010 [6]. Although mast events (increased seed production of various trees) seem to be of importance in triggering hantavirus epidemics, it should be remembered that only hantavirus epidemics in Atlantic and continental western Europe are mast-driven (although this seems not entirely true for Germany as in some years the country experienced very regional outbreaks), while other mechanisms drive these events in northern and eastern Europe [1,2].
The bank vole (M. glareolus), the principle vertebrate host for PUUV, is a generalist polyphagous species, i.e. eating seeds and fruits and occasionally invertebrates. It only acquires 50% of its energy intake from hard fruits and this only in the winter months. The yellownecked field mouse (A. flavicollis), the principle vertebrate host for DOBV, is predominantly a seed eater, but the invertebrate portion of its diet can be considerably higher than for the bank vole. The diet of both M. glareolus and A. flavicollis varies considerably in different regions in Europe: in Atlantic western Europe (Belgium, France) oak and beech seed crops are instrumental [10,11], while in continental Europe (the Białowieża Primeval forest in Poland, for instance) oak (Quercus petraea) and hornbeam (Carpinus betulus) seed crops regulate population sizes of both species [12,13]. Both the bank vole and the yellow-necked field mouse prefer a forest environment. The third rodent species of interest, the striped field mouse (A. agrarius, carrier of SAAV), is typical of a mixed habitat of agricultural fields and forest, and its population dynamics thus relate not only to forest conditions but also to anthropogenic factors [14].
For all three species, winter survival is related to food availability in the preceding summer and autumn, spring numbers are dependent on winter mortality, which according to the rodent catchers is estimated to reach on average 70% of autumn numbers for voles and 85% for mice, and summer/autumn numbers are primarily related to vegetation biomass and temperature.
The hantavirus activity peaks indicated by our data did not in all years correlate with mast cycles. Although mast events are supposed to occur over large areas and even on sub-continental level, hantavirus epidemics in western Europe can probably not be related solely to mast events of one tree species, given the highly different levels of hantavirus activity in neighbouring countries in 2005, 2007, 2008 and 2010 (see Table 2) where mast events occur simultaneously. Unfortunately, detailed information about seed crops of the different endemic tree species, e.g. beech (Fagus sylvatica), oak (Quercus sp.) and hornbeam (C. betulus), that can significantly influence rodent winter survival rates are not always available in most countries.
At present, all European countries dispose of the same range of diagnostic tools (for a recent review, see [15]), i.e. IgG and IgM IFA and ELISA, classical or real-time RT-PCR methods targeting specific hantaviral sero-/ genotypes followed by sequence analysis of the amplicons in order to study the molecular epidemiology of the circulating strains. Neutralisation tests are, due to the special requirements of these tests, only available in a few countries and are in general only used for research purposes.

Conclusions
Hantavirus infections continue to be a risk in the European Union. To our knowledge, notification systems have not changed in the past decade. In the past 10 years the annual number of diagnosed cases has significantly increased but it remains unclear whether this is due to higher awareness and better diagnostic tools or to a real increase in acquired infections. Epidemics occur locally and in foci, i.e. in regions where climatic, biotic and abiotic conditions pave the way for the carrier species to become abundant and humans to come in contact with the virus.
Infections caused by PUUV remain the most prevalent in Europe, and in regions where the virus is circulating, the number of infected individuals can reach hundreds or thousands per year, DOBV infections on the other hand are much less frequent, and important outbreaks are scarce. Incidence data on hantavirus infections are unfortunately not available.
The 2010 PUUV outbreak in Germany seems to be an isolated incident and is currently closely monitored by the local authorities. Further longitudinal studies are needed in Europe to better understand the factors that drive the oscillation of human cases on a local, regional and continental scale including a combination of landscape and land use, habitat, climate and geographical parameters.