Markus Brummer-Korvenkontio

Rapid diagnosis of hantavirus disease with an IgG-avidity assay

Nephropathia epidemica (NE), due to Puumala virus, is endemic in eastern Europe and Scandinavia. Serodiagnosis of NE relies on conventional indirect immunofluorescence to detect IgG against Puumala virus. However, in conventional serology, most patients with acute NE have raised but stable non-diagnostic antibody titres. For better serodiagnosis of NE, we have developed a test that measures the avidity (functional affinity) of IgG antibodies against Puumala virus. This new assay was highly specific and sensitive; a diagnosis of NE could be confirmed or excluded rapidly from an early single serum sample. With this test we have now verified the diagnosis of NE in more than 1300 Finnish patients during 22 months in 1989-91. Our findings point to an incidence of confirmed hantavirus disease much higher than previously shown.

Nephropathia epidemica in Finland: a retrospective study of 126 cases.

A total of 126 (99 males, 27 females) serologically confirmed hospital-treated adult cases of nephropathia epidemica (NE) were studied. The initial diagnosis suggested by the referring physician was correct in only 28%. Some rare clinical manifestations of NE were observed; acute myopericarditis in 3 patients and encephalitis in 1. Pulmonary involvement due to vascular congestion was observed in 16% and liver involvement in 34% of the patients. Thrombocytopenia was present in 75%, leukocytosis in 50% and anemia in 50%. Erythrocyte sedimentation rate (ESR) was 2-108 (mean 38) mm/h and C-reactive protein (CRP) 0-126 (mean 52) mg/l. Proteinuria was observed in 94%, hematuria in 58% and pyuria in 28%. Electrolyte abnormalities (hyponatremia, hypokalemia, hypocalcemia, hyperphosphatemia) were all common but rarely serious. Serum lipid changes caused by the acute infection and renal failure included very low total and HDL-cholesterol as well as high triglyceride levels. Renal function was transiently impaired in 94% of the patients and 7 needed transient dialysis therapy. All recovered.

Cloning and sequencing of Puumala virus Sotkamo strain S and M RNA segments: evidence for strain variation in hantaviruses and expression of the nucleocapsid protein

The prototype Puumala virus (PV) Sotkamo strain small (S) and medium (M) RNA genome segments were amplified by the polymerase chain reaction (PCR), cloned and sequenced. The S segment is 1830 nucleotides long with an open reading frame coding for 433 amino acids. The identity to the PV Hällnäs strain was 83% at the nucleotide and 96% at the amino acid level. The M segment in the Sotkamo strain is 3616 nucleotides long and contains one open reading frame of 1148 amino acids with 83% nucleotide and 94% amino acid identity to the Hällnäs strain. Most amino acid changes were conservative and the five predicted glycosylation sites are identical. The amino acid identity to the prototype hantavirus, Hantaan virus, was 62 and 54% for S and M segments, respectively. The coding region of the S segment was further amplified by PCR, ligated to pEX vectors and expressed in Escherichia coli as a beta-galactosidase fusion protein and was seen to be specifically detected by nephropathia epidemica sera in immunoblotting.

Epidemiological Study of Nephropathia epidemica in Finland 1989-96

This study presents data on 33,000 serum samples studied from July 1989 to June 1996 in Finland, with 6,701 serologically confirmed Puumala virus (PUU) infections. In addition, a PUU serosurvey of 8,000 sera from Finland is presented. On average, 957 PUU infections were detected annually, resulting in an incidence of 19/100,000; mortality was less than 0.1%. The infection was most common in the district of Ita-Savo with an incidence of 90/100,000. The seasonal peak was in November-December; however, the urban population had its incidence peak in August. Local epidemics mirrored bank vole densities, with 3-4-y cycles. Males contracted the disease at a mean age of 40 y, females at 44 y (male:female ratio 2:1). The disease was relatively rare in children and elderly people. The nationwide PUU antibody prevalence for women entering Finnish maternity clinics was 3%, suggesting 5% for the total population. The highest prevalences (7% for young women) were encountered in eastern Finland. In the district with the highest clinical alert, approximately 30% of all PUU infections were estimated to lead to clinical disease with serological confirmation.

Susceptibility of human cells to Puumala virus infection

Nephropathia epidemica involves several organs including kidney, lung, liver and brain. To investigate the susceptibility of putative target cells to the agent responsible, Puumala virus, we screened established human cell lines of lung (WI-38, A-427, CCD-11Lu), kidney (A-704), liver (Hep G2), pharynx (Detroit 562), submaxillary gland (A-253) and neural (SK-N-MC, SH-SY5Y) origin as well as primary human kidney glomerular cells, endothelial cells and peripheral blood monocytes/macrophages. Propagation of the Sotkamo strain of Puumala virus was also tested in the primary kidney, spleen and lung cells of bank voles (the natural host of the virus). All of the primary cells and most of the established cell lines expressed viral protein, synthesized viral RNA and secreted infectious virus, except the neural SK-N-MC and SH-SY5Y cells. None of the tested cell types except the primary bank vole kidney cells could propagate the virus as efficiently as the Vero E6 cells. The observed host cell range is wide and consistent with a multiorgan involvement of Puumala virus. No cytopathic effects were seen in any of the infected cell cultures.

Genetic variation of wild Puumala viruses within the serotype, local rodent populations and individual animal

Reverse transcriptase polymerase chain reaction cloning and sequencing were used to determine the range of S gene/N protein variability in wild Puumala virus (PUU) strains and to study phylogenetic relationships between two groups of strains which originated from Finland and from European Russia. Analyses of the nucleotide and predicted amino acid sequences showed: (1) all PUU strains shared a common ancient ancestor; and (2) the more recent ancestors were different for the Finnish branch and the Russian branch of PUU strains. A cluster of amino acid substitutions in the N protein of Finnish strains was found; this cluster was located within a highly variable region of the molecule carrying B-cell epitopes (Vapalahti et al., J. Med. Virol., 1995, in press). Different levels of S gene/N protein diversity of PUU were revealed supporting the view of geographical clustering of genetic variants. Puumala virus from individual voles was found to be a complex mixture of closely related variants-quasispecies. The ratio of non-silent to silent nucleotide mutations registered in the S genes/N proteins of PUU quasispecies was 4- to 16-fold higher than that in Puumala virus strains, resulting in a more wide range of quasispecies N protein sequence diversity.

Four Fatal Cases of nephropathia epidemica

Four serologically confirmed fatal cases of nephropathia epidemica (NE), the mild form of hemorrhagic fever with renal syndrome (HFRS) are described. All the patients had disseminated intravascular coagulation. Autopsies revealed hemorrhage and necrotic areas of their pituitary glands, myocarditis, venous congestion and hemorrhage of the kidneys as well as pulmonary edema and hemorrhage of the lungs in all patients. This report provides new evidence that NE can be a fatal disease.

Epidemiology of Sindbis virus infections in Finland 1981-96: possible factors explaining a peculiar disease pattern.

Pogosta disease (PD), an epidemic rash-arthritis occurring in late summer is caused by Sindbis virus (SINV) and is transmitted to humans by mosquitoes. Altogether 2183 PD cases were serologically confirmed 1981-96 in Finland, with an annual incidence of 2.7/100000 (18 in the most endemic area of Northern Karelia). The annual average was 136 (varying from 1 to 1282) with epidemics occurring in August-September with a 7-year interval. Studies on 6320 patients with suspected rubella (1973-89) revealed 107 PD cases. The depth of snow cover and the temperature in May-July seemed to predict the number of cases. The morbidity was highest in 45- to 65-year-old females and lowest in children. Subclinical SINV infections were 17 times more common than the clinical ones. The SINV-antibody prevalence in fertile-age females was 0.6% in 1992; the estimated seroprevalence in Finland is about 2%. Among game animals the tetraonids (black grouse and capercaillie) had the highest seroprevalence (65%) in the epidemic year of 1981.

ÆTIOLOGICAL RELATION BETWEEN KOREAN HÆMORRHAGIC FEVER AND NEPHROPATHIA EPIDEMICA

A close aetiological relation between Korean haemorrhagic fever and nephropathia epidemica of Scandinavia has often been suggested because of similarities in their clinical picture and epidemiology. This relation has now been confirmed serologically; raised titres of antibody to Korean haemorrhagic fever agent and seroconversions have been found by the indirect fluorescent-antibody technique in 20 patients with nephropathia epidemica.

Lipids of cultured mosquito cells (Aedes albopictus). Comparison with cultured mammalian fibroblasts (BHK 21 cells).

Abstract The lipids of an established cell line of a mosquito, Aedes albopictus were analyzed by Chromatographie and degradative methods. The lipid classes and their hydro-carbon chains resembled those of several Diptera species that have been analyzed from whole insects. The mosquito cells differed profoundly from cultured baby hamster kidney cells (BHK 21 cells) in their membrane lipids. As these two cells are suitable hosts for Semliki Forest virus, they provide us with good model systems for the study of lipid-protein interactions in the virus.