Puumala Virus RNA Research Articles

Positive Correlation between Serum Tryglycerides and Pro-Inflammatory Cytokines in Hemorrhagic Fever with Renal Syndrome

Hemorrhagic fever with renal syndrome (HFRS) is endemic in the Republic of Tatarstan. Clinically, it is characterized by kidney insufficiency and hemorrhagic disorders. Elevated serum lipase as well as increased cholesterol, total phospholipids and triglycerides has been documented in some HFRS cases suggesting the role of lipid metabolism in the disease pathogenesis. However, little is known about association between serum lipids and cytokine activation in HFRS.Materials and Methods: Serum samples from HFRS patients admitted to Republican Clinical Hospital for Infectious Disease named after Agafonov were collected along with healthy controls matched for the region. The Institutional Review Board of the Kazan Federal University approved this study and informed consent was obtained from each study subject according to the guidelines approved under this protocol (article 20, Federal Law “Protection of Health Rights of Citizens of Russian Federation” N323- FZ, 11.21.2011). Serum cytokines were probed using Bio Plex Pro Human Cytokine 27-plex Panel and Bio Plex Human Cytokine 21-plex Panels (Bio-Rad, Hercules, CA, USA) multiplex magnetic bead-based antibody detection kits following manufacturer's instructions. Data collected was analyzed using MasterPlex CT control software and MasterPlex QT analysis software (MiraiBio, San Bruno, CA, USA). Standard curves for each analytes were generated using standards provided by manufacturer.Levels of total cholesterol, tryglycerides and HDCL were determined using Novochol-200 kit, Triglyceride-Novo kit (Vektor-Best, Russia) and HDCL-Cholesterol-Novo-A kit according the manufacturer's instructions. Puumala virus RNA was detected using nested PCR.Statistical analysis was conducted using R language for statistical computing (www.R-project.org), RStudio (www.rstudio.com) and packages “tableone” (https://CRAN.R-project.org/package=tableone) and “reshape2” (Wickham, 2007). Illustrations were generated using “ggplot2” package (Wickham, 2009). Comparisons were carried out using Mann-Whitney test for continuous variables. Spearman's rank correlation coefficients were plotted as a heatmap. Differences were considered significant at p<0.05.Results: One hundred and ninety two HFRS patients (156 males and 36 females; median age of 37 years) were recruited into this study. Median duration of hospitalization was 8 days. Twenty controls were also recruited (8 males and 12 females; median age of 26.5). Analysis of HFRS serum lipids showed increased levels of VDCL and triglycerides (p<0.001, Table 1). We also detected significant upregulation of pro-inflammatory cytokines, growth factors and chemoattractants as compared to controls, while levels of TNFβ, IL-1α, CCL27, CXCL1 and CXCL12α were lower (Table 2).Weak to moderate significant positive correlations between cytokines and lipids were revealed in HFRS cases. Correlations were found between serum triglycerides and IL-4, IL-12(p70), IL-13, IL-1α, IL-2RA, IL-3, IL12(p40), IL-16 and IL-18 (Fig. 1A); IFNα-2 (Fig. 1C); growth factors SCF and β-NGF (Fig. 1D); leukocyte migration factors G-SSCF, CCL27, CCL7, CXCL9 and CXCL12α (Fig. 1E). Cholesterol correlated only with IL-4 and G-SCF, while HDCL was correlated with 8 analytes: IL-1α, IL-16, IFNα-2, SCF, β-NGF, CCL27, CCL7 and CXCL12α. We also observed correlation between VDCL and 6 cytokines: IL-1 α, IFNα-2, β-NGF, CCL27, CCL7 along with CXCL12α. LDCL was correlated with IL-1 β, IL-7, TNFα and bFGF (Fig. 1).Lack of significant correlation was found between: serum levels of LDCL and factors involved in leukocyte migration as well as interferons (Fig.1C, E); VDCL and TNFs (Fig.1B); cholesterol and IFNs and growth factors (Fig. 1B-D); triglycerides and TNFs (Fig.1B).Weak to moderate significant negative correlations between cytokines and lipids were also revealed in HFRS cases. Interestingly, significant negative correlation was found between HDCL and 8 cytokines (IL-1β, IL-5, IL-17, TNFα, IFNγ, PDGF-BB, bFGF, CCL5). Out of these 8 cytokines, three (IL-5, IL-17, IFNγ) were also negatively correlated with serum levels of VDCL.Conclusion: Our data demonstrates correlation between serum lipid and cytokines in HFRS. Strong significant correlations were found between serum triglycerides and cytokines, suggesting that these lipids affect HFRS pathogenesis.This study was supported by RSF grant 15-14-00016. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Real-Time RT-PCR for Diagnostic Use in Detection of Puumala Virus.

Puumala virus (PUUV) is the most common cause of hantavirus infection in Europe, with thousands of cases occurring particularly in Northern, Central and Eastern Europe and Russia. It causes a mild form of hemorrhagic fever with renal syndrome also known as nephropathia epidemica (NE) with clinical picture ranging from mild to severe. Currently, the laboratory diagnosis of NE is mainly based on serology. Here, we evaluated a real-time one-step qRT-PCR (PUUV-qRT-PCR) for detection of PUUV with 238 consecutive diagnostic serum samples from patients with suspected PUUV infection. The PUUV-qRT-PCR was both specific and sensitive for PUUV RNA. The analytical sensitivity (limit of detection) was estimated to be four copies of PUUV per reaction. Altogether 28 out of 30 (93%) PUUV IgM positive samples were positive also for PUUV RNA. No false positives were detected and the specificity was thus 100%. Interestingly, one sample was found positive in PUUV-qRT-PCR prior to subsequent IgM and IgG seroconversion. PUUV-qRT-PCR could be used for diagnostics in the early phase of NE infection and might be helpful especially in the rare severe cases when the patient’s condition may deteriorate rapidly.

Genetic Characterization of Small (s)-Segment Genome Puumala Virus Strain Kazan

The Republic of Tatarstan is one of the most active endemic regions for nephropathia epidemica (NE) in the Russian Federation. Annually, over 1000 cases of NE are registered, with an average mortality rate of 0.43 %. NE is a zoonosis where human become infected by inhaling a hantavirus-contaminated aerosol. Puumala virus is commonly detected in NE cases. Although NE cases have been registered in the region since 1958, little is known about the genetic variability of Puumala virus circulating in Tatarstan. We conducted phylogenetic analysis of the full length (1828 nt) small (S) segment genome sequence of Puumala virus RNA isolated from four bank voles (Myodes glareolus) captured during the 2014 outbreak. These virus sequences were compared to known sequences of Puumala viruses from nearby regions such as Samara and the Republic of Bashkortostan, as well as to European isolates. We found that there was over 89 % nucleotide identity between Puumala virus sequences isolated from the bank voles captured in Tatarstan. Sequence identity between Puumala viruses from the Tatarstan bank voles and the sequences from Republic of Bashkortostan and the Samara region were 90–95 %. Less similarity was found between Tatarstan sequences and Puumala strains circulating in Europe (79.7–87.7 %). Taken together, these data suggest that Puumala viruses circulating within the bank vole population in the Republic of Tatarstan are phylogenetically closer to the viruses circulating in neighboring regions of Russia.

Association of low serum TGF-β level in hantavirus infected patients with severe disease.

BackgroundHantaviruses are emerging zoonotic pathogens which cause hemorrhagic fever with renal syndrome, an immune-mediated pathogenesis is discussed. The aim of the present study was to investigate the role of TGF-β expression in acute hantavirus infection.ResultsWe retrospectively studied 77 patients hospitalised with acute Puumala infection during a hantavirus epidemic in Germany in 2012. Hantavirus infection was confirmed by positive anti-Puumala hantavirus IgG and IgM. Plasma levels of transforming growth factor (TGF)-β1 and TGF-β2 were analysed. Based on glomerular filtration rate on admission, patients were divided in mild and severe course of disease. Puumala virus RNA was detected by PCR amplification of the viral L segment gene. Out of 77 Puumala virus infected patients, 52 (68%) were male. A seasonal distribution was detected in our cohort with a peak in summer 2012, the highest incidence was observed in the age group of 30–39 years. Puumala virus RNA was detectable in 4/77 cases. Patients with severe disease had a significant longer hospital stay than patients with mild disease (6.2 vs 3.6 days). Thrombocyte count (186 vs 225 per nl), serum TGF-β1 (74 vs 118 ng/l) and TGF-β2 (479 vs 586 pg/l) were significantly lower in severe compared to mild disease. However, C-reactive protein (CRP) was significantly higher in patients with severe disease (62 vs 40 mg/l). TGF-β1/Cr was the most sensitive and specific marker associated with renal dysfunction.ConclusionHigh serum CRP and low serum TGF-β in the early phase of hantavirus infection is associated with a severe course of disease. Our results support the hypothesis of an immune-mediated pathogenesis in hantavirus infection.

First Molecular Evidence for Puumala Hantavirus in Poland

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

Multiple Synchronous Outbreaks of Puumala Virus, Germany, 2010

To investigate 2,017 cases of hantavirus disease in Germany, we compared 38 new patient-derived Puumala virus RNA sequences identified in 2010 with bank vole–derived small segment RNA sequences. The epidemic process was driven by outbreaks of 6 Puumala virus clades comprising strains of human and vole origin. Each clade corresponded to a different outbreak region.

Von Mäusen auf Menschen – Erkenntnisse aus der Hantavirus-Epidemie 2010

With 2017 notified cases the largest hantavirus epidemic in Germany has occurred in 2010. We report on two interesting cases illustrating the wide range of the individual clinical course and the diagnostic problems in hantavirus disease. The first patient was a seriously ill 44-year-old man who needed dialysis after an onset of flu-like symptoms with oliguria. An initially negative result of a hantavirus serology focused attention on rapidly progressive glomerulonephritis. The second patient, a 22-year-old man, presented with severe neurological symptoms with seizures. Pathological examination of the renal-biopsy specimen in Case 1 reportedly showed the typical pattern of tubulointerstitial damage in the renal cortex and the outer medulla as in hantavirus infection. In a repeated analysis Puumala virus RNA as a marker of acute infection was found. After dialysis and administration of higher-dose systemic glucocorticoids the patient slowly recovered. In Case 2 the severe neurological symptoms caused a complete neurological diagnostic with lumbar puncture and MRI before the detection of specific antibodies and Puumala virus RNA showed that nephropathia epidemica was the disease. The patient recovered after 10 days. Because of the variability of symptoms and the extrarenal manifestations of the disease the nephropathia epidemica can occasionally cause problems of differential diagnosis. A rapid diagnosis is important because of the urgent differentiation of other renal diseases with bad prognosis.

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

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

Hantavirus Infection in Istanbul, Turkey

To the Editor: More than 20 serotypes of hantavirus have been identified, and 11 infect humans. Puumala virus (PUUV), Dobrava virus (DOBV), and Seoul virus cause different forms of hemorrhagic renal syndrome (1,2). DOBV is endemic to Turkey and countries in the Balkan region. Approximately 10,000–12,000 cases of infection with PUUV and DOBV occur in European Russia each year (3). Initial case reports identified a hantavirus epidemic (laboratory confirmed) in February 2009 that involved 12 persons in Bartin and Zonguldak in western Turkey near the Black Sea. The hantavirus responsible for this epidemic was a PUUV subtype (4). We report a man infected with DOBV in Turkey who died 2 days after admission to an intensive care unit (ICU). The patient was a 22-year-old man who lived near Istanbul, Turkey. He was admitted to the Silivri State Hospital in March 2010 because of fatigue, diffuse pain, nausea, and vomiting. Approximately 2 hours after admission, ecchymotic rashes developed on his upper extremities and spread to other areas. His general condition worsened, and 15 hours later, he was transferred to the ICU of the Emergency Service of Gulhane Military Medical Academy Haydarpasa Training Hospital. His medical history did not include exposure to rodents or any travel. At admission to the ICU, his general condition was poor, and his speech was garbled and incoherent. He had a body temperature of 37.2°C, a pulse of 140 beats/min, an arterial blood pressure of 90/60 mm Hg, diffuse hemorrhagic foci, and a disseminated ecchymotic rash. Laboratory test results showed the following: 13,200 leukocytes/mm3, 92% polymorphonuclear leukocytes, hemoglobin 11.6 mg/dL, 385,000 platelets/mm3, alanine aminotransferase 62 IU/mL, aspartate aminotransferase 170 IU/mL, creatine phosphokinase 2,115 IU/L, lactate dehydrogenase 1,109 IU/L, urea 65 mg/dL, creatinine 3.78 mg/dL, prothrobin time 24.8 s, activated partial thromboplastin time 116.3 s, potassium 2.9 mEq/L, C-reactive protein 326 mg/dL, and erythrocyte sedimentation rate 132 mm/h. Subsequently, urinary output decreased and respiratory functions worsened. He then lost consciousness and was subjected to mechanic ventilation. Lumbar puncture was not performed because of risk for bleeding (high international normalized ratio values for blood coagulation and thrombocytopenia). Cranial computed tomographic scan did not show any pathologic changes. Treatment with ceftriaxone, 4 g/day intravenously, was initiated, and the dose was adjusted according to creatinine clearance because of suspected meningococcemia. A single dose of prednisolone, 80 mg intravenously, was given concomitantly. Bacterial growth was not observed in cultures of urine and blood samples. The Hanta Profile 1 EUROLINE Test (Euroimmun, Luebeck, Germany) was used to detect immunoglobulin (Ig) G and IgM against 3 hantavirus serotypes (PUUV, DOBV, and Hantaan virus). Results of a hantavirus IgM immunoblot test were positive for DOBV. The QIAamp viral RNA Mini Extraction Kit (QIAGEN, Hilden, Germany) was used for extraction of viral RNA. PUUV and DOBV RNA in serum and urine samples were investigated by using an in-house real-time PCR (Rotorgene; QIAGEN). DOBV RNA was detected in urine samples by PCR (Table). Table Detection of Dobrava virus in 22-year-old patient, Turkey* Meningococcemia, acute hemorrhagic fever, and Crimean-Congo hemorrhagic fever were considered in the differential diagnosis for the patient. Other diseases were excluded by biochemical, serologic, and microbiologic test results. Hantavirus infection was diagnosed in this patient on the basis of criteria recommended by the European Network for Diagnostics of Imported Viral Diseases (5). On the second day of treatment, the patient died of cardiopulmonary arrest. The patient had worked as a security guard in a new prison located in an area that had contained oak and hornbeam forests. DOBV is carried by rodents (Apodemus flavicollis), and the habitat of this rodent in Europe is open oak or beech forest. In a field study performed in rural areas of Turkey near the Black Sea and Aegean Sea, hantavirus was detected in Microtus spp. voles (6). In another study performed in regions near the Aegean Sea, DOBV was detected in 7 (3.5%) of 200 patients with acute or chronic renal failure (7). However, information about specific regions in Turkey in which hantavirus is endemic is limited. Hantavirus infections, which were first identified in northwestern Turkey in 2009 and subsequently in Istanbul, should be considered in the diagnosis of patients who have fever and bleeding. Because of recent emergence of hantavirus in Turkey, areas to which this virus is endemic and where risk for infection is highest have not been identified. Therefore, all inhabitants at high risk for infection (forest workers, military personnel, farmers, persons living in or near a forest, persons handling wood) should be informed about this risk.

Hantavirus RNA in Saliva from Patients with Hemorrhagic Fever with Renal Syndrome

Hantaviruses cause 2 zoonotic diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome. Infection is usually initiated after inhalation of virus-contaminated rodent excreta. In addition to the zoonotic infection route, growing evidence suggests person-to-person transmission of Andes virus. For this reason, we studied whether saliva from HFRS patients contained hantavirus. During an outbreak in northern Sweden of nephropathia epidemica (NE), a milder form of hemorrhagic fever with renal syndrome, we collected saliva and plasma from 14 hospitalized NE patients with verified Puumala virus (PUUV) infection. PUUV RNA was detected in saliva from 10 patients (range 1,530-121,323 PUUV RNA copies/mL) by quantitative reverse transcription-PCR. The PUUV S-segment sequences from saliva and plasma of the same patients were identical. Our data show that hantavirus RNA could be detected in human saliva several days after onset of disease symptoms and raise the question whether interhuman transmission of hantavirus may occur through saliva.

Puumala virus RNA in cerebrospinal fluid in a patient with uncomplicated nephropathia epidemica

a Department of Medical Microbiology, University of Oulu, Oulu, Finland b Clinical Microbiology Laboratory, Oulu University Hospital, Finland c Department of Virology, Haartman Institute, University of Helsinki, Finland d Department of Diagnostic Radiology, Oulu University Hospital, Finland e Department of Ophthalmology, Oulu University Hospital, Finland f Department of Clinical Microbiology, University of Kuopio and Immunogenetics Laboratory, University of Turku, Finland g Department of Internal Medicine, Oulu University Hospital, Finland

Hantavirus Disease Outbreak in Germany: Limitations of Routine Serological Diagnostics and Clustering of Virus Sequences of Human and Rodent Origin

In Europe, hemorrhagic fever with renal syndrome results mainly from infection with Puumala virus (PUUV) or Dobrava virus. For 31 patients from a hantavirus disease outbreak in Lower Bavaria, a district in southeast Germany, serodiagnosis was undertaken by enzyme-linked immunosorbent assay, immunofluorescence assay, and immunoblot analysis. In a few of these cases, however, PUUV-specific typing of antibodies by these standard assays failed and a virus neutralization assay under biosafety level 3 conditions was required to verify the infection by this virus type. PUUV RNA was amplified by reverse transcription-PCR from acute-phase sera of three patients and was found to be very closely related to virus sequences obtained from bank voles (Clethrionomys glareolus) trapped in the same area. These findings link the outbreak with a novel PUUV lineage, "Bavaria," circulating in the local rodent population. The Bavaria lineage associated with the outbreak is only distantly related to other PUUV lineages from Germany.

Puumala Virus RNA in Patient with Multiorgan Failure

To the Editor: The hantaviruses (genus Hantavirus, family Bunyaviridae) include human pathogens and occur worldwide (1). In Western and Central Europe, the predominant serotype is Puumala virus (PUUV), which causes epidemic nephropathy. We report the first Austrian patient with reverse transcription–polymerase chain reaction (RT-PCR)–confirmed PUUV infection and, to our knowledge, the first detection of PUUV-specific RNA in bone marrow. On April 27, 2004, a previously healthy 52-year-old bus driver stopped his bus because of visual disturbance, dizziness, headache, and weakness in his legs; he then lost consciousness for a few minutes. He was seen at the neurology emergency service and subsequently admitted to the university hospital in Graz. He smoked tobacco, drank beer on the weekends, and cleaned his bus in the garage daily. The patient showed slight paresis of the right leg, nystagmus, cognitive deficit, and retrograde amnesia. Laboratory tests showed increases in (normal values are shown in parentheses) C-reactive protein (CRP) 40 mg/L ( 3,000 ng/mL, aspartate aminotransferase 368 U/L (<35), alanine aminotransferase 96 U/L (<45), γ-glutamyl transpeptidase 182 U/L (<55), erythrocytes 3.76 × 109/mL, leukocytes 9.09 × 106/mL, thrombocytopenia of 9.2 × 104 platelets/mL, and lymphoplasmacytoid cells on peripheral blood smear. Serum electrophoresis and immunofixation showed an increased γ-globulin fraction with oligoclonal immunoglobulin G (IgG) λ and IgGκ components. A bone marrow biopsy showed hypercellularity and 15% lymphoid cells with plasmocytoid features. Fluorescence-activated cell sorter testing showed 3% reactive B- and T-cell blasts but no signs of a malignant hematologic disease. Culture of bronchoalveolar lavage for bacteria and fungi was negative. Urinary antigen tests for Legionella spp. and pneumococci were negative. Serum antibody tests for Leptospira spp. were negative, but IgM against PUUV was detected by POC Puumala rapid test (Erilab Ltd, Kuopio, Finland) and recomLine Bunyavirus IgG/IgM test (Mikrogen, Martinsried, Germany). PUUV RNA was detectable in serum and in bone marrow by RT-PCR (2). PUUV was confirmed with a bootstrap probability of 99% on phylogenetic analysis (2). On May 1, status epilepticus developed and was treated with clonazepam. On May 2, renal function deteriorated and progressed to a maximum serum creatinine concentration of 4 mg/dL (0.6–1.3) and urea of 244 mg/dL (10–45), which required hemodialysis. CRP increased to 360 mg/L, and blood pressure decreased to 95/65 mmHg. The patient received intensive supportive care including dopamine and norepinephrine. After improvement, the patient was extubated on May 9. Eight days later, fever (temperature up to 40°C), Enterococcus faecalis bacteremia, nosocomial pneumonia from methicillin-resistant Staphylococcus aureus, respiratory failure requiring mechanic ventilation, and renal failure developed in the patient. Despite antimicrobial drug therapy with linezolid, the patient died 19 days after reintubation. In Austria, before this case, PUUV RNA had only been detected by RT-PCR in rodents (2). We report the first Austrian patient with RT-PCR–confirmed PUUV infection. Furthermore, PUUV-specific RNA had never been detected in bone marrow. In animal studies, PUUV induces production of proinflammatory cytokines, such as interleukin (IL)-6 and IL-10 (3). IL-6 constitutes a major growth factor for myeloma and plasma cells, induces immunoglobulin production, and is an active factor in B-cell differentiation (4,5). IL-10 is a differentiation factor for plasma cell formation and immunoglobulin secretion. Since we detected a clear increase of IL-6, IL-10, and tumor necrosis factor α (TNFα) during the acute phase of infection (IL-6 133.0 pg/dL, IL-10 218.0 U/mL, and TNFα 29.7 pg/mL), we assume that lymphoplasmacytoid cells in bone marrow and peripheral blood of our patient and his production of oligoclonal γ-globulins were due to PUUV-induced cytokine release. Epidemic nephropathy usually takes a benign course, but multiorgan failure with cerebral involvement developed in our patient. Whereas neurologic symptoms such as headache (97% of patients), blurred vision (40%), and vomiting (31%) are common in patients infected with PUUV, only a few cases have been reported with severe central nervous system involvement (i.e., meningitis, epileptiform seizures) (6,7). Our patient had visual disturbances, slight paresis of the right leg, nystagmus, cognitive deficit, retrograde amnesia, and status epilepticus. We want to draw attention to the severe course PUUV infections can rarely take. The presence of PUUV in bone marrow explains the marked hematologic changes with lymphoplasmacytoid cells in marrow and peripheral blood.

Polymerase chain reaction detection of Puumala virus RNA in formaldehyde-fixed biopsy material

Infections with hantaviruses, mainly Clethrionomys-derived Puumala viruses, are known causes of acute renal failure [hemorrhagic fever with renal syndrome (HFRS)] in western Europe. Laboratory diagnosis is primarily based on serology. At the time of clinical symptoms, viral RNA can hardly be detected in the blood or urine, indicating that polymerase chain reaction (PCR) is of little diagnostic value for these infections. Biopsy material is usually formaldehyde-fixed and, thus, regarded as poor quality for PCR applications. The aim of this study was to establish a technique to retrieve such material for laboratory diagnostic. Formaldehyde-fixed, paraffin-embedded kidney biopsies of 14 patients with renal failure either clinically suspected for HFRS (7 cases) or caused by unknown (2 cases) or known other causes (drugs, sarcoidosis; 5 cases) were histologically investigated. An established S segment-specific PCR assay was applied to RNA isolated from the biopsies, and amplification products were verified by direct sequence determination. Investigations revealed a typical histopathological appearance for hantavirus infections in all seven suspected HFRS cases and one case of unknown cause. With five of the suspected HFRS cases, hantavirus-specific RNA was detected. Sequence comparison revealed a close relationship to corresponding nucleoproteins of known Puumala viruses. The established technique provides a simple and powerful tool that expands the diagnostic possibilities, especially for otherwise unidentified or retrospective cases. It further allows insight into the molecular epidemiology of HFRS-causing agents.

Detection of nephropathia epidemica virus RNA in patient samples using a nested primer-based polymerase chain reaction.

A nested primer-based polymerase chain reaction was constructed for the detection of Puumala virus RNA in patient samples. Puumala virus RNA was detected in cells from the urinary and the respiratory tracts and in peripheral blood mononuclear cells of patients with nephropathia epidemica. After inoculation with nephropathia epidemica patient material on Vero E6 cells and propagation for nine passages (4 months), Puumala virus RNA was detected at every passage. Hybridization under high-stringency conditions revealed that the overall nucleotide homology between the different patient isolates and the prototype strain (Puumala) is high. Using cDNA from Hällnäs B1 strain as a probe, hybridization occurred only under low-stringency conditions.