The Hantaan virus glycoprotein precursor is cleaved at the conserved pentapeptide WAASA.

The medium segment of the tripartite negative-stranded RNA genome of hantaviruses encodes for the predicted glycoprotein precursor GPC. We have demonstrated here the expression of the glycoprotein precursor of Hantaan virus following transfection of mammalian cells. The cleavage of the precursor into the glycoproteins G1 and G2 followed the rules for signal peptides and seemed to occur directly at the pentapeptide motif "WAASA." Our data indicate that the signal peptidase complex is responsible for the proteolytic processing of the precursor GPC of Hantaan virus. The comparison of this region of the glycoprotein precursor, including the absolutely conserved WAASA motif, suggests a similar cleavage event for all hantavirus glycoproteins.

A new Clethrionomys-derived hantavirus from Germany: evidence for distinct genetic sublineages of Puumala viruses in Western Europe.

Puumala (PUU) viruses are the predominant etiologic agents of hantavirus infections in Europe. The most important reservoir is the bank vole, Clethrionomys glareolus (Cg), belonging to the subfamily Arvicolinae of the Muridae family. Here we report on the molecular characterization of the first rodent-derived sequence (PUU/Cg-Erft) from Germany. Comparison of the S and M segment coding regions revealed 92.5 and 92.8% identity, respectively, with PUU/H-9013, a human isolate from France. However, only 83.1% identity was found with the S segment of a previously reported PUU sequence from a German HFRS case (PUU/H-Berkel) indicating the co-existence of two distinct sublineages in Germany. Phylogenetic and alignment analyses of S and M segment coding regions enabled us to assign PUU viruses/sequences to at least six distinct genetic sublineages. Membership was defined by nucleotide sequence differences of < 8%, whereas a diversity of > 14% clearly outgrouped a virus/sequence. Based on S segment sequences the sublineage represented by Clethrionomys rufocanus-derived viruses from Japan diverged at a well supported node from the clade harbouring all Clethrionomys glareolus-derived European PUU viruses. A correlation between genetic relationship and geographic origin of PUU viruses was observed which may support a co-evolution of PUU viruses with distinct subspecies of their reservoir host.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Nuclear translocation of mutagenized forms of human cytomegalovirus glycoprotein B (gpUL55).

To define structural elements involved in translocation of human cytomegalovirus (HCMV) glycoprotein B (gB) to the inner nuclear membrane (INM) compartment, mutagenized gB derivatives with deletions of the potential membrane anchor domains or of portions of the cytoplasmic tail were stably expressed in human astrocytoma cells. Subcellular localization examined by immunofluorescence and cell fractionation suggested that all gB derivatives reached the INM; however, reduced amounts were found after deletion of the extreme carboxy terminus [amino acids 856-906; gB(Del3)]. Pulse-chase analysis revealed accumulation in nuclear fractions of all gB derivatives during the chase, except for gB(Del3), which exhibited impaired nuclear retention. A carboxy-terminal nucleoplasmin-like signal localized within the respective deletion may thus be involved in nuclear transport and retention of HCMV gB. Immunoprecipitation after 32P-radiolabelling of the gB transfectants verified that the gB molecule is phosphorylated at a carboxy-terminal consensus motif for casein kinase II.