A New Picornavirus Isolated from Bank Voles (Clethrionomys glareolus)

AbstractA previously unknown picornavirus was isolated from bank voles (Clethrionomys glareolus). Electron microscopy images and sequence data of the prototype isolate, named Ljungan virus, showed that it is a picornavirus. The amino acid sequences of predicted Ljungan virus capsid proteins VP2 and VP3 were closely related to the human pathogen echovirus 22 (approximately 70% similarity). A partial 5′ noncoding region sequence of Ljungan virus showed the highest degree of relatedness to cardioviruses. Two additional isolates were serologically and molecularly related to the prototype

Genetic and antigenic characterisation of Puumala virus in voles and man

The present study has focused on the genetic and antigenic characteristics of Puumala (PUU) virus,the causative agent of nephropathia epidemica (NE). An assay for detection of neutralising antibodiesto PUU virus was developed. In sequentially bled NE patients, neutralising antibodies were detectableat the onset of disease, and the titers continued to rise during the following two years. Equally hightiters were seen in NE-convalescents even after more than 10-20 years. The test was subsequently used in production of neutralising human monoclonal antibodies (MAbs), and for cloning of antibody neutralisation escape virus mutants. To investigate if PUU virus could be detected in NE patients, highly sensitive and specific methods for detection of viral RNA and antigen were established. PUU virus RNA was detected by polymerase chain reaction (PCR) in acute NE patients. By direct nucleotide sequencing of the generated PCR products, the genetic correlation between PUU virus in NE patients and bank voles was demonstrated. Sequence analysis of several PUU strains of different geographical origins demonstrated significant genetic heterogeneity, and showed that Swedish PUU strains were similar to each other but different from all other strains. To further characterise Swedish PUU viruses, the S and M segments of strain 83-L20 were subjected to sequence analysis. By phylogenetic analysis it was shown that 83-L20 was more closely related to PUU strains from Finland than to strains from Russia or Central Europe. Two hantavirus strains, isolated from Microtus fortis trapped in far east Russia, were characterised by cross-neutralisation test and partial sequencing of the S and M segments. The viruses were shown tobe closely related to PUU virus, while forming a novel serotype within the Hantavirus genus. This newlydefined serotype was proposed to be named Khabarovsk. To investigate the genetic variation in PUU strains in Sweden, bank voles trapped at different locations were subjected to PCR and nucleotide sequencing. It was found that the PUU strains in northern Sweden were substantially different from the strains in central Sweden. By analysis of bankvole mitochondrial DNA it was demonstrated that the two distinct PUU virus lineages correlated to the distribution of two distinct bank vole populations. These two populations have been proposed to have arisen from post-glacial emigration to Sweden from two directions, from Finland to the northern parts, and from the European continent to the southern and central parts. PUU virus specific human spleen B cells were pre-selected using a novel method based on bank voleMAbs, magnetic beads and viral antigen. By transformation of selected cells with Epstein-Barr virusand subsequent fusion with a human-mouse hybridorna cell line, four clones could be established. The clones were found to be stable and to excrete IgG antibodies to PUU virus envelope glycoprotein G2. The MAbs were shown to react with an epitope conserved in all examined PUU virus strains from Finland, Russia, Sweden and Belgium. By using a combination of the neutralisation test and the antigen ELISA, two virus mutants escaping from a human and a bank vole MAb, specific for G2 and G1, respectively, could be rescued. The mutants were verified to lack the corresponding epitopes by several serological techniques. The neutralising epitopes, previously found to be immuno-dominant in humans, were mapped by sequence analysis. Peptides covering the defined regions were synthesised and analysed for immunogenicity. The studies of the escape mutants provided additional clues to the mechanisms of neutralisation