Bayesian Binary Mixture Models as a Flexible Alternative to Cut-Off Analysis of ELISA Results, a Case Study of Seoul Orthohantavirus.

Serological assays, such as the enzyme-linked immunosorbent assay (ELISA), are popular tools for establishing the seroprevalence of various infectious diseases in humans and animals. In the ELISA, the optical density is measured and gives an indication of the antibody level. However, there is variability in optical density values for individuals that have been exposed to the pathogen of interest, as well as individuals that have not been exposed. In general, the distribution of values that can be expected for these two categories partly overlap. Often, a cut-off value is determined to decide which individuals should be considered seropositive or seronegative. However, the classical cut-off approach based on a putative threshold ignores heterogeneity in immune response in the population and is thus not the optimal solution for the analysis of serological data. A binary mixture model does include this heterogeneity, offers measures of uncertainty and the direct estimation of seroprevalence without the need for correction based on sensitivity and specificity. Furthermore, the probability of being seropositive can be estimated for individual samples, and both continuous and categorical covariates (risk-factors) can be included in the analysis. Using ELISA results from rats tested for the Seoul orthohantavirus, we compared the classical cut-off method with a binary mixture model set in a Bayesian framework. We show that it performs similarly or better than cut-off methods, by comparing with real-time quantitative polymerase chain reaction (RT-qPCR) results. We therefore recommend binary mixture models as an analysis tool over classical cut-off methods. An example code is included to facilitate the practical use of binary mixture models in everyday practice.

Human infection with Seoul orthohantavirus in Korea, 2019.

Of various rodent-borne hantaviruses, Seoul orthohantavirus (SEOV) causes haemorrhagic fever with renal syndrome (HFRS), as does Hantaan orthohantavirus (HTNV). Given global-scale of cases of human infection with SEOV, it is of great clinical importance to distinguish SEOV from other HFRS-causing hantaviruses. In May 2019, a middle-aged patient who had lived in a suburban area of Chungcheong Province, Republic of Korea and enjoyed outdoor activities was transferred to Asan Medical Center in Seoul, Republic of Korea with HFRS; his symptoms included high fever and generalized myalgia. The rapid diagnostic test performed immediately after his transfer detected HTNV-specific antibodies, and the patient was treated accordingly. However, two consecutive IFAs performed at ten-day intervals showed no HTNV-specific immunoglobulin (Ig) G. During continuous supportive care, next-generation sequencing successfully identified viral genomic sequences in the patient's serum, which were SEOV and not HTNV. Phylogenetic analysis grouped the L, M, and S genes of this SEOV strain together with those of rat- or human-isolated Korean strains reported previously. Given global outbreaks and public health threats of zoonotic hantaviruses, a causative pathogen of hantavirus HFRS should be identified correctly at the time of diagnosis and by point-of-care testing.

Seoul Virus Infection and Spread in United States Home-Based Ratteries: Rat and Human Testing Results From a Multistate Outbreak Investigation.

BACKGROUND: During 2017, a multistate outbreak investigation occurred after the confirmation of Seoul virus (SEOV) infections in people and pet rats. A total of 147 humans and 897 rats were tested. METHODS: In addition to immunoglobulin (Ig)G and IgM serology and traditional reverse-transcription polymerase chain reaction (RT-PCR), novel quantitative RT-PCR primers/probe were developed, and whole genome sequencing was performed. RESULTS: Seventeen people had SEOV IgM, indicating recent infection; 7 reported symptoms and 3 were hospitalized. All patients recovered. Thirty-one facilities in 11 US states had SEOV infection, and among those with ≥10 rats tested, rat IgG prevalence ranged 2%-70% and SEOV RT-PCR positivity ranged 0%-70%. Human laboratory-confirmed cases were significantly associated with rat IgG positivity and RT-PCR positivity (P = .03 and P = .006, respectively). Genomic sequencing identified >99.5% homology between SEOV sequences in this outbreak, and these were >99% identical to SEOV associated with previous pet rat infections in England, the Netherlands, and France. Frequent trade of rats between home-based ratteries contributed to transmission of SEOV between facilities. CONCLUSIONS: Pet rat owners, breeders, and the healthcare and public health community should be aware and take steps to prevent SEOV transmission in pet rats and to humans. Biosecurity measures and diagnostic testing can prevent further infections.

Identification and validation of specific B-cell epitopes of hantaviruses associated to hemorrhagic fever and renal syndrome.

BACKGROUND: Orthohantavirus infection is a neglected global health problem affecting approximately 200,000 people/year, spread by rodent hosts and associated to fatal human diseases, such as hemorrhagic fever with renal syndrome (HFRS) and orthohantavirus cardiopulmonary syndrome (HCPS). Circulation of HFRS-associated orthohantaviruses, such as Seoul, Gou, Amur, Dobrava and Hantaan, are supposed to be restricted to Eurasian countries even though their hosts can be a worldwide distribution. Few confirmed HFRS orthohantavirus infections in humans have been reported in American countries, but due to lower medical awareness of the symptoms of this zoonosis, it could be associated to viral underreporting or to misdiagnosis with several tropical hemorrhagic diseases. Serological evidence of orthohantavirus infections, using enzyme-linked immunosorbent assay for the presence of immunoglobulin M and G against recombinant nucleoprotein protein, remains as an essential assay for viral surveillance. In this study, we aimed to identify in silico immunogenic B-cell linear epitopes present on orthohantavirus nucleoprotein that are exclusive to HFRS-related species. METHODOLOGY/PRINCIPAL FINDINGS: In silico analysis were performed using Seoul orthohantavirus nucleoprotein (SHNP) sequence as a model. Linear B-cell-epitopes on SHNP and its immunogenicity were predicted by BepiPred-2.0 and Vaxijen algorithms, respectively. The conservancy of predicted epitopes was compared with the most clinically relevant HFRS or HCPS-associated orthohantavirus, aiming to identify specific sequences from HFRS-orthohantavirus. Peptide validation was carried out by ELISA using Balb/c mice sera immunized with purified recombinant rSHNP. Peptides cross-reactivity against HCPS orthohantavirus were evaluated using immunized sera from mice injected with recombinant Juquitiba orthohantavirus nucleoprotein (rJHNP). CONCLUSION/SIGNIFICANCE: In silico analysis revealed nine potential immunogenic linear B-cell epitopes from SHNP; among them, SHNP(G72-D110) and SHNP(P251-D264) showed a high degree of sequence conservation among HFRS-related orthohantavirus and were experimentally validated against rSHNP-IMS and negatively validated against rJHNP-IMS. Taken together, we identified and validated two potential antigenic B-cell epitopes on SHNP, which were conserved among HFRS-associated orthohantavirus and could be applied to the development of novel immunodiagnostic tools for orthohantavirus surveillance.

Orthohantaviruses belonging to three phylogroups all inhibit apoptosis in infected target cells.

Orthohantaviruses, previously known as hantaviruses, are zoonotic viruses that can cause hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS) in humans. The HPS-causing Andes virus (ANDV) and the HFRS-causing Hantaan virus (HTNV) have anti-apoptotic effects. To investigate if this represents a general feature of orthohantaviruses, we analysed the capacity of six different orthohantaviruses - belonging to three distinct phylogroups and representing both pathogenic and non-pathogenic viruses - to inhibit apoptosis in infected cells. Primary human endothelial cells were infected with ANDV, HTNV, the HFRS-causing Puumala virus (PUUV) and Seoul virus, as well as the putative non-pathogenic Prospect Hill virus and Tula virus. Infected cells were then exposed to the apoptosis-inducing chemical staurosporine or to activated human NK cells exhibiting a high cytotoxic potential. Strikingly, all orthohantaviruses inhibited apoptosis in both settings. Moreover, we show that the nucleocapsid (N) protein from all examined orthohantaviruses are potential targets for caspase-3 and granzyme B. Recombinant N protein from ANDV, PUUV and the HFRS-causing Dobrava virus strongly inhibited granzyme B activity and also, to certain extent, caspase-3 activity. Taken together, this study demonstrates that six different orthohantaviruses inhibit apoptosis, suggesting this to be a general feature of orthohantaviruses likely serving as a mechanism of viral immune evasion.

Comparison of serological assays to titrate Hantaan and Seoul hantavirus-specific antibodies.

BACKGROUND: Hantaan and Seoul viruses, in the Hantavirus genus, are known to cause hemorrhagic fever with renal syndrome (HFRS). The plaque reduction neutralization test (PRNT), as conventional neutralization test for hantaviruses, is laborious and time-consuming. Alternatives to PRNT for hantaviruses are required. METHODS: In this study, the methods for Hantaan and Seoul viruses serological typing including microneutralization test (MNT), pseudoparticle neutralization test (PPNT) and immunofluorescence assay based on viral glycoproteins (IFA-GP) were developed and compared with PRNT using a panel of 74 sera including 44 convalescent sera of laboratory confirmed HFRS patients and 30 patients sera of non-hantavirus infection. Antibody titres and serotyping obtained with different methods above were analyzed by paired-t, linear correlation, McNemar χ2 and Kappa agreement tests. RESULTS: Antibody titres obtained with MNT50, PPNT50 and IFA-GP were significantly correlated with that obtained with PRNT50 (p < 0.001). GMT determined by PPNT50 was statistically higher than that determined by PRNT50 (p < 0.001), while GMT determined by MNT50 and IFA-GP were equal with (p > 0.05) and less than (p < 0.001) that obtained with PRNT50 respectively. Serotyping obtained with MNT50 and PRNT50, PPNT50 and PRNT50 were highly consistent (p < 0.001), whereas that obtained with IFA-GP and PRNT50 were moderately consistent (p < 0.001). There were no significant differences for serotyping between PRNT50 and MNT50, as well as PRNT50 and PPNT50 (p > 0.05). IFA-GP was less sensitive than PRNT50 and MNT50 for serotyping of hantaviruses infection (p < 0.05). However, for 79.5% (35/44) samples, serotyping determined by IFA-GP and PRNT50 were consistent. CONCLUSIONS: MNT50 and PPNT50 both can be used as simple and rapid alternatives to PRNT50, and MNT50 is more specific while PPNT50 is more sensitive than other assays for neutralizing antibody determination. So far, this work has been the most comprehensive comparison of alternatives to PRNT.

Molecular and serological evidence for Seoul virus in rats (Rattus norvegicus) in Zhangmu, Tibet, China.

We report the detection of a virus, tentatively identified as Seoul virus (SEOV), from a rat (Rattus norvegicus) collected in the city of Zhangmu, Tibet. SEOV RNA was detected in lung tissue by reverse transcription (RT)-PCR, followed by sequencing. Serum samples collected from Zhangmu were positive for SEOV-specific antibodies (indirect fluorescent antibody test that used SEO antigen). Sequencing and phylogenetic analysis of partial L and S sequences together with serology results suggest that the Zhangmu01 hantavirus is an isolate of SEOV, that hantaviruses circulate in Tibet, and that rats may act as natural reservoirs for the virus.

Influence of HLA-DRB alleles on haemorrhagic fever with renal syndrome in a Chinese Han population in Hubei Province, China.

Specific human leucocyte antigen (HLA) alleles are considered a genetic risk factor for the progression of haemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses. The aim of this study was to establish whether HLA-DRB alleles are associated with the severity of HFRS caused by different types of hantaviruses in a Chinese Han population from Hubei Province of central China. Twenty-two specific HLA-DRB alleles were analysed by sequence-specific primer-polymerase chain reaction (SSP-PCR) in 100 HFRS patients and 213 healthy volunteers. Associations of HLA-DRB alleles with the severity and clinical parameters of HFRS caused by Hantaan virus (HTNV) or Seoul virus (SEOV) infection were evaluated. Six alleles (HLA-DRB1*0401-0411, HLA-DRB1*1001, HLA-DRB1*1101-1105, HLA-DRB1*1201-1202, HLA-DRB1*1305 and DRB5*0101-0201) demonstrated strong associations with HFRS caused by HTNV and SEOV infections. Further comparison of these HLA-DRB1 allele frequencies between HFRS patients with differing severities and healthy controls demonstrated that the HLA-DRB1*0401-0411, HLA-DRB1*1001 and DRB1*1305 alleles were more frequent in the moderate course of HTNV-infected HFRS. Meanwhile, the DRB1*1101-1105 allele was more frequently observed in the severe course of HTNV-infected HFRS. We also found that the HLA-DRB1*1201-1202 allele frequency was higher in the moderate course of SEOV-infected HFRS, whereas the DRB5*0101-0201 allele may play a protective role in moderate HFRS caused by both HTNV and SEOV infections. These results provide evidence of the influence of HLA-DRB on the severity of HFRS and confirm the effect of HLA-DRB on HFRS during different types of hantavirus infection in a Chinese Han population in Hubei Province, China.

Comment on Jameson et al.: Prevalence of antibodies against hantaviruses in serum and saliva of adults living or working on farms in Yorkshire, United Kingdom.

This British hantavirus IgG prevalence study, aimed at 119 asymptomatic farmers in England, and using indirect immunofluorescence assay (IFA) as screening technique, concluded that rat-transmitted Seoul virus (SEOV) might be the main suspect as hantaviral pathogen in the UK. Exactly the same conclusion, using the same IFA screening technique, resulted from a 1994 serosurvey in the same country, and in 627 clinical cases plus 100 healthy controls. SEOV-positive study subjects were also mainly farmers with heavy rat-exposure, but residing in Northern-Ireland, a region where all other known rodent reservoirs for pathogenic hantaviruses are known to be absent, except the wild rat. A rodent capture action in and around the farms of eight seropositives confirmed SEOV seropositivity in 21.6% of 51 rats. All SEOV seropositives were patients, hospitalized with an acute feverish condition, a majority of which having the clinical picture of hantavirus-induced nephropathy, known as hemorrhagic fever with renal syndrome (HFRS). Leptospirosis, often mimicking perfectly HFRS, was serologically excluded. Thus, SEOV was established as a human hantaviral pathogen in the UK and in Europe 20 years ago.

Prevalence of antibodies against hantaviruses in serum and saliva of adults living or working on farms in Yorkshire, United Kingdom.

Hantaviruses are an established cause of haemorrhagic fever with renal syndrome (HFRS) in Europe. Following a confirmed case of HFRS in the UK, in an individual residing on a farm in North Yorkshire and the Humber, a tidal estuary on the east coast of Northern England, and the subsequent isolation of a Seoul hantavirus from rats trapped on the patient's farm, it was considered appropriate to further investigate the public health risk of this virus in the region. Of a total 119 individuals tested, nine (7.6%) were seropositive for hantavirus antibodies. Seven of the seropositive samples showed a stronger reaction to Seoul and Hantaan compared to other clinically relevant hantaviruses. Observation of rodents during the day, in particular mice, was associated with a reduced risk of seropositivity. In addition to one region known to be at risk following an acute case, five further potential risk areas have been identified. This study supports recently published evidence that hantaviruses are likely to be of public health interest in the region.

Infections by Leptospira interrogans, Seoul virus, and Bartonella spp. among Norway rats (Rattus norvegicus) from the urban slum environment in Brazil.

Norway rats (Rattus norvegicus) are reservoir hosts for zoonotic pathogens that cause significant morbidity and mortality in humans. Studies evaluating the prevalence of zoonotic pathogens in tropical Norway rat populations are rare, and data on co-infection with multiple pathogens are nonexistent. Herein, we describe the prevalence of leptospiral carriage, Seoul virus (SEOV), and Bartonella spp. infection independently, in addition to the rates of co-infection among urban, slum-dwelling Norway rats in Salvador, Brazil, trapped during the rainy season from June to August of 2010. These data were complemented with previously unpublished Leptospira and SEOV prevalence information collected in 1998. Immunofluorescence staining of kidney impressions was used to identify Leptospira interrogans in 2010, whereas isolation was used in 1998, and western blotting was used to detect SEOV antibodies in 2010, whereas enzyme-linked immunosorbent assay (ELISA) was used in 1998: in 2010, Bartonella spp. were isolated from a subsample of rats. The most common pathogen in both years was Leptospira spp. (83%, n=142 in 1998, 63%, n=84 in 2010). SEOV was detected in 18% of individuals in both 1998 and 2010 (n=78 in 1998; n=73 in 2010), and two species of Bartonella were isolated from 5 of 26 rats (19%) tested in 2010. The prevalence of all agents increased significantly with rat mass/age. Acquisition of Leptospira spp. occurred at a younger mass/age than SEOV and Bartonella spp. infection, suggesting differences in the transmission dynamics of these pathogens. These data indicate that Norway rats in Salvador serve as reservoir hosts for all three of these zoonotic pathogens and that the high prevalence of leptospiral carriage in Salvador rats poses a high degree of risk to human health.

Seoul virus-infected rat lung endothelial cells and alveolar macrophages differ in their ability to support virus replication and induce regulatory T cell phenotypes.

Hantaviruses cause a persistent infection in reservoir hosts that is attributed to the upregulation of regulatory responses and downregulation of proinflammatory responses. To determine whether rat alveolar macrophages (AMs) and lung microvascular endothelial cells (LMVECs) support Seoul virus (SEOV) replication and contribute to the induction of an environment that polarizes CD4(+) T cell differentiation toward a regulatory T (Treg) cell phenotype, cultured primary rat AMs and LMVECs were mock infected or infected with SEOV and analyzed for viral replication, cytokine and chemokine responses, and expression of cell surface markers that are related to T cell activation. Allogeneic CD4(+) T cells were cocultured with SEOV-infected or mock-infected AMs or LMVECs and analyzed for helper T cell (i.e., Treg, Th17, Th1, and Th2) marker expression and Treg cell frequency. SEOV RNA and infectious particles in culture media were detected in both cell types, but at higher levels in LMVECs than in AMs postinfection. Expression of Ifnß, Ccl5, and Cxcl10 and surface major histocompatibility complex class II (MHC-II) and MHC-I was not altered by SEOV infection in either cell type. SEOV infection significantly increased Tgfß mRNA in AMs and the amount of programmed cell death 1 ligand 1 (PD-L1) in LMVECs. SEOV-infected LMVECs, but not AMs, induced a significant increase in Foxp3 expression and Treg cell frequency in allogeneic CD4(+) T cells, which was virus replication and cell contact dependent. These data suggest that in addition to supporting viral replication, AMs and LMVECs play distinct roles in hantavirus persistence by creating a regulatory environment through increased Tgfß, PD-L1, and Treg cell activity.

Immunogenicity of a multi-epitope DNA vaccine against hantavirus.

Hemorrhagic fever with renal syndrome (HFRS) is a severe epidemic disease caused by hantaviruses including Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV) and Puumala virus. Three of the four HFRS hantaviruses, HTNV, SEOV, and PUUV are found in China. Currently, there is no effective strategy available to reduce infection risk. In this study, we constructed a multi-epitope chimeric DNA vaccine that encodes expressing 25 glycoprotein epitopes from SEOV, HTNV and PUUV (designated as SHP chimeric gene). Vaccination of BALb/c mice with SHP multi-epitope chimeric DNA vaccine led to a dramatic augmentation of humoral and cellular responses. The SHP vaccine DNA was detected in many organs but not for more than 60 d. There was no risk of mutation due to integration. Thus, the SHP multi-epitope chimeric DNA vaccine is a potential effective and safe DNA vaccine against infection by SEOV, HTNV, and PUUV.

Induction of protective immunity in a Syrian hamster model against a cytopathogenic strain of Andes virus.

Andes virus (ANDV) is responsible for the Hantavirus Pulmonary Syndrome cases in Argentina and neighboring countries, with moderate to high case-fatality rates. ANDV has some particular features, which make it unique among other members of the Hantavirus genus such as person-to-person transmission and causing a disease similar to Hantavirus Pulmonary Syndrome in the hamster as an animal model. The kinetics of replication in Vero E6 cells of an ANDV strain isolated in Argentina, called Andes/ARG, was studied. Cytopathic effect and the formation of clear plaques were observed and therefore Andes/ARG could be quantified by classic plaque assay. The Andes/ARG strain was found to be highly lethal in Syrian hamsters allowing experiments to demonstrate the protective potential of vaccines. A recombinant nucleocapsid protein of ANDV induced a long lasting antibody response and protective immunity against a homologous challenge, but to a lower extent against heterologous challenge by the Seoul virus.

Prevalence of hantavirus infection in wild rodents from five provinces in Korea, 2007.

Hemorrhagic fever with renal syndrome (HFRS) is an infectious disease caused by hantaviruses of the family Bunyaviridae. Among them, Hantaan virus (HTNV) is most widely distributed in Korea. The striped field mouse, Apodemus agrarius, is the natural host of HTNV in rural Korea. We trapped 766 small mammals of three species (1 Eothenomys regulus, 13 Crocidura suaveolens, and 752 Apodemus agrarius) in five provinces in Korea from January to December 2007. We tested 542 rodent sera for HTNV antibodies by an indirect immunofluorescent assay (IFA), finding antibody prevalences of 4 to 29% among the five provinces. Peaks in monthly antibody prevalence occurred in spring and fall. Antibody prevalence during the second peak coincided increased HFRS incidence in autumn. We used multiplex reverse transcription polymerase chain reaction (RT-PCR) to detect the partial S segment of Hantaan, Seoul, and Puumala viruses in 766 lung samples of all captured animals and found HTNV RNA in 25 A. agrarius. Two isolates of HTNV were obtained from PCR-positive A. agrarius by cultivation in Vero E6 cells. This first systemic survey of monthly antibody prevalence in hantavirus hosts in wide regions in Korea could provide useful information for other researchers studying environmental and ecological factors affecting HFRS.

A single immunization with a recombinant canine adenovirus type 2 expressing the seoul virus Gn glycoprotein confers protective immunity against seoul virus in mice.

Seoul virus (SEOV), a member of hantavirus genus, is one of the causative agents of hemorrhagic fever with renal syndrome (HFRS) and afflicts tens of thousands of people annually. In this paper, we evaluate the immune response induced by a replication-competent recombinant canine adenovirus type 2 expressing the Gn protein of SEOV (rCAV-2-Gn) in BALB/c mice. Sera from immunized mice contained neutralizing antibodies that could specifically recognize SEOV and neutralize its infectivity in vitro. Moreover, the recombinant virus induced complete protection against a lethal challenge with the highly virulent SEOV strain CC-2. Protective level neutralizing antibodies were maintained for at least 20 weeks. The efficacy of the recombinant was similar to that induced by a currently available inactivated HFRS vaccine. This recombinant virus is therefore a potential alternative to the inactivated vaccine.

Hantaviruses in rodents and humans, Inner Mongolia Autonomous Region, China.

Surveys were carried out in 2003-2006 to better understand the epidemiology of hantaviruses in the Inner Mongolia Autonomous Region of China (Inner Mongolia). Hemorrhagic fever with renal syndrome (HFRS) was first reported in this region in 1955 and has been an important public health problem here since then. During 1955-2006, 8,309 persons with HFRS were reported in Inner Mongolia (average incidence rate 0.89/100,000), and 261 (3.14%) died. Before the 1990s, all HFRS cases occurred in northeastern Inner Mongolia. Subsequently, HFRS cases were registered in central (1995) and western (1999) Inner Mongolia. In this study, hantaviral antigens were identified in striped field mice (Apodemus agrarius) from northeastern Inner Mongolia and in Norway rats (Rattus norvegicus) from middle and western Inner Mongolia. Phylogenetic analysis of hantaviral genome sequences suggests that HFRS has been caused mainly by Hantaan virus in northeastern Inner Mongolia and by Seoul virus in central and western Inner Mongolia.

Seoul virus and hantavirus disease, Shenyang, People's Republic of China.

An outbreak of hemorrhagic fever with renal syndrome (HFRS) occurred among students in Shenyang Pharmaceutical University in 2006. We conducted a study to characterize etiologic agents of the outbreaks and clarify the origin of hantaviruses causing infections in humans and laboratory animals. Immunoglobulin (Ig) M or IgG antibodies against Seoul virus (SEOV) were detected in the serum samples of all 8 patients. IgG antibodies against hantavirus were also identified in laboratory rats, which were used by these students for their scientific research. Phylogenetic analysis showed that partial small segment sequences recovered from humans, laboratory rats, and local wild rats belonged to SEOV. Hantavirus sequences recovered from humans and laboratory rats clustered within 1 of 3 lineages of SEOV circulating among local wild rats in Shenyang. These results suggest that the HFRS outbreak in Shenyang was caused by SEOV that was circulating among local wild rats and had also infected the laboratory rats.

Serological and genetic evidence for the presence of Seoul hantavirus in Rattus norvegicus in Flanders, Belgium.

Seoul hantavirus (SEOV), carried by Rattus rattus (black rat) and R. norvegicus (Norway, brown rat), was reported to circulate as well as cause HFRS cases in Asia. As Rattus sp. are present worldwide, SEOV has the potential to cause human disease worldwide. In Europe however, only SEOV prevalence in rats from France was reported and no confirmed cases of SEOV infection were published. We here report genetic and serological evidence for the presence of SEOV virus in brown rat populations in Belgium. We also serologically screened an at-risk group that was in contact with R. norvegicus on a daily basis and found no evidence for SEOV infection.