Species-specific responses during Seoul orthohantavirus infection in human and rat lung microvascular endothelial cells.

Seoul orthohantavirus (SEOV) is a rat-borne zoonotic virus that is transmitted via inhalation of aerosolized infectious excreta, and can cause hemorrhagic fever with renal syndrome (HFRS) in humans worldwide. In rats, SEOV predominantly exists as a persistent infection in the absence of overt clinical signs. Lack of disease in rats is attributed to downregulation of pro-inflammatory and upregulation of regulatory host responses. As lung microvascular endothelial cells (LMECs) represent a primary target of infection in both human and rats, infections in these cells provide a unique opportunity to study the central role of LMECs in the dichotomy between pathogenicity in both species. In this study, host responses to SEOV infection in primary human and rat LMECs were directly compared on a transcriptional level. As infection of rat LMECs was more efficient than human LMECs, the majority of anti-viral defense responses were observed earlier in rat LMECs. Most prominently, SEOV-induced processes in both species included responses to cytokine stimulus, negative regulation of innate immune responses, responses to type I and II interferons, regulation of pattern recognition receptor signaling and MHC-I signaling. However, over time, in the rat LMECs, responses shifted from an anti-viral state towards a more immunotolerant state displayed by a PD-L1, B2M-, JAK2-focused interaction network aiding in negative regulation of cytotoxic CD8-positive T cell activation. This suggests a novel mechanism by which species-specific orthohantavirus-induced endothelium and T cell crosstalk may play a crucial role in the development of acute disease in humans and persistence in rodents.

Comparison of Three Detection Methods for Seoul Virus Causing Hemorrhagic Fever with Renal Syndrome.

BACKGROUND: Seoul virus (SEOV) is a significant causative pathogen of hemorrhagic fever with renal syndrome (HFRS). Accurate discrimination of SEOV infection from other viral or bacterial infections holds vital clinical importance. METHODS: Our study utilized quantitative real-time PCR (qRT-PCR), metagenomic next-generation sequencing (mNGS), and immunological assays to identify the pathogen causing HFRS. RESULTS: For the case, mNGS identified SEOV and suspected host or environmental microorganisms at 5 days from symptom onset. qRT-PCR detected SEOV between 5 to 8 days from symptom onset. Anti-hantavirus IgM antibodies reached positive criteria at 7 days and IgG antibodies at 9 days from symptom onset. CONCLUSIONS: qRT-PCR, mNGS, and immunological assays each have merits and drawbacks. Optimal selection depends on laboratory conditions and clinical requirements.

Hantavirus Disease Cluster Caused by Seoul Virus, Germany.

A cluster of 3 persons in Germany experienced hantavirus disease with renal insufficiency. Reverse transcription PCR-based genotyping revealed infection by Seoul hantavirus transmitted from pet rats. Seoul virus could be responsible for disease clusters in Europe, and infected pet rats should be considered a health threat.

Genetic diversity and molecular evolution of Seoul virus in Hebei province, China.

Seoul virus (SEOV) is a major pathogen which causes hemorrhagic fever with renal syndrome (HFRS), and is present all over the world. However, there are currently few long-term systematic studies of SEOV's phylogenetic and evolutionary mechanisms in epidemic areas. Thus, in this study, we used RT-PCR combined with NGS to obtain the genomes of six SEOV viruses from 1993, as well as 56 Hebei province-specific tissue samples from 1999 to 2022. Phylogenetic analysis showed that the SEOV samples could be divided into seven groups and showed geographic clustering. The geographic region may be the main factor affecting the genetic diversity of SEOV. We also found that SEOV was subject to strong overall purifying selection and positive selection at certain sites during evolution. Recombination events and high nucleotide substitution rates were also shown to accelerate SEOV's evolution. Evolutionary feature of the L segment is more representative of complete genome. Our detailed analysis provides a deeper understanding of the genetic diversity and evolutionary drivers of SEOV within its primary epidemic areas. It will be important to further monitor epidemiological trends and drivers of variation to help increase our understanding of the pathogenicity of SEOV infections.

Orthohantavirus infections in humans and rodents in the Yichun region, China, from 2016 to 2021.

BACKGROUND: Rodents are the predominant natural hosts of orthohantavirus and the source of human infection, hemorrhagic fever with renal syndrome (HFRS) caused by orthohantavirus is a severe public health problem in the Yichun region, Jiangxi Province, China. However, little information is known about the infection of orthohantavirus in humans and rodents, and the genetic characteristics of the epidemic orthohantavirus in the region. METHODS: The clinical data of HFRS cases in 2016-2021 was analyzed. Virus infection in rodents was analyzed by orthohantavirus antigen detection using immunofluorescent assay, and the species of orthohantaviruses in rodents and patients were identified by real-time RT-PCR and gene sequencing. The S and M segments of orthohantaviruses from rodents and patients were recovered and analyzed. RESULTS: A total of 1,573 HFRS cases were reported in the Yichun region from 2016 to 2021, including 11 death cases. HFRS cases peaked twice each year: in winter from November to January and early summer from May to June. Farmers constituted the predominant population suffering from HFRS. The orthohantavirus antigen was identified in five species of rodents: Apodemus agrarius (A. agrarius), Rattus norvegicus (R. norvegicus), Sorex araneus, Rattus losea (R. losea), and Niviventer confucianus (N. confucianus). The real-time RT-PCR test and genetic analysis results showed that Hantaan orthohantavirus (HTNV), Seoul orthohantavirus (SEOV), and Dabieshan orthohantavirus (DBSV) were circulated in the rodents. HTNV, SEOV, and DBSV from the rodents were distantly related to other known orthohantaviruses and belonged to novel genetic lineages. SEOV and HTNV were found in HFRS patients, but 97.8% (90/92) of the infections were caused by HTNV. Winter and early summer peaks were both caused by HTNV. The HTNV sequences recovered from HFRS cases were closely related to those from A. agrarius. CONCLUSIONS: In the Yichun region, the orthohantaviruses transmitted in rodents include HTNV, SEOV, and DBSV, which have obvious genetic characteristics and high genetic diversity. At the same time, this region is an HFRS mixed epidemic area dominated by HTNV, with two peaks every year, which deserves our high attention.

A Development of Rapid Whole-Genome Sequencing of Seoul orthohantavirus Using a Portable One-Step Amplicon-Based High Accuracy Nanopore System.

Whole-genome sequencing provides a robust platform for investigating the epidemiology and transmission of emerging viruses. Oxford Nanopore Technologies allows for real-time viral sequencing on a local laptop system for point-of-care testing. Seoul orthohantavirus (Seoul virus, SEOV), harbored by Rattus norvegicus and R. rattus, causes mild hemorrhagic fever with renal syndrome and poses an important threat to public health worldwide. We evaluated the deployable MinION system to obtain high-fidelity entire-length sequences of SEOV for the genome identification of accurate infectious sources and their genetic diversity. One-step amplicon-based nanopore sequencing was performed from SEOV 80-39 specimens with different viral copy numbers and SEOV-positive wild rats. The KU-ONT-SEOV-consensus module was developed to analyze SEOV genomic sequences generated from the nanopore system. Using amplicon-based nanopore sequencing and the KU-ONT-consensus pipeline, we demonstrated novel molecular diagnostics for acquiring full-length SEOV genome sequences, with sufficient read depth in less than 6 h. The consensus sequence accuracy of the SEOV small, medium, and large genomes showed 99.75-100% (for SEOV 80-39 isolate) and 99.62-99.89% (for SEOV-positive rats) identities. This study provides useful insights into on-site diagnostics based on nanopore technology and the genome epidemiology of orthohantaviruses for a quicker response to hantaviral outbreaks.

Investigation of a subunit protein vaccine for HFRS based on a consensus sequence between envelope glycoproteins of HTNV and SEOV.

Due to the global resurgence of hemorrhagic fever with renal syndrome (HFRS), more attention is being focused on this dangerous illness. In China and Korea, the only vaccines available are the virus-inactivated vaccine against Hantaan virus (HTNV) or Seoul virus (SEOV), but their efficacy and safety are inadequate. Therefore, it is important to develop new vaccines that are safer and more efficient to neutralize and regulate areas with a high prevalence of HFRS. We employed bioinformatics methods to design a recombinant protein vaccine based on conserved regions of protein consensus sequences in HTNV and SEOV membranes. The S2 Drosophila expression system was utilized to enhance protein expression, solubility and immunogenicity. After the Gn and Gc proteins of HTNV and SEOV were successfully expressed, mice were immunized, and the humoral immunity, cellular immunity, and in vivo protection of the HFRS universal subunit vaccine were systematically evaluated in mouse models. These results indicated that the HFRS subunit vaccine generated elevated levels of binding and neutralizing antibodies, particularly IgG1, compared to that of the traditional inactivated HFRS vaccine. Additionally, the spleen cells of immunized mice secreted IFN-r and IL-4 cytokines effectively. Moreover, the HTNV-Gc protein vaccine successfully protected suckling mice from HTNV infection and stimulated GC responses. In this research, a new scientific approach is investigated to develop a universal HFRS subunit protein vaccine that is capable of producing effective humoral and cellular immunity in mice. The results suggest that this vaccine could be a promising candidate for preventing HFRS in humans.

Development of a novel minigenome and recombinant VSV expressing Seoul hantavirus glycoprotein-based assays to identify anti-hantavirus therapeutics.

Seoul virus (SEOV) is an emerging global health threat that can cause hemorrhagic fever with renal syndrome (HFRS), which results in case fatality rates of ∼2%. There are no approved treatments for SEOV infections. We developed a cell-based assay system to identify potential antiviral compounds for SEOV and generated additional assays to characterize the mode of action of any promising antivirals. To test if candidate antivirals targeted SEOV glycoprotein-mediated entry, we developed a recombinant reporter vesicular stomatitis virus expressing SEOV glycoproteins. To facilitate the identification of candidate antiviral compounds targeting viral transcription/replication, we successfully generated the first reported minigenome system for SEOV. This SEOV minigenome (SEOV-MG) screening assay will also serve as a prototype assay for discovery of small molecules inhibiting replication of other hantaviruses, including Andes and Sin Nombre viruses. Ours is a proof-of-concept study in which we tested several compounds previously reported to have activity against other negative-strand RNA viruses using our newly developed hantavirus antiviral screening systems. These systems can be used under lower biocontainment conditions than those needed for infectious viruses, and identified several compounds with robust anti-SEOV activity. Our findings have important implications for the development of anti-hantavirus therapeutics.

The Power of We.

"The Power of We" is a personal tribute to the individuals and organizations that collaborated in the discovery and advancement of knowledge of the hantaviruses following the original isolation of Hantaan virus by Ho Wang Lee. It focuses on the work done primarily at the United States Army Medical Research Institute of Infectious Diseases during the decade of the 1980s under the leadership of Joel Dalrymple, who worked in close partnership with Ho Wang Lee. These early studies helped define the global distribution of Seoul virus and provided seminal information on its maintenance and transmission among urban rats. Other collaborations involved partners in Europe, Asia, and Latin America and resulted in the isolation of novel hantaviruses, a better understanding of their global distribution, and validation of diagnostics and therapeutic interventions for treatment of human diseases. By working in partnership, scientists from around the world made critical discoveries that led to a better understanding of the hantaviruses. "The Power of We" demonstrates that we all benefit when we work together with a shared vision, a common commitment to excellence, and mutual respect.

Pet Rats as the Likely Reservoir for Human Seoul Orthohantavirus Infection.

Seoul orthohantavirus (SEOV) is a rat-associated zoonotic pathogen with an almost worldwide distribution. In 2019, the first autochthonous human case of SEOV-induced hemorrhagic fever with renal syndrome was reported in Germany, and a pet rat was identified as the source of the zoonotic infection. To further investigate the SEOV reservoir, additional rats from the patient and another owner, all of which were purchased from the same vendor, were tested. SEOV RNA and anti-SEOV antibodies were found in both of the patient's rats and in two of the three rats belonging to the other owner. The complete coding sequences of the small (S), medium (M), and large (L) segments obtained from one rat per owner exhibited a high sequence similarity to SEOV strains of breeder rat or human origin from the Netherlands, France, the USA, and Great Britain. Serological screening of 490 rats from breeding facilities and 563 wild rats from Germany (2007-2020) as well as 594 wild rats from the Netherlands (2013-2021) revealed 1 and 6 seropositive individuals, respectively. However, SEOV RNA was not detected in any of these animals. Increased surveillance of pet, breeder, and wild rats is needed to identify the origin of the SEOV strain in Europe and to develop measures to prevent transmission to the human population.

First study of Seoul virus (SEOV) in urban rodents from newly urbanized areas of Gran La Plata, Argentina.

Alterations of ecosystems have deep effects on the distribution of parasites. Big cities of Argentina present structural features that favor the presence of synanthropic species, acting as source of zoonotic diseases, for example in urban rodents: the Norway rat (Rattus norvegicus) and the black rat (R. rattus). One of the important zoonotic pathogens related are the RNA virus Hantavirus, with high prevalence rates in South America. The aim of this study was to explore and identify the presence of Hantavirus in urban rodents from Gran La Plata, Argentina. The presence of anti-hantavirus IgG antibodies was determined by the Enzyme-Linked Immunosorbent Assay. Six samples turned out positive for Seoul virus (SEOV, p = 14.3%). These are the first records of SEOV in urban rodents in Gran La Plata. It represents the first report in R. rattus in Argentina, and in America. This situation underscores the inequality and historical forgetfulness of a portion of society, calling for urgent action to be taken in this regard.

Role of Seaports and Imported Rats in Seoul Hantavirus Circulation, Africa.

Seoul orthohantavirus (SEOV) is not considered a major public health threat on the continent of Africa. However, Africa is exposed to rodentborne SEOV introduction events through maritime traffic after exponential growth of trade with the rest of the world. Serologic studies have already detected hantavirus antibodies in human populations, and recent investigations have confirmed circulation of hantavirus, including SEOV, in rat populations. Thus, SEOV is a possible emerging zoonotic risk in Africa. Moreover, the range of SEOV could rapidly expand, and transmission to humans could increase because of host switching from the usual brown rat (Rattus norvegicus) species, which is currently invading Africa, to the more widely installed black rat (R. rattus) species. Because of rapid economic development, environmental and climatic changes, and increased international trade, strengthened surveillance is urgently needed to prevent SEOV dissemination among humans in Africa.

Screening and identification of HTNVpv entry inhibitors with high-throughput pseudovirus-based chemiluminescence.

Hantaviruses, such as Hantaan virus (HTNV) and Seoul virus, are the causative agents of Hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS), and are important zoonotic pathogens. China has the highest incidence of HFRS, which is mainly caused by HTNV and Seoul virus. No approved antiviral drugs are available for these hantaviral diseases. Here, a chemiluminescence-based high-throughput-screening (HTS) assay was developed and used to screen HTNV pseudovirus (HTNVpv) inhibitors in a library of 1813 approved drugs and 556 small-molecule compounds from traditional Chinese medicine sources. We identified six compounds with in vitro anti-HTNVpv activities in the low-micromolar range (EC50 values of 0.1-2.2 â€‹µmol/L; selectivity index of 40-900). Among the six selected compounds, cepharanthine not only showed good anti-HTNVpv activity in vitro but also inhibited HTNVpv-fluc infection in Balb/c mice 5 â€‹h after infection by 94% (180 â€‹mg/kg/d, P â€‹< â€‹0.01), 93% (90 â€‹mg/kg/d, P â€‹< â€‹0.01), or 92% (45 â€‹mg/kg/d, P â€‹< â€‹0.01), respectively, in a bioluminescent imaging mouse model. A time-of-addition analysis suggested that the antiviral mechanism of cepharanthine involves the membrane fusion and entry phases. Overall, we have established a HTS method for antiviral drugs screening, and shown that cepharanthine is a candidate for HCPS and HFRS therapy. These findings may offer a starting point for the treatment of patients infected with hantaviruses.

Central nervous system infection with Seoul Orthohantavirus in a child after hematopoietic stem cell transplantation: a case report.

BACKGROUND: Patients with allogeneic hematopoietic stem cell transplantation (allo-HSCT) are prone to complicate viral infection. Central nervous system (CNS) involvement caused by the viruses is rare but with poor prognosis. Hantavirus, which usually cause hemorrhagic fever with renal syndrome (HFRS), and none case has been reported about these infection in allo-HSCT patients. CASE PRESENTATION: In August 2021, a 13-year-old male child developed intermittent fever and refractory hypotension after allo-HSCT. Magnetic resonance imaging of the head revealed abnormal signal foci in the left midbrain cerebral peduncle and bilateral thalamus. His family reported traces of mouse activity in the patient's home kitchen. HFRS was suspected, but with no significant kidney damage. The specific immunoglobulin (Ig) G and M of hantavirus were negative. The metagenomic next-generation sequencing (mNGS) detected Seoul Orthohantavirus (SEOV) sequences directly in cerebrospinal fluid and blood. CONCLUSIONS: Allo-HSCT patients are a high-risk group for infection. Usually the causative agent of infection is difficult to determine, and sometimes the site of infection is concealed. This report highlights the importance of suspecting hantavirus infection in allo-HSCT patients with CNS symptoms despite the absence of renal syndromes. The mNGS is a powerful tool for detecting pathogens. CNS infection with Seoul orthohantavirus in transplant patients is rare but possible as demonstrated in this case. To the best of our knowledge, this is the first reported case employing mNGS to diagnose SEOV caused CNS infection in an allo-HSCT patient.

Survey of rodent-borne pathogens in Singapore reveals the circulation of Leptospira spp., Seoul hantavirus, and Rickettsia typhi.

Rodents living alongside humans increases the probability of encounter and also the transmission of rodent-borne diseases. Singapore's cosmopolitan urban landscape provides a perfect setting to study the prevalence of four rodent-borne pathogens: Seoul hantavirus (SEOV), Leptospira species, Rickettsia typhi and Yersinia pestis, and identify the potential risk factors which may influence rodent density and transmission of rodent-borne diseases. A total of 1143 rodents were trapped from 10 unique landscape structures throughout Singapore. Real-time quantitative Polymerase Chain Reactions were used to detect pathogenic and intermediate Leptospira spp. and Yersinia pestis, whereas the seroprevalence of SEOV and R. typhi were analysed by Enzyme-Linked Immunosorbent Assay and Immunofluorescence Assay respectively. Multivariable logistic regression analysis was used to evaluate the association between prevalence of infection in rodent reservoirs and risk factors. Most of the rodents were caught in public residential developments (62.2%). Among the tested rodents, 42.4% were infected with Leptospira spp., while 35.5% and 32.2% were seropositive for SEOV and R. typhi respectively, whereas Yersinia pestis was not detected. Furthermore, risk factors including habitat, species, gender, and weight of rodents, influenced prevalence of infection to a varying extent. This study highlights the presence of Leptospira spp., SEOV and R. typhi in Singapore's rodent population, suggesting the need for effective rodent management and sanitation strategies to prevent further circulation and transmission to humans.

Seoul Virus Associated with Pet Rats, Scotland, UK, 2019.

We describe a case of hemorrhagic fever with renal syndrome caused by Seoul virus in a woman in Scotland, UK. Whole-genome sequencing showed the virus belonged to a lineage characterized by recent international expansion, probably driven by trade in pet rats.

Genetic Characterization of Seoul Virus in the Seaport of Cotonou, Benin.

Seoul virus is a zoonotic pathogen carried by the brown rat Rattus norvegicus. Information on its circulation in Africa is limited. In this study, the virus was detected in 37.5% of brown rats captured in the Autonomous Port of Cotonou, Benin. Phylogenetic analyses place this virus in Seoul virus lineage 7.

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.

Immunological Responses to Seoul Orthohantavirus in Experimentally and Naturally Infected Brown Rats (Rattus norvegicus).

To clarify the mechanism of Seoul orthohantavirus (SEOV) persistence, we compared the humoral and cell-mediated immune responses to SEOV in experimentally and naturally infected brown rats. Rats that were experimentally infected by the intraperitoneal route showed transient immunoglobulin M (IgM) production, followed by an increased anti-SEOV immunoglobulin G (IgG) antibody response and maturation of IgG avidity. The level of SEOV-specific cytotoxic T lymphocytes (CTLs) peaked at 6 days after inoculation and the viral genome disappeared from serum. In contrast, naturally infected brown rats simultaneously had a high rate of SEOV-specific IgM and IgG antibodies (28/43). Most of the IgM-positive rats (24/27) had the SEOV genome in their lungs, suggesting that chronic SEOV infection was established in those rats. In female rats with IgG avidity maturation, the viral load in the lungs was decreased. On the other hand, there was no relationship between IgG avidity and viral load in the lungs in male rats. A CTL response was not detected in naturally infected rats. The difference between immune responses in the experimentally and naturally infected rats is associated with the establishment of chronic infection in natural hosts.