Molecular genetics and evolution of Puumala hantavirus

Puumala virus (PUUV) is the causative agent of nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome. Finland has the highest documented incidence of NE with around 1000 cases diagnosed annually. PUUV is also found in other Scandinavian countries, Central Europe and the European part of Russia. PUUV belongs to the genus Hantavirus in the family Bunyaviridae. Hantaviruses are rodent-borne viruses each carried by a specific host that is persistently and asymptomatically infected by the virus. PUUV is carried by the bank voles (Myodes glareolus, previously known as Clethrionomys glareolus). Hantaviruses have co-evolved with their carrier rodents for millions of years and these host animals are the evolutionary scene of hantaviruses. In this study, PUUV sequences were recovered from bank voles captured in Denmark and Russian Karelia to study the evolution of PUUV in Scandinavia. Phylogenetic analysis of these strains showed a geographical clustering of genetic variants following the presumable migration pattern of bank voles during the recolonization of Scandinavia after the last ice age approximately 10 000 years ago. The currently known PUUV genome sequences were subjected to in-depth phylogenetic analyses and the results showed that genetic drift seems to be the major mechanism of PUUV evolution. In general, PUUV seems to evolve quite slowly following a molecular clock. We also found evidence for recombination in the evolution of some genetic lineages of PUUV. Viral microevolution was studied in controlled virus transmission in colonized bank voles and changes in quasispecies dynamics were recorded as the virus was transmitted from one animal to another. We witnessed PUUV evolution in vivo, as one synonymous mutation became repeatedly fixed in the viral genome during the experiment. The detailed knowledge on the PUUV diversity was used to establish new sensitive and specific detection methods for this virus. Direct viral invasion of the hypophysis was demonstrated for the first time in a lethal case of NE. PUUV detection was done by immunohistochemistry, in situ hybridization and RT-nested-PCR of the autopsy tissue samples.

Accumulation of point mutations and reassortment of genomic RNA segments are involved in the microevolution of Puumala hantavirus in a bank vole (Myodes glareolus) population

"The genetic diversity of Puumala hantavirus (PUUV) was studied in a local population of its natural host, the bank vole (Myodes glareolus). The trapping area (2.5x2.5 km) at Konnevesi, Central Finland, included 14 trapping sites, at least 500 m apart; altogether, 147 voles were captured during May and October 2005. Partial sequences of the S, M and L viral genome segments were recovered from 40 animals. Seven, 12 and 17 variants were detected for the S, M and L sequences, respectively; these represent new wild-type PUUV strains that belong to the Finnish genetic lineage. The genetic diversity of PUUV strains from Konnevesi was 0.2-4.9% for the S segment, 0.2-4.8% for the M segment and 0.2-9.7% for the L segment. Most nucleotide substitutions were synonymous and most deduced amino acid substitutions were conservative, probably due to strong stabilizing selection operating at the protein level. Based on both sequence markers and phylogenetic clustering, the S, M and L sequences could be assigned to two groups, 'A' and 'B'. Notably, not all bank voles carried S, M and L sequences belonging to the same group, i.e. SAMALA or SBMBLB.. A substantial proportion (8/40, 20%) of the newly characterized PUUV strains possessed reassortant genomes such as SBMALA, SAMBLB or SBMALB. These results suggest that at least some of the PUUV reassortants are viable and can survive in the presence of their parental strains."

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 Pallasjarvi, 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 Pallasjarvi 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 Fermoscandia."

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.

Complete genome of a Puumala virus strain from Central Europe

Puumala virus (PUUV) is one of the predominant hantavirus species in Europe causing mild to moderate cases of haemorrhagic fever with renal syndrome. Parts of Lower Saxony in north-western Germany are endemic for PUUV infections. In this study, the complete PUUV genome sequence of a bank vole-derived tissue sample from the 2007 outbreak was determined by a combined primer-walking and RNA ligation strategy. The S, M and L genome segments were 1,828, 3,680 and 6,550 nucleotides in length, respectively. Sliding-window analyses of the nucleotide sequences of all available complete PUUV genomes indicated a non-homogenous distribution of variability with hypervariable regions located at the 3′-ends of the S and M segments. The overall similarity of the coding genome regions to the other PUUV strains ranged between 80.1 and 84.7 % at the level of the nucleotide sequence and between 89.5 and 98.1 % for the deduced amino acid sequences. In comparison to the phylogenetic trees of the complete coding sequences, trees based on partial segments revealed a general drop in phylogenetic support and a lower resolution. The Astrup strain S and M segment sequences showed the highest similarity to sequences of strains from geographically close sites in the Osnabrück Hills region. In conclusion, a primer-walking-mediated strategy resulted in the determination of the first complete nucleotide sequence of a PUUV strain from Central Europe. Different levels of variability along the genome provide the opportunity to choose regions for analyses according to the particular research question, e.g., large-scale phylogenetics or within-host evolution.

Spatiotemporal dynamics of Puumala hantavirus associated with its rodent host, Myodes glareolus

Many viruses significantly impact human and animal health. Understanding the population dynamics of these viruses and their hosts can provide important insights for epidemiology and virus evolution. Puumala virus (PUUV) is a European hantavirus that may cause regional outbreaks of hemorrhagic fever with renal syndrome in humans. Here, we analyzed the spatiotemporal dynamics of PUUV circulating in local populations of its rodent reservoir host, the bank vole (Myodes glareolus) during eight years. Phylogenetic and population genetic analyses of all three genome segments of PUUV showed strong geographical structuring at a very local scale. There was a high temporal turnover of virus strains in the local bank vole populations, but several virus strains persisted through multiple years. Phylodynamic analyses showed no significant changes in the local effective population sizes of PUUV, although vole numbers and virus prevalence fluctuated widely. Microsatellite data demonstrated also a temporally persisting subdivision between local vole populations, but these groups did not correspond to the subdivision in the virus strains. We conclude that restricted transmission between vole populations and genetic drift play important roles in shaping the genetic structure and temporal dynamics of PUUV in its natural host which has several implications for zoonotic risks of the human population.

Other Side of This Life : Death, Value, and Social Being in Thomas Pynchon's Fiction

This dissertation analyzes the interrelationship between death, the conditions of (wo)man s social being, and the notion of value as it emerges in the fiction of the American novelist Thomas Pynchon (1937 ). Pynchon s present work includes six novels V. (1963), The Crying of Lot 49 (1966), Gravity s Rainbow (1973), Vineland (1990), Mason & Dixon (1997), Against the Day (2006) and several short stories. Death constitues a central thematic in Pynchon s work, and it emerges through recurrent questions of mortality, suicide, mass destruction, sacrifice, afterlife, entropy, the relationship between the animate and the inanimate, and the limits of representation. In Pynchon, death is never a mere biological given (or event); it is always determined within a certain historical, cultural, and ideological context. Throughout his work, Pynchon questions the strict ontological separation of life and death by showing the relationship between this separation and social power. Conceptual divisions also reflect the relationship between society and its others, and death becomes that through which lines of social demarcation are articulated. Determined as a conceptual and social "other side", death in Pynchon forms a challenge to modern culture, and makes an unexpected return: the dead return to haunt the living, the inanimate and the animate fuse, and technoscientific attempts at overcoming and controlling death result in its re-emergence in mass destruction and ecological damage. The questioning of the ontological line also affects the structuration of Pynchon's prose, where the recurrent narrated and narrative desire to reach the limits of representation is openly associated with death. Textualized, death appears in Pynchon's writing as a sudden rupture within the textual functioning, when the "other side", that is, the bare materiality of the signifier is foregrounded. In this study, Pynchon s cultural criticism and his poetics come together, and I analyze the subversive role of death in his fiction through Jean Baudrillard s genealogy of the modern notion of death from L échange symbolique et la mort (1976). Baudrillard sees an intrinsic bond between the social repression of death in modernity and the emergence of modern political economy, and in his analysis economy and language appear as parallel systems for generating value (exchange value/ sign-value). For Baudrillard, the modern notion of death as negativity in relation to the positivity of life, and the fact that death cannot be given a proper meaning, betray an antagonistic relation between death and the notion of value. As a mode of negativity (that is, non-value), death becomes a moment of rupture in relation to value-based thinking in short, rationalism. Through this rupture emerges a form of thinking Baudrillard labels the symbolic, characterized by ambivalence and the subversion of conceptual opposites.

Hantavirus glycoprotein GN in virion assembly and regulation of interferon response

Hantaviruses have a tri-segmented negative-stranded RNA genome. The S segment encodes the nucleocapsid protein (N), M segment two glycoproteins, Gn and Gc, and the L segment the RNA polymerase. Gn and Gc are co-translationally cleaved from a precursor and targeted to the cis-Golgi compartment. The Gn glycoprotein consists of an external domain, a transmembrane domain and a C-terminal cytoplasmic domain. In addition, the S segment of some hantaviruses, including Tula and Puumala virus, have an open reading frame (ORF) encoding a nonstructural potein NSs that can function as a weak interferon antagonist. The mechanisms of hantavirus-induced pathogenesis are not fully understood but it is known that both hemorrhagic fever with renal syndrome (HFRS) and hantavirus (cardio) pulmonary syndrome (HCPS) share various features such as increased capillary permeability, thrombocytopenia and upregulation of TNF-. Several hantaviruses have been reported to induce programmed cell death (apoptosis), such as TULV-infected Vero E6 cells which is known to be defective in interferon signaling. Recently reports describing properties of the hantavirus Gn cytoplasmic tail (Gn-CT) have appeared. The Gn-CT of hantaviruses contains animmunoreceptor tyrosine-based activation motif (ITAM) which directs receptor signaling in immune and endothelial cells; and contain highly conserved classical zinc finger domains which may have a role in the interaction with N protein. More functions of Gn protein have been discovered, but much still remains unknown. Our aim was to study the functions of Gn protein from several aspects: synthesis, degradation and interaction with N protein. Gn protein was reported to inhibit interferon induction and amplication. For this reason, we also carried out projects studying the mechanisms of IFN induction and evasion by hantavirus. We first showed degradation and aggresome formation of the Gn-CT of the apathogenic TULV. It was reported earlier that the degradation of Gn-CT is related to the pathogenicity of hantavirus. We found that the Gn-CT of the apathogenic hantaviruses (TULV, Prospect Hill virus) was degraded through the ubiquitin-proteasome pathway, and TULV Gn-CT formed aggresomes upon treatment with proteasomal inhibitor. Thus the results suggest that degradation and aggregation of the Gn-CT may be a general property of most hantaviruses, unrelated to pathogenicity. Second, we investigated the interaction of TULV N protein and the TULV Gn-CT. The Gn protein is located on the Golgi membrane and its interaction with N protein has been thought to determine the cargo of the hantaviral ribonucleoprotein which is an important step in virus assembly, but direct evidence has not been reported. We found that TULV Gn-CT fused with GST tag expressed in bacteria can pull-down the N protein expressed in mammalian cells; a mutagenesis assay was carried out, in which we found that the zinc finger motif in Gn-CT and RNA-binding motif in N protein are indispensable for the interaction. For the study of mechanisms of IFN induction and evasion by Old World hantavirus, we found that Old World hantaviruses do not produce detectable amounts of dsRNA in infected cells and the 5 -termini of their genomic RNAs are monophosphorylated. DsRNA and tri-phosphorylated RNA are considered to be critical activators of innate immnity response by interacting with PRRs (pattern recognition receptors). We examined systematically the 5´-termini of hantavirus genomic RNAs and the dsRNA production by different species of hantaviruses. We found that no detectable dsRNA was produced in cells infected by the two groups of the old world hantaviruses: Seoul, Dobrava, Saaremaa, Puumala and Tula. We also found that the genomic RNAs of these Old World hantaviruses carry 5´-monophosphate and are unable to trigger interferon induction. The antiviral response is mainly mediated by alpha/beta interferon. Recently the glycoproteins of the pathogenic hantaviruses Sin Nombre and New York-1 viruses were reported to regulate cellular interferon. We found that Gn-CT can inhibit the induction of IFN activation through Toll-like receptor (TLR) and retinoic acid-inducible gene I-like RNA helicases (RLH) pathway and that the inhibition target lies at the level of TANK-binding kinase 1 (TBK-1)/ IKK epislon complex and myeloid differentiation primary response gene (88) (MyD88) / interferon regulatory factor 7 (IRF-7) complex.

Glycoprotein Interactions in the Assembly of Hantaviruses

Hantaviruses are one of the five genera of the vector-borne virus family Bunyaviridae. While other members of the family are transmitted via arthropods, hantaviruses are carried and transmitted by rodents and insectivores. Occasional transmission to humans occurs via inhalation of aerosolized rodent excreta. When transmitted to man hantaviruses cause hemorrhagic fever with renal syndrome (HFRS, in Eurasia, mortality ~10%) and hantavirus cardiopulmonary syndrome (HCPS, in the Americas, mortality ~40%). The single-stranded, negative-sense RNA genome of hantaviruses is in segments S, M and L that respectively encode for nucleocapsid (N), glycoproteins Gn and Gc, and RNA-dependent RNA-polymerase (RdRp or L protein). The genome segments, encapsidated by N protein to form ribonucleoprotein (RNP), are enclosed inside a lipid envelope decorated by spikes formed of Gn and Gc. The focus of this study was to understand the mechanisms and interactions through which the virion is formed and maintained. We observed that when extracted from virions both Gn and Gc favor homo- over hetero-oligomerization. The minimal glycoprotein complexes extracted from virion by detergent were observed, by using ultracentrifugation and gel filtration, to be tetrameric Gn and homodimeric Gc. These results led us to suggest a model where tetrameric Gn complexes are interconnected through homodimeric Gc units to form the grid-like surface architecture described for hantaviruses. This model was found to correlate with the three-dimensional (3D) reconstruction of virion surface created using cryo-electron tomography (cryo-ET). The 3D-density map showed the spike complex formed of Gn and Gc to be 10 nm high and to display a four-fold symmetry with dimensions of 15 nm times 15 nm. This unique square-shaped complex on a roughly round virion creates a hitch for the assembly, since a sphere cannot be broken into rectangles. Thus additional interactions are likely required for the virion assembly. In cryo-ET we observed that the RNP makes occasional contacts to the viral membrane, suggesting an interaction between the spike and RNP. We were able to demonstrate this interaction using various techniques, and showed that both Gn and Gc contribute to the interaction. This led us to suggest that in addition to the interactions between Gn and Gc, also the interaction between spike and RNP is required for assembly. We found galectin-3 binding protein (referred to as 90K) to co-purify with the virions and showed an interaction between 90K and the virion. Analysis of plasma samples taken from patients hospitalized for Puumala virus infection showed increased concentrations of 90K in the acute phase and the increased 90K level was found to correlate with several parameters that reflect the severity of acute HFRS. The results of these studies confirmed, but also challenged some of the dogmas on the structure and assembly of hantaviruses. We confirmed that Gn and RNP do interact, as long assumed. On the other hand we demonstrated that the glycoproteins Gn and Gc exist as homo-oligomers or appear in large hetero-oligomeric complexes, rather than form primarily heterodimers as was previously assumed. This work provided new insight into the structure and assembly of hantaviruses.

Hantavirus infection: Insights into entry, assembly and pathogenesis

Hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped viruses incorporating a segmented, negative-sense RNA genome. Each hantavirus is carried by its specific host, either a rodent or an insectivore (shrew), in which the infection is asymptomatic and persistent. In humans, hantaviruses cause Hemorrhagic fever with renal syndrome (HFRS) in Eurasia and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. In Finland, Puumala virus (genus Hantavirus) is the causative agent of NE, a mild form of HFRS. The HFRS-type diseases are often associated with renal failure and proteinuria that might be mechanistically explained by infected kidney tubular cell degeneration in patients. Previously, it has been shown that non-pathogenic hantavirus, Tula virus (TULV), could cause programmed cell death, apoptosis, in cell cultures. This suggested that the infected kidney tubular degeneration could be caused directly by virus replication. In the first paper of this thesis the molecular mechanisms involved in TULV-induced apoptosis was further elucidated. A virus replication-dependent down-regulation of ERK1/2, concomitantly with the induced apoptosis, was identified. In addition, this phenomenon was not restricted to TULV or to non-pathogenic hantaviruses in general since also a pathogenic hantavirus, Seoul virus, could inhibit ERK1/2 activity. Hantaviruses consist of membrane-spanning glycoproteins Gn and Gc, RNA-dependent RNA polymerase (L protein) and nucleocapsid protein N, which encapsidates the viral genome, and thus forms the ribonucleoprotein (RNP). Interaction between the cytoplasmic tails of viral glycoproteins and RNP is assumed to be the only means how viral genetic material is incorporated into infectious virions. In the second paper of this thesis, it was shown by immunoprecipitation that viral glycoproteins and RNP interact in the purified virions. It was further shown that peptides derived from the cytoplasmic tails (CTs) of both Gn and Gc could bind RNP and recombinant N protein. In the fourth paper the cytoplamic tail of Gn but not Gc was shown to interact with genomic RNA. This interaction was probably rather unspecific since binding of Gn-CT with unrelated RNA and even single-stranded DNA were also observed. However, since the RNP consists of both N protein and N protein-encapsidated genomic RNA, it is possible that the viral genome plays a role in packaging of RNPs into virions. On the other hand, the nucleic acid-binding activity of Gn may have importance in the synthesis of viral RNA. Binding sites of Gn-CT with N protein or nucleic acids were also determined by peptide arrays, and they were largely found to overlap. The Gn-CT of hantaviruses contain a conserved zinc finger (ZF) domain with an unknown function. Some viruses need ZFs in entry or post-entry steps of the viral life cycle. Cysteine residues are required for the folding of ZFs by coordinating zinc-ions, and alkylation of these residues can affect virus infectivity. In the third paper, it was shown that purified hantavirions could be inactivated by treatment with cysteine-alkylating reagents, especially N-ethyl maleimide. However, the effect could not be pin-pointed to the ZF of Gn-CT since also other viral proteins reacted with maleimides, and it was, therefore, impossible to exclude the possibility that other cysteines besides those that were essential in the formation of ZF are required for hantavirus infectivity.

Savolax lähn : Sysmä, Hardola, Hirvensalmi, St Michels, Jockas, Sulkava, Menduharju, Christina, Pumala socknar och capeller

1 kartta : käsinpiirros, vär. ; 56,5 x 103 cm. Kuvailu kääntöpuolelta. Mittakaava laskettu janamittakaavasta. Kankaalle liimattu.

Multiple infections of rodents with zoonotic pathogens in Austria

Rodents are important reservoirs for a large number of zoonotic pathogens. We examined the occurrence of 11 viral, bacterial, and parasitic agents in rodent populations in Austria, including three different hantaviruses, lymphocytic choriomeningitis virus, orthopox virus, Leptospira spp., Borrelia spp., Rickettsia spp., Bartonella spp., Coxiella burnetii, and Toxoplasma gondii. In 2008, 110 rodents of four species (40 Clethrionomys glareolus, 29 Apodemus flavicollis, 26 Apodemus sylvaticus, and 15 Microtus arvalis) were trapped at two rural sites in Lower Austria. Chest cavity fluid and samples of lung, spleen, kidney, liver, brain, and ear pinna skin were collected. We screened selected tissue samples for hantaviruses, lymphocytic choriomeningitis virus, orthopox viruses, Leptospira, Borrelia, Rickettsia, Bartonella spp., C. burnetii, and T. gondii by RT-PCR/PCR and detected nucleic acids of Tula hantavirus, Leptospira spp., Borrelia afzelii, Rickettsia spp., and different Bartonella species. Serological investigations were performed for hantaviruses, lymphocytic choriomeningitis virus, orthopox viruses, and Rickettsia spp. Here, Dobrava-Belgrade hantavirus-, Tula hantavirus-, lymphocytic choriomeningitis virus-, orthopox virus-, and rickettsia-specific antibodies were demonstrated. Puumala hantavirus, C. burnetii, and T. gondii were neither detected by RT-PCR/PCR nor by serological methods. In addition, multiple infections with up to three pathogens were shown in nine animals of three rodent species from different trapping sites. In conclusion, these results show that rodents in Austria may host multiple zoonotic pathogens. Our observation raises important questions regarding the interactions of different pathogens in the host, the countermeasures of the host's immune system, the impact of the host–pathogen interaction on the fitness of the host, and the spread of infectious agents among wild rodents and from those to other animals or humans.