Pathogenic hantaviruses selectively inhibit beta3 integrin directed endothelial cell migration.

Hantaviruses cause two diseases of man, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Pathogenic and non-pathogenic hantaviruses use beta3 and beta1 integrins, respectively, to enter endothelial cells. Beta3 integrins were recently reported to bind receptors that regulate vascular permeability suggesting that hantavirus beta3 integrin interactions may regulate endothelial cell function and contribute to viral pathogenesis. In this study we investigated the ability of pathogenic and non-pathogenic hantaviruses to regulate beta3 and beta1 integrin directed endothelial cell functions. We found that pathogenic NY-1, SNV, HTN, SEO and PUU viruses blocked endothelial cell migration on beta3, but not beta1, integrin ligands. Migration is similarly inhibited by antibodies to beta3 integrins which selectively block vitronectin directed endothelial cell migration. As a result, the ability of endothelial cells to migrate on integrin ligands was selectively inhibited by only pathogenic hantaviruses. Infection by NY-1 virus inhibited endothelial cell migration as early as 24-48 h post-infection. In contrast, non-pathogenic PH and TUL viruses had no effect on the ability of endothelial cells to migrate on either beta3 or beta1 integrin ligands from 1 to 5 days post-infection. These findings indicate that only hantaviruses which use beta3 integrins, and are associated with HPS and HFRS diseases, functionally dysregulate endothelial cell migration. These findings further demonstrate that hantaviruses regulate only beta3 integrin directed endothelial cell functions and have no effect on beta1 integrin functions. Since beta3 integrins are linked to changes in vascular permeability and the maintenance of vascular integrity, these findings suggest a means by which hantavirus usage and regulation of beta3 integrins may contribute to hantavirus pathogenesis.

Genetic and serotypic characterization of Sin Nombre-like viruses in Canadian Peromyscus maniculatus mice.

In Canada, hantavirus infected deer mice (Peromyscus maniculatus) have been collected from British Columbia to Newfoundland. Partial sequencing of G1 and N protein encoding regions from Canadian Peromyscus maniculatus-borne hantaviruses demonstrated the existence of significant genotypic divergence among strains. Phylogenetic analysis showed that Sin Nombre (SN)-like viruses from eastern and western Canadian deer mice can be divided into at least two broad-based genogroups. Sequencing of mitochondrial DNA from infected deer mice originating from various eastern and western provinces showed that SN-like virus genogroups appeared to be associated with distinct haplotypes of mice. Sera from deer mice infected with eastern and western viral genotypes neutralized the Sin Nombre virus strain, Convict Creek 107, but not the New York 1 hantavirus. Despite the genetic heterogeneity of Canadian SN-like strains these hantaviruses do not appear to define unique hantavirus serotypes.

Cellular receptors and hantavirus pathogenesis.

Hantaviruses cause two potentially lethal diseases, HPS and HFRS, and both diseases result in defects in vascular permeability and platelet function. Human beta 3 integrins confer cellular susceptibility to HPS- and HFRS-causing hantaviruses, a fact directly linking platelets, endothelial cells, and hantavirus diseases to the use of [figure: see text] cellular receptors that maintain capillary integrity and regulate platelet function. The role of vitronectin, PAI-1, uPAR, and complement cascades in hantavirus pathogenesis are unstudied but may contribute to specific disease syndromes effected by hantaviruses. The divergence of hantavirus surface glycoproteins and common beta 3-integrin usage provides further insight into the interaction of hantaviruses with cells. G1 and G2 glycoprotein variation is likely to contribute to additional interactions that determine pathogenic responses to individual viruses. beta 3-integrin usage also suggests that common elements exist on G1 or the more highly conserved G2 surface glycoprotein, which mediate viral attachment to integrins. Although there is currently no data defining the virion attachment protein, the development of antibodies that recognize the hantavirus attachment protein and block integrin interactions is of interest since it is likely to provide an additional point for therapeutic intervention and vaccine development. There are a plethora of effects that could be elicited by hantavirus regulation of cellular beta 3 integrins and their ligands that are consistent with hantavirus diseases. Since beta 3 integrins are critical adhesive receptors on platelets and endothelial cells and regulate both vascular permeability and platelet activation and adhesion, the use of these receptors by hantaviruses is likely to be fundamental to hantavirus pathogenesis. The lack of an animal model for hantavirus pathogenesis has prevented a systematic analysis of immune and cellular responses to hantavirus infections, and it impedes our ability to study protective or therapeutic approaches to hantavirus diseases. However, recent findings suggest that human beta 3 integrins within transgenic mice may provide animal models of hantavirus pathogenesis and have the potential to radically alter the ability to investigate hantavirus disease.

Cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by beta3 integrins.

Hantaviruses replicate primarily in the vascular endothelium and cause two human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). In this report, we demonstrate that the cellular entry of HFRS-associated hantaviruses is facilitated by specific integrins expressed on platelets, endothelial cells, and macrophages. Infection of human umbilical vein endothelial cells and Vero E6 cells by the HFRS-causing hantaviruses Hantaan (HTN), Seoul (SEO), and Puumala (PUU) is inhibited by antibodies to alphavbeta3 integrins and by the integrin ligand vitronectin. The cellular entry of HTN, SEO, and PUU viruses, but not the nonpathogenic Prospect Hill (PH) hantavirus (i.e., a virus with no associated human disease), was also mediated by introducting recombinant alphaIIbbeta3 or alphavbeta3 integrins into beta3-integrin-deficient CHO cells. In addition, PH infectivity was not inhibited by alphavbeta3-specific sera or vitronectin but was blocked by alpha5beta1-specific sera and the integrin ligand fibronectin. RGD tripeptides, which are required for many integrin-ligand interactions, are absent from all hantavirus G1 and G2 surface glycoproteins, and GRGDSP peptides did not inhibit hantavirus infectivity. Further, a mouse-human hybrid beta3 integrin-specific Fab fragment, c7E3 (ReoPro), also inhibited the infectivity of HTN, SEO, and PUU as well as HPS-associated hantaviruses, Sin Nombre (SN) and New York-1 (NY-1). These findings indicate that pathogenic HPS- and HFRS-causing hantaviruses enter cells via beta3 integrins, which are present on the surfaces of platelets, endothelial cells, and macrophages. Since beta3 integrins regulate vascular permeability and platelet function, these findings also correlate beta3 integrin usage with common elements of hantavirus pathogenesis.

New York 1 and Sin Nombre viruses are serotypically distinct viruses associated with hantavirus pulmonary syndrome.

New York 1 virus (NY-1) and Sin Nombre virus (SN) are associated with hantavirus pulmonary syndrome (HPS). NY-1 and SN are derived from unique mammalian hosts and geographic locations but have similar G1 and G2 surface proteins (93 and 97% identical, respectively). Focus reduction neutralization assays were used to define the serotypic relationship between NY-1 and SN. Sera from NY-1-positive Peromyscus leucopus neutralized NY-1 and SN at titers of >/=1/3,200 and 16-fold-lower neutralizing titers to NY-1 than to SN. Reference sera to Hantaan, Seoul, and Prospect Hill viruses also failed to neutralize NY-1. These results indicate that SN and NY-1 define unique hantavirus serotypes and implicate the presence of additional HPS-associated hantavirus serotypes in the Americas.

Dynamics of Puumala hantavirus infection in naturally infected bank voles (Clethrinomys glareolus).

Specific features of hantavirus infection in bank vole (Clethrionomys glareolus) were studied in the endemic area of hemorrhagic fever with renal syndrome (HFRS) in the foothills of the Ural mountains, using long-term observations on living animals by the capture-mark-recapture (CMR) method. The results demonstrated that the infection naturally circulating in the voles is chronic (lasting for up to 15 months) and asymptomatic, with a peak of Puumala virus accumulation and release from the organism during the first month after infection. It was shown that the bank vole population includes young animals with maternal immunity, which remain resistant to the Puumala virus infection for 3-3.5 months. The infection rate in voles depended on the age and sexual maturity of animals. The greatest proportion of seropositive animals was observed among overwintered males. Seroconversion in voles was more frequent during the period of high reproductive activity.

beta3 Integrins mediate the cellular entry of hantaviruses that cause respiratory failure.

Newly emerged hantaviruses replicate primarily in the pulmonary endothelium, cause acute platelet loss, and result in hantavirus pulmonary syndrome (HPS). We now report that specific integrins expressed on platelets and endothelial cells permit the cellular entry of HPS-associated hantaviruses. Infection with HPS-associated hantaviruses, NY-1 and Sin Nombre virus (SNV), is inhibited by antibodies to beta3 integrins and by the beta3-integrin ligand, vitronectin. In contrast, infection with the nonpathogenic (no associated human disease) Prospect Hill virus was inhibited by fibronectin and beta1-specific antibodies but not by beta3-specific antibodies or vitronectin. Transfection with recombinant alphaIIb beta3 or alphav beta3 integrins rendered cells permissive to NY-1 and SNV but not Prospect Hill virus infection, indicating that alphaIIb beta3 and alphav beta3 integrins mediate the entry of NY-1 and SNV hantaviruses. Furthermore, entry is divalent cation independent, not blocked by arginine-glycine-aspartic acid peptides and still mediated by, ligand-binding defective, alphaIIb beta3-integrin mutants. Hence, NY-1 and SNV entry is independent of beta3 integrin binding to physiologic ligands. These findings implicate integrins as cellular receptors for hantaviruses and indicate that hantavirus pathogenicity correlates with integrin usage.

Histopathology of Peromyscus leucopus naturally infected with pathogenic NY-1 hantaviruses: pathologic markers of HPS viral infection in mice.

Hantavirus research is impeded by the absence of animal models of viral pathogenesis. We have studied the histopathology of mice (P. leucopus) naturally infected with the NY-1 hantavirus on Shelter Island, New York. Five mice were determined to be seropositive in Western blotting to Four Corners Virus nucleocapsid protein and had serum antibodies to Seoul and Puumala hantavirus antigens by immunofluorescence assay. Hantavirus gene segments of the NY-1 hantavirus were identified in these mice and shown to be 99% identical to hantavirus genes isolated from the Rhode Island patient with hantavirus pulmonary syndrome. In ultrastructural examinations, we identified hantavirus particles in pulmonary endothelial cells. Morphologically, these mice demonstrate lymphohistocytic infiltrates in hepatic portal zones and slightly increased numbers of immunoblasts in splenic red pulp. Additionally, the alveolar septa in the lungs of infected mice are edematous with hyperplasia of type 1 pneumocytes. Naturally infected P. leucopus may serve as potentially useful animal models of hantavirus pulmonary syndrome disease.

Sequence analysis of the complete S genomic segment of a newly identified hantavirus isolated from the white-footed mouse (Peromyscus leucopus): phylogenetic relationship with other sigmodontine rodent-borne hantaviruses.

Four Corners (FC) or Sin Nombre virus, a hantavirus harbored by the deer mouse (Peromyscus maniculatus), is the principal etiologic agent of hantavirus pulmonary syndrome (HPS). Recently, a hantavirus, designated New York (NY) virus, isolated from a white-footed mouse (Peromyscus leucopus) captured on Shelter Island, New York, was molecularly linked to a fatal case of HPS occurring in the northeastern United States. To clarify the genetic and phylogenetic relationship between NY and FC viruses and other sigmodontine rodent-borne hantaviruses, we amplified and sequenced the entire S genomic segment of NY virus. The S segment of NY virus was 2078 nucleotides long, with an open reading frame of 1284 nucleotides in the virus complementary strand, capable of encoding a protein of 428 amino acids, and with a 752-nucleotide long 3'-noncoding region, comprised of numerous imperfect repeats. Pairwise analysis indicated that NY virus was more similar to FC virus than to other sigmodontine rodent-borne hantaviruses, differing from strains of FC virus by 16.6-17.8% and 7.0-8.2% at the nucleotide and amino acid levels, respectively. As determined by the maximum parsimony and neighbor-joining methods, NY virus formed a separate lineage from FC virus and was phylogenetically distinct from hantaviruses harbored by other sigmodontine rodents. Whether or not NY and FC viruses represent distinct viral species is unclear. Further analyses of hantaviruses harbored by white-footed mice are needed to clarify the genetic diversity and evolution of Peromyscus-borne hantaviruses.

Molecular linkage of hantavirus pulmonary syndrome to the white-footed mouse, Peromyscus leucopus: genetic characterization of the M genome of New York virus.

The complete M segment sequences of hantaviruses amplified from tissues of a patient with hantavirus pulmonary syndrome in the northeastern United States and from white-footed mice, Peromyscus leucopus, from New York were 99% identical and differed from those of Four Corners virus by 23%. The serum of this patient failed to recognize a conserved, immunodominant epitope of the Four Corners virus G1 glycoprotein. Collectively, these findings indicate that P. leucopus harbors a genetically and antigenically distinct hantavirus that causes hantavirus pulmonary syndrome.

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.

Sequences of wild Puumala virus genes show a correlation of genetic variation with geographic origin of the strains.

An experimental scheme was developed for direct sequence analysis of Puumala virus-containing specimens from wild rodents (Clethrionomys glareolus). Total RNA isolated from rodent lung tissues was reverse-transcribed in the presence of a universal 11 nucleotide primer complementary to all three viral RNA segments followed by amplification in a PCR with gene-specific primers. A full-length PCR product of approximately 1800 bp from the S segment encoding the viral nucleoprotein and a product of approximately 900 bp from the M segment (encoding the C-terminal two-thirds of the G2 protein and including the 3' non-coding region) of Puumala virus (from C. glareolus trapped in Udmurtia) were prepared and sequenced. No pronounced differences to Vero cell-grown viruses were seen. The Udmurtia/894Cg/91 strain was more closely related to the Bashkiria/CG18-20/84 strain than to the Finnish prototype strain of Puumala virus, Sotkamo/V-2969/81. Thus there is a correlation with the geographic origin of the three strains. The results indicate the occurrence of genetic drift and different selection pressures leading to (i) clustering of mutations, (ii) a lower frequency of nucleotide substitutions in the coding than in the 3' noncoding regions and (iii) a higher frequency of amino acid substitutions in G2 than in the N protein.

IgG avidity assay for estimation of the time after onset of hantavirus infection in colonized and wild bank voles.

An immunoglobulin G avidity assay was used to determine recent and past hantavirus infection in bank voles (Clethrionomys glareolus). Sera of experimentally infected bank voles were studied at different time intervals. The avidity of specific IgG increased over time after infection. This experimental data were used to estimate the time after onset of hantavirus infection in naturally infected bank voles caught in an endemic area. The possibility to discriminate between recently infected animals and animals infected some time ago is important since the proportion of recently infected bank voles represents the intensity of the epizootic which in turn correlates to the risk of humans to contract the disease.

Seroepidemiology of haemorrhagic fever with renal syndrome in Bulgaria.

During the period of 1954-1986, 399 cases of haemorrhagic fever with renal syndrome (HFRS) were registered in Bulgaria with 63 (15.7%) deaths. Three hundred serum samples from 214 patients who had contracted the disease from 1957 to 1986 were investigated by indirect fluorescent antibody test (IF-AT). As antigen Vero-E6 cells infected with the Asian Hantaan virus were used, as well as lung sections from bank voles (Clethrionomys glareolus) infected with strains Udmurt and Kazan 6-Cg from the European part of the U.S.S.R. Specific antibodies were detected in 194 sera, i.e. in 90.6% of persons investigated: in 131 single serum samples and in 63 paired sera. The results of the serological studies which covered 53.6% of all known local cases showed the territorial distribution of the natural foci in this country and the aetiologic relationship with the HFRS virus from the European part of the U.S.S.R.

Features of circulation of hemorrhagic fever with renal syndrome (HFRS) virus among small mammals in the European U.S.S.R.

The use of indirect fluorescent antibody testing (IFAT) and enzyme linked immunosorbant assay (ELISA) procedures allowed the hemorrhagic fever with renal syndrome (HFRS) virus antigen to be detected not only in the known reservoir host, Clethrionomys glareolus, but also in 7 other species of small mammals in European foci of the U.S.S.R. Marked viscerotropism of HFRS virus and the participation of brown fat in maintaining the infection in rodents were demonstrated. The frequency of detection of circulating antigen and antibody to HFRS virus in rodents is indicative of the high level of activity of the virus in its epizootic foci.

Adaptation to laboratory and wild animals of the haemorrhagic fever with renal syndrome virus present in the foci of European U.S.S.R. Brief report.

Four strains of haemorrhagic fever with renal syndrome virus (HFRSV) from rodents or patients in European U.S.S.R. foci of HFRS were isolated in laboratory bred C. glareolus. The sensitivity of these animals to HFRSV was compared with that of five other laboratory and wild animals.

Detection of hemorrhagic fever with renal syndrome (HFRS) virus in the lungs of bank voles Clethrionomys glareolus and redbacked voles Clethrionomys rutilus trapped in HFRS foci in the European part of U.S.S.R., and serodiagnosis of this infection in man.

The antigen of HFRS virus was demonstrated by means of the indirect fluorescent antibody procedure in the lung tissue of bank and redbacked voles (Clethrionomys glareolus, Cl. rutilus) trapped in HFRS foci in the European part of USSR. This antigen has been used satisfactorily for serodiagnosis of HFRS in several European and Asian regions of the USSR where HFRS had been found to be endemic.