Population, environmental, and community effects on local bank vole (Myodes glareolus) Puumala virus infection in an area with low human incidence

In this study, the distribution of Puumala hantavirus (PUUV) infection in local bank vole Myodes glareolus populations in an area with low human PUUV infection (nephropathia epidemica [NE]) incidence in northern Belgium was monitored for 2 consecutive years. Bank voles were trapped in preferred habitat and tested for anti-PUUV IgG. Infection data were related to individual bank vole features, population demography, and environmental variables. Rare occurrence of PUUV infection was found and PUUV prevalence was low compared with data from the high NE incidence area in southern Belgium. Small-scale climatic differences seemed to play a role in PUUV occurrence, vegetation index and deciduous forest patch size both influenced PUUV prevalence and number of infected voles in a positive way. The data suggested a density threshold in vole populations below which PUUV infection does not occur. This threshold may vary between years, but the abundance of bank voles does not seem to affect the degree of PUUV seroprevalence further. We found indications for a dilution effect on PUUV prevalence, dependent on the relative proportion of nonhost wood mice Apodemus sylvaticus in a study site. In conclusion, we regard the combination of a dilution effect, a possible threshold density that depends on local conditions, and a higher fragmentation of suitable bank vole habitat in our study area as plausible explanations for the sparse occurrence of PUUV infection and low prevalence detected. Thus, beside human activity patterns, local environmental conditions and rodent community structure are also likely to play a role in determining PUUV infection risk for humans.     

Habitat factors associated with bank voles (Clethrionomys glareolus) and concomitant hantavirus in northern Sweden

Puumala virus (PUUV), genus hantavirus, causes nephropathia epidemica, a mild form of hemorrhagic fever with renal syndrome in humans. In this study, bank voles, the natural reservoir of PUUV, were captured at locations of previous human PUUV exposure and paired controls within a region of high incidence in northern Sweden. The aim of the study was to evaluate the influence of environmental factors on the abundance of bank voles and the occurrence of PUUV. The total number of voles and the number of PUUV-infected voles did not differ between locations of previous human PUUV exposure and paired controls. The number of bank voles expressing antibodies to PUUV infection increased linearly with total bank vole abundance implying density independent transmission. Using principal component and partial correlation analysis, we found that particular environmental characteristics associated with old-growth moist forests (i.e., those dominated by Alectoria spp., Picea abies, fallen wood, and Vaccinium myrtillus) were also associated with increased abundance of bank vole and hence the number of PUUV-infected bank voles, whereas there were no correlations with factors associated with dry environments (i.e., Pinus sylvestris and V. vitis-idea). This suggests that circulation and persistence of PUUV within bank vole populations was influenced by habitat factors. Future modeling of risk of exposure to hantavirus and transmission of PUUV within vole populations should include the influence of these factors.     

First Molecular Evidence for Puumala Hantavirus in Poland

Puumala virus (PUUV) causes mild to moderate cases of haemorrhagic fever with renal syndrome (HFRS), and is responsible for the majority of hantavirus infections of humans in Fennoscandia, Central and Western Europe. Although there are relatively many PUUV sequences available from different European countries, little is known about the presence of this virus in Poland. During population studies in 2009 a total of 45 bank voles were trapped at three sites in north-eastern Poland, namely islands on Dejguny and Dobskie Lakes and in a forest near Mikołajki. S and M segment-specific RT-PCR assays detected PUUV RNA in three animals from the Mikołajki site. The obtained partial S and M segment sequences demonstrated the highest similarity to the corresponding segments of a PUUV strain from Latvia. Analysis of chest cavity fluid samples by IgG ELISA using a yeast-expressed PUUV nucleocapsid protein resulted in the detection of two seropositive samples, both being also RT-PCR positive. Interestingly, at the trapping site in Mikołajki PUUV-positive bank voles belong to the Carpathian and Eastern genetic lineages within this species. In conclusion, we herein present the first molecular evidence for PUUV in the rodent reservoir from Poland.     

Hantavirus infection in bank voles in the natural reservoir. Part 1. Characteristics of an infection process in bank voles]

The specific features of hantavirus infection in naturally infected bank voles (Clethrionomys glareolus), the principal host of hantavirus of the serotype Puumala, were studied during long-term observation of individually marked animals in the active focus of hemorrhagic fever with renal syndrome (HFRS) in the south of Udmurtia. The infection time in the bank voles was defined by paired serum seroconversion tests. In the natural focus, hantavirus was shown to cause asymptomatic persistent infection in the bank voles with the body's peak accumulation of the virus and its environmental discharge within the first month of infection. In this period the animals present the greatest epidemic and epizootic hazards. Hantavirus infection has no negative impact on the viability of bank voles.     

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.     

Complete Genome and Phylogeny of Puumala Hantavirus Isolates Circulating in France

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

Temporal variation in individual factors associated with hantavirus infection in bank voles during an epizootic: implications for Puumala virus transmission dynamics

Puumala virus (PUUV), the causal agent of nephropathia epidemica in humans, is one of the many hantaviruses included in the list of emerging pathogens. Hantavirus infection is not distributed evenly among PUUV reservoir hosts (i.e., bank voles [Myodes glareolus]). Besides environmental factors and local population features, individual characteristics play an important role in vole PUUV infection risk. Identifying the relative importance of these individual characteristics can provide crucial information on PUUV transmission processes. In the present study, bank voles were monitored during the nephropathia epidemica outbreak of 2005 in Belgium. Vole sera were tested for presence of immunoglobulin G against PUUV, and a logistic mixed model was built to investigate the temporal variation in individual characteristics and their relative importance to PUUV infection risk in bank voles. Relative risk calculations for individual vole characteristics related to PUUV infection in the reservoir host show that reproductive activity dominates infection risk. The gender effect is only found in reproductively active voles, where reproductively active males have the highest infection risk. Results also revealed a clear seasonal variation in the importance of reproductive activity linked to PUUV infection. In contrast to the main effect found in other trapping sessions, no difference in infection risk ratio was found between reproductively active and nonactive voles in the spring period. Combined with increased infection risk for the reproductively nonactive group at that time, these results indicate a shift in the transmission process due to changes in bank vole behavior, physiology, or climate conditions. Hence, our results suggest that mathematical models should take into account seasonal shifts in transmission mechanisms. When these results are combined with the seasonal changes in population structure during the epizootic period, we identify vole reproductive activity and length of the breeding season as potential drivers of PUUV epizootics in west-central European regions.     

Genetic Diversity of Puumala orthohantavirus in Rodents and Human Patients in Austria, 2012–2019

Puumala orthohantavirus (PUUV) has a wide distribution throughout Europe. Distinctive temporal patterns of spillover into the human population are related to population dynamics of the reservoir host, the bank vole (Clethrionomys glareolus). As the rodent host is tied to specific habitats with small individual ranges, PUUV genetic diversity is also highly correlated with geographic distance. Using sequenced portions of viral S and M segments, we determined whether geographic clusters were supported. Human cases of PUUV infections are concentrated in southeastern Austria. We detected four distinct genotypes: two genotypes of the Alpe-Adria (ALAD) lineage typically associated with southeast Europe, and two sublineages of the Central Europe (CE) lineage. One cluster of CE genotypes represents a phylogenetically distinct sublineage compared to previously reported CE clades, and extends the boundary of the CE lineage further south than previously reported.     

Characterization of Hantavirus N Protein Intracellular Dynamics and Localization

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation, and several other crucial processes during hantavirus infection. In this study, we generated fluorescently tagged N protein constructs derived from Puumalavirus (PUUV), the dominant hantavirus species in Central, Northern, and Eastern Europe. We comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We observed formation of large, fibrillar PUUV N protein aggregates, rapidly coalescing from early punctate and spike-like assemblies. Moreover, we found significant spatial correlation of N with vimentin, actin, and P-bodies but not with microtubules. N constructs also co-localized with Gn and Gc albeit not as strongly as the glycoproteins associated with each other. Finally, we assessed oligomerization of N constructs, observing efficient and concentration-dependent multimerization, with complexes comprising more than 10 individual proteins.     

Identification of FactorsInfluencing the Puumala Virus Seroprevalence within Its Reservoir in aMontane Forest Environment

Puumala virus (PUUV) is a major cause of mild to moderate haemorrhagic fever with renal syndrome and is transmitted by the bank vole (Myodes glareolus). There has been a high cumulative incidence of recorded human cases in South-eastern Germany since 2004 when the region was first recognized as being endemic for PUUV. As the area is well known for outdoor recreation and the Bavarian Forest National Park (BFNP) is located in the region, the increasing numbers of recorded cases are of concern. To understand the population and environmental effects on the seroprevalence of PUUV in bank voles we trapped small mammals at 23 sites along an elevation gradient from 317 to 1420m above sea level. Generalized linear mixed effects models(GLMEM) were used to explore associations between the seroprevalence of PUUV in bank voles and climate and biotic factors. We found that the seroprevalence of PUUV was low (6%–7%) in 2008 and 2009, and reached 29% in 2010. PUUV seroprevalence was positively associated with the local species diversity and deadwood layer, and negatively associated with mean annual temperature, mean annual solar radiation, and herb layer. Based on these findings, an illustrative risk map for PUUV seroprevalence prediction in bank voles was created for an area of the national park. The map will help when planning infrastructure in the national park (e.g., huts, shelters, and trails).     

Immunogenetic Factors Affecting Susceptibility of Humans and Rodents to Hantaviruses and the Clinical Course of Hantaviral Disease in Humans

We reviewed the associations of immunity-related genes with susceptibility of humans and rodents to hantaviruses, and with severity of hantaviral diseases in humans. Several class I and class II HLA haplotypes were linked with severe or benign hantavirus infections, and these haplotypes varied among localities and hantaviruses. The polymorphism of other immunity-related genes including the C4A gene and a high-producing genotype of TNF gene associated with severe PUUV infection. Additional genes that may contribute to disease or to PUUV infection severity include non-carriage of the interleukin-1 receptor antagonist (IL-1RA) allele 2 and IL-1β (-511) allele 2, polymorphisms of plasminogen activator inhibitor (PAI-1) and platelet GP1a. In addition, immunogenetic studies have been conducted to identify mechanisms that could be linked with the persistence/clearance of hantaviruses in reservoirs. Persistence was associated during experimental infections with an upregulation of anti-inflammatory responses. Using natural rodent population samples, polymorphisms and/or expression levels of several genes have been analyzed. These genes were selected based on the literature of rodent or human/hantavirus interactions (some Mhc class II genes, Tnf promoter, and genes encoding the proteins TLR4, TLR7, Mx2 and β3 integrin). The comparison of genetic differentiation estimated between bank vole populations sampled over Europe, at neutral and candidate genes, has allowed to evidence signatures of selection for Tnf, Mx2 and the Drb Mhc class II genes. Altogether, these results corroborated the hypothesis of an evolution of tolerance strategies in rodents. We finally discuss the importance of these results from the medical and epidemiological perspectives.     

Severity Biomarkers in Puumala Hantavirus Infection

Annually, over 10,000 cases of hemorrhagic fever with renal syndrome (HFRS) are diagnosed in Europe. Puumala hantavirus (PUUV) causes most of the European HFRS cases. PUUV causes usually a relatively mild disease, which is rarely fatal. However, the severity of the infection varies greatly, and factors affecting the severity are mostly unrevealed. Host genes are known to have an effect. The typical clinical features in PUUV infection include acute kidney injury, thrombocytopenia, and increased vascular permeability. The primary target of hantavirus is the endothelium of the vessels of different organs. Although PUUV does not cause direct cytopathology of the endothelial cells, remarkable changes in both the barrier function of the endothelium and the function of the infected endothelial cells occur. Host immune or inflammatory mechanisms are probably important in the development of the capillary leakage. Several immunoinflammatory biomarkers have been studied in the context of assessing the severity of HFRS caused by PUUV. Most of them are not used in clinical practice, but the increasing knowledge about the biomarkers has elucidated the pathogenesis of PUUV infection.     

Epidemiology of nephropathia epidemica in Sweden

A comprehensive study of nephropathia epidemica (NE), caused by Puumala virus, was conducted in Sweden. Human sera from residents of various regions of Sweden were examined for antibody to Puumala virus, and the incidence of NE was determined. Small mammals were captured at locations throughout Sweden and were examined for antibody to Puumala virus and for antigen. The human serosurvey found the highest prevalence rates and the highest incidence rates in northern Sweden. The bank vole was found to be the most-abundant rodent, as well as the species most frequently positive for antibody. Antibody-positive voles were restricted to the northern two-thirds of the country, an area corresponding to that where most human disease was noted. These results suggest that the bank vole is the principal host of Puumala virus in Sweden and clearly establish the region near 64 degrees N as highly endemic for NE in humans.     

Adaptive property of hantaviruses during their isolation from cultured VERO-E6 cells in relation to the type of the ecological host

As a result of virological studies, 185 lung tissue specimens from 4 rodent species caught near Khabarovsk were isolated and fixed in the passages of cultured Vero-6 cells of 68 hantavirus strains. The capacity of the strains to adapt to the cells was assessed by using the adaptive index involving the mean rates of successful isolation, its duration, hantavirus antigen titer in the material used for infection. The strains of hantavirus serotypes were noted for the highest adaptive properties, which are ecologically associated with rodents of the family Mus, such as field and East-Asiatic mice. Lower adaptive capacities were established for the strains of hantavirus serotypes, which are ecologically related to rodents of the family Cricetidae, such as large and large-toothed redback voles. The differences found in the adaptive capacities of hantavirus strains cultured in Vero-E6 cells reflect the degree of specialization of some hantavirus serotypes to particular host rodent species during their long-term coevolution.     

ABO and Rhesus Blood Groups in Acute Puumala Hantavirus Infection

Puumala hantavirus (PUUV) causes hemorrhagic fever with renal syndrome. We aimed to evaluate whether ABO and rhesus blood groups associate with the susceptibility or the severity of PUUV infection. We analyzed blood groups in 289 adult patients treated in Tampere University hospital due to PUUV infection during the years 1982–2017. Patients’ blood group distribution was compared to that of healthy, voluntary blood donors living in the Tampere University Hospital responsibility area (n = 21,833). The severity of PUUV infection, as judged by the severity of acute kidney injury (AKI), thrombocytopenia, inflammation, capillary leakage, and the length of hospital care, was analyzed across the groups. The ABO and rhesus blood group distributions did not differ between the patients and blood donors. Patients with non-O blood groups had lower systolic blood pressure compared to patients with blood group O, but there was no difference in other markers of capillary leakage or in the severity of AKI. Minor deviations in the number of platelets and leukocytes were detected between the O and non-O blood groups. To conclude, patients with blood group O may be less susceptible to hypotension, but otherwise blood groups have no major influences on disease susceptibility or severity during acute PUUV infection.     

Alcohol Consumption and Its Influence on the Clinical Picture of Puumala Hantavirus Infection

Puumala hantavirus (PUUV) causes hemorrhagic fever with renal syndrome. Characteristic clinical findings include acute kidney injury (AKI), thrombocytopenia, and capillary leakage. Smoking increases the risk of severe AKI, but it is not known whether alcohol consumption predisposes patients to a more severe infection. Liver and pancreatic enzymes, as well as biomarkers of alcohol consumption (gamma-glutamyl transferase, GGT; carbohydrate-deficient transferrin, CDT; GGT-CDT combination; and ethyl glucuronide, EtG), were measured from 66 patients with acute PUUV infection during hospitalization and at the convalescence phase. Alcohol consumption was present in 41% of the study population, 15% showing signs of heavy drinking. Alcohol use did not affect the severity of PUUV induced AKI nor the overall clinical picture of the infection. Liver enzyme levels (GGT or alanine aminotransferase, ALT) were elevated in 64% of the patients, but the levels did not associate with the markers reflecting the severity of the disease. Serum amylase activities at the convalescent stage were higher than those at the acute phase (p < 0.001). No cases with acute pancreatitis were found. In conclusion, our findings indicate that alcohol consumption does not seem to affect the clinical course of an acute PUUV infection.     

Innate and adaptive immune responses against human Puumala virus infection: immunopathogenesis and suggestions for novel treatment strategies for severe hantavirus‐associated syndromes

Two related hyperinflammatory syndromes are distinguished following infection of humans with hantaviruses: haemorrhagic fever with renal syndrome (HFRS) seen in Eurasia and hantavirus pulmonary syndrome (HPS) seen in the Americas. Fatality rates are high, up to 10% for HFRS and around 35%–40% for HPS. Puumala virus (PUUV) is the most common HFRS‐causing hantavirus in Europe. Here, we describe recent insights into the generation of innate and adaptive cell‐mediated immune responses following clinical infection with PUUV. First described are studies demonstrating a marked redistribution of peripheral blood mononuclear phagocytes (MNP) to the airways, a process that may underlie local immune activation at the site of primary infection. We then describe observations of an excessive natural killer (NK) cell activation and the persistence of highly elevated numbers of NK cells in peripheral blood following PUUV infection. A similar vigorous CD8 Tcell response is also described, though Tcell responses decline with viraemia. Like MNPs, many NK cells and CD8 T cells also localize to the lung upon acute PUUV infection. Following this, findings demonstrating the ability of hantaviruses, including PUUV, to cause apoptosis resistance in infected target cells, are described. These observations, and associated inflammatory cytokine responses, may provide new insights into HFRS and HPS disease pathogenesis. Based on similarities between inflammatory responses in severe hantavirus infections and other hyperinflammatory disease syndromes, we speculate whether some therapeutic interventions that have been successful in the latter conditions may also be applicable in severe hantavirus infections.     

Muju Virus,Harbored by Myodes regulus in Korea,Might Represent a Genetic Variant of Puumala Virus,the Prototype Arvicolid Rodent-Borne Hantavirus

The genome of Muju virus (MUJV), identified originally in the royal vole (Myodes regulus) in Korea, was fully sequenced to ascertain its genetic and phylogenetic relationship with Puumala virus (PUUV), harbored by the bank vole (My. glareolus), and a PUUV-like virus, named Hokkaido virus (HOKV), in the grey red-backed vole (My. rufocanus) in Japan. Whole genome sequence analysis of the 6544-nucleotide large (L), 3652-nucleotide medium (M) and 1831-nucleotide small (S) segments of MUJV, as well as the amino acid sequences of their gene products, indicated that MUJV strains from different capture sites might represent genetic variants of PUUV, the prototype arvicolid rodent-borne hantavirus in Europe. Distinct geographic-specific clustering of MUJV was found in different provinces in Korea, and phylogenetic analyses revealed that MUJV and HOKV share a common ancestry with PUUV. A better understanding of the taxonomic classification and pathogenic potential of MUJV must await its isolation in cell culture.