Tarja Sironen

Uncovering the mysteries of hantavirus infections

Hantaviruses are negative-sense single-stranded RNA viruses that infect many species of rodents, shrews, moles and bats. Infection in these reservoir hosts is almost asymptomatic, but some rodent-borne hantaviruses also infect humans, causing either haemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS). In this Review, we discuss the basic molecular properties and cell biology of hantaviruses and offer an overview of virus-induced pathology, in particular vascular leakage and immunopathology.

Hantavirus infections in Europe and their impact on public health

Hantaviruses (genus Hantavirus, family Bunyaviridae) are enveloped tri‐segmented negative‐stranded RNA viruses each carried by a specific rodent or insectivore host species. Several different hantaviruses known to infect humans circulate in Europe. The most common is Puumala (PUUV) carried by the bank vole; another two important, genetically closely related ones are Dobrava–Belgrade (DOBV) and Saaremaa viruses (SAAV) carried by Apodemus mice (species names follow the International Committee on Taxonomy of Viruses nomenclature). Of the two hantaviral diseases, hemorrhagic fever with renal syndrome (HFRS) and hantaviral cardiopulmonary syndrome, the European viruses cause only HFRS: DOBV with often severe symptoms and a high case fatality rate, and PUUV and SAAV more often mild disease. More than 10,000 HFRS cases are diagnosed annually in Europe and in increasing numbers. Whether this is because of increasing recognition by the medical community or due to environmental factors such as climate change, or both, is not known. Nevertheless, in large areas of Europe, the population has a considerable seroprevalence but only relatively few HFRS cases are reported. Moreover, no epidemiological data are available from many countries. We know now that cardiac, pulmonary, ocular and hormonal disorders are, besides renal changes, common during the acute stage of PUUV and DOBV infection. About 5% of hospitalized PUUV and 16%–48% of DOBV patients require dialysis and some prolonged intensive‐care treatment. Although PUUV–HFRS has a low case fatality rate, complications and long‐term hormonal, renal, and cardiovascular consequences commonly occur. No vaccine or specific therapy is in general use in Europe. We conclude that hantaviruses have a significant impact on public health in Europe. Copyright

Molecular Evolution of Puumala Hantavirus

ABSTRACT Puumala virus (PUUV) is a negative-stranded RNA virus in the genusHantavirus, family Bunyaviridae. In this study, detailed phylogenetic analysis was performed on 42 complete S segment sequences of PUUV originated from several European countries, Russia, and Japan, the largest set available thus far for hantaviruses. The results show that PUUV sequences form seven distinct and well-supported genetic lineages; within these lineages, geographical clustering of genetic variants is observed. The overall phylogeny of PUUV is star-like, suggesting an early split of genetic lineages. The individual PUUV lineages appear to be independent, with the only exception to this being the Finnish and the Russian lineages that are closely connected to each other. Two strains of PUUV-like virus from Japan form the most ancestral lineage diverging from PUUV. Recombination points within the S segment were searched for and evidence for intralineage recombination events was seen in the Finnish, Russian, Danish, and Belgian lineages of PUUV. Molecular clock analysis showed that PUUV is a stable virus, evolving slowly at a rate of 0.7 × 10−7 to 2.2 × 10−6 nt substitutions per site per year.

Hypophyseal hemorrhage and panhypopituitarism during Puumala Virus Infection: Magnetic Resonance Imaging and detection of viral antigen in the hypophysis.

We describe 3 cases of nephropathia epidemica (NE) that confirm that Puumala virus infection may cause hypophyseal injury. Autopsy revealed a hemorrhagic hypophysis positive for Puumala virus antigen in both neuroendocrine stromal and vascular endothelial cells in 1 patient, and 2 patients developed hypophyseal hemorrhage (diagnosed with magnetic resonance imaging) during or shortly after acute NE, both of whom developed panhypopituitarism.

Isolation, Identification, and Characterization of Novel Arenaviruses, the Etiological Agents of Boid Inclusion Body Disease

ABSTRACT 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 Kochs 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.

Pathology of Puumala Hantavirus Infection in Macaques

Hantaviruses are globally important human pathogens that cause hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Capillary leakage is central to hantaviral diseases, but how it develops, has remained unknown. It has been hypothesized that the pathogenesis of hantavirus infection would be a complex interplay between direct viral effects and immunopathological mechanisms. Both of these were studied in the so far best model of mild hemorrhagic fever with renal syndrome, i.e. cynomolgus macaques infected with wild-type Puumala hantavirus. Viral RNA detected by in situ hybridization and nucleocapsid protein detected by immunohistochemical staining were observed in kidney, spleen and liver tissues. Inflammatory cell infiltrations and tubular damage were found in the kidneys, and these infiltrations contained mainly CD8-type T-cells. Importantly, these results are consistent with those obtained from patients with hantaviral disease, thus showing that the macaque model of hantavirus infection mimics human infection also on the tissue level. Furthermore, both the markers of viral replication and the T-cells appeared to co-localize in the kidneys to the sites of tissue damage, suggesting that these two together might be responsible for the pathogenesis of hantavirus infection.

Tick-borne encephalitis virus in ticks in Finland, Russian Karelia and Buryatia.

Tick-borne encephalitis (TBE) is a central nervous system infection caused by a flavivirus [tick-borne encephalitis virus (TBEV)], transmitted by Ixodes ticks and endemic in a large region in Eurasia. We collected 2411 ticks from Finland and Russia in 2003-2008, screened them for TBEV by RT-PCR and isolated and analysed eight strains belonging to all three TBEV subtypes; in addition, we obtained two European-subtype strains from human serum samples. TBEV RNA prevalence in unengorged ticks was approximately 1 % both in the northernmost TBE-endemic areas of Europe in Finland and Russian Karelia, and in Siberia in Buryatia. In Finland, both Ixodes ricinus and Ixodes persulcatus ticks were found from distinct areas and, in Russian Karelia, were overlapping in the same study site. TBEV E and NS3 gene sequences obtained showed a variability of 0-4 % within European-subtype strains, 2-9 % for Siberian-subtype strains and 3-13 % for Far Eastern-subtype strains.

European subtype tick-borne encephalitis virus in Ixodes persulcatus ticks.

To the Editor: The northernmost tick-borne encephalitis (TBE) focus is in Simo, Finnish Lapland. Four TBE cases were confirmed during 2008–2009. Tick-borne encephalitis virus (TBEV) is transmitted by Ixodes spp. ticks and is endemic to Eurasia from central Europe to the Far East. The virus has 3 subtypes: European (TBEV-Eur), Siberian (TBEV-Sib), and Far Eastern (TBEV-FE). TBEV-Eur is mainly transmitted by I. ricinus ticks (sheep ticks) and the 2 other subtypes by I. persulcatus ticks (taiga ticks). The range of I. ricinus ticks covers most of continental Europe and the British Isles; I. persulcatus ticks are distributed throughout eastern Europe and Asia to the People’s Republic of China and Japan. The transmission cycle of at least TBEV-Eur in nature is fragile and depends on microclimatic conditions. Thus, within the I. ricinus distribution area, TBE is endemic merely focally (1,2). In Finland, TBE foci are located by the sea or large lakes (Figure A1). Both vector tick species are found: I. ricinus ticks in the southern and central parts of the country, but I. persulcatus ticks are in scattered foci along the western coast, including the Kokkola archipelago and Narpio municipality, where they carry TBEV-Sib (3,4) (Figure A1). The first human TBE cases from Simo in Lapland (65°40′N, 24°54′E; Figure A1) were reported during 2008 (n = 2) and 2009 (n = 2). On the basis of interviews with the 2 patients from 2008, we collected 97 ticks and 17 bank voles from the 2 probable sites of infection during June 2009. From the rodents, we extracted blood from the heart and performed TBEV-antibody tests by immunofluorescence assay. The ticks were placed in 51 pools (1–3 ticks/pool). We isolated RNA from tick pools and rodent lungs and brains by TriPure Isolation Reagent (Roche Diagnostics, Indianapolis, IN, USA) and performed real-time reverse transcription–PCR (5) to detect TBEV RNA. For the positive tick pools, we confirmed the identification species by Ixodes mtDNA sequencing (6). Six of 51 tick pools (with a total of 97 I. persulcatus ticks) were positive for TBEV in real-time reverse transcription–PCR, resulting in 6% TBEV RNA prevalence. At least 1 organ was positive for TBEV RNA in as many as 15/17 bank voles, in line with our finding that TBEV RNA persists in rodents for months (7); 4 rodents had antibodies to TBEV. The TBEV RNA prevalence among ticks and rodents was relatively high, as is the incidence among humans (0.57 cases/year/1,000 inhabitants) in Simo, indicating a focus with high activity. We isolated 6 TBEV strains from suckling mice (experimental animal permit ESLH-2008–06558/Ym-23): 2 from I. persulcatus tick pools (Simo-38 and Simo-48; pools of 2 and 3 ticks, respectively), and 4 from TBEV antibody– and RNA-positive rodent lung–brain suspensions (Simo-2, -5, -7 and -9). Partial envelope (E) and nonstructural protein 3 genes (4) of the isolated TBEV strains were sequenced (accession nos. HQ228014–HQ228024, GenBank) and subjected to phylogenetic analysis (Figure A1). Within the 1208 nt from the E gene, Simo-38 and Simo-48 from ticks and Simo-9 from a bank vole were identical. Other sequences differed for 1 nt and Simo-2 for 1 aa compared with the others. All strains were monophyletic and belonged to the TBEV-Eur subtype. The partial nonstructural protein 3 gene sequences were identical, and the phylogenetic tree showed similar topography as for the E gene (not shown). The only tick species found in Simo was I. persulcatus, further widening its known distribution along the western coast of Finland (Figure A1). However, the virus subtype found in Simo was TBEV-Eur strain, the main vector of which is the I. ricinus tick. TBEV-Eur strains are commonly very closely related to each other and do not form clear geographic clusters (4). Thus, it is difficult to deduce the origin of the virus. The nearest TBE-endemic focus is the Kokkola archipelago, ≈200 km south (Figure A1), but there I. persulcatus ticks carry the TBEV-Sib strain (3). The nearest areas to which the TBEV-Eur strain is endemic are in southern Finland where only I. ricinus ticks have been found. Cattle serum samples were negative for antibodies to TBEV in the Simo area in the 1960s (8). The first human TBE cases from Simo were identified during 2008 and 2009. We isolated TBEV strains from ticks and rodents in 2009. Simo appears to be a recently established, and the northernmost, TBE focus known. TBEV may have been introduced to Simo from a geographically distinct location recently, likely within the past 50 years. TBE seems to be moving northward in Europe (9) and shifting upward to higher elevations in the mountains (10), apparently influenced by climate change. An altered microclimate favoring TBE circulation (1), in addition to introduction of the virus, could also explain the recent emergence of TBE in Simo. In conclusion, Simo in Finnish Lapland is a new TBE-endemic focus demonstrating northward movement of foci and an unusual combination of the TBEV-Eur strain and I. persulcatus ticks in an area with no evidence of cocirculation of tick species or TBEV subtypes.

Analysis of Puumala hantavirus in a bank vole population in northern Finland: evidence for co-circulation of two genetic lineages and frequent reassortment between strains

In this study, for the first time, two distinct genetic lineages of Puumala virus (PUUV) were found within a small sampling area and within a single host genetic lineage (Ural mtDNA) at Pallasjärvi, northern Finland. Lung tissue samples of 171 bank voles (Myodes glareolus) trapped in September 1998 were screened for the presence of PUUV nucleocapsid antigen and 25 were found to be positive. Partial sequences of the PUUV small (S), medium (M) and large (L) genome segments were recovered from these samples using RT-PCR. Phylogenetic analysis revealed two genetic groups of PUUV sequences that belonged to the Finnish and north Scandinavian lineages. This presented a unique opportunity to study inter-lineage reassortment in PUUV; indeed, 32 % of the studied bank voles appeared to carry reassortant virus genomes. Thus, the frequency of inter-lineage reassortment in PUUV was comparable to that of intra-lineage reassortment observed previously (Razzauti, M., Plyusnina, A., Henttonen, H. & Plyusnin, A. (2008). J Gen Virol 89, 1649-1660). Of six possible reassortant S/M/L combinations, only two were found at Pallasjärvi and, notably, in all reassortants, both S and L segments originated from the same genetic lineage, suggesting a non-random pattern for the reassortment. These findings are discussed in connection to PUUV evolution in Fennoscandia.

Central nervous system-related symptoms and findings are common in acute Puumala hantavirus infection.

Abstract Background. Puumala hantavirus (PUUV) causes a hemorrhagic fever with renal syndrome (HFRS) also called nephropathia epidemica (NE). Recent case reports and retrospective studies suggest that NE may damage the pituitary gland. Based on these observations, our goal was to explore the nature of this complication prospectively. Methods. A total of 58 hospitalized patients with acute NE volunteered to participate. Central nervous system (CNS) symptoms were recorded, cerebrospinal fluid (CSF) samples were collected, human leukocyte antigen (HLA) haplotype was analyzed, brain magnetic resonance imaging (MRI) was acquired, and electroencephalography (EEG) was recorded. Patients with abnormal pituitary MRI finding were examined by an endocrinologist. Results. Most patients experienced CNS symptoms, and half of the CSF samples were positive for PUUV IgM, elevated protein level, or leukocyte count. CSF of patients negative for DR15(2)-DQ6 haplotype was less frequently affected. MRI revealed pituitary hemorrhage in two patients; these two patients suffered sudden loss of vision associated with headache, and they both developed hypopituitarism. Only one patient required long-term hormonal replacement therapy. Conclusion. CNS-related symptoms and inflammation in the CSF are common in acute NE. Genetic properties of the host may predispose to CNS involvement. It does seem that pituitary injury and subsequent hormonal insufficiency may complicate the recovery.