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.

Human immune response to Puumala virus glycoproteins and nucleocapsid protein expressed in mammalian cells.

Puumala hantavirus (PUUV) glycoproteins G1 and G2 and nucleocapsid protein (N) were expressed in BHK-21 cells by transfection of a plasmid producing a recombinant alphavirus replicon. Coexpression of G1 and G2 from separate constructs seemed to be important for the optimal folding of the glycoproteins, as evaluated by a panel of MAbs detecting conformational epitopes. To evaluate the human antibody response against recombinant G1, G2 and N, several panels of sera were tested by immunofluorescence assay (IFA). Also human sera showed the best reactivity towards G1 and G2 coexpressed from separate transcripts (G1 + G2). Notably, only 2% of the acute sera (total number = 133) contained IgG antibodies against G1 + G2, whereas of old-immunity sera (total number = 100) 87% were G1 + G2 positive. Analysis of a panel of serial patient sera showed that as the immunity matured, IgG antibodies against the recombinant glycoproteins appeared and the titers increased in the course of time, while antibodies against the recombinant N were present already in the acute phase in high titers. The granular fluorescence pattern in PUUV IgG-IFA, associated with the acute phase of immunity, was linked to the presence of antibodies against N, whereas the diffuse fluorescence pattern associated with old-immunity, was linked to the development of antibodies against G1 + G2. The granular fluorescence pattern in PUUV IgG-IFA had a predictive value of 100% for acute PUUV infection. Weak cross-reaction with PUUV glycoproteins was observed in 36% of old-immunity DOBV-specific human sera.

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.

Puumala virus infections in Finland: increased occupational risk for farmers.

Puumala hantavirus, transmitted by bank voles (Clethrionomys glareolus), causes a mild-type hemorrhagic fever with renal syndrome. The disease is common in Finland and is considered an occupational hazard for farmers, but the actual risk has not been assessed by analytical studies. Data on 5,132 serologically confirmed Puumala virus infections during 1989-1994 were analyzed, and cases among farmers and the population living in similar conditions were compared. The farmers contracted the disease earlier and more often than did the comparison group. In the province of Mikkeli with the highest incidence (70/100,000), the risk ratio was 5.1 (95% confidence interval (CI) 3.0-8.4) for 20- to 29-year-old farmers; in the older age groups, the risk was still increased but the risk ratios were lower. The peak incidence in the comparison group was 10 years later (age group 30-39 years). For the whole country, the result was similar although less marked. The average risk ratio adjusted by age, sex, and geographic variation was 1.7 (95% CI 1.5-1.8) for the whole country and 1.9 (95% CI 1.5-2.3) for the Mikkeli province, where 80% of Puumala virus infections among young farmers could be estimated to be attributable to occupation.

Detection of mosquito saliva-specific IgE antibodies by capture ELISA.

We developed an IgE-capture ELISA and measured mosquito saliva-specific IgE antibodies in 27 children sensitive to mosquito bites. Children with large 15-min bite wheals had significantly higher (P < 0.0005) mosquito saliva-specific IgE levels than children with small wheals. In the latter group, the saliva-specific IgE level was significantly higher (P = 0.031) than the levels of six infants never exposed to mosquitoes. A positive correlation (r = 0.65; P = 0.0002) was found between the size of the 15-min wheal and the mosquito saliva-specific IgE antibody levels. These results further support the role of mosquito saliva-specific IgE antibodies in the pathogenesis of mosquito-bite whealing. Compared to immunoblotting, IgE-capture ELISA provides a quantitative method to measure mosquito saliva-specific IgE antibodies.

Inkoo and Tahyna, the European California serogroup bunyaviruses: sequence and phylogeny of the S RNA segment.

Inkoo (INK) and Tahyna (TAH) viruses, European representatives of the California serogroup (CAL), genus Bunyavirus, family Bunyaviridae, are transmitted by mosquitoes and frequently infect man. The S segments of INK and TAH prototype strains were amplified, cloned and sequenced. INK S consists of 986 and TAH S of 977 nucleotides (nt) coding for a nucleocapsid protein of 235 amino acids (aa) and, in an overlapping reading frame, for a nonstructural protein of 92 or 97 aa, respectively. By S segment sequences and phylogenetic analysis INK was seen to be most closely related to Jamestown Canyon virus, isolated in the USA (92.4% nt and 96.6% aa identity), which is currently classified in a different subcomplex within the CAL viruses. TAH was genetically closest to Lumbo virus, isolated in Mozambique (89.0% nt and 94.1% aa identity). The data suggest that genetic variation within the CAL viruses is less related to geographical distance than to similarity in ecological cycles.

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.

Human B-cell epitopes of Puumala virus nucleocapsid protein, the major antigen in early serological response.

Puumala virus (PUU) is a member of the Hantavi rus genus in the family Bunyaviridae and the etiologic agent of nephropathia epidemica (NE), a form of haemorrhagic fever with renal syndrome (HFRS). In this study we compared the immunofluorescence patterns of NE sera and antibodies raised against recombinant PUU proteins and confirm that the nucleocapsid protein is the major target in the early IgG response of NE patients and provides the molecular basis for simple and rapid differentiation between acute illness and old immunity by granular vs. diffuse fluorescence staining in the indirect immunofluorescence test. The differential kinetics of B-cell responses to PUU nucleocapsid vs. envelope proteins was emphasized further by the endpoint titres of IgG antibodies to N, G1 and G2 proteins in NE patients. The granular fluorescence correlated with low IgG avidity in 99.8%, and diffuse fluorescence with high avidity in 100% of 617 NE sera studied. Epitope scanning with overlapping 14-mer peptides covering the whole nucleocapsid protein by a shift of 3 amino acids revealed six major antigenic epitopes recognized by sera from acute-phase NE patients. The epitopes clustered mainly in the hydrophilic regions, and two of them in a highly variable region which could probably serve as an antigen to distinguish serologically between infections of closely related hantaviruses, some apparently apathogenic, some causing lethal infections. The anti-peptide epitope pattern varied between different individuals and a collection of several pin-bound peptides was needed to be recognised by most NE sera studied.

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.

Tula virus: a newly detected hantavirus carried by European common voles.

A novel hantavirus has been discovered in European common voles, Microtus arvalis and Microtus rossiaemeridionalis. According to sequencing data for the genomic RNA S segment and nucleocapsid protein and data obtained by immunoblotting with a panel of monoclonal antibodies, the virus, designated Tula virus, is a distinct novel member of the genus Hantavirus. Phylogenetic analyses of Tula virus indicate that it is most closely related to Prospect Hill, Puumala, and Muerto Canyon viruses. The results support the view that the evolution of hantaviruses follows that of their primary carriers. Comparison of strains circulating within a local rodent population revealed a genetic drift via accumulation of base substitutions and deletions or insertions. The Tula virus population from individual animals is represented by quasispecies, indicating the potential for rapid evolution of the agent.

Clinical picture of nephropathia epidemica in children.

The clinical picture of nephropathia epidemica (NE) among children is poorly understood. We made a retrospective analysis of 32 patients aged 4-15 years treated in hospital for serologically verified recent NE. The most common clinical findings were high fever (100%), nausea (81%), vomiting (72%), tenderness in the kidney area (63%), abdominal pains (59%) and headache (59%). A peculiar symptom of NE, transient visual abnormalities, was found in 25% of patients. Four children had clinical bleeding and 1 had encephalitis. 44% were transiently hypertensive. Renal function was impaired in 84%, proteinuria was present in 97%, hematuria in 73% and leukocyturia in 44%. Other common laboratory findings were thrombocytopenia (87%), leukocytosis (41%), elevated ERS (74%, up to 76 mm/h) and CRP level (89%, up to 97 mg/l), elevated liver enzymes (53%) and hypoalbuminemia (50%). No child needed dialysis therapy and all recovered. NE seems to be less severe in children than in adults.

Renal biopsy findings and clinicopathologic correlations in nephropathia epidemica.

A series of 126 adult patients with a serologically confirmed diagnosis of nephropathia epidemica (NE) were studied during the acute phase of the disease. In 86 cases, renal biopsy was performed. The severity of renal failure correlated slightly with blood inflammatory parameters and the degree of hematuria but not with the amount of proteinuria. The degree of hematuria correlated inversely with the level of thrombocytopenia. The most common histopathologic lesion was acute tubulointerstitial nephritis. Interstitial edema and inflammatory cell infiltrations were most usually present, followed by tubular epithelial and luminal alterations. Slight glomerular mesangial changes were present in 25% of the biopsy specimens. Except for hemorrhage in the outer renal medulla, the histologic lesions were relatively mild and unspecific. Interstitial hemorrhage should remind a pathologist of the possibility of NE. Tubular, interstitial and glomerular histologic damage were but vascular lesions were not associated with the clinical severity of renal failure. Glomerular alterations did not relate to the amount of urine protein excretion. Correlations, however, were so weak that in clinical work renal biopsy is usually not indicated for determination of the severity of renal failure in NE. Intrinsic renal events are probably important in the development of renal failure in NE.

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.

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.

Puumala virus antibody and immunoglobulin G avidity assays based on a recombinant nucleocapsid antigen.

Puumala virus is the causative agent of nephropathia epidemica (NE), a hantavirus infection which occurs widely in northern and central Europe and is generally diagnosed by the indirect immunofluorescence (IF) method. We have now expressed the Puumala virus Sotkamo strain nucleocapsid (N) protein-coding S genome segment as a beta-galactosidase fusion protein in Escherichia coli by using the pEX2 expression vector. The recombinant protein was purified by cutting the protein band from an agarose gel, melting the agarose, and removing the protein by freezing, incubation on ice, and centrifugation. The recovery was about 1 to 5 mg/200 ml of bacterial suspension, sufficient for coating 100 to 500 enzyme immunoassay microtiter plates. In a study of 312 IF-positive and 233 IF-negative serum samples from NE patients, the recombinant-N-protein enzyme immunoassay detected immunoglobulin G antibodies to Puumala virus with 97.8% sensitivity and 98.5% specificity compared with the IF test results. In addition, an immunoglobulin G avidity enzyme immunoassay was developed and used successfully to diagnose acute NE from a single serum sample. The results demonstrate that the bioengineered antigen is suitable for use in routine diagnostic assays for Puumala virus immunity and recent infection.