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Rat-borne Seoul hantaviruses are only just being recognized globally as important human pathogens, even though they have been studied and isolated on 4 different continents (Asia, Europe, the Americas, and Africa) since the early 1980s. Recent reports from Europe and the USA described Seoul hantavirus infection in pet rats and their breeders or owners, suggesting the potential emergence of a “new” public health problem. Wild and laboratory rat-induced Seoul hantavirus infections have, however, been described since the early eighties, due to the omnipresence of the rodent reservoir, the brown rat Rattus norvegicus. Recent studies showed no fundamental differences between pathogenicity and phylogeny of pet rat-Seoul hantaviruses and their formerly described wild or laboratory rat counterparts. The paucity of diagnosed Seoul virus-induced disease in the West is in striking contrast to the thousands of cases recorded since the 1980s in the Far-East, particularly in China. This review of four continents (Asia, Europe, America, and Africa) puts this “emerging infection” in historic perspective, concluding there is an urgent need for greater medical awareness of Seoul virus-induced human pathology in many parts of the world. Given the mostly milder and atypical clinical presentation, sometimes with preserved normal kidney function, the importance of simple urine examination is stressed, since initial, massive but transient proteinuria and microhematuria are rarely lacking. With increasingly sophisticated technology and enhanced clinical awareness, many specific hantaviruses are now recognized; however, SEOV remains the globally most widely distributed hantavirus.&nbsp

Recent reports from Europe and the USA described Seoul orthohantavirus infection in pet rats and their breeders/owners, suggesting the potential emergence of a "new" public health problem. Wild and laboratory rat-induced Seoul infections have, however, been described since the early eighties, due to the omnipresence of the rodent reservoir, the brown rat Rattus norvegicus. Recent studies showed no fundamental differences between the pathogenicity and phylogeny of pet rat-induced Seoul orthohantaviruses and their formerly described wild or laboratory rat counterparts. The paucity of diagnosed Seoul virus-induced disease in the West is in striking contrast to the thousands of cases recorded since the 1980s in the Far East, particularly in China. This review of four continents (Asia, Europe, America, and Africa) puts this "emerging infection" into a historical perspective, concluding there is an urgent need for greater medical awareness of Seoul virus-induced human pathology in many parts of the world Given the mostly milder and atypical clinical presentation, sometimes even with preserved normal kidney function, the importance of simple but repeated urine examination is stressed, since initial but transient proteinuria and microhematuria are rarely lacking. ispartof: VIRUSES-BASEL vol:11 issue:7 ispartof: location:Switzerland status: published

Transient proteinuria and acute kidney injury (AKI) are characteristics of Puumala virus (PUUV) infection. Albuminuria peaks around the fifth day and associates with AKI severity. To evaluate albuminuria disappearance rate, we quantified albumin excretion at different time points after the fever onset. The study included 141 consecutive patients hospitalized due to acute PUUV infection in Tampere University Hospital, Finland. Timed overnight albumin excretion (cU-Alb) was measured during the acute phase in 133 patients, once or twice during the convalescent phase within three months in 94 patients, and at six months in 36 patients. During hospitalization, 30% of the patients had moderately increased albuminuria (cU-Alb 20&ndash, 200 &mu, g/min), while 57% presented with severely increased albuminuria (cU-Alb &gt, g/min). Median cU-Alb was 311 &mu, g/min (range 2.2&ndash, 6460) &le, 7 days after fever onset, 235 &mu, g/min (range 6.8&ndash, 5479) at 8&ndash, 13 days and 2.8 &mu, g/min (range 0.5&ndash, 18.2) at 14&ndash, 20 days. After that, only one of the measurements showed albuminuria (35.4 &mu, g/min at day 44). At six months, the median cU-Alb was 2.0 &mu, g/min (range 0.6&ndash, 14.5). Albuminuria makes a flash-like appearance in PUUV infection and returns rapidly to normal levels within 2&ndash, 3 weeks after fever onset. In the case of AKI, this is a unique phenomenon.

Genetic diversity, attributable to the low fidelity of reverse transcription, recombination and mutation, is an important feature of infectious retroviruses. Under selective pressure, such as that imposed by superinfection interference, gammaretroviruses commonly adapt their envelope proteins to use alternative receptors to overcome this entry block. The first characterized koala retroviruses KoRV subgroup A (KoRV-A) were remarkable in their absence of envelope genetic variability. Once it was determined that KoRV-A was present in all koalas in US zoos, regardless of their disease status, we sought to isolate a KoRV variant whose presence correlated with neoplastic malignancies. More than a decade after the identification of KoRV-A, we isolated a second subgroup of KoRV, KoRV-B from koalas with lymphomas. The envelope proteins of KoRV-A and KoRV-B are sufficiently divergent to confer the ability to bind and employ distinct receptors for infection. We have now obtained a number of additional KoRV envelope variants. In the present studies we report these variants, and show that they differ from KoRV-A and KoRV-B envelopes in their host range and superinfection interference properties. Thus, there appears to be considerable variation among KoRVs envelope genes suggesting genetic diversity is a factor following the KoRV-A infection process.

Background: Puumala hantavirus (PUUV) induces an acute tubulointerstitial nephritis and acute kidney injury (AKI). Our aim was to evaluate the prognosis of severe AKI associated with PUUV infection. Methods: We examined 556 patients who were treated at Tampere University Hospital during 1982-2013 for acute, serologically confirmed PUUV infection. Plasma creatinine was measured during hospitalization, convalescence, and 1, 2, and 5 years after the acute infection. Results: Plasma creatinine concentration was elevated (>100 μmol/l) in 459 (83%) patients, while altogether 189 patients (34%) had severe AKI defined as Kidney Disease: Improving Global Outcomes (KDIGO) stage 3, that is, plasma creatinine ≥353.6 μmol/l (4.0 mg/dl) or need of dialysis. There were no fatal cases during the hospitalization or the following 3 months. Fatality rate during the years following PUUV infection did not differ between patients who had suffered from severe AKI versus those without severe AKI. Post-hospitalization plasma creatinine values were available for 188 (34%) patients. One month after the acute infection, patients with prior severe AKI had higher median plasma creatinine concentration (82 µmol/l, range 54-184) than patients without severe AKI (74 µmol/l, range 55-109, p = 0.005). After 1 year, no significant difference existed in median plasma creatinine concentrations between patients with (71 µmol/l, range 36-123) and without prior severe AKI (72 µmol/l, range 34-116, p = 0.711). After 5 years all but 1 patient had normal creatinine levels. Conclusions: In contrast to the worldwide well-accepted KDIGO criteria, severe AKI associated with PUUV infection is not associated with excess fatality but has a very good prognosis, both in the short and long terms.

The European prototype of hantavirus, Puumala virus (PUUV), isolated from a common wild rodent, the bank vole (Myodes glareolus), causes nephropathia epidemica (NE). NE can perfectly mimic haemolytic-uraemic syndrome (HUS), progressing from an aspecific flu-like syndrome to acute kidney injury with thrombocytopaenia, and presenting with some signs of haemolytic anaemia and/or coagulopathy. Moreover, both NE and HUS can occur in local outbreaks. We report an isolated case of NE, initially referred for plasmapheresis for suspected HUS, although signs of overt haemolysis were lacking. Early suspicion of hantavirus infection, later confirmed by serology and reverse transcription polymerase chain reaction (RT-PCR), prevented subsequent excessive treatment modalities. ispartof: European Journal of Clinical Microbiology & Infectious Diseases vol:37 issue:1 pages:135-140 ispartof: location:Germany status: published

Dobrava-Belgrade virus (DOBV) is a human pathogen that has evolved in, and is hosted by, mice of several species of the genus Apodemus. We propose a subdivision of the species Dobrava-Belgrade virus into four related genotypes – Dobrava, Kurkino, Saaremaa, and Sochi – that show characteristic differences in their phylogeny, specific host reservoirs, geographical distribution, and pathogenicity for humans.

To the editor: The recent confirmation by RT-PCR of a case of haemorrhagic fever with renal syndrome (HFRS), induced by Tula virus (TULV) in France [1], confirms the pathogenicity of this arvicolid hantavirus, a fact not generally acknowledged yet, or at least still contested [2]. The clinical presentation of the demonstrated TULV HFRS case was, however, unusual: besides the classic fever with thrombocytopenia and elevated transaminases, leukopenia instead of leukocytosis with left shift was found, and the renal function remained strictly within normal limits. However, renal involvement was nevertheless indicated by transient microscopic haematuria. Regrettably, transient but massive and unselective proteinuria, the renal hallmark in probably all hantavirus infections, was once more not discussed. Interestingly, a false-positive serological screening result for another arvicolid hantavirus, Puumala virus (PUUV), was obtained in three assays of two different formats (immunofluorescence assay (IFA) and ELISA), but could not be confirmed by routine RT-PCR, i.e. by using PUUV-specific primers [1]. As subsequently shown by Reynes et al., TULV and PUUV are two closely related, yet genetically distinct hantavirus species, both carried by distinct voles of the Arvicolinae, a subfamily of the Cricetidae rodent family [1].

Impacts • Dynamic data-based model generates stochastic forecasts of the occurrence of nephropathia epidemica (NE) epidemics 3 months ahead. • We have demonstrated that NE outbreaks can be accurately predicted using climatic and vegetation data or bank voles' population dynamics with a dynamic data-based model. • Such a modelling approach can be used to develop new tools to help prevent future NE outbreaks. Summary Wildlife-originated zoonotic diseases in general are a major contributor to emerging infectious diseases. Hantaviruses more specifically cause thousands of human disease cases annually worldwide, while understanding and predicting human hantavirus epidemics pose numerous unsolved challenges. Nephropathia epidemica (NE) is a human infection caused by Puumala virus, which is natu- rally carried and shed by bank voles (Myodes glareolus). The objective of this study was to develop a method that allows model-based predicting 3 months ahead of the occurrence of NE epidemics. Two data sets were utilized to develop and test the models. These data sets were concerned with NE cases in Finland and Belgium. In this study, we selected the most relevant inputs from all the available data for use in a dynamic linear regression (DLR) model. The number of NE cases in Finland were modelled using data from 1996 to 2008. The NE cases were predicted based on the time series data of average monthly air tem- perature (� C) and bank voles' trapping index using a DLR model. The bank voles' trapping index data were interpolated using a related dynamic harmonic regression model (DHR). Here, the DLR and DHR models used time-varying parameters. Both the DHR and DLR models were based on a unified state-space estimation framework. For the Belgium case, no time series of the bank voles' population dynamics were available. Several studies, however, have suggested that the population of bank voles is related to the variation in seed production of beech and oak trees in Northern Europe. Therefore, the NE occurrence pat- tern in Belgium was predicted based on a DLR model by using remotely sensed phenology parameters of broad-leaved forests, together with the oak and beech seed categories and average monthly air temperature (� C) using data from 2001 to 2009. Our results suggest that even without any knowledge about hantavirus dynamics in the host population, the time variation in NE outbreaks in Finland

The connection between nephropathia epidemica (NE) and vegetation dynamics has been emphasized in recent studies. Changing climate has been suggested as a triggering factor of recently observed epidemiologic peaks in reported NE cases. We have investigated whether there is a connection between the NE occurrence pattern in Belgium and specific trends in remotely sensed phenology parameters of broad-leaved forests. The analysis of time series of the MODIS Enhanced Vegetation Index revealed that changes in forest phenology, considered in literature as an effect of climate change, may affect the mechanics of NE transmission. ispartof: International Journal of Environmental Research and Public Health vol:7 issue:6 pages:2486-2500 ispartof: location:Switzerland status: published

Hantaviruses, members of the family Bunyaviridae, are the causative agents of hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Hantaviruses are currently demarcated into species based on the guidelines provided by the International Committee on Taxonomy of Viruses (ICTV). These guidelines however, are often ignored by the descriptors of novel hantaviruses. With this study we attempted to refine the second ICTV guideline for hantavirus species demarcation by phylogenetically analyzing all in Genbank available complete sequences derived from the S, M or L segments of hantaviruses. S and M segment amino acid sequence comparison allowed clear and unequivocal distinction between different hantavirus species, and lead us to propose additional criteria for the demarcation of hantavirus species (S segment amino acid distance >10% or M segment amino acid distance >12%) and hantavirus groups (S segment amino distance >24% or M segment amino acid distance >32%). With this study, we propose to adjust the second rule of the ICTV classification guidelines ("a 7% difference in amino acid identity when comparing the complete S segment and M segment sequences") to a more appropriate rule, "a 10% difference in S segment similarity and a 12% difference in M segment similarity based on complete amino acid sequences" in accordance with the current situation in the hantavirus field.

Hantaviruses, which are mainly rodent-borne viruses, cause hemorrhagic fever with renal syndrome in the Old World, and hantavirus pulmonary syndrome in the New World. A neutralization test based on quantitative RT-PCR, the replication reduction neutralization test (RRNT), was developed for efficient detection of hantavirus-neutralizing antibodies. The effectiveness of the RRNT was evaluated by examining several hantaviruses and hantavirus-specific convalescent human serum samples. All convalescent serum samples tested by RRNT caused significant decreases in hantavirus genomes with only one specific hantavirus species, which allowed a straightforward identification of the related hantavirus. The results obtained by RRNT were completely comparable with the results obtained by focus reduction neutralization test (FRNT). The RRNT approach is a reliable and rapid alternative for FRNT, hitherto considered as the gold standard for hantavirus serology.

(2007). ACUTE KIDNEY INJURY IN EMERGING, NON-TROPICAL INFECTIONS. Acta Clinica Belgica: Vol. 62, No. 6, pp. 387-395.

SUMMARYTwo global (re-)emerging zoonoses, leptospirosis and hantavirus infections, are clinically indistinguishable. Thirty-one patients, hospitalized in Sri Lanka for acute severe leptospirosis, were after exclusion of other potentially involved pathogens, prospectively screened with IgM ELISA for both pathogens. Of these, nine (29·0%) were positive for leptospirosis only, one (3·2%) for hantavirus only, seven (22·5%) for both pathogens concomitantly, whereas 13 (41·9%) remained negative for both. Moreover, in a retrospective study of 23 former patients, serologically confirmed for past leptospirosis, six (26·0%) were also positive in two different IgG ELISA hantavirus formats. Surprisingly, European Puumala hantavirus (PUUV) results were constantly higher, although statistically not significantly different, than Asian Hantaan virus (HTNV), suggesting an unexplained cross-reaction, since PUUV is considered absent throughout Asia. Moreover, RT–PCR on all hantavirus IgM ELISA positives was negative. Concomitant leptospirosis-hantavirus infections are probably heavily underestimated worldwide, compromising epidemiological data, therapeutical decisions, and clinical outcome.

Nephropathia epidemica (NE) is a human infection caused by Puumala virus (PUUV), which is naturally carried and shed by bank voles (Myodes glareolus). The objective was to develop a dynamic model of the NE cases and the bank vole population in both Finland and Belgium by defining the periodic components with a dynamic harmonic regression (DHR) model. The defined periodic components can be further used to adapt mechanistic Susceptible and Infective (SI) models regionally. Despite the difference in bank vole population dynamics and NE cases between the Western European temperate zone and boreal zones the DHR model was able to quantify the dynamics of NE cases in Belgium and Central Finland with a coefficient of determination (R2) of 0.70 and 0.82 respectively and to quantify the dynamics of bank vole population in Belgium and Central Finland with R2 of 0.80 and 0.98 respectively. DHR identified 18 month cycles in the bank vole population in Belgium. This approach demonstrated two year cycles in Belgian NE outbreaks. DHR identified three year cycles in Finnish bank vole populations which in turn cause three year cycles in the NE outbreaks in Central Finland. Because the bank vole population data in Finland was contemporary with the data of NE cases, the DHR showed a three month delay between the NE cases and the bank vole population in Central Finland. This approach may help us in our understanding of the spatial and temporal dynamics of NE cases and the bank vole populations in different regions.

The bank vole (Myodes glareolus) is the natural host of Puumala virus (PUUV) in vast areas of Europe. PUUV is one of the hantaviruses which are transmitted to humans by infected rodents. PUUV causes a general mild form of hemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica (NE). Vector-borne and zoonotic diseases generally display clear spatial patterns due to different space-dependent factors. Land cover influences disease transmission by controlling both the spatial distribution of vectors or hosts, as well as by facilitating the human contact with them. In this study the use of ecological niche modelling (ENM) for predicting the geographical distribution of bank vole population on the basis of spatial climate information is tested. The Genetic Algorithm for Rule-set Prediction (GARP) is used to model the ecological niche of bank voles in Western Europe. The meteorological data, land cover types and geo-referenced points representing the locations of the bank voles (latitude/longitude) in the study area are used as the primary model input value. The predictive accuracy of the bank vole ecologic niche model was significant (training accuracy of 86%). The output of the GARP models based on the 50% subsets of points used for testing the model showed an accuracy of 75%. Compared with random models, the probability of such high predictivity was low (χ2 tests, p &lt, 10−6). As such, the GARP models were predictive and the used ecologic niche model indeed indicates the ecologic requirements of bank voles. This approach successfully identified the areas of infection risk across the study area. The result suggests that the niche modelling approach can be implemented in a next step towards the development of new tools for monitoring the bank vole’s population.

The antiviral activity of water-soluble dextrans derivatized with varying percentages of carboxymethyl, benzylamide, and sulfonate groups was evaluated. Several of the polymers exhibited potent antiviral activity against a variety of enveloped viruses, but not against non-enveloped viruses, and only when present during virus adsorption. The mechanism of activity against retroviruses [i.e. human immunodeficiency virus (HIV)] and herpes viruses (i.e. human cytomegalovirus) could be ascribed to inhibition of virus binding to the cells. An absolute requirement for anti-HSV activity appeared to be a sufficiently high percentage of benzylamide and benzylamide sulfonate groups. This did not, however, apply for human cytomegalovirus, respiratory syncytial virus, and HIV. The sensitivity of the latter viruses appeared to be influenced by factors other than the global chemical composition, which leads us to assume that physical factors such as the distribution and sequence of the substituents on the sugar backbone play an important role in the antiviral activity of the derivatized dextrans.

Hantavirus (HTV) is recently discovered "hemorrhagic fever virus" belonging to the Bunyaviridae family, which is spread throughout the world by wild rodents and/or laboratory rats. During an epidemic in the Belgian-French Ardennes in 1993, more than 200 acute cases were recorded of the milder European form of HTV-illness, otherwise known as Nephropathia epidemica (NE). This variant may be recognized by the sudden onset of fever, acute renal failure, thrombocytopenia and sometimes by ophthalmologic complications. The symptomatology is rather aspecific and diagnosis can only be confirmed by serologic tests, of which the best option nowadays seems to be: screening by IgG EIA, followed by IgM confirmation with a mu-capture EIA test. Some of the tests described allow an evaluation of the causative serotype or even the moment of infection. Next to the "classic" serologic assays for detection of specific viral antibodies, we describe briefly our own experience with newer tests such as "high density particle agglutination" and "line immuno assay". Polymerase chain reaction for viral RNA genome typing and immunohistochemical colouring of the viral antigen in tissues seem to offer promising alternatives for the immediate future.

The strong correlation between environmental conditions and abundance and spatial spread of the tick Ixodes ricinus is widely documented. I. ricinus is in Europe the main vector of the bacterium Borrelia burgdorferi, the pathogen causing Lyme borreliosis (LB). Humidity in vegetated systems is a major factor in tick ecology and its effects might translate into disease incidence in humans. Time series of two remotely sensed indices with sensitivity to vegetation greenness and moisture were tested as explanatory variables of LB incidence. Wavelet-based multiresolution analysis allowed the examination of these signals at different temporal scales in study sites in Belgium, where increases in LB incidence were reported in recent years. The analysis showed the potential of the tested indices for disease monitoring, the usefulness of analyzing the signal in different time frames and the importance of local characteristics of the study area for the selection of the vegetation index.

Since, to our knowledge, no clinically documented cases of haemorrhagic fever with renal syndrome (HFRS) have been reported in Northern Ireland, a sero-epidemiological study was carried out to assess the degree of Hantavirus immunity in a group of 627 Northern Irish patients presenting with symptoms suggestive of HFRS and 100 healthy controls. IFA screening for IgG Hantavirus specific antibodies was carried out with a panel of up to 9 different Hantaviral antigens. IgM screening was performed using a commercially available ji-capture ELISA based upon two recombinant Hantaviral nucleocapsid antigens. A seropositivity of 2.1% (15/727) was recorded, with an almost exclusive reaction against a rat-derived R22VP30 strain of the Seoul serotype. Sole reliance upon non-rat-derived classic screening antigens Hantaan (HTN 76–118) and Puumala (CG 18–20) would have resulted in the detection of only 2/15 (13.3%) of cases in IgG IFA, and 8/14 (57.1%) of the cases in IgM ELISA. The findings indicate that for the first time in Europe, and more specifically in N. Ireland, non-laboratory outbreaks of HFRS may be caused by wild rats acting as a reservoir for a Seoul-like Hantavirus. Conventional Hantavirus serology using Hantaan and Puumala as screening antigens does not appear sufficient for the detection of such cases of HFRS. Hence, we propose the addition of a rat derived Hantaviral antigen to the antigen screening panel as a means of improving the specificity of the detection methods. © 1994 Wiley-Liss, Inc.

Sera from Dutch and Belgium individuals who suffered from nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome (HFRS), were tested for the distribution of classes and subclasses of Hantavirus (HV)-specific antibodies at different times after the onset of the disease, with class- and subclass-specific Ig capture enzyme-linked immunosorbent assays (ELISAs). In the acute, early convalescent, and convalescent phases, predominantly specific IgA, IgM, and IgG3 antibodies were detected. Specific IgG2 antibodies were only detected at low levels in the early convalescent and convalescent phases. In the late convalescent phase specific IgG1 and IgG3 antibodies were found, whereas in the late postconvalescent phase only specific IgG1 antibodies proved to be present. Specific IgG4 antibodies were not detected in any of the respective phases. These data show that the simultaneous determination of classes and subclasses of HV specific serum antibodies allows the estimation of the time elapsed after the onset of NE.

Hantaviruses were first described in Korea in 1978, and are an important group of “new” pathogens, now constituting the most widely distributed zoonotic (i.e. transmitted from vertebrated animals to humans) viruses on earth. Hantaviruses are “emerging” viruses, and are now confirmed as being the only viral haemorrhagic fever agents with a worldwide distribution, including even the temperate Northern hemisphere (Clement et al. 2007a). They are spread by wild rodents (and perhaps also by insectivores), infecting man via their aerosolized but infectious excreta. So far, at least 23 different hantavirus species are officially recognized, each with its own main rodent reservoir, and hence specific geographical spread (Maes et al, 2009). The most important pathogens are Hantaan and Seoul virus in Asia, Puumala and Dobrava virus in Europe and West-Russia, and Sin Nombre and Andes virus in the Americas. American hantaviruses, discovered only in 1993, affect mainly the human lung and cause the “hantavirus cardiopulmonary syndrome” (HCPS), with a fatality rate of about 35%. By contrast, all Old World hantaviruses are targeted mainly to the human kidney resulting in the “haemorrhagic fever with renal syndrome” (HFRS), an often epidemic form of acute kidney injury (AKI), resulting in a rapidly progressive, but ultimately self-remitting acute renal failure (Clement et al., 2007a, 2007b). Among hantaviral pathogens, the European Puumala virus (PUUV) is the least severe. It causes an infection, aptly named “nephropathia epidemica” (NE), which in fact is a mild form of HFRS, with a very low fatality rate of 0.5-0.1 %. However, more severe cases do occur, resulting in multiorgan involvement, necessitating sometimes live-saving acute haemodialysis and/or mechanical lung ventilation in intensive care settings (Clement et al., 1994a, 2007a, 2007b).

Nephropathia epidemica (NE) is a human infection caused by Puumala virus (PUUV), which is naturally carried and shed by bank voles (Myodes glareolus). Population dynamics and infectious diseases in general, such as NE, have often been modelled with mechanistic SIR (Susceptible, Infective and Remove with immunity) models. Precipitation and temperature have been found to be indicators of NE, however most SIR models do not take them into account. The objective of this paper was to develop a dynamic model of incidences of NE in Belgium by taking into account climatological data. A multiple–input, single-output (MISO) transfer function was used to model the incidence of NE. In a first step, the NE cases were modelled based on data from 1996 until 2003 with an R T 2 of 0.68. In the next step the MISO model was validated for incidences of NE in Belgium from 2003 to 2008 ( R T 2 of 0.54). The output of the MISO models was the number of NE cases in Belgium over the time period 1996 until 2008 and the inputs were average measured monthly precipitation (mm), and temperature (°C) as well as estimated carrying capacity (vole ha−1). The monthly values of carrying capacity (K) were estimated for the whole period by using an existing mechanistic SIR model. K is related to the variation in seed production in Northern Europe, which has an effect on the population of bank voles. In the future, such modelling approaches may be used to predict and monitor forthcoming NE cases based on climate and vegetation data as a tool for prevention of NE cases.

An enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of serum antibodies against group-specific epitopes of the glycoproteins (G1, G2) and nucleoprotein (NP) of the genus Hantavirus. This assay was used to study the kinetics of the development of serum antibodies after natural infection with Puumala-like virus in humans. To this end a panel of 34 serum samples collected from individuals at different times after natural infection was tested by the ELISA. The samples were also tested for specific IgM and IgG levels against Puumala-like virus, which provided confirmatory data about the presumed timing of infection. It was shown that serum antibodies against the G1 epitope were present in the acute and early convalescent period just before antibodies to the NP epitope could be demonstrated. In contrast, antibodies to two G2 epitopes were present not earlier than in the convalescent and late convalescent period. Since all these categories of antibodies seem to persist for long periods, antibodies against the G1 epitope and the NP epitope may be of specific diagnostic value. Furthermore, levels of G1-specific antibodies and antibodies to either NP or G2 may allow estimation of the time elapsed following initial infection.

We report a case of hantavirus infection (nephropathia epidemica) diagnosed in a Belgian backpacker returning from a trekking expedition in Ecuador, after likely heavy exposure to rodents. Because of epidemiological inconsistency, molecular investigation was performed and revealed a Puumala infection acquired during very limited exposure in Belgium upon return.

Rodent-borne hantaviruses are associated with two main clinical disorders in humans: hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Although hantavirus diseases can be life threatening and numerous research efforts are focused on the development of hantavirus prevention, no specific antiviral therapy is yet available and, at this time, no WHO-approved vaccine has gained widespread acceptance. This review will summarize the current knowledge and recent progress as well as new speculative approaches in the development of hantavirus vaccines.

In Europe, Puumala virus and Dobrava virus are the major hantaviruses that cause hemorrhagic fever with renal syndrome in humans. As hantaviruses can cause diseases with high morbidity and mortality rates, and as to date there is no specific treatment, efforts are concentrated on the development of vaccines. In this study we characterized the immunogenicity of recombinant nucleocapsid proteins of Puumala virus (PUUV) linked to a carrier protein corresponding to the outer membrane protein A from Klebsiella pneumoniae (rP40). The rP40 molecule is a novel carrier protein that facilitates exogenous antigen uptake by dendritic cells. We cloned and expressed the recombinant PUUV proteins in the E. coli mutant ICONE 200 using the tryptophan promoter-controlled pTEXmp18 expression vector. All recombinant PUUV proteins were found to be highly immunogenic in NMRI mice after three immunizations of 10 microg each of the protein. Only the truncated construct, P40-Puu118, gave high antibody titers after two vaccinations of 0.2 microg each. Likewise in the challenge experiments in NMRI mice, only the truncated construct P40-Puu118 resulted in 100% protection after three immunizations of 10 microg each. The results suggest that P40-Puu118 in particular is a good candidate for a recombinant vaccine against Puumala virus. All recombinant proteins linked to rP40 induced high antibody responses, indicating that rP40 is a carrier protein with potential for use in other vaccines.

To the Editor: In a recent letter (1), dos Santos et al. described 3 cases of hantavirus pulmonary syndrome (HPS) from Juquitiba and stated that “the first human cases of symptomatic infection by hantaviruses were reported from Brazil in 1993.” However, we described 8 cases of symptomatic hemorrhagic fever with renal syndrome (HFRS) in Recife, Brazil, 5 months before the initial May 1993 report of Sin Nombre virus (SNV)–induced HPS in the United States (2). Our report was therefore the first published account of symptomatic hantavirus infections, not just in Brazil but anywhere in the Americas (3). Serum samples from our Brazilian HFRS cases, collected in 1990, were screened by an immunofluorescence assay (IFA) and ELISA for immunoglobulin G, as were the current Brazilian HPS cases (1). Two of our patients had an increased immunoglobulin M titer by ELISA (2). Rat-transmitted Seoul virus (SEOV) was considered most likely because this was the only hantavirus strain showing clear positive results in IFA (2,3). All the Recife cases in 1990 had reported likely rat contacts and were initially diagnosed as leptospirosis with acute renal failure and thrombocytopenia, clinical hallmarks of both HFRS and leptospirosis (3). We also subsequently found evidence of SEOV infection in 31 (15%) of 201 leptospirosis-suspected acute renal failure cases from Belem, Brazil, confirmed in 1 case with highly specific neutralization tests (4). Moreover, as we predicted (3), some of the 133 (41%) of 326 urban cases of acute renal failure from Salvador, Brazil, which appeared nonconfirmed for leptospirosis (5), were later shown to be caused by SEOV (unpub. data). Finally, of 379 schoolchildren from Salvador at high risk for frequent rat exposure, 13.2% were IFA positive for the Korean prototype Hantaan virus (HTNV) but none for the American SNV (6). Because both HTNV and its rodent reservoir are absent from the American biotope, HTNV seroreactivity should be considered a cross-reaction to another related murine antigen; that is to say, the ratborne SEOV. Wild rats (Rattus rattus and R. norvegicus ) are the only Old World rodents ubiquitous in the New World and thus a potential source of SEOV infection in the Americas (3,7). Moreover, the first hantavirus characterized in South America was SEOV, isolated as long ago as 1984 from a rat caught in Belem (7). Furthermore, the first 3 clinical cases of hantavirus infection in the United States were SEOV-induced (Baltimore rat virus) HFRS cases and not HPS (8). The clinical syndromes of HFRS and HPS can appear identical, with pulmonary edema, shock, and renal insufficiency with marked proteinuria and thrombocytopenia (9). Moreover, worldwide ELISA testing with a single antigen such as SNV or Puumala virus (PUUV) can result in misleading cross-reactions, since both viruses are genetically related. Although this SNV-PUUV cross-reactivity enabled the first recognition of HPS cases in the New World 13 years ago, this may now lead to the wrong clinical diagnosis and reinforces the need for specific tests such as neutralization tests or reverse transcription–PCR. Although not as lethal and probably not so frequent as HPS, SEOV-induced HFRS may still be greatly underestimated in the Americas, or misdiagnosed as leptospirosis.

Severe human infection with Hantavirus is characterized by high fever, cold chills, thrombocytopenia, arterial hypotension, acute renal failure, and/or adult respiratory distress syndrome (ARDS)-like pulmonary involvement, but the clinical course varies greatly between individuals. We investigated whether genetically determined differences in tumor necrosis factor (TNF)-alpha production can influence the severity of Hantavirus disease. We studied a TNF-alpha single-nucleotide promoter polymorphism (SNP) at position -238 (a guanine [G]-to-adenine [A] transition) and ex vivo TNF-alpha production in a recall study of 36 Belgian patients who had a serologically proven form of Puumala virus-induced Hantavirus infection with the kidney as main target organ. In our study, the highest creatinine levels were found in patients with the lowest ex vivo TNF-alpha production. Creatinine levels correlated inversely with TNF-alpha production (R = -0.35, p < 0.05). The number of thrombocytes was significantly lower in patients with the GA-238 genotype (low TNF-alpha producers) compared with patients with the GG-238 genotype. In our study, genetically determined low production of TNF-alpha was associated with some parameters indicating a more severe clinical course of Puumala Hantavirus infection in humans, possibly by impaired activation of TNF-alpha-dependent antiviral mechanisms, which could in turn result in decreased clearance of Hantavirus.

Puumala virus, a hantavirus belonging to the Bunyaviridae family, causes a human disease known as nephropathia epidemica, a mild form of hemorrhagic fever with renal syndrome. The implementation of effective decontamination procedures is critical in hantavirus research to minimize the risk of personnel exposure. This study investigated the efficacy of Clidox((R)), Dettol((R)), ethanol, Halamid-d((R)), peracetic acid, sodium hypochloride and Virkon((R))S for inactivating Puumala virus. A real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to quantify Puumala virus before and after treatment with these products. Inactivation of Puumala virus was effective after 10min with all products except ethanol. Inactivation with absolute ethanol was effective only after 30min. Using the qRT-PCR method, this study has shown that the commercially available products Clidox((R)), Halamid-d((R)) and Virkon((R))S in particular represent a rapid and safe way to decontaminate surfaces with possible Puumala virus contamination. These products can be used in solutions of 1-2%, with contact times greater than 10min, for inactivating effectively Puumala virus.

Publisher Summary Hantavirus is a genus in the Bunyaviridae family, comprising more than 30 different hantavirus (HTV) species. HTV is transmitted to humans by rodent excretions and aerosols thereof, consisting of infectious respiratory and urinary droplets from apparently healthy rodent carriers. Infected rodents excrete infectious virus in saliva, urine, faeces, and from their lungs, for the duration of their lives. Person-to-person transmission of HTV appears to be extremely rare. There are no effective treatments for HTV infections except ribavirin, a nucleoside analogue, and that should be given only early and intravenously in the clinical course. Thus, its use is limited to quickly recognized (for example, during an epidemic) and severe forms of disease.

We describe the cloning and expression of Dobrava hantavirus (DOBV) nucleocapsid proteins and a truncated form consisting of the first 118 N-terminal amino acids, and the capacity of these E. coli ICONE 200-expressed recombinant proteins (rNp) to induce a protective immune response against DOBV in mice. As an alternative carrier protein, the outer membrane protein A derived from Klebsiella pneumoniae (rP40) has been coupled to different rNp constructs. All recombinant proteins were found to be highly immunogenic after three immunizations of rNp. The immunizations resulted in the induction of a strong Np-specific IgG response with a predominance of IgG1 over IgG2b and IgG2a, suggesting a mixed Th1/Th2 cell involvement. A specific IgG3 response could not be detected. Mice immunized with recombinant DOBV rNp without rP40 showed lower nucleocapsid-specific antibody responses in comparison with the rP40-conjugated constructs, but all mice were found to be protected against DOBV challenge. Our results indicate that the rNp constructs coupled to rP40, represent promising vaccine candidates.

A 37-year-old Belgian patient presented with acute nephropathia epidemica (NE) shortly after a camping holiday in southern France. Unusual symptoms were initial noncardiogenic lung involvement, followed by severe acute renal failure, acute acalculous cholecystitis, presence of immunoblasts in the bone marrow, and hemolytic anemia, presenting as hemolytic uremic syndrome. Positive immunoglobulin (Ig) A and rising IgG titers against Puumala hantavirus (PUUV) were detected, but IgM remained negative on days 8 and 20. The results of reverse-transcriptase-polymerase chain reaction performed on day 8 were positive for PUUV. This is the first report of an iatrogenically IgM-negative hantavirus case due to the selective removal of heavy-weight molecules during plasma exchange via the centrifugation technique. This is also the first report of proven NE from the Mediterranean part of France.

Puumala is the most common hantavirus serotype in Europe and is spread mainly by the red bank vole. Between 1 July 1992 and 31 January 1994, an outbreak of Puumala virus-induced nephropathia epidemica (NE) occurred in the Belgian Ardennes. Serologically confirmed cases (n = 41) were compared with two groups of asymptomatic seronegative controls. Risks identified included sighting of living rodents, exposure to rodent droppings, and trapping rodents during the 4 weeks preceding onset of symptoms. Activities during this 4-week period that presented the greatest risk were woodcutting, reopening of a nonaerated room, and strenuous physical effort. This is the first case-control study on risk factors for NE in Europe. In comparison with the American form of hantavirus pulmonary syndrome, which is spread by deer mice, professional activity appears to be a more important risk factor for acquisition of hantavirus in Europe.

Dear Editor, We read with great interest the paper of C. Strady et al. on leptospirosis and nephropathia epidemica (NE) in NorthEastern-France [1]. Herein, and as questioned already before [2], the authors refer once more to the French Institut Pasteur Humain or IPH strain 90-13 for screening Puumala virus (PUUV)-induced NE cases in France with IFA and/or ELISA. Isolation of this strain, announced as the first human PUUV isolate in France and in West-Europe, was published in 1995, and complete sequencing of its medium (M) RNA segment, respectively small (S) RNA segment was introduced in GenBank under respective accession numbers U22418 and U 22423 [3]. From the moment of its reported isolation in 1990, IPH 90-13 was acknowledged being more sensitive than other, nonautochthonous PUUV strains for picking up local French NE cases in IFA format [4]. However, a remarkable similarity was noted with CH 13891, a Belgian rodent strain already in use [4]. CH 13891 had been sent in 1987 by one of us (G vd G) to Pasteur Institute, Paris, in the frame of a Franco-Belgian collaboration to study emerging NE, and had been used successfully since then for serodiagnosing French NE cases, likewise in an IFA format [5]. This strain had been isolated in 1985 from a bank vole captured in the early ‘80s around Turnhout, North-Belgium [6], thus constituting in fact the earliest West-European PUUV isolate, available one year after Sotkamo, the prototype PUUV strain from Finland, and Hallnas from Sweden, both isolated in 1984. Moreover, the very first (1983) Belgian NE cases had been already serodiagnosed in IFA with this antigen, then called “Turnhout HFRS antigen” [7]. However, and as with so many other early hantavirus isolates (including also the Finnish Sotkamo) from the ‘80s pioneer times, CG 13891 was introduced in GenBank only years after a first characterisation, in this case finally in 1999. The Belgian rodent strain, isolated 5 years prior to the putative French human strain, appeared to have a 100% identical S and M RNA sequence pattern. The French and American authors of the original IPH 90-13 report concluded duly that a contamination with the Belgian CG 13891 had occurred earlier in the Paris lab, and published two successive Errata [8, 9]. They also corrected the GenBank situation, re-stating under accession numbers U22418 and U 22423 that “Sequence entry was originally reported as strain 90-13, but later found to be a laboratory contamination of strain CG 13891”. Nevertheless, many French clinicians and even serological labs continue nowadays to use the IPH 90-13 denomination for their serological diagnosis, thus referring in fact to a strain that is proven since 1999 to be non-existent [2]. In summary, and as confirmed again in the current study [1], the Paris and subsequently the Lyon reference labs for hemorrhagic fevers have been using from 1987 on CG 13891 in an IFA format, before adding from 2000 on an ELISA format, and later even a recombinant ELISA form, all derived from the same Belgian strain. In a technical paper describing the J. Clement (*) : P. Maes :M. Van Ranst Hantavirus Reference Centre, Laboratory of Clinical Virology & Rega Institute for Medical Research, University of Leuven, U.Z. Gasthuisberg, Herestraat, 49, B-3000 Leuven, Belgium e-mail: jan.clement@uzleuven.be

Several fox vaccination campaigns against rabies have been undertaken in Belgium by using a vaccinia-rabies recombinant virus distributed in baits in the field. However, foxes and other wild animals that may ingest the baits could be infected at the same time by another orthopoxvirus, such as cowpox virus, which circulates in wildlife. Recombination between the two viruses could therefore occur. A serological survey for antibodies to orthopoxvirus, and particularly to cowpox virus, was undertaken in foxes and in several other wild species. Antibodies were detected only in two rodent species, in 16 of 25 bank voles (64 per cent) and in two of 29 woodmice (7 per cent). The risk of virus recombination in wildlife can therefore be considered to be extremely low.

The significance of IgG antibody levels determined by a binding assay (ELISA) was investigated as a surrogate marker for the presence of neutralizing and hemagglutination inhibiting antibodies in sera from individuals vaccinated against tick-borne encephalitis (TBE). To assess the extent of interference by flavivirus cross-reactive antibodies, sera from persons with a proven or suspected history of other flavivirus infections and/or vaccinations were also examined. An excellent and highly significant correlation was found between ELISA IgG units and the antibody titers obtained by the hemagglutination inhibition (HI) as well as by the neutralization test (NT), provided that there was no other exposure to flavivirus antigens except TBE vaccination. Yellow fever vaccination and/or dengue virus infections induced significant levels of antibodies reactive in the TBE ELISA and HI test, which did not exhibit, however, neutralizing activity against TBE virus. The phenomenon and problem of "original antigenic sin" was demonstrated in a TBE vaccinee with a history of previous flavivirus infections. TBE vaccination first induced a booster reaction resulting in a rise in the level of cross-reactive antibodies only, whereas TBE virus-neutralizing antibodies became detectable only after the third vaccination. It is concluded that the level of IgG antibodies determined by ELISA is a good marker for predicting the presence of neutralizing antibodies after TBE vaccination, but only in the absence of flavivirus cross-reactive antibodies. Otherwise, a neutralization assay is necessary for assessing immunity.

SummaryA serological survey for the prevalence of hantavirus infections in The Netherlands was carried out on > 10000 sera, from selected human populations, and different feral and domestic animal species. Hantavirus-specific antibodies were found in about 1% of patients suspected of acute leptospirosis, 10% of patients with acute nephropathia, and in less than 0·1% haemodialysis and renal transplant patients. Among individuals with a suspected occupational risk, 6% of animal trappers, 4% of forestry workers, 2% of laboratory workers and 0·4% of farmers were seropositive. The majority of the seropositive individuals lived in rural and forested areas. The main animal reservoir of the infection was shown to be the red bank vole (Clethrionomys glareolus). Epidemiological, clinical and laboratory findings seen in serologically confirmed human cases were similar to those associated with nephropathia epidemica.

Serum nitrate levels, a measure of nitric oxide (NO) production in vivo, were very high (95 +/- 14 microM) in 13 patients infected with Puumala virus, the European variant of Hantavirus (HTV), as compared to those in healthy subjects (33 +/- 3 microM). Serum nitrate levels showed a high and significant correlation with scores on the Acute Physiological And Chronic Health Evaluation (APACHE II) scale and with serum creatinine, and an inverse correlation with platelet counts. Serial serum measurements of nitrate in 2 severe cases showed very high levels at the onset of arterial hypotension and acute renal failure. We conclude that excessive amounts of NO are produced in patients infected with Puumala virus and that this reactive nitrogen intermediate could play an important role in the pathogenesis of the disease.

In Europe, the TBE-flavivirus (particularly the Western subtype CEE or Central European encephalitis-virus) and the Borrelia burgdorferi (Bb)-spirochete are maintained in the same wild rodent reservoirs (Clethrionomys glareolus [red bank vole], Apodemus sylvaticus [woodmouse] and Apodemus flavicollis [yellow necked field mouse] (de Boer et al, 1993),(Rodhain et al, 1987). These viral or bacterial entities are also transmitted to man by the same vector, namely a tick (Ixodes ricinus). Persons involved in open field activities, particularly in forested regions are at potential risk of infection with both of these illnesses since contact with ticks is more probable.

Introduction Nephropathia epidemica is a Rodent-borne disease. Changing climate has been suggested as a triggering factor of recently observed epidemiologic peaks in reported (NE). We aimed at investigating whether there is a connection between the temporal pattern in NE occurrence in Belgium and specific trends in remotely sensed phaenology parameters of broad-leaved forests together with the oak and beech seed categories and the North Atlantic Oscillation (NAO). Methodology The NE cases are higher in summer time than in winter time and it has a clear seasonal component. In order to the two environmental variables to quantify the dynamics of NE cases we used a dynamic harmonic Regression (DHR) model of the time series of monthly NE cases from 1996 to 2008. The selected variables were then used in a next step as inputs in multiple-inputs single-output (MISO) transfer function model to describe the NE dynamics as a function of climate and vegetation dynamics. Results Based on the seasonal and cyclic component of NE the NAO index and oak and beach seed production categories were selected as an inputs for the MISO model. The MISO model was built to describe the incidence of NE cases from 2005 to 2008 (R 2 of 0.89). Conclusion The results of the present study support defining the significant environmental factors explaining the spread and dynamics of the disease. In this way, a first step is made towards a tool that allows monitoring and predicting the NE cases.

Serum samples were collected from 27 individuals who had been infected with a member of the genus Hantavirus in the Netherlands or Belgium during the last 15 years. These samples were tested in an immunofluorescence assay (IFA) and two enzyme-linked immunosorbent assay (ELISA) systems, using different virus strains that represented each of the four recently proposed serotypes of this genus. The serum samples from 11 individuals who had been infected through contacts with laboratory rats showed the highest reactivities with Hantaan virus (serotype I) and SR-11 (serotype II) in the IFA and ELISA systems. The samples of 16 individuals who had probably been infected through contacts with wild rodents showed the highest reactivities with Hallnas virus (serotype III) in the IFA. All except two of these also showed the highest reactivity with Hallnas virus in the two different ELISA systems.

Puumala hantavirus is the most common human hantavirus infection in Europe. 1 2 It is transmitted to humans by inhalation or contamination of skin breaches by urine or faeces of infected bank voles. Infection ranges from subclinical to a severe influenza-like illness progressing to acute renal failure.3 We carried out a case-control study in an endemic area in France and Belgium to estimate knowledge of hantavirus and identify possible risk factors for infection. National reference laboratories in each country identified cases for the study. A case was defined as someone with laboratory confirmed IgM positive Puumala hantavirus infection between 1 April 1996 and 31 July 1996 in the French departments Nord, Ardennes, and Aisne and Belgian provinces of Hainaut, Namur, and Luxembourg. Controls were matched by sex, community (village), and age group. They were randomly selected from the telephone book. Interviews were carried out by telephone using a standardised questionnaire covering knowledge …