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1.  The fundamental role of endothelial cells in hantavirus pathogenesis 
Hantavirus, a genus of rodent- and insectivore-borne viruses in the family Bunyaviridae, is a group of emerging zoonotic pathogens. Hantaviruses cause hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome in man, often with severe consequences. Vascular leakage is evident in severe hantavirus infections, and increased permeability contributes to the pathogenesis. This review summarizes the current knowledge on hantavirus interactions with hematopoietic and endothelial cells, and their effects on the increased vascular permeability.
doi:10.3389/fmicb.2014.00727
PMCID: PMC4273638  PMID: 25566236
hantavirus; endothelial dysfunction; HFRS; HCPS; bunyavirus
2.  A Protein L -Based Immunodiagnostic Approach Utilizing Time-Resolved Förster Resonance Energy Transfer 
PLoS ONE  2014;9(9):e106432.
Chelated lanthanides such as europium (Eu) have uniquely long fluorescence emission half-lives permitting their use in time-resolved fluorescence (TRF) assays. In Förster resonance energy transfer (FRET) a donor fluorophore transfers its emission energy to an acceptor fluorophore if in sufficiently close proximity. The use of time-resolved (TR) FRET minimizes the autofluorescence of molecules present in biological samples. In this report, we describe a homogenous immunoassay prototype utilizing TR-FRET for detection of antibodies in solution. The assay is based on labeled protein L, a bacterial protein that binds to immunoglobulin (Ig) light chain, and labeled antigen, which upon association with the same Ig molecule produce a TR-FRET active complex. We show that the approach is functional and can be utilized for both mono- and polyvalent antigens. We also compare the assay performance to that of another homogenous TR-FRET immunoassay reported earlier. This novel assay may have wide utility in infectious disease point-of-care diagnostics.
doi:10.1371/journal.pone.0106432
PMCID: PMC4152265  PMID: 25181527
4.  Identification of linear human B-cell epitopes of tick-borne encephalitis virus 
Virology Journal  2014;11:115.
Background
Tick-borne encephalitis (TBE) is a central nervous system infection transmitted to humans by ticks. The causative agent, tick-borne encephalitis virus (TBEV), belongs to the genus Flavivirus (family Flaviviridae), which includes globally important arthropod-borne viruses, such as dengue, Yellow fever, Japanese encephalitis and West Nile viruses. Flaviviruses are highly cross-reactive in serological tests that are currently based on viral envelope proteins. The envelope (E) protein is the major antigenic determinant and it is known to induce neutralizing antibody responses.
Methods
We synthesized the full-length TBEV proteome as overlapping synthetic 18-mer peptides to find dominant linear IgG epitopes. To distinguish natural TBEV infections from responses to TBE immunization or other flavivirus infections, the peptides were probed with sera of patients infected with TBEV, West Nile virus (WNV) or dengue virus (DENV), sera from TBE vaccinees and negative control sera by SPOT array technique.
Results
We identified novel linear TBEV IgG epitopes in the E protein and in the nonstructural protein 5 (NS5).
Conclusions
In this study, we screened TBEV structural and nonstructural proteins to find linear epitopes specific for TBEV. We found 11 such epitopes and characterized specifically two of them to be potential for differential diagnostics. This is the first report of identifying dominant linear human B-cell epitopes of the whole TBEV genome. The identified peptide epitopes have potential as antigens for diagnosing TBEV and to serologically distinguish flavivirus infections from each other.
doi:10.1186/1743-422X-11-115
PMCID: PMC4078944  PMID: 24946852
Epitope; Flavivirus; Tick-borne encephalitis virus
5.  Immunogenetic Factors Affecting Susceptibility of Humans and Rodents to Hantaviruses and the Clinical Course of Hantaviral Disease in Humans 
Viruses  2014;6(5):2214-2241.
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.
doi:10.3390/v6052214
PMCID: PMC4036553  PMID: 24859344
hantavirus; Puumala virus; interaction; hosts; reservoirs; rodents; immunity-related genes
6.  Isolation, Identification, and Characterization of Novel Arenaviruses, the Etiological Agents of Boid Inclusion Body Disease 
Journal of Virology  2013;87(20):10918-10935.
Boid inclusion body disease (BIBD) is a progressive, usually fatal disease of constrictor snakes, characterized by cytoplasmic inclusion bodies (IB) in a wide range of cell types. To identify the causative agent of the disease, we established cell cultures from BIBD-positive and -negative boa constrictors. The IB phenotype was maintained in cultured cells of affected animals, and supernatants from these cultures caused the phenotype in cultures originating from BIBD-negative snakes. Viruses were purified from the supernatants by ultracentrifugation and subsequently identified as arenaviruses. Purified virus also induced the IB phenotype in naive cells, which fulfilled Koch's postulates in vitro. One isolate, tentatively designated University of Helsinki virus (UHV), was studied in depth. Sequencing confirmed that UHV is a novel arenavirus species that is distinct from other known arenaviruses including those recently identified in snakes with BIBD. The morphology of UHV was established by cryoelectron tomography and subtomographic averaging, revealing the trimeric arenavirus spike structure at 3.2-nm resolution. Immunofluorescence, immunohistochemistry, and immunoblotting with a polyclonal rabbit antiserum against UHV and reverse transcription-PCR (RT-PCR) revealed the presence of genetically diverse arenaviruses in a large cohort of snakes with BIBD, confirming the causative role of arenaviruses. Some snakes were also found to carry arenavirus antibodies. Furthermore, mammalian cells (Vero E6) were productively infected with UHV, demonstrating the potential of arenaviruses to cross species barriers. In conclusion, we propose the newly identified lineage of arenaviruses associated with BIBD as a novel taxonomic entity, boid inclusion body disease-associated arenaviruses (BIBDAV), in the family Arenaviridae.
doi:10.1128/JVI.01123-13
PMCID: PMC3807292  PMID: 23926354
7.  Polymorphisms of PAI-1 and platelet GP Ia may associate with impairment of renal function and thrombocytopenia in Puumala hantavirus infection 
Thrombosis research  2011;129(5):10.1016/j.thromres.2011.11.007.
Introduction
Puumala virus (PUUV) infection is a viral hemorrhagic fever with renal syndrome (HFRS) characterized by thrombocytopenia and acute impairment of renal function. We aimed to assess whether genetic polymorphisms of platelet antigens together with those of von Willebrand factor (VWF) and plasminogen activator inhibitor (PAI-1) correlate with disease severity.
Patients and methods
172 consecutive hospital-treated patients with serologically confirmed acute PUUV infection were included. Platelet glycoprotein (GP) IIIa T>C (rs5918), GP Ia T>C (rs1126643), GP Ib C>T (rs6065), GP VI T>C (rs1613662), VWF A>G (rs1063856) and PAI-1 A>G (rs2227631) were genotyped. The associations of the rarer alleles with variables reflecting the severity of the disease were analyzed.
Results
PAI-1 G-carriers had higher maximum creatinine level compared with the non-carriers (median 213 μmol/l, range 60–1499 μmol/l vs. median 122 μmol/l, range 51–1156 μmol/l, p=0.01). The GG-genotypes had higher creatinine levels than GA- and AA-genotypes (medians 249 μmol/l, 204 μmol/l and 122 μmol/l, respectively, p=0.03). Polymorphisms of GP VI and VWF associated with lower creatinine levels during PUUV infection. The minor C-allele of GP Ia associated with lower platelet counts (median 44×109/l, range 20–90×109/l vs median 64×109/l, range 3–238×109/l; p=0.02).
Conclusions
Polymorphism of PAI-1, a major regulator of fibrinolysis, has an adverse impact on the outcome of kidney function in PUUV-HFRS. Platelet collagen receptor GP Ia polymorphism associates with lower platelet count.
doi:10.1016/j.thromres.2011.11.007
PMCID: PMC3879723  PMID: 22133274
Coagulation; Fibrinolysis; Hantavirus; HPA; Platelet; Polymorphism
8.  High Activity of Indoleamine 2,3-Dioxygenase Is Associated With Renal Insufficiency in Puumala Hantavirus Induced Nephropathia Epidemica 
Journal of medical virology  2011;83(4):10.1002/jmv.22018.
Nephropathia epidemica (NE) is a hemorrhagic fever with renal syndrome caused by Puumala hantavirus. The severity of NE varies greatly. The aim of the present study was to evaluate whether serum indoleamine 2,3-dioxygenase (IDO) activity is associated with the severity of NE. A prospectively collected cohort of 102 consecutive patients with acute serologically confirmed NE was examined. Serum kynurenine, tryptophan, creatinine, CRP, and blood cell count were measured for up to 5 consecutive days after admission. The kynurenine to tryptophan (kyn/trp) ratio reflecting IDO activity was calculated. A maximum kyn/trp ratio >202 μmol/mmol had a sensitivity of 85% and a specificity of 75% for detecting maximum serum creatinine values >250 μmol/L by receiver operating characteristic (ROC) analysis. A maximum kyn/trp ratio >202 μmol/mmol (high IDO level) was also associated with other parameters reflecting the severity of the disease and renal impairment. Patients with high IDO levels had higher maximum serum creatinine (379 vs. 102 μmol/L, P < 0.001), plasma C-reactive protein (104.1 vs. 72.1 mg/L, P = 0.029), and blood leukocyte values (11.9 vs. 9.0 × 109/L, P < 0.001) compared to patients with kyn/trp ratio ≤202 μmol/mmol. They also had lower minimum urinary output (1,100 vs. 1,900 ml/day, P < 0.001) and longer hospital stays (8 vs. 5 days, P < 0.001). In conclusion, high serum IDO activity was associated with increased disease severity and renal impairment in NE.
doi:10.1002/jmv.22018
PMCID: PMC3874118  PMID: 21328391
Puumala hantavirus; nephropathia epidemica; kynurenine; tryptophan; indoleamine 2,3-dioxygenase
9.  The Three Subtypes of Tick-Borne Encephalitis Virus Induce Encephalitis in a Natural Host, the Bank Vole (Myodes glareolus) 
PLoS ONE  2013;8(12):e81214.
Tick-borne encephalitis virus (TBEV) infects bank voles (Myodes glareolus) in nature, but the relevance of rodents for TBEV transmission and maintenance is unclear. We infected colonized bank voles subcutaneously to study and compare the infection kinetics, acute infection, and potential viral persistence of the three known TBEV subtypes: European (TBEV-Eur), Siberian (TBEV-Sib) and Far Eastern (TBEV-FE). All strains representing the three subtypes were infective and highly neurotropic. They induced (meningo)encephalitis in some of the animals, however most of the cases did not present with apparent clinical symptoms. TBEV-RNA was cleared significantly slower from the brain as compared to other organs studied. Supporting our earlier findings in natural rodent populations, TBEV-RNA could be detected in the brain for up to 168 days post infection, but we could not demonstrate infectivity by cell culture isolation. Throughout all time points post infection, RNA of the TBEV-FE was detected significantly more often than RNA of the other two strains in all organs studied. TBEV-FE also induced prolonged viremia, indicating distinctive kinetics in rodents in comparison to the other two subtypes. This study shows that bank voles can develop a neuroinvasive TBEV infection with persistence of viral RNA in brain, and mount an anti-TBEV IgG response. The findings also provide further evidence that bank voles can serve as sentinels for TBEV endemicity.
doi:10.1371/journal.pone.0081214
PMCID: PMC3862475  PMID: 24349041
10.  Plasma Levels of Soluble Urokinase-Type Plasminogen Activator Receptor Associate with the Clinical Severity of Acute Puumala Hantavirus Infection 
PLoS ONE  2013;8(8):e71335.
Objectives
Urokinase-type plasminogen activator receptor is a multifunctional glycoprotein, the expression of which is increased during inflammation. It is known to bind to β3-integrins, which are elementary for the cellular entry of hantaviruses. Plasma soluble form of the receptor (suPAR) levels were evaluated as a predictor of severe Puumala hantavirus (PUUV) infection and as a possible factor involved in the pathogenesis of the disease.
Design
A single-centre prospective cohort study.
Subjects and Methods
Plasma suPAR levels were measured twice during the acute phase and once during the convalescence in 97 patients with serologically confirmed acute PUUV infection using a commercial enzyme-linked immunosorbent assay (ELISA).
Results
The plasma suPAR levels were significantly higher during the acute phase compared to the control values after the hospitalization (median 8.7 ng/ml, range 4.0–18.2 ng/ml vs. median 4.7 ng/ml, range 2.4–12.2 ng/ml, P<0.001). The maximum suPAR levels correlated with several variables reflecting the severity of the disease. There was a positive correlation with maximum leukocyte count (r = 0.475, p<0.001), maximum plasma creatinine concentration (r = 0.378, p<0.001), change in weight during the hospitalization (r = 0.406, p<0.001) and the length of hospitalization (r = 0.325, p = 0.001), and an inverse correlation with minimum platelet count (r = −0.325, p = 0.001) and minimum hematocrit (r = −0.369, p<0.001).
Conclusion
Plasma suPAR values are markedly increased during acute PUUV infection and associate with the severity of the disease. The overexpression of suPAR possibly activates β3-integrin in PUUV infection, and thus might be involved in the pathogenesis of the disease.
doi:10.1371/journal.pone.0071335
PMCID: PMC3749226  PMID: 23990945
11.  Elevated Cerebrospinal Fluid Neopterin Concentration Is Associated with Disease Severity in Acute Puumala Hantavirus Infection 
Nephropathia epidemica (NE) caused by Puumala hantavirus (PUUV) is the most common hemorrhagic fever with renal syndrome (HFRS) in Europe. The infection activates immunological mechanisms that contribute to the pathogenesis and characteristics of the illness. In this study we measured cerebrospinal fluid (CSF) neopterin concentration from 23 acute-phase NE patients. We collected data on kidney function, markers of tissue permeability, haemodynamic properties, blood cell count, length of hospitalisation, inflammatory parameters, and ophthalmological properties. The neopterin levels were elevated (>5.8 nmol/L) in 22 (96%) NE-patients (mean 45.8 nmol/L); these were especially high in patients with intrathecal PUUV-IgM production (mean 58.2 nmol/L, P = 0.01) and those with elevated CSF protein concentrations (mean 63.6 nmol/L, P < 0.05). We also observed a correlation between the neopterin and high plasma creatinine value (r = 0.66, P = 0.001), low blood thrombocyte count (r = −0.42, P < 0.05), and markedly disturbed refractory properties of an eye (r = 0.47, P < 0.05). Length of hospitalisation correlated with the neopterin (r = 0.42, P < 0.05; male patients r = 0.69, P < 0.01). Patients with signs of tissue oedema and increased permeability also had high neopterin concentrations. These results reinforce the view that PUUV-HFRS is a general infection that affects the central nervous system and the blood-brain barrier.
doi:10.1155/2013/634632
PMCID: PMC3747491  PMID: 23983770
12.  Time-Resolved FRET -Based Approach for Antibody Detection – A New Serodiagnostic Concept 
PLoS ONE  2013;8(5):e62739.
Förster resonance energy transfer (FRET) is a phenomenon widely utilized in biomedical research of macromolecular interactions. In FRET energy is transferred between two fluorophores, the donor and the acceptor. Herein we describe a novel approach utilizing time-resolved FRET (TR-FRET) for the detection of antibodies not only in a solution-phase homogenous assay but also in single- and two-step solid-phase assays. Our method is based on the principle that the Y-shaped immunoglobulin G molecule is able to simultaneously bind two identical antigen molecules. Hence, if a specific IgG is mixed with donor- and acceptor-labeled antigens, the binding of antigens can be measured by TR-FRET. Using donor- and acceptor-labeled streptavidins (SAs) in conjunction with a polyclonal and a monoclonal anti-SA antibody we demonstrate that this approach is fully functional. In addition we characterize the immune complexes responsible for the TR-FRET signal using density gradient ultracentrifugation and solid-phase immunoassays. The homogenous TR-FRET assay described provides a rapid and robust tool for antibody detection, with a wide potential in medical diagnostics.
doi:10.1371/journal.pone.0062739
PMCID: PMC3647052  PMID: 23667515
13.  Molecular Epidemiology of Outbreak-Associated and Wild-Waterfowl-Derived Newcastle Disease Virus Strains in Finland, Including a Novel Class I Genotype 
Journal of Clinical Microbiology  2012;50(11):3664-3673.
Newcastle disease (ND) is a highly contagious, severe disease of poultry caused by pathogenic strains of Newcastle disease virus (NDV; or avian paramyxovirus-1). NDV is endemic in wild birds worldwide and one of the economically most important poultry pathogens. Most of the published strains are outbreak-associated strains, while the apathogenic NDV strains that occur in wild birds, posing a constant threat to poultry with their capability to convert into more virulent forms, have remained less studied. We screened for NDV RNA in cloacal and oropharyngeal samples from wild waterfowl in Finland during the years 2006 to 2010: 39 of 715 birds were positive (prevalence, 5.5%). The partial or full-length F genes of 37 strains were sequenced for phylogenetic purposes. We also characterized viruses derived from three NDV outbreaks in Finland and discuss the relationships between these outbreak-associated and the wild-bird-associated strains. We found that all waterfowl NDV isolates were lentogenic strains of class I or class II genotype I. We also isolated a genetically distinct class I strain (teal/Finland/13111/2008) grouping phylogenetically together with only strain HIECK87191, isolated in Northern Ireland in 1987. Together they seem to form a novel class I genotype genetically differing from other known NDVs by at least 12%.
doi:10.1128/JCM.01427-12
PMCID: PMC3486212  PMID: 22972821
14.  Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses 
In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n = 97); SEOV (China, n = 5); DOBV (Romania, n = 7); SNV (Canada, n = 23); ANDV (Argentina and Chile, n = 52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n = 177; 96%) and/or IgM (n = 131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).
Author Summary
Hantaviruses are the causative agents of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) — serious emerging diseases, with case-fatality rates of up to 15% and about 35%, respectively. So far, over 21 human pathogenic serotypes have been described, which are classified into New World (circulating in the Americas) and Old World (Asia and Europe) hantaviruses. The prodromal phase of hantavirus infections — fever, myalgia, headache and gastrointestinal symptoms — is indistinguishable from those of many other viral infections. The cardiopulmonary phase of HFRS and diuretic phase of HFRS mimic the acute respiratory distress syndrome and renal failure, respectively. In this context, clinical diagnosis has to be confirmed by laboratory testing, which is predominantly based on serology. Although there is an increasing awareness of hantaviruses, infections are still underdiagnosed, in part due to a lack of available standardized serological assays. This study evaluated a commercial multiparametric indirect immunofluorescence assay for the simultaneous detection of antibodies against clinically important Old World (Hantaan, Puumala, Seoul, Saaremaa and Dobrava) and New World (Sin Nombre and Andes) hantaviruses. Test performance was found to be comparable to established highly sensitive and specific in-house assays.
doi:10.1371/journal.pntd.0002157
PMCID: PMC3617148  PMID: 23593524
15.  Analysis of Complete Puumala Virus Genome, Finland 
Emerging Infectious Diseases  2012;18(12):2070-2072.
Puumala virus causes nephropathia epidemica, a rodent-borne zoonosis that is endemic to Europe. We sequenced the complete Puumala virus genome that was directly recovered from a person who died and compared it with those of viruses from local bank voles. The virus strain involved was neither a unique nor rare genetic variant.
doi:10.3201/eid1812.120747
PMCID: PMC3557877  PMID: 23171600
HFRS; nephropathia epidemica; hantavirus; Puumala virus; viruses; Finland; zoonoses; rodents
16.  Complex evolution and epidemiology of Dobrava-Belgrade hantavirus: definition of genotypes and their characteristics 
Archives of Virology  2012;158(3):521-529.
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.
doi:10.1007/s00705-012-1514-5
PMCID: PMC3586401  PMID: 23090188
17.  Environmental Change and Disease Dynamics: Effects of Intensive Forest Management on Puumala Hantavirus Infection in Boreal Bank Vole Populations 
PLoS ONE  2012;7(6):e39452.
Intensive management of Fennoscandian forests has led to a mosaic of woodlands in different stages of maturity. The main rodent host of the zoonotic Puumala hantavirus (PUUV) is the bank vole (Myodes glareolus), a species that can be found in all woodlands and especially mature forests. We investigated the influence of forest age structure on PUUV infection dynamics in bank voles.
Over four years, we trapped small mammals twice a year in a forest network of different succession stages in Northern Finland. Our study sites represented four forest age classes from young (4 to 30 years) to mature (over 100 years) forests. We show that PUUV-infected bank voles occurred commonly in all forest age classes, but peaked in mature forests. The probability of an individual bank vole to be PUUV infected was positively related to concurrent host population density. However, when population density was controlled for, a relatively higher infection rate was observed in voles trapped in younger forests. Furthermore, we found evidence of a “dilution effect” in that the infection probability was negatively associated with the simultaneous density of other small mammals during the breeding season.
Our results suggest that younger forests created by intensive management can reduce hantaviral load in the environment, but PUUV is common in woodlands of all ages. As such, the Fennoscandian forest landscape represents a significant reservoir and source of hantaviral infection in humans.
doi:10.1371/journal.pone.0039452
PMCID: PMC3380007  PMID: 22745755
18.  The Degree of Leukocytosis and Urine GATA-3 mRNA Levels Are Risk Factors for Severe Acute Kidney Injury in Puumala Virus Nephropathia Epidemica 
PLoS ONE  2012;7(4):e35402.
Puumala hantavirus (PUUV) infection, also known as nephropathia epidemica, is the most common cause of hemorrhagic fever with renal syndrome (HFRS) in Europe. The pathogenesis of PUUV nephropathia epidemica is complex and multifactorial, and the risk factors for severe acute kidney injury (AKI) during acute PUUV infection are not well defined. We conducted a prospective study of hospitalized patients with PUUV infection in Tampere, Finland to identify acute illness risk factors for HFRS severity. Serial daily blood and urine samples were collected throughout acute illness and at 2 week and 6 month convalescent visits. By univariate analyses, the maximum white blood cell count during acute illness was a risk factor for severe AKI. There were no significant associations between PUUV-induced AKI severity and platelet counts, C-reactive protein, or alanine aminotransferase levels. Maximum plasma interleukin (IL)-6, urine IL-6, and urine IL-8 concentrations were positively associated with PUUV-induced AKI. Finally, the maximum urinary sediment GATA-3 mRNA level was positively correlated with the peak fold-change in serum creatinine, regardless of AKI severity classification. By multivariate analyses, we found that the maximum levels of leukocytes and urinary sediment GATA-3 mRNA during acute illness were independent risk factors for severe PUUV-induced AKI. We have identified novel acute illness risk factors for severe PUUV-induced AKI.
doi:10.1371/journal.pone.0035402
PMCID: PMC3327672  PMID: 22523590
19.  Searching for Cellular Partners of Hantaviral Nonstructural Protein NSs: Y2H Screening of Mouse cDNA Library and Analysis of Cellular Interactome 
PLoS ONE  2012;7(4):e34307.
Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.
doi:10.1371/journal.pone.0034307
PMCID: PMC3323627  PMID: 22506017
20.  Plasma Cell-Free DNA Levels Are Elevated in Acute Puumala Hantavirus Infection 
PLoS ONE  2012;7(2):e31455.
Introduction
Puumala hantavirus (PUUV) causes a hemorrhagic fever with renal syndrome called nephropathia epidemica (NE). The aim of the present study was to evaluate plasma cell-free DNA (cf-DNA) levels and urinary cf-DNA excretion in acute NE as well as their associations with the severity of the disease.
Methods
Total plasma cf-DNA was quantified directly in plasma of 61 patients and urine of 20 patients with acute NE. We also carried out a qualitative high-sensitivity lab-on-a-chip DNA assay in 20 patients to elucidate the appearance of cf-DNA in plasma and urine.
Results
The maximum plasma cf-DNA values taken during acute NE were significantly higher than the control values taken after the hospitalization period (median 1.33 µg/ml, range 0.94–3.29 µg/ml vs. median 0.77 µg/ml, range 0.55–0.99 µg/ml, P<0.001). The maximum plasma cf-DNA levels correlated positively with maximum blood leukocyte count (r = 0.388, P = 0.002) and the length of hospital stay (r = 0.376, P = 0.003), and inversely with minimum blood platelet count (r = −0.297, P = 0.020). Qualitative analysis of plasma cf-DNA revealed that in most of the patients cf-DNA displayed a low-molecular weight appearance, corresponding to the size of apoptotic DNA (150–200 bp). The visually graded maximum cf-DNA band intensity correlated positively with the maximum quantity of total plasma cf-DNA (r = 0.513, P = 0.021). Maximum urinary excretion of cf-DNA in turn was not markedly increased during the acute phase of NE and did not correlate with any of the variables reflecting severity of the disease or with the maximum plasma cf-DNA level.
Conclusions
The plasma levels of cf-DNA are elevated during acute PUUV infection and correlate with the apoptotic cf-DNA-band intensity. The plasma cf-DNA concentration correlates with some variables reflecting the severity of the disease. The urinary excretion of cf-DNA does not reflect the degree of inflammation in the kidney.
doi:10.1371/journal.pone.0031455
PMCID: PMC3274523  PMID: 22347483
21.  Hantavirus infections in fluctuating host populations: the role of maternal antibodies 
Infected females may transfer maternal antibodies (MatAbs) to their offspring, which may then be transiently protected against infections the mother has encountered. However, the role of maternal protection in infectious disease dynamics in wildlife has largely been neglected. Here, we investigate the effects of Puumala hantavirus (PUUV)-specific MatAbs on PUUV dynamics, using 7 years' data from a cyclic bank vole population in Finland. For the first time to our knowledge, we partition seropositivity data from a natural population into separate dynamic patterns for MatAbs and infection. The likelihood of young of the year carrying PUUV-specific MatAbs during the breeding season correlated positively with infection prevalence in the overwintered parent population in the preceding spring. The probability of PUUV infection varied between seasons (highest in spring, lowest in late summer) and depended on population structure, but was also, in late autumn, notably, negatively related to summer MatAb prevalence, as well as to infection prevalence earlier in the breeding season. Hence, our results suggest that high infection prevalence in the early breeding season leads to a high proportion of transiently immune young individuals, which causes delays in transmission. This suggests, in turn, that MatAb protection has the potential to affect infection dynamics in natural populations.
doi:10.1098/rspb.2010.1022
PMCID: PMC2992709  PMID: 20591866
bank vole; infection transmission; maternal antibody; Puumala hantavirus; transient immunity
22.  Intracerebral Borna Disease Virus Infection of Bank Voles Leading to Peripheral Spread and Reverse Transcription of Viral RNA 
PLoS ONE  2011;6(8):e23622.
Bornaviruses, which chronically infect many species, can cause severe neurological diseases in some animal species; their association with human neuropsychiatric disorders is, however, debatable. The epidemiology of Borna disease virus (BDV), as for other members of the family Bornaviridae, is largely unknown, although evidence exists for a reservoir in small mammals, for example bank voles (Myodes glareolus). In addition to the current exogenous infections and despite the fact that bornaviruses have an RNA genome, bornavirus sequences integrated into the genomes of several vertebrates millions of years ago. Our hypothesis is that the bank vole, a common wild rodent species in traditional BDV-endemic areas, can serve as a viral host; we therefore explored whether this species can be infected with BDV, and if so, how the virus spreads and whether viral RNA is transcribed into DNA in vivo.
We infected neonate bank voles intracerebrally with BDV and euthanized them 2 to 8 weeks post-infection. Specific Ig antibodies were detectable in 41%. Histological evaluation revealed no significant pathological alterations, but BDV RNA and antigen were detectable in all infected brains. Immunohistology demonstrated centrifugal spread throughout the nervous tissue, because viral antigen was widespread in peripheral nerves and ganglia, including the mediastinum, esophagus, and urinary bladder. This was associated with viral shedding in feces, of which 54% were BDV RNA-positive, and urine at 17%. BDV nucleocapsid gene DNA occurred in 66% of the infected voles, and, surprisingly, occasionally also phosphoprotein DNA. Thus, intracerebral BDV infection of bank vole led to systemic infection of the nervous tissue and viral excretion, as well as frequent reverse transcription of the BDV genome, enabling genomic integration. This first experimental bornavirus infection in wild mammals confirms the recent findings regarding bornavirus DNA, and suggests that bank voles are capable of bornavirus transmission.
doi:10.1371/journal.pone.0023622
PMCID: PMC3174072  PMID: 21935357
23.  Young male patients are at elevated risk of developing serious central nervous system complications during acute Puumala hantavirus infection 
BMC Infectious Diseases  2011;11:217.
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.
doi:10.1186/1471-2334-11-217
PMCID: PMC3166934  PMID: 21838931
hantavirus; encephalitis; hypopituitarism
24.  European Subtype Tick-borne Encephalitis Virus in Ixodes persulcatus Ticks 
Emerging Infectious Diseases  2011;17(2):323-325.
doi:10.3201/eid1702.101487
PMCID: PMC3376769  PMID: 21291624
vector-borne infections; tick-borne encephalitis virus; viruses; TBEV; Ixodes; Finnish Lapland; letter
25.  Tick-borne Encephalitis Virus in Wild Rodents in Winter, Finland, 2008–2009 
Emerging Infectious Diseases  2011;17(1):72-75.
Rodents might maintain tick-borne encephalitis virus (TBEV) in nature through latent persistent infections. During 2 subsequent winters, 2008 and 2009, in Finland, we detected RNA of European and Siberian subtypes of TBEV in Microtus agrestis and Myodes glareolus voles, respectively. Persistence in rodent reservoirs may contribute to virus overwintering.
doi:10.3201/eid1701.100051
PMCID: PMC3204619  PMID: 21192857
Tick-borne encephalitis virus; rodent; persistence; Finland; TBEV-Eur; TBEV-Sib; Myodes glareolus; Microtus agrestis; viruses; dispatch

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