MAIT cell activation is associated with disease severity markers in acute hantavirus infection.

Hantaviruses are zoonotic RNA viruses that cause severe acute disease in humans. Infected individuals have strong inflammatory responses that likely cause immunopathology. Here, we studied the response of mucosal-associated invariant T (MAIT) cells in peripheral blood of individuals with hemorrhagic fever with renal syndrome (HFRS) caused by Puumala orthohantavirus, a hantavirus endemic in Europe. We show that MAIT cell levels decrease in the blood during HFRS and that residual MAIT cells are highly activated. This activation correlates with HFRS severity markers. In vitro activation of MAIT cells by hantavirus-exposed antigen-presenting cells is dependent on type I interferons (IFNs) and independent of interleukin-18 (IL-18). These findings highlight the role of type I IFNs in virus-driven MAIT cell activation and suggest a potential role of MAIT cells in the disease pathogenesis of viral infections.

Molecular rationale for antibody-mediated targeting of the hantavirus fusion glycoprotein.

The intricate lattice of Gn and Gc glycoprotein spike complexes on the hantavirus envelope facilitates host-cell entry and is the primary target of the neutralizing antibody-mediated immune response. Through study of a neutralizing monoclonal antibody termed mAb P-4G2, which neutralizes the zoonotic pathogen Puumala virus (PUUV), we provide a molecular-level basis for antibody-mediated targeting of the hantaviral glycoprotein lattice. Crystallographic analysis demonstrates that P-4G2 binds to a multi-domain site on PUUV Gc and may preclude fusogenic rearrangements of the glycoprotein that are required for host-cell entry. Furthermore, cryo-electron microscopy of PUUV-like particles in the presence of P-4G2 reveals a lattice-independent configuration of the Gc, demonstrating that P-4G2 perturbs the (Gn-Gc)4 lattice. This work provides a structure-based blueprint for rationalizing antibody-mediated targeting of hantaviruses.

Isolation and characterization of new Puumala orthohantavirus strains from Germany.

Orthohantaviruses are re-emerging rodent-borne pathogens distributed all over the world. Here, we report the isolation of a Puumala orthohantavirus (PUUV) strain from bank voles caught in a highly endemic region around the city Osnabrück, north-west Germany. Coding and non-coding sequences of all three segments (S, M, and L) were determined from original lung tissue, after isolation and after additional passaging in VeroE6 cells and a bank vole-derived kidney cell line. Different single amino acid substitutions were observed in the RNA-dependent RNA polymerase (RdRP) of the two stable PUUV isolates. The PUUV strain from VeroE6 cells showed a lower titer when propagated on bank vole cells compared to VeroE6 cells. Additionally, glycoprotein precursor (GPC)-derived virus-like particles of a German PUUV sequence allowed the generation of monoclonal antibodies that allowed the reliable detection of the isolated PUUV strain in the immunofluorescence assay. In conclusion, this is the first isolation of a PUUV strain from Central Europe and the generation of glycoprotein-specific monoclonal antibodies for this PUUV isolate. The obtained virus isolate and GPC-specific antibodies are instrumental tools for future reservoir host studies.

Analytical Performance of the RIDASCREEN® Hantavirus Puumala IgG/IgM ELISA Assay.

The National Reference Center for Hantavirus in Belgium is currently using the Hantavirus IgM/IgG ELISA Progen kit (Heidelberg, Germany) for the detection of the most prevalent Hantavirus in Western Europe, Puumala virus (PUUV). Two commercially available PUUV kits were compared: Progen and RIDASCREEN® Hantavirus Puumala IgM/IgG ELISA assay (Darmstadt, Germany). METHODS: The sensitivity was evaluated with a panel of 68 samples from patients with an acute infection (n = 44) or a past infection (n = 24). Specificity was evaluated with a panel of 62 samples from patients with potentially false borderline results (n = 7) (no seroconversion), seronegative samples (n = 25) and potentially cross reacting samples (n = 30). Discordances were resolved by immunoblot. Substantial agreement was calculated using Cohen kappa coefficient. RESULTS: The RIDASCREEN® kit showed a higher specificity (IgM: 94.3%; IgG: 94.4%) than the Progen kit (IgM: 77.0% IgG: 93.0%). The sensitivity for IgM ELISA was 100% for both assays. IgG sensitivity was, respectively, 98.3% and 100% for Progen and RIDASCREEN®. A Cohen kappa coefficient of 0.76 and 0.90 was found between Puumala IgM and IgG, respectively. CONCLUSIONS: This study showed a higher specificity for the RIDASCREEN® kit than the Progen kit, while the sensitivity was as good as for the Progen kit.

Puumala and Tula Virus Differ in Replication Kinetics and Innate Immune Stimulation in Human Endothelial Cells and Macrophages.

Old world hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) upon zoonotic transmission to humans. In Europe, the Puumala virus (PUUV) is the main causative agent of HFRS. Tula virus (TULV) is also widely distributed in Europe, but there is little knowledge about the pathogenicity of TULV for humans, as reported cases are rare. We studied the replication of TULV in different cell types in comparison to the pathogenic PUUV and analyzed differences in stimulation of innate immunity. While both viruses replicated to a similar extent in interferon (IFN)-deficient Vero E6 cells, TULV replication in human lung epithelial (A549) cells was slower and less efficient when compared to PUUV. In contrast to PUUV, no replication of TULV could be detected in human microvascular endothelial cells and in macrophages. While a strong innate immune response towards PUUV infection was evident at 48 h post infection, TULV infection triggered only a weak IFN response late after infection of A549 cells. Using appropriate in vitro cell culture models for the orthohantavirus infection, we could demonstrate major differences in host cell tropism, replication kinetics, and innate immune induction between pathogenic PUUV and the presumably non- or low-pathogenic TULV that are not observed in Vero E6 cells and may contribute to differences in virulence.

Urine and Free Immunoglobulin Light Chains as Analytes for Serodiagnosis of Hantavirus Infection.

Rapid point-of-care testing is a megatrend in infectious disease diagnosis. We have introduced a homogeneous immunoassay concept, which is based on the simultaneous binding of antigen and protein L to a given immunoglobulin molecule. The complex formation is detected utilizing time-resolved Förster resonance energy transfer between antigen-attached donor and acceptor-labeled protein L, hence the name LFRET. Here, we demonstrate that urine can be used as a sample matrix in LFRET-based serodiagnostics. We studied urine samples collected during the hospitalization and recovery of patients with acute Puumala orthohantavirus (PUUV) infection. We compared PUUV antibody-specific LFRET signals in urine to those in plasma, and found excellent correlation in the test outcomes The LFRET test from urine was positive in 40/40 patients with acute PUUV infection. PUUV causes a mild form of hemorrhagic fever with renal syndrome, characterized by acute kidney injury and proteinuria. Immunofluorescence and western blotting demonstrated PUUV-IgG and -IgA in urine, however, the presence of intact immunoglobulins did not fully explain the LFRET signals. We purified free light chains (FLCs) from both urine and serum of healthy volunteers and patients with acute PUUV infection, and verified the presence of antigen-specific FLCs. Antigen-specific FLCs provide a new means for non-invasive antibody detection and disease diagnosis.

Orthohantaviruses belonging to three phylogroups all inhibit apoptosis in infected target cells.

Orthohantaviruses, previously known as hantaviruses, are zoonotic viruses that can cause hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS) in humans. The HPS-causing Andes virus (ANDV) and the HFRS-causing Hantaan virus (HTNV) have anti-apoptotic effects. To investigate if this represents a general feature of orthohantaviruses, we analysed the capacity of six different orthohantaviruses - belonging to three distinct phylogroups and representing both pathogenic and non-pathogenic viruses - to inhibit apoptosis in infected cells. Primary human endothelial cells were infected with ANDV, HTNV, the HFRS-causing Puumala virus (PUUV) and Seoul virus, as well as the putative non-pathogenic Prospect Hill virus and Tula virus. Infected cells were then exposed to the apoptosis-inducing chemical staurosporine or to activated human NK cells exhibiting a high cytotoxic potential. Strikingly, all orthohantaviruses inhibited apoptosis in both settings. Moreover, we show that the nucleocapsid (N) protein from all examined orthohantaviruses are potential targets for caspase-3 and granzyme B. Recombinant N protein from ANDV, PUUV and the HFRS-causing Dobrava virus strongly inhibited granzyme B activity and also, to certain extent, caspase-3 activity. Taken together, this study demonstrates that six different orthohantaviruses inhibit apoptosis, suggesting this to be a general feature of orthohantaviruses likely serving as a mechanism of viral immune evasion.

Relationship between circulating vascular endothelial growth factor and its soluble receptor in patients with hemorrhagic fever with renal syndrome.

Hemorrhagic fever with renal syndrome (HFRS) is characterized by endothelial dysfunction with capillary leakage without obvious cytopathology in the capillary endothelium. The aim of the study was to analyze the kinetics of vascular endothelial growth factor (VEGF) and its soluble receptor (sVEGFR-2) in HFRS patients infected with Dobrava (DOBV) or Puumala virus (PUUV). VEGF and sVEGFR-2 levels were measured in daily plasma and urine samples of 73 patients with HFRS (58 with PUUV, 15 with DOBV) and evaluated in relation to clinical and laboratory variables. In comparison with the healthy controls, initial samples (obtained in the first week of illness) from patients with HFRS had higher plasma and urine VEGF levels, whereas sVEGFR-2 levels were lower in plasma but higher in urine. VEGF levels did not differ in relation to hantavirus species, viral load, or the severity of HFRS. The comparison of VEGF dynamics in plasma and urine showed the pronounced secretion of VEGF in urine. Significant correlations were found between daily VEGF/sVEGFR-2 levels and platelet counts, as well as with diuresis: the correlations were positive for plasma VEGF/sVEGFR-2 levels and negative for urine levels. In addition, patients with hemorrhagic manifestations had very high plasma and urine VEGF, together with high urine sVEGFR-2. Measuring the local secretion of sVEGFR-2 in urine might be a useful biomarker for identifying HFRS patients who will progress to severe disease.

Bank vole immunoheterogeneity may limit Nephropatia Epidemica emergence in a French non-endemic region.

Ecoevolutionary processes affecting hosts, vectors and pathogens are important drivers of zoonotic disease emergence. In this study, we focused on nephropathia epidemica (NE), which is caused by Puumala hantavirus (PUUV) whose natural reservoir is the bank vole, Myodes glareolus. We questioned the possibility of NE emergence in a French region that is considered to be NE-free but that is adjacent to a NE-endemic region. We first confirmed the epidemiology of these two regions and we demonstrated the absence of spatial barriers that could have limited dispersal, and consequently, the spread of PUUV into the NE-free region. We next tested whether regional immunoheterogeneity could impact PUUV chances to circulate and persist in the NE-free region. We showed that bank voles from the NE-free region were sensitive to experimental PUUV infection. We observed high levels of immunoheterogeneity between individuals and also between regions. Antiviral gene expression (Tnf and Mx2) reached higher levels in bank voles from the NE-free region. During experimental infections, anti-PUUV antibody production was higher in bank voles from the NE-endemic region. These results indicated a lower susceptibility to PUUV for bank voles from this NE-free region, which might limit PUUV persistence and therefore, the risk of NE.

Experimental infections of wild bank voles (Myodes glareolus) from nephropatia epidemica endemic and non-endemic regions revealed slight differences in Puumala virological course and immunological responses.

In Europe, the occurrence of nephropathia epidemica (NE), a human disease caused by Puumala virus (PUUV), exhibits considerable geographical heterogeneity despite the continuous distribution of its reservoir, the bank vole Myodes glareolus. To better understand the causes of this heterogeneity, wild voles sampled in two adjacent NE endemic and non-endemic regions of France were infected experimentally with PUUV. The responses of bank voles to PUUV infection, based on the levels of anti-PUUV IgG and viral RNA, were compared. Slight regional differences were highlighted despite the high inter-individual variability. Voles from the NE non-endemic region showed greater immune responsiveness to PUUV infection, but lower levels of RNA in their organs than voles from the endemic region. These results suggest the existence of regional variations in the sensitivity of bank voles that could contribute to the apparent absence of PUUV circulation among voles and the absence of NE in the non-endemic region.

Indoleamine 2,3-dioxygenase activity is associated with regulatory T cell response in acute Puumala hantavirus infection.

High indoleamine 2,3-dioxygenase (IDO) activity is associated with clinically severe acute infection caused by Puumala hantavirus. The immunoregulatory effects of IDO can be mediated either through metabolic control of effector T cells, caused by depletion of the essential amino acid tryptophan, or intercellular signaling and activation of regulatory T cell responses. Here, we have studied 24 patients with acute Puumala hantavirus infection to distinguish between these possibilities. Maximum IDO activity showed a significant positive correlation with FOXP3 expression levels in regulatory T cells, a quantitative surrogate marker for suppressive capability. In contrast, IDO activity did not correlate with the frequency of CD8+ effector cells in cell cycle. The data suggest that in Puumala infection, the mechanism responsible for the suppressive effect of IDO is not metabolic control of effector cells but rather the signaling mediated by tryptophan breakdown products, such as kynurenine.

Human Puumala hantavirus infection in northern Sweden; increased seroprevalence and association to risk and health factors.

BACKGROUND: The rodent borne Puumala hantavirus (PUUV) causes haemorrhagic fever with renal syndrome in central and northern Europe. The number of cases has increased and northern Sweden has experienced large outbreaks in 1998 and 2006-2007 which raised questions regarding the level of immunity in the human population. METHODS: A randomly selected population aged between 25 and 74 years from northern Sweden were invited during 2009 to participate in a WHO project for monitoring of trends and determinants in cardiovascular disease. Health and risk factors were evaluated and sera from 1,600 participants were available for analysis for specific PUUV IgG antibodies using a recombinant PUUV nucleocapsid protein ELISA. RESULTS: The overall seroprevalence in the investigated population was 13.4 %, which is a 50 % increase compared to a similar study only two decades previously. The prevalence of PUUV IgG increased with age, and among 65-75 years it was 22 %. More men (15.3 %) than women (11.4 %) were seropositive (p < 0.05). The identified risk factors were smoking (OR = 1.67), living in rural areas (OR = 1.92), and owning farmland or forest (OR = 2.44). No associations were found between previous PUUV exposure and chronic lung disease, diabetes, hypertension, renal dysfunction, stroke or myocardial infarction. CONCLUSIONS: PUUV is a common infection in northern Sweden and there is a high life time risk to acquire PUUV infection in endemic areas. Certain risk factors as living in rural areas and smoking were identified. Groups with increased risk should be targeted for future vaccination when available, and should also be informed about appropriate protection from rodent secreta.

Hantavirus cardiopulmonary syndrome due to Puumala virus in Germany.

In Germany Puumala virus (PUUV), known to cause mild forms of hemorrhagic fever with renal syndrome (HFRS), is the predominating endemic hantavirus. We herein describe an unusually severe case of a PUUV infection that occurred in summer 2015 in South Eastern Germany in a region known to be endemic for PUUV since over ten years. A 54-year-old female gardener was admitted to hospital with fever, cough and dyspnea. Within 48hours the patient developed a rapid progressive adult respiratory distress syndrome (ARDS) with circulatory failure and required ECMO (extracorporeal membrane oxygenation) treatment. Serological and molecular biological examinations of serum samples confirmed an infection with PUUV. Partial sequences of the S- and M-segment clustered to a strain previously described in South Eastern Germany. Our reported case highlights, that in rare incidents PUUV can cause hantavirus cardiopulmonary syndrome, a syndrome that is usually found after infections with New World hantaviruses, and neurological symptoms.

A Molecular-Level Account of the Antigenic Hantaviral Surface.

Hantaviruses, a geographically diverse group of zoonotic pathogens, initiate cell infection through the concerted action of Gn and Gc viral surface glycoproteins. Here, we describe the high-resolution crystal structure of the antigenic ectodomain of Gn from Puumala hantavirus (PUUV), a causative agent of hemorrhagic fever with renal syndrome. Fitting of PUUV Gn into an electron cryomicroscopy reconstruction of intact Gn-Gc spike complexes from the closely related but non-pathogenic Tula hantavirus localized Gn tetramers to the membrane-distal surface of the virion. The accuracy of the fitting was corroborated by epitope mapping and genetic analysis of available PUUV sequences. Interestingly, Gn exhibits greater non-synonymous sequence diversity than the less accessible Gc, supporting a role of the host humoral immune response in exerting selective pressure on the virus surface. The fold of PUUV Gn is likely to be widely conserved across hantaviruses.

Serological survey in the Finnish human population implies human-to-human transmission of Ljungan virus or antigenically related viruses.

Ljungan virus (LV) is a picornavirus related to human parechoviruses (HPeV). The virus has been found in bank voles (Myodes glareolus) and several other rodent species, and suggested to have zoonotic potential. Thus far, seroepidemiological data on LV infections in humans are scarce. In this study, we aimed to characterize the demographic and geographical distribution of LV-reactive antibodies in Finland, and to investigate its occurrence in patients suspected of having a rodent-borne disease, nephropathia epidemica (NE) caused by Puumala hantavirus (PUUV). Using an immunofluorescence assay (LV strain 145SLG), we screened human sera (n = 1378) and found LV-reactive antibodies in 36% of samples. The probability of possessing LV-reactive antibodies peaked at age of 14 years, suggesting that most infections occur in childhood. The prevalence of LV-reactive antibodies was significantly higher in the urbanized area surrounding Helsinki than in more rural Central Finland. These findings are uncharacteristic of a rodent-borne pathogen, and therefore we consider human-to-human transmission of one or several Ljungan-like viruses as a likely cause for most of the observed antibody responses.

A multi-head intradermal electroporation device allows for tailored and increased dose DNA vaccine delivery to the skin.

The identification of an effective and tolerable delivery method is a necessity for the success of DNA vaccines in the clinic. This article describes the development and validation of a multi-headed intradermal electroporation device which would be applicable for delivering multiple DNA vaccine plasmids simultaneously but spatially separated. Reporter gene plasmids expressing green and red fluorescent proteins were used to demonstrate the impact of spatial separation on DNA delivery to increase the number of transfected cells and avoid interference through visible expression patterns. To investigate the impact of plasmid interference on immunogenicity, a disease target was investigated where issues with multi-valent vaccines had been previously described. DNA-based Hantaan and Puumala virus vaccines were delivered separately or as a combination and the effect of multi-valence was determined by appropriate assays. While a negative impact was observed for both antigenic vaccines when delivered together, these effects were mitigated when the vaccine was delivered using the multi-head device. We also demonstrate how the multi-head device facilitates higher dose delivery to the skin resulting in improved immune responses. This new multi-head platform device is an efficient, tolerable and non-invasive method to deliver multiple plasmid DNA constructs simultaneously allowing the tailoring of delivery sites for combination vaccines. Additionally, this device would allow the delivery of multi-plasmid vaccine formulations without risk of impacted immune responses through interference. Such a low-cost, easy to use device platform for the delivery of multi-agent DNA vaccines would have direct applications by the military and healthcare sectors for mass vaccination purposes.

Serological survey of Seewis virus antibodies in patients suspected for hantavirus infection in Finland; a cross-reaction between Puumala virus antiserum with Seewis virus N protein?

Puumala virus (PUUV, carried by Myodes glareolus) co-circulates with Seewis virus (SWSV, carried by Sorex araneus) in Finland. While PUUV causes 1000-3000 nephropathia epidemica (NE) cases annually, the pathogenicity of SWSV to man is unknown. To study the prevalence of SWSV antibodies in hantavirus fever-like patients' sera, we used recombinant SWSV nucleocapsid (N) protein as the antigen in ELISA, immunofluorescence assay (IFA) and immunoblotting. While characterizing the recombinant SWSV N protein, we observed that a polyclonal rabbit antiserum against PUUV N protein cross-reacted with SWSV N protein and vice versa. We initially screened 486 (450 PUUV-seronegative and 36 PUUV-seropositive) samples sent to Helsinki University Hospital Laboratory for PUUV serodiagnosis during 2002 and 2007 in an SWSV N protein IgG ELISA. In total, 4.2 % (19/450) of the PUUV-seronegative samples were reactive in the SWSV N protein IgG ELISA and none of the tested samples [43 PUUV-seronegative (weakly reactive in the SWSV IgG ELISA) and 15 random] were reactive in the SWSV N protein IgM ELISA. None of the IgG reactions could be confirmed by IFA or immunoblotting. Furthermore, among the 36 PUUV-seropositive samples three were reactive in SWSV N protein IgG and ten in SWSV N protein IgM ELISA. One PUUV-seropositive sample reacted with SWSV N protein in IFA and four in immunoblotting. Finally, we applied competitive ELISA to confirm that the observed reactivity was due to cross-reactivity rather than a true SWSV response. In conclusion, no evidence of SWSV infection was found among the 486 samples studied; however, we did demonstrate that PUUV antiserum cross-reacted with shrew-borne hantavirus N protein.

Rapid homogeneous immunoassay based on time-resolved Förster resonance energy transfer for serodiagnosis of acute hantavirus infection.

We recently introduced a homogeneous immunoassay based on time-resolved Förster resonance energy transfer (TR-FRET) elicited by fluorophore-labeled antigen and fluorophore-labeled protein L, bound by an immunoglobulin. As the first clinical application, we employ this approach (LFRET) in serodiagnosis of Puumala hantavirus (PUUV) infection. A reference panel containing serum from individuals with acute (n = 21) or past (n = 17) PUUV infection and from PUUV-seronegative individuals (n = 20) was used to define the parameters. The clinical assay performance was evaluated with a prospectively collected serum panel (panel 2; n = 153). Based on the results for panel 1, the threshold for positivity was set at a signal level that was 3-fold over background, while those with a signal <3-fold over the background level were considered PUUV seronegative. With panel 1, 20/21 acute- and 7/10 past-infection samples induced positive signals, compared to 0/20 seronegatives. With panel 2, a positive signal was obtained in 39/40 acute- and 4/10 past-infection samples, as opposed to 7/103 seronegatives. However, after IgG depletion, 58/61 acute-infection samples were LFRET positive, while all past-infection and seronegative samples were negative, corresponding to 100% specificity and 95% sensitivity in detection of acute PUUV infection. We demonstrate that the novel immunoassay is a promising tool for rapid serodiagnosis of acute Puumala virus infection.

A multi-head intradermal electroporation device allows for tailored and increased dose DNA vaccine delivery to the skin.

The identification of an effective and tolerable delivery method is a necessity for the success of DNA vaccines in the clinic. This manuscript describes the development and validation of a multi-headed intradermal electroporation device which would be applicable for delivering multiple DNA vaccine plasmids simultaneously but spatially separated. Reporter gene plasmids expressing green and red fluorescent proteins were used to demonstrate the impact of spatial separation on DNA delivery to increase the number of transfected cells and avoid interference through visible expression patterns. To investigate the impact of plasmid interference on immunogenicity, a disease target was investigated where issues with multi-valent vaccines had been previously described. DNA-based Hantaan and Puumala virus vaccines were delivered separately or as a combination and the effect of multi-valence was determined by appropriate assays. While a negative impact was observed for both antigenic vaccines when delivered together, these effects were mitigated when the vaccine was delivered using the multi-head device. We also demonstrate how the multi-head device facilitates higher dose delivery to the skin resulting in improved immune responses. This new multi-head platform device is an efficient, tolerable and non-invasive method to deliver multiple plasmid DNA constructs simultaneously allowing the tailoring of delivery sites for combination vaccines. Additionally, this device would allow the delivery of multi-plasmid vaccine formulations without risk of impacted immune responses through interference. Such a low-cost, easy to use device platform for the delivery of multi-agent DNA vaccines would have direct applications by the military and healthcare sectors for mass vaccination purposes.