Phylogenetic analysis of variants of the Puumala virus (Hantaviridae: Orthohantavirus) circulating in the Saratov region.

The objective is to determine the complete nucleotide sequence and conduct a phylogenetic analysis of genome variants of the Puumala virus isolated in the Saratov region. MATERIALS AND METHODS: The samples for the study were field material collected in the Gagarinsky (formerly Saratovsky), Engelssky, Novoburassky and Khvalynsky districts of the Saratov region in the period from 2019 to 2022. To specifically enrich the Puumala virus genome in the samples, were used PCR and developed a specific primer panel. Next, the resulting PCR products were sequenced and the fragments were assembled into one sequence for each segment of the virus genome. To construct phylogenetic trees, the maximum parsimony algorithm was used. RESULTS: Genetic variants of the Puumala virus isolated in the Saratov region have a high degree of genome similarity to each other, which indicates their unity of origin. According to phylogenetic analysis, they all form a separate branch in the cluster formed by hantaviruses from other subjects of the Volga Federal District. The virus variants from the Republics of Udmurtia and Tatarstan, as well as from the Samara and Ulyanovsk regions, are closest to the samples from the Saratov region. CONCLUSION: The data obtained show the presence of a pronounced territorial confinement of strains to certain regions or areas that are the natural biotopes of their carriers. This makes it possible to fairly accurately determine the territory of possible infection of patients and/or the circulation of carriers of these virus variants based on the sequence of individual segments of their genome.

Hybrid capture-based next-generation sequencing of new and old world Orthohantavirus strains and wild-type Puumala isolates from humans and bank voles.

Orthohantaviruses, transmitted primarily by rodents, cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome in the Americas. These viruses, with documented human-to-human transmission, exhibit a wide case-fatality rate, 0.5-40 %, depending on the virus species, and no vaccine or effective treatment for severe Orthohantavirus infections exists. In Europe, the Puumala virus (PUUV), carried by the bank vole Myodes glareolus, causes a milder form of HFRS. Despite the reliance on serology and PCR for diagnosis, the three genomic segments of Swedish wild-type PUUV have yet to be completely sequenced. We have developed a targeted hybrid-capture method aimed at comprehensive genomic sequencing of wild-type PUUV isolates and the identification of other Orthohantaviruses. Our custom-designed panel includes >11,200 probes covering the entire Orthohantavirus genus. Using this panel, we sequenced complete viral genomes from bank vole lung tissue, human plasma samples, and cell-cultured reference strains. Analysis revealed that Swedish PUUV isolates belong to the Northern Scandinavian lineage, with nucleotide diversity ranging from 2.8 % to 3.7 % among them. Notably, no significant genotypic differences were observed between the viral sequences from reservoirs and human cases except in the nonstructural protein. Despite the high endemicity of PUUV in Northern Sweden, these are the first complete Swedish wild-type PUUV genomes and substantially increase our understanding of PUUV evolution and epidemiology. The panel's sensitivity enables genomic sequencing of human samples with viral RNA levels reflecting the natural progression of infection and underscores our panel's diagnostic value, and could help to uncover novel Orthohantavirus transmission routes.

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.

Prevalence of Hantaviruses Harbored by Murid Rodents in Northwestern Ukraine and Discovery of a Novel Puumala Virus Strain.

In Europe, two species of hantaviruses, Puumala orthohantavirus (PUUV) and Dobrava orthohantavirus (DOBV), cause hemorrhagic fever with renal syndrome in humans. The rodent reservoirs for these viruses are common throughout Ukraine, and hence, the goal of this study was to identify the species and strains of hantaviruses circulating in this region. We conducted surveillance of small rodent populations in a rural region in northwestern Ukraine approximately 30 km from Poland. From the 424 small mammals captured, we identified nine species, of which the most abundant were Myodes glareolus, the bank vole (45%); Apodemus flavicollis, the yellow-necked mouse (29%); and Apodemus agrarius, the striped field mouse (14.6%) Using an indirect immunofluorescence assay, 15.7%, 20.5%, and 33.9% of the sera from M. glareolus, A. glareolus, and A. flavicollis were positive for hantaviral antibodies, respectively. Additionally, we detected antibodies to the hantaviral antigen in one Microtus arvalis, one Mus musculus, and one Sorex minutus. We screened the lung tissue for hantaviral RNA using next-generation sequencing and identified PUUV sequences in 25 small mammals, including 23 M. glareolus, 1 M. musculus, and 1 A. flavicollis, but we were unable to detect DOBV sequences in any of our A. agrarius specimens. The percent identity matrix and Bayesian phylogenetic analyses of the S-segment of PUUV from 14 M. glareolus lungs suggest the highest similarity (92-95% nucleotide or 99-100% amino acid) with the Latvian lineage. This new genetic information will contribute to future molecular surveillance of human cases in Ukraine.

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.

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.

Molecular and epidemiological characteristics of human Puumala and Dobrava-Belgrade hantavirus infections, Germany, 2001 to 2017.

IntroductionTwo hantavirus species, Puumala (PUUV) and Dobrava-Belgrade (DOBV) virus (genotype Kurkino), are endemic in Germany. Recent PUUV outbreaks raised questions concerning increasing frequency of outbreaks and expansion of PUUV endemic areas.AimsTo describe the epidemiology of human PUUV and DOBV infections in Germany.MethodsWe conducted an observational retrospective study analysing national hantavirus surveillance data notified to the national public health institute and hantavirus nucleotide sequences from patients collected at the national consultation laboratory between 2001 and 2017. Matching molecular sequences with surveillance data, we conducted epidemiological, phylogenetic and phylogeographic analyses.ResultsIn total, 12,148 cases of symptomatic hantavirus infection were notified 2001-17 (mean annual incidence: 0.87/100,000; range: 0.09-3.51). PUUV infections showed a highly variable space-time disease incidence pattern, causing large outbreaks every 2-3 years with peaks in early summer and up to 3,000 annually reported cases. Sex-specific differences in disease presentation were observed. Of 202 PUUV nucleotide sequences obtained from cases, 189 (93.6%) fall into well-supported phylogenetic clusters corresponding to different endemic areas in Germany. DOBV infections caused few, mostly sporadic cases in autumn and winter in the north and east of Germany.ConclusionsThe frequency of PUUV outbreaks increased between 2001 and 2017 but our data does not support the suggested expansion of endemic areas. The epidemiology of PUUV and DOBV-Kurkino infections differs in several aspects. Moreover, the latter are relatively rare and combining efforts and data of several countries to identify risk factors and develop specific recommendations for prevention could be worthwhile.

Post-infectious fatigue following Puumala virus infection.

Background: Puumala virus infection or nephropathia epidemica (NE) is common in northern Sweden. NE causes haemorrhagic fever with renal syndrome. Most patients make a full recovery, but a convalescent phase with fatigue has been reported. Although post-infectious fatigue has been demonstrated for other viral infections, it is not well studied in relation to NE. This study assessed recovery time and levels of fatigue in former NE patients, as compared to the general population. Methods: NE patients diagnosed in northern Sweden between 2007 and 2011, together with a comparison sample from the general population, answered a questionnaire on demographic and health-related factors, including the Fatigue Severity Scale (FSS), and characteristics of NE infection. Self-reported recovery time was assessed, and fatigue levels were compared across the two groups by multiple linear regression, stratified by gender. Results: In total, 1132 NE patients and 915 comparison group subjects participated. Time to complete recovery was reported to exceed 3 months for 47% and 6 months for 32% of the NE patients. Recovery time differed by gender and age. NE patients had significantly higher FSS scores than the comparison group. Differences were greater among women than men, and adjustments for current illness, body mass index, smoking and current residence only slightly modified the estimates. Conclusions: Individuals with previous NE infection show higher fatigue scores than non-infected individuals, even 5 years following the infection. Full recovery takes half a year or longer for a substantial proportion of former NE patients.

[First case of nephropathia epidemica in southern Sweden]. / Första fallet av skånsk sorkfeber - Ytterst sällsynt söder om Mälardalen ­ men nu tycks Puumalavirus spridas till nya områden i Sverige.

Nephropathia epidemica (NE) is the European version of hemorrhagic fever with renal syndrome. NE is caused by the Puumala type of Hantavirus with the bank vole (Clethrionomys glareolus) as the natural reservoir. Endemic regions include northern Sweden with an incidence of 53 to 569 cases per year, depending on the cyclic variation of rodent populations. In southern Sweden, south of the Mälardalen region, no cases of NE have been described without a historical account of travel to endemic areas in the weeks beforehand. This case report describes the course of disease of a patient diagnosed with NE, who had only stayed in the southernmost region of Sweden: Skåne. This may indicate the spread of NE to more southern regions of Sweden. NE should consequently be considered as a differential diagnosis at emergency and infectious disease departments throughout Sweden.

Severe Sinus Bradycardia in Puumala virus infection.

Puumala orthohantavirus (PUUV) is the most prevalent of the four species of zoonotic hantaviruses found in Europe, causing nephropathia epidemica, a mild form of hemorrhagic fever with acute kidney injury that presents with elevated serum creatinine level, proteinuria and hematuria. The febrile phase of the infection begins with flu-like syndrome and visual disturbance. Laboratory results can show thrombocytopenia. The oliguric phase with elevated serum creatinine level then occurs. Cardiac involvement is sometimes observed, especially ECG abnormality: transient T-waves inversion, generally in the lateral or inferior leads. Marked bradycardia has been exceptionally described. We report the case of a 36-year-old woman with acute PUUV infection. Two days after admission, the patient presented a sinus bradycardia at 25/min. The bradycardia was asymptomatic, persisted one week and resolved spontaneously. Cardiac involvement in Puumala virus infection seems not to be associated with a bad prognosis. Bradycardia in the course of an influenza-like illness in endemic areas should suggest several pathogens such as legionella, Q fever or PUUV virus infection.

Puumala Hantavirus Genotypes in Humans, France, 2012-2016.

The analysis of the nucleoprotein gene of 77 Puumala hantavirus strains detected in human samples in France during 2012-2016 showed that all belonged to the Central European lineage. We observed 2 main clusters, geographically structured; one included strains with the Q64 signature and the other strains with the R64 signature.

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.

Acute hantavirus infection presenting as haemolytic-uraemic syndrome (HUS): the importance of early clinical diagnosis.

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

Circulation and diagnostics of Puumala virus in Norway: nephropatia epidemica incidence and rodent population dynamics.

Hantaviruses pose a public health concern worldwide causing haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Puumala virus (PUUV) is the most prevalent hantavirus in Central and Northern Europe, and causes a mild form of HFRS, also known as nephropathia epidemica (NE). In nature, the main host of PUUV is the bank vole (Myodes glareolus), and transmission to humans occurs through inhalation of aerosols from rodent excreta. Nephropathia epidemica is particularly prevalent in Nordic countries, however, few studies of PUUV have been performed in Norway. The aim of this study was to analyse the dynamics of PUUV in Norway and compare with bank vole population dynamics, and also to complement the current diagnostic methodology of NE in Norway. Our results showed a significant seasonal and geographical variation of NE, and a general parallel peak trend between bank vole population densities and human NE incidence. A real-time and a nested PCR were successfully established as an invaluable diagnostic tool, with detection and sequencing of PUUV in a human serum sample for the first time in Norway. Phylogenetic analysis showed clustering of the obtained human sample with previous Norwegian bank vole isolates.

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.

Insufficient efficacy and safety of intravenous ribavirin in treatment of haemorrhagic fever with renal syndrome caused by Puumala virus.

BACKGROUND: Intravenous ribavirin has been reported to be an effective treatment for haemorrhagic fever with renal syndrome (HFRS) caused by Hantaan virus in Asia. However, its therapeutic benefits for HFRS caused by Puumala virus (PUUV) in Europe are still unknown. METHODS: A randomized, open-label study of efficacy and safety of intravenous ribavirin in the treatment of HFRS was conducted in the European part of Russia. Seventy-three patients with suspected HFRS within 4 d of the onset of the disease were randomized to receive either intravenous ribavirin (33 mg/kg, followed by 16 mg/kg given every 6 h for 4 d and by 8 mg/kg given every 8 h for 3 d) plus standard therapy (n = 37) or standard therapy alone (n = 36). The primary outcome was the average change from baseline in viral load over time estimated as area under the viral load curve minus baseline (AUCMB). Fifty-five patients with HFRS confirmed by nested reverse transcriptase - polymerase chain reaction (PCR) assay were included in the assessment of the efficacy. All patients entered into the clinical trial were included in the assessment of the safety. RESULTS: PUUV was detected in all cases of confirmed HFRS. Viral load kinetics were similar in both treatment groups. Significantly more patients receiving ribavirin than standard therapy experienced low haemoglobin level (95% vs 36%), hyperbilirubinemia (81% vs 3%), sinus bradycardia (43% vs 14%), and rash (19% vs 0%). CONCLUSIONS: Results of the study showed insufficient efficacy and safety of intravenous ribavirin in the treatment of HFRS caused by PUUV.

Puumala hantavirus infections in bank vole populations: host and virus dynamics in Central Europe.

BACKGROUND: In Europe, bank voles (Myodes glareolus) are widely distributed and can transmit Puumala virus (PUUV) to humans, which causes a mild to moderate form of haemorrhagic fever with renal syndrome, called nephropathia epidemica. Uncovering the link between host and virus dynamics can help to prevent human PUUV infections in the future. Bank voles were live trapped three times a year in 2010-2013 in three woodland plots in each of four regions in Germany. Bank vole population density was estimated and blood samples collected to detect PUUV specific antibodies. RESULTS: We demonstrated that fluctuation of PUUV seroprevalence is dependent not only on multi-annual but also on seasonal dynamics of rodent host abundance. Moreover, PUUV infection might affect host fitness, because seropositive individuals survived better from spring to summer than uninfected bank voles. Individual space use was independent of PUUV infections. CONCLUSIONS: Our study provides robust estimations of relevant patterns and processes of the dynamics of PUUV and its rodent host in Central Europe, which are highly important for the future development of predictive models for human hantavirus infection risk.

Clinical and virological characteristics of hantavirus infections in a 2014 Croatian outbreak.

INTRODUCTION: Croatia is endemic for hemorrhagic fever with renal syndrome (HFRS), with both Puumala (PUUV) and Dobrava virus (DOBV) documented. Several large outbreaks were recorded in 1995, 2002, and 2012. We analyzed demographic, clinical, laboratory, and virological characteristics of HFRS cases detected in three geographically close natural foci (Ogulin, Slunj, and the Plitvice Lakes surroundings) during the 2014 outbreak. METHODOLOGY: From January to December 2014, 122 patients with suspected HFRS were tested for hantavirus IgM/IgG antibodies using an indirect immunofluorescence assay (IFA). Cross-reactive samples were further tested using a western blot (WB). For hospitalized patients from Ogulin area, clinical and laboratory data were analyzed. RESULTS: Acute infection was documented in 57 (46.7%) patients, of whom 75.4% were hospitalized. Ten (8.2%) patients were found to be IgG seropositive. Patients were 15-69 years of age and predominantly male (74.5%). The outbreak started in winter months, with most cases recorded from May to July (80.7%). The most frequently reported symptoms were fever (96.3%), chills/shivering (62.9%), and lumbar pain (48.1%). Mild clinical form was found in 66.7% patients, moderate in 18.5%, and severe in 14.8% patients (all but one infected with PUUV). One patient died. Using IFA, 48.8% patients showed monotypic antibody response, while in 51.2%, cross-reactive antibodies were found. PUUV was confirmed in 94.7% and DOBV in 5.3% HFRS cases by WB. CONCLUSIONS: Central mountainous Croatian regions are still highly endemic areas for HFRS. A higher percentage of severe PUUV infections could be at least partly associated with a patient's immune status.

A highly divergent Puumala virus lineage in southern Poland.

Puumala virus (PUUV) represents one of the most important hantaviruses in Central Europe. Phylogenetic analyses of PUUV strains indicate a strong genetic structuring of this hantavirus. Recently, PUUV sequences were identified in the natural reservoir, the bank vole (Myodes glareolus), collected in the northern part of Poland. The objective of this study was to evaluate the presence of PUUV in bank voles from southern Poland. A total of 72 bank voles were trapped in 2009 at six sites in this part of Poland. RT-PCR and IgG-ELISA analyses detected three PUUV positive voles at one trapping site. The PUUV-infected animals were identified by cytochrome b gene analysis to belong to the Carpathian and Eastern evolutionary lineages of bank vole. The novel PUUV S, M and L segment nucleotide sequences showed the closest similarity to sequences of the Russian PUUV lineage from Latvia, but were highly divergent to those previously found in northern Poland, Slovakia and Austria. In conclusion, the detection of a highly divergent PUUV lineage in southern Poland indicates the necessity of further bank vole monitoring in this region allowing rational public health measures to prevent human infections.