Inhibition of interferon I induction by non-structural protein NSs of Puumala virus and other vole-associated orthohantaviruses: phenotypic plasticity of the protein and potential functional domains.

The orthohantavirus Puumala virus (PUUV), which is transmitted by bank voles (Clethrionomys glareolus), and other vole-borne hantaviruses contain in their small (S) genome segment two overlapping open reading frames, coding for the nucleocapsid protein and the non-structural protein NSs, a putative type I interferon (IFN-I) antagonist. To investigate the role of NSs of PUUV and other orthohantaviruses, the expression pattern of recombinant NSs constructs and their ability to inhibit human IFN-I promoter activity were investigated. The NSs proteins of PUUV and related cricetid-borne orthohantaviruses showed strong inhibition of IFN-I promoter induction. We identified protein products originating from three and two methionine initiation codons in the NSs ORF of PUUV during transfection and infection, respectively. The three putative start codons are conserved in all PUUV strains analysed. Translation initiation at these start codons influenced the inhibitory activity of the NSs products, with the wild-type (wt) construct expressing two proteins starting at the first and second methionine and showing strong inhibition activity. Analysis of in vitro-generated variants and naturally occurring PUUV NSs proteins indicated that amino acid variation in the NSs protein is well tolerated, suggesting its phenotypic plasticity. The N-terminal 20-amino-acid region of the NSs protein was found to be associated with strong inhibition and to be highly vulnerable to amino acid exchanges and tag fusions. Infection studies using human, bank vole, and Vero E6 cells did not show obvious differences in the replication capacity of PUUV Sotkamo wt and a strain with a truncated NSs protein (NSs21Stop), showing that the lack of a full-length NSs might be compensated by its N-terminal peptide, as seen in transfection experiments. These results contribute to our understanding of virus-host interactions and highlight the importance of future innate immunity studies in reservoir hosts.

Circulating Extracellular Vesicle Tissue Factor Activity During Orthohantavirus Infection Is Associated With Intravascular Coagulation.

BACKGROUND: Puumala orthohantavirus (PUUV) causes hemorrhagic fever with renal syndrome (HFRS). Patients with HFRS have an activated coagulation system with increased risk of disseminated intravascular coagulation (DIC) and venous thromboembolism (VTE). The aim of the study was to determine whether circulating extracellular vesicle tissue factor (EVTF) activity levels associates with DIC and VTE (grouped as intravascular coagulation) in HFRS patients. METHODS: Longitudinal samples were collected from 88 HFRS patients. Patients were stratified into groups of those with intravascular coagulation (n = 27) and those who did not (n = 61). We measured levels of circulating EVTF activity, fibrinogen, activated partial prothrombin time, D-dimer, tissue plasminogen activator (tPA), plasminogen activator inhibitor 1 (PAI-1), and platelets. RESULTS: Plasma EVTF activity was transiently increased during HFRS. Levels of EVTF activity were significantly associated with plasma tPA and PAI-1, suggesting that endothelial cells could be a potential source. Patients with intravascular coagulation had significantly higher peak EVTF activity levels compared with those who did not, even after adjustment for sex and age. The peak EVTF activity value predicting intravascular coagulation was 0.51 ng/L with 63% sensitivity and 61% specificity with area under the curve = 0.63 (95% confidence interval, 0.51-0.76) and P = .046. CONCLUSIONS: Plasma EVTF activity during HFRS is associated with intravascular coagulation.

Host-Associated Absence of Human Puumala Virus Infections in Northern and Eastern Germany.

Human hantavirus disease cases, caused by Puumala virus (PUUV), are mainly recorded in western and southern areas of Germany. This bank vole reservoir survey confirmed PUUV presence in these regions but its absence in northern and eastern regions. PUUV occurrence is associated with the presence of the Western bank vole phylogroup.

Spatial prediction and validation of zoonotic hazard through micro-habitat properties: where does Puumala hantavirus hole - up?

BACKGROUND: To predict the risk of infectious diseases originating in wildlife, it is important to identify habitats that allow the co-occurrence of pathogens and their hosts. Puumala hantavirus (PUUV) is a directly-transmitted RNA virus that causes hemorrhagic fever in humans, and is carried and transmitted by the bank vole (Myodes glareolus). In northern Sweden, bank voles undergo 3-4 year population cycles, during which their spatial distribution varies greatly. METHODS: We used boosted regression trees; a technique inspired by machine learning, on a 10 - year time-series (fall 2003-2013) to develop a spatial predictive model assessing seasonal PUUV hazard using micro-habitat variables in a landscape heavily modified by forestry. We validated the models in an independent study area approx. 200 km away by predicting seasonal presence of infected bank voles in a five-year-period (2007-2010 and 2015). RESULTS: The distribution of PUUV-infected voles varied seasonally and inter-annually. In spring, micro-habitat variables related to cover and food availability in forests predicted both bank vole and infected bank vole presence. In fall, the presence of PUUV-infected voles was generally restricted to spruce forests where cover was abundant, despite the broad landscape distribution of bank voles in general. We hypothesize that the discrepancy in distribution between infected and uninfected hosts in fall, was related to higher survival of PUUV and/or PUUV-infected voles in the environment, especially where cover is plentiful. CONCLUSIONS: Moist and mesic old spruce forests, with abundant cover such as large holes and bilberry shrubs, also providing food, were most likely to harbor infected bank voles. The models developed using long-term and spatially extensive data can be extrapolated to other areas in northern Fennoscandia. To predict the hazard of directly transmitted zoonoses in areas with unknown risk status, models based on micro-habitat variables and developed through machine learning techniques in well-studied systems, could be used.

Interferons Induce STAT1-Dependent Expression of Tissue Plasminogen Activator, a Pathogenicity Factor in Puumala Hantavirus Disease.

Hantaviruses are zoonotic viruses that show various degrees of vasculopathy in humans. In this study, we analyzed the regulation of 2 fibrinolytic parameters, tissue plasminogen activator (tPA) and its physiological inhibitor, plasminogen activator inhibitor 1 (PAI-1), in Puumala hantavirus (PUUV)-infected patients and in human microvascular endothelial cells. We detected strong upregulation of tPA in the acute phase of illness and in PUUV-infected macaques and found the tPA level to positively correlate with disease severity. The median levels of PAI-1 during the acute stage did not differ from those during the recovery phase. In concordance, hantaviruses induced tPA but not PAI-1 in microvascular endothelial cells, and the induction was demonstrated to be dependent on type I interferon. Importantly, type I and II interferons directly upregulated tPA through signal transducer and activator of transcription 1 (STAT1), which regulated tPA gene expression via a STAT1-responsive enhancer element. These results suggest that tPA may be a general factor in the immunological response to viruses.

Smoking is associated with aggravated kidney injury in Puumala hantavirus-induced haemorrhagic fever with renal syndrome.

BACKGROUND: Previous studies indicate that smoking affects the outcome of some infections and is a risk factor for Puumala virus (PUUV) infection. The aim of this study was to assess the effect of smoking on the clinical severity of PUUV infection and the prevalence of smoking in patients with PUUV infection. METHODS: A questionnaire on smoking habits was sent to 494 patients in 2012, who had been treated in Tampere University Hospital, Finland, for serologically confirmed PUUV infection during years 1982-2012. RESULTS: Of all patients, 357 (72%) participated. Maximum plasma creatinine level measured during acute illness was significantly higher in current smokers than in non-smokers (median: 273 versus 184 µmol/L, P < 0.001). Current smokers had a higher maximum blood leucocyte count than non-smokers (median: 10.8 versus 8.9 × 10(9)/L, P < 0.001) and they were younger than non-smokers (38 versus 45 years, P < 0.001). There were no differences between current smokers and non-smokers in the other variables reflecting the severity of PUUV infection. Altogether 51% were current smokers at the time of onset of the illness, 57% of males and 36% of females. During these years in Finland, smoking among males in the same aged population has decreased from 33 to 22% and among females, smoking has varied between 14 and 20%. CONCLUSIONS: Smoking is common in patients with PUUV infection. Current smokers suffer from more severe acute kidney injury (AKI) and they have higher leucocyte count than non-smokers in PUUV infection. Smoking cessation decreases the risk of severe AKI to the same level as observed in never-smokers.

Urinary Neutrophil Gelatinase-Associated Lipocalin (NGAL) and proteinuria predict severity of acute kidney injury in Puumala virus infection.

BACKGROUND: Nephropathia epidemica (NE) is a mild form of hemorrhagic fever with renal syndrome (HFRS) that is caused by the Puumala virus. Periodic outbreaks have been described in endemic areas, with a substantial number of previously healthy individuals developing acute kidney injury (AKI). There is a considerable diversity in the clinical course of the disease, and few patients require renal replacement therapy. METHODS: We tested whether urinary neutrophil gelatinase associated lipocalin (uNGAL), urine albumin/creatinine ratio (uACR), urine protein/creatinine ratio (uPCR), urine dipstick protein, C-reactive protein, procalcitonin, leukocyte and platelet count, determined on admission to the hospital, can predict the severity of AKI. Sixty-one patients were analyzed during admission in the emergency department. RESULTS: The variables most strongly associated with peak plasma creatinine concentration were uNGAL (ß = 0.70, p <0.0001), uPCR (ß = 0.64, p = 0.001), uACR (ß = 0.61, p = 0.002), and dipstick proteinuria (ß = 0.34, p = 0.008). The highest AUC-ROC to predict stage 3 AKI according to the acute kidney injury network's (AKIN) classification was seen for uNGAL (0.81, p = 0.001). CONCLUSION: uNGAL accurately predicts the severity of AKI in NE. This could help emergency room physicians predict disease severity and allow for initial risk stratification.

Landscape features and helminth co-infection shape bank vole immunoheterogeneity, with consequences for Puumala virus epidemiology.

Heterogeneity in environmental conditions helps to maintain genetic and phenotypic diversity in ecosystems. As such, it may explain why the capacity of animals to mount immune responses is highly variable. The quality of habitat patches, in terms of resources, parasitism, predation and habitat fragmentation may, for example, trigger trade-offs ultimately affecting the investment of individuals in various immunological pathways. We described spatial immunoheterogeneity in bank vole populations with respect to landscape features and co-infection. We focused on the consequences of this heterogeneity for the risk of Puumala hantavirus (PUUV) infection. We assessed the expression of the Tnf-α and Mx2 genes and demonstrated a negative correlation between PUUV load and the expression of these immune genes in bank voles. Habitat heterogeneity was partly associated with differences in the expression of these genes. Levels of Mx2 were lower in large forests than in fragmented forests, possibly due to differences in parasite communities. We previously highlighted the positive association between infection with Heligmosomum mixtum and infection with PUUV. We found that Tnf-α was more strongly expressed in voles infected with PUUV than in uninfected voles or in voles co-infected with the nematode H. mixtum and PUUV. H. mixtum may limit the capacity of the vole to develop proinflammatory responses. This effect may increase the risk of PUUV infection and replication in host cells. Overall, our results suggest that close interactions between landscape features, co-infection and immune gene expression may shape PUUV epidemiology.

[A hantavirus killed an Israeli researcher: hazards while working with wild animals].

An Israeli researcher working in Finland with Bank Voles, contracted an infectious viral disease and died. This was a rare event, but it is important to learn about this class of viruses and to be aware of the hazards while working in the field in close contact with wild animals. The virus termed Puumala belongs to the genus Hanta from the Bunyaviridae family. The natural reservoir is rodents, mice, rats and Bank Votes for the Puuamala strain. The disease is termed HFRS (hemorrhagic fever with renal syndrome), is prevalent in Asia and Europe, affecting 200,000 people a year, with 5-15% percent mortality (although in Finland mortality rate is 0.1%). The New World strains cause HPS (hemorrhagic pulmonary syndrome) affecting 200 people a year with 40% mortality. Virus is present in all rodents excretions, and route of infection is by aerosols, hand to mucus membranes contamination, by rodents bites and by contaminated food or water. More than 226 work related infections were documented. Treatment with Ribavirin helps in HFRS but not in HPS. The virus is stable in the environment for long periods, and research must be carried out at biosafety level 3. Working outdoors in rodent infested area, should be carried out using protective clothing, gloves, googles and face mask whenever aerosol producing tasks are performed. Both indoor and outdoor, it is important to adhere to self-hygienic procedures, especially hand washing.

[Variants of the immunoreactivity and infectious process in bank vole (Myodes glareolus) experimentally infected with the hantavirus Puumala (PUUV)].

As a result of a longitudinal study of the Puumala hantavirus (PUUV) in the experimentally infected bank voles (Myodes glareolus), we revealed three groups of the voles differing in the immunoreactivity and viral antigen concentration in the organs. The close correlation between these parameters suggested the existence of various mechanisms of the hantavirus persistence in the host.

Puumala virus infections associated with cardiovascular causes of death.

We studied the causes of death of patients in Sweden with diagnoses of hemorrhagic fever with renal syndrome (HFRS) during 1997-2009. Cardiovascular disorders were a common cause of death during acute-phase HFRS and were the cause of death for >50% of those who died during the first year after HFRS.

High pentraxin-3 plasma levels associate with thrombocytopenia in acute Puumala hantavirus-induced nephropathia epidemica.

Our aim was to investigate whether plasma levels of the long pentraxin-3 (PTX3) associate with the severity of Puumala hantavirus-induced nephropathia epidemica (NE). Sixty-one prospectively identified consecutively hospitalized NE patients were examined. Plasma PTX3, interleukin (IL)-6, terminal complement complex SC5b-9, complement component C3, C-reactive protein (CRP), creatinine, sodium, kynurenine, and tryptophan levels, as well as the blood cell count, were determined for up to five consecutive days after hospitalization. Receiver operating characteristic (ROC) analysis revealed that the maximum PTX3 level >101.6 ng/ml (high PTX3) showed a sensitivity of 71% and a specificity of 89% for detecting platelet level <50 × 10(9)/l, with an area under the curve (AUC) value of 0.78 (95% confidence interval [CI] 0.63-0.94). High PTX3 level was also associated with several other variables reflecting the severity of the disease: patients with high PTX3 level had higher maximum blood leukocyte (16.1 vs. 9.7 × 10(9)/l, p < 0.001), plasma IL-6 (16.9 vs. 9.0 pg/ml, p = 0.007), and creatinine (282 vs. 124 µmol/l, p = 0.007) levels than patients with low maximum PTX3 level. They also had longer hospital stays (8 vs. 5 days, p = 0.015) compared to patients with low PTX3 level. High plasma PTX3 levels are associated with thrombocytopenia and the overall severity of NE.

Signs of general inflammation and kidney function are associated with the ocular features of acute Puumala hantavirus infection.

BACKGROUND: Puumala hantavirus (PUUV) causes nephropathia epidemica (NE), a type of viral haemorrhagic fever with renal syndrome (HFRS). This febrile infection may affect the kidneys, central nervous system (CNS), and the eye. Acute illness is associated with increased tissue permeability and tissue oedema, and many patients experience reduced vision. The aim of this study was to explore the physiological events associated with the ocular features of acute NE. METHODS: This was a prospective study of 46 NE patients who were examined during the acute infection and 1 month after hospitalization. Visual acuity, refraction, intraocular pressure (IOP), and ocular dimensions were evaluated. Cerebrospinal fluid and blood samples were collected, brain magnetic resonance imaging and electroencephalography were recorded, and HLA haplotype was analyzed. The degrees of tissue oedema and fluid imbalance were evaluated. RESULTS: CNS examinations did not reveal the source of the ocular changes in acute NE. The plasma C-reactive protein concentration correlated with the lens thickness and the IOP. The plasma creatinine level was associated with the change in anterior chamber depth. However, oliguric and polyuric patients displayed similar ocular findings. Patients positive for the DR3-DQ2 haplotype experienced the least diminished visual acuity. CONCLUSIONS: The level of systemic inflammation rather than CNS involvement appears to account for the ocular changes during acute PUUV infection, and the severity of kidney dysfunction may also have a significant role. In addition, the genetic properties of the host may well explain the ocular features of acute hantavirus infection.

[Genetic determinancy of the change in the VE-cadherin expression and intensified vessel deendothelisation at hemorrhagic fever with renal syndrome].

The goal of this work was to determine a correlation between the VE-cadherin and circulating endothelial cells (CECs) blood levels at hemorrhagic fever with renal syndrome (HFRS) of different severity and research association between the VE-cadherin gene c. 1550T>C missense mutation and HRFS severity. Significant decreasing of the VE-cadherin and increasing of CECs blood levels in the course of the disease in all studied groups was established. Most prominent changes were found at severe type with complications. There was found a strong negative correlation between these two indexes. There was significant high frequency of homozygotic genotype *T/*T at severe type with complications. It was concluded that there was increased endothelium desquamation due to the VE-cadherin internalization at moderate and severe uncomplicated types of HFRS and as a result ofVE-cadherin gene c. 1550T>C missense mutation at severe type with complications.

[Clinical and morphological parallels in lung lesions associated with hemorrhagic fever with renal syndrome].

The authors report the results of clinical examination of the respiratory, organs of 97 patients with hemorrhagic fever and renal syndrome (HFRS) with confirmed Puumala serotype. Retrospective analysis of 16 fatal cases is presented. It is shown that patients with moderately severe and severe forms of the disease exhibit early clinical and X-ray signs of pulmonary lesions and respiratory insufficiency Some of them suffer acute respiratory distress syndrome confirmed by morphological findings at autopsy.

Cytokine expression during early and late phase of acute Puumala hantavirus infection.

BACKGROUND: Hantaviruses of the family Bunyaviridae are emerging zoonotic pathogens which cause hemorrhagic fever with renal syndrome (HFRS) in the Old World and hantavirus pulmonary syndrome (HPS) in the New World. An immune-mediated pathogenesis is discussed for both syndromes. The aim of our study was to investigate cytokine expression during the course of acute Puumala hantavirus infection. RESULTS: We retrospectively studied 64 patients hospitalised with acute Puumala hantavirus infection in 2010 during a hantavirus epidemic in Germany. Hantavirus infection was confirmed by positive anti-hantavirus IgG/IgM. Cytokine expression of IL-2, IL-5, IL-6, IL-8, IL-10, IFN-γ, TNF-α and TGF-ß1 was analysed by ELISA during the early and late phase of acute hantavirus infection (average 6 and 12 days after onset of symptoms, respectively). A detailed description of the demographic and clinical presentation of severe hantavirus infection requiring hospitalization during the 2010 hantavirus epidemic in Germany is given. Acute hantavirus infection was characterized by significantly elevated levels of IL-2, IL-6, IL-8, TGF-ß1 and TNF-α in both early and late phase compared to healthy controls. From early to late phase of disease, IL-6, IL-10 and TNF-α significantly decreased whereas TGF-ß1 levels increased. Disease severity characterized by elevated creatinine and low platelet counts was correlated with high pro-inflammatory IL-6 and TNF-α but low immunosuppressive TGF-ß1 levels and vice versa . CONCLUSION: High expression of cytokines activating T-lymphocytes, monocytes and macrophages in the early phase of disease supports the hypothesis of an immune-mediated pathogenesis. In the late phase of disease, immunosuppressive TGF-ß1 level increase significantly. We suggest that delayed induction of a protective immune mechanism to downregulate a massive early pro-inflammatory immune response might contribute to the pathologies characteristic of human hantavirus infection.

Concomitant influence of helminth infection and landscape on the distribution of Puumala hantavirus in its reservoir, Myodes glareolus.

BACKGROUND: Puumala virus, the agent of nephropathia epidemica (NE), is the most prevalent hantavirus in Europe. The risk for human infection seems to be strongly correlated with the prevalence of Puumala virus (PUUV) in populations of its reservoir host species, the bank vole Myodes glareolus. In humans, the infection risks of major viral diseases are affected by the presence of helminth infections. We therefore proposed to analyse the influence of both helminth community and landscape on the prevalence of PUUV among bank vole populations in the Ardennes, a PUUV endemic area in France. RESULTS: Among the 313 voles analysed, 37 had anti-PUUV antibodies. Twelve gastro-intestinal helminth species were recorded among all voles sampled. We showed that PUUV seroprevalence strongly increased with age or sexual maturity, especially in the northern forests (massif des Ardennes). The helminth community structure significantly differed between this part and the woods or hedgerows of the southern cretes pre-ardennaises. Using PUUV RNA quantification, we identified significant coinfections between PUUV and gastro-intestinal helminths in the northern forests only. More specifically, PUUV infection was positively associated with the presence of Heligmosomum mixtum, and in a lesser extent, Aonchotheca muris-sylvatici. The viral load of PUUV infected individuals tended to be higher in voles coinfected with H. mixtum. It was significantly lower in voles coinfected with A. muris-sylvatici, reflecting the influence of age on these latter infections. CONCLUSIONS: This is the first study to emphasize hantavirus--helminth coinfections in natural populations. It also highlights the importance to consider landscape when searching for such associations. We have shown that landscape characteristics strongly influence helminth community structure as well as PUUV distribution. False associations might therefore be evidenced if geographic patterns of helminths or PUUV repartition are not previously identified. Moreover, our work revealed that interactions between helminths and landscape enhance/deplete the occurrence of coinfections between PUUV and H. mixtum or A. muris-sylvatici. Further experimental analyses and long-term individual surveys are now required to confirm these correlative results, and to ascertain the causal links between helminth and PUUV infection risks.

Landscape genetics highlights the role of bank vole metapopulation dynamics in the epidemiology of Puumala hantavirus.

Rodent host dynamics and dispersal are thought to be critical for hantavirus epidemiology as they determine pathogen persistence and transmission within and between host populations. We used landscape genetics to investigate how the population dynamics of the bank vole Myodes glareolus, the host of Puumala hantavirus (PUUV), vary with forest fragmentation and influence PUUV epidemiology. We sampled vole populations within the Ardennes, a French PUUV endemic area. We inferred demographic features such as population size, isolation and migration with regard to landscape configuration. We next analysed the influence of M. glareolus population dynamics on PUUV spatial distribution. Our results revealed that the global metapopulation dynamics of bank voles were strongly shaped by landscape features, including suitable patch size and connectivity. Large effective size in forest might therefore contribute to the higher observed levels of PUUV prevalence. By contrast, populations from hedge networks highly suffered from genetic drift and appeared strongly isolated from all other populations. This might result in high probabilities of local extinction for both M. glareolus and PUUV. Besides, we detected signatures of asymmetric bank vole migration from forests to hedges. These movements were likely to sustain PUUV in fragmented landscapes. In conclusion, our study provided arguments in favour of source-sink dynamics shaping PUUV persistence and spread in heterogeneous, Western European temperate landscapes. It illustrated the potential contribution of landscape genetics to the understanding of the epidemiological processes occurring at this local scale.

Young male patients are at elevated risk of developing serious central nervous system complications during acute Puumala hantavirus infection.

BACKGROUND: Our aim was to characterize clinical properties and laboratory parameters in patients with or without cerebrospinal fluid (CSF) findings suggestive of central nervous system (CNS) involvement, and especially those who developed serious CNS complications during acute nephropathia epidemica (NE) caused by Puumala hantavirus (PUUV) infection. METHODS: A prospective cohort of 40 patients with acute NE and no signs of major CNS complications was analyzed. In addition, 8 patients with major CNS complications associated with NE were characterized. We collected data of CNS symptoms, CSF analysis, brain magnetic resonance imaging (MRI) results, electroencephalography (EEG) recordings, kidney function, and a number of laboratory parameters. Selected patients were evaluated by an ophthalmologist. RESULTS: Patients with a positive CSF PUUV IgM finding or major CNS complications were more often males (p < 0.05) and they had higher plasma creatinine values (p < 0.001) compared to those with negative CSF PUUV IgM. The degree of tissue edema did not explain the CSF findings. Patients with major CNS complications were younger than those with negative CSF PUUV IgM finding (52.9 vs. 38.5 years, p < 0.05). Some patients developed permanent neurological and ophthalmological impairments. CONCLUSIONS: CNS and ocular involvement during and after acute NE can cause permanent damage and these symptoms seem to be attributable to true infection of the CNS rather than increased tissue permeability. The possibility of this condition should be borne in mind especially in young male patients.

Central Nervous System and Ocular Manifestations in Puumala Hantavirus Infection.

Puumala hantavirus (PUUV), carried and spread by the bank vole (Myodes glareolus), causes a mild form of hemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica (NE). Acute high fever, acute kidney injury (AKI), thrombocytopenia, and hematuria are typical features of this syndrome. In addition, headache, blurred vision, insomnia, vertigo, and nausea are commonly associated with the disease. This review explores the mechanisms and presentations of ocular and central nervous system involvement in acute NE.