Innate Immunity to Orthohantaviruses: Could Divergent Immune Interactions Explain Host-specific Disease Outcomes?

The genus Orthohantavirus (family Hantaviridae, order Bunyavirales) consists of numerous genetic and pathologically distinct viral species found within rodent and mammalian insectivore populations world-wide. Although reservoir hosts experience persistent asymptomatic infection, numerous rodent-borne orthohantaviruses cause severe disease when transmitted to humans, with case-fatality rates up to 40%. The first isolation of an orthohantavirus occurred in 1976 and, since then, the field has made significant progress in understanding the immune correlates of disease, viral interactions with the human innate immune response, and the immune kinetics of reservoir hosts. Much still remains elusive regarding the molecular mechanisms of orthohantavirus recognition by the innate immune response and viral antagonism within the reservoir host, however. This review provides a summary of the last 45 years of research into orthohantavirus interaction with the host innate immune response. This summary includes discussion of current knowledge involving human, non-reservoir rodent, and reservoir innate immune responses to viruses which cause hemorrhagic fever with renal syndrome and hantavirus cardio-pulmonary syndrome. Review of the literature concludes with a brief proposition for the development of novel tools needed to drive forward investigations into the molecular mechanisms of innate immune activation and consequences for disease outcomes in the various hosts for orthohantaviruses.

Impact of Predator Exclusion and Habitat on Seroprevalence of New World Orthohantavirus Harbored by Two Sympatric Rodents within the Interior Atlantic Forest.

Understanding how perturbations to trophic interactions influence virus-host dynamics is essential in the face of ongoing biodiversity loss and the continued emergence of RNA viruses and their associated zoonoses. Herein, we investigated the role of predator exclusion on rodent communities and the seroprevalence of hantaviruses within the Reserva Natural del Bosque Mbaracayú (RNBM), which is a protected area of the Interior Atlantic Forest (IAF). In the IAF, two sympatric rodent reservoirs, Akodon montensis and Oligoryzomys nigripes, harbor Jaborá and Juquitiba hantavirus (JABV, JUQV), respectively. In this study, we employed two complementary methods for predator exclusion: comprehensive fencing and trapping/removal. The goal of exclusion was to preclude the influence of predation on small mammals on the sampling grids and thereby potentially reduce rodent mortality. Following baseline sampling on three grid pairs with different habitats, we closed the grids and began predator removal. By sampling three habitat types, we controlled for habitat-specific effects, which is important for hantavirus-reservoir dynamics in neotropical ecosystems. Our six-month predator exclusion experiment revealed that the exclusion of terrestrial mammalian predators had little influence on the rodent community or the population dynamics of A. montensis and O. nigripes. Instead, fluctuations in species diversity and species abundances were influenced by sampling session and forest degradation. These results suggest that seasonality and landscape composition play dominant roles in the prevalence of hantaviruses in rodent reservoirs in the IAF ecosystem.

Polyclonal alpaca antibodies protect against hantavirus pulmonary syndrome in a lethal Syrian hamster model.

The use of antibody-based therapies for the treatment of high consequence viral pathogens has gained interest over the last fifteen years. Here, we sought to evaluate the use of unique camelid-based IgG antibodies to prevent lethal hantavirus pulmonary syndrome (HPS) in Syrian hamsters. Using purified, polyclonal IgG antibodies generated in DNA-immunized alpacas, we demonstrate that post-exposure treatments reduced viral burdens and organ-specific pathology associated with lethal HPS. Antibody treated animals did not exhibit signs of disease and were completely protected. The unique structures and properties, particularly the reduced size, distinct paratope formation and increased solubility of camelid antibodies, in combination with this study support further pre-clinical evaluation of heavy-chain only antibodies for treatment of severe respiratory diseases, including HPS.

Hantavirus Research in Finland: Highlights and Perspectives.

Finland has the highest incidence of hantavirus infections globally, with a significant impact on public health. The large coverage of boreal forests and the cyclic dynamics of the dominant forest rodent species, the bank vole Myodes glareolus, explain most of this. We review the relationships between Puumala hantavirus (PUUV), its host rodent, and the hantavirus disease, nephropathia epidemica (NE), in Finland. We describe the history of NE and its diagnostic research in Finland, the seasonal and multiannual cyclic dynamics of PUUV in bank voles impacting human epidemiology, and we compare our northern epidemiological patterns with those in temperate Europe. The long survival of PUUV outside the host and the life-long shedding of PUUV by the bank voles are highlighted. In humans, the infection has unique features in pathobiology but rarely long-term consequences. NE is affected by specific host genetics and risk behavior (smoking), and certain biomarkers can predict the outcome. Unlike many other hantaviruses, PUUV causes a relatively mild disease and is rarely fatal. Reinfections do not exist. Antiviral therapy is complicated by the fact that when symptoms appear, the patient already has a generalized infection. Blocking vascular leakage measures counteracting pathobiology, offer a real therapeutic approach.

Longitudinal Assessment of Cytokine Expression and Plasminogen Activation in Hantavirus Cardiopulmonary Syndrome Reveals Immune Regulatory Dysfunction in End-Stage Disease.

Pathogenic New World orthohantaviruses cause hantavirus cardiopulmonary syndrome (HCPS), a severe immunopathogenic disease in humans manifested by pulmonary edema and respiratory distress, with case fatality rates approaching 40%. High levels of inflammatory mediators are present in the lungs and systemic circulation of HCPS patients. Previous studies have provided insights into the pathophysiology of HCPS. However, the longitudinal correlations of innate and adaptive immune responses and disease outcomes remain unresolved. This study analyzed serial immune responses in 13 HCPS cases due to Sin Nombre orthohantavirus (SNV), with 11 severe cases requiring extracorporeal membrane oxygenation (ECMO) treatment and two mild cases. We measured viral load, levels of various cytokines, urokinase plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1). We found significantly elevated levels of proinflammatory cytokines and PAI-1 in five end-stage cases. There was no difference between the expression of active uPA in survivors' and decedents' cases. However, total uPA in decedents' cases was significantly higher compared to survivors'. In some end-stage cases, uPA was refractory to PAI-1 inhibition as measured by zymography, where uPA and PAI-1 were strongly correlated to lymphocyte counts and IFN-γ. We also found bacterial co-infection influencing the etiology and outcome of immune response in two cases. Unsupervised Principal Component Analysis and hierarchical cluster analyses resolved separate waves of correlated immune mediators expressed in one case patient due to a sequential co-infection of bacteria and SNV. Overall, a robust proinflammatory immune response, characterized by an imbalance in T helper 17 (Th17) and regulatory T-cells (Treg) subsets, was correlated with dysregulated inflammation and mortality. Our sample size is small; however, the core differences correlated to survivors and end-stage HCPS are instructive.

Hantavirus infection-induced B cell activation elevates free light chains levels in circulation.

In humans, orthohantaviruses can cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). An earlier study reported that acute Andes virus HPS caused a massive and transient elevation in the number of circulating plasmablasts with specificity towards both viral and host antigens suggestive of polyclonal B cell activation. Immunoglobulins (Igs), produced by different B cell populations, comprise heavy and light chains; however, a certain amount of free light chains (FLCs) is constantly present in serum. Upregulation of FLCs, especially clonal species, associates with renal pathogenesis by fibril or deposit formations affecting the glomeruli, induction of epithelial cell disorders, or cast formation in the tubular network. We report that acute orthohantavirus infection increases the level of Ig FLCs in serum of both HFRS and HPS patients, and that the increase correlates with the severity of acute kidney injury in HFRS. The fact that the kappa to lambda FLC ratio in the sera of HFRS and HPS patients remained within the normal range suggests polyclonal B cell activation rather than proliferation of a single B cell clone. HFRS patients demonstrated increased urinary excretion of FLCs, and we found plasma cell infiltration in archival patient kidney biopsies that we speculate to contribute to the observed FLC excreta. Analysis of hospitalized HFRS patients' peripheral blood mononuclear cells showed elevated plasmablast levels, a fraction of which stained positive for Puumala virus antigen. Furthermore, B cells isolated from healthy donors were susceptible to Puumala virus in vitro, and the virus infection induced increased production of Igs and FLCs. The findings propose that hantaviruses directly activate B cells, and that the ensuing intense production of polyclonal Igs and FLCs may contribute to acute hantavirus infection-associated pathological findings.

Orthohantavirus Survey in Indigenous Lands in a Savannah-Like Biome, Brazil.

In Brazil, the first confirmed cases of hantavirus cardiopulmonary syndrome in Indigenous populations occurred in 2001. The purpose of this study was to determine the seroprevalence of orthohantavirus infections in the Utiariti Indigenous land located in the southeastern region of the Brazilian Amazon. In December 2014 and 2015, a survey was conducted using an enzyme-linked immunosorbent assay in nine villages belonging to the Haliti-Paresí Indigenous communities. A total of 301 participants were enrolled in the study. Of the two study cohorts, the one from 2014 showed a prevalence of 12.4%, whereas the one from 2015 had a serum prevalence of 13.4%. Analysis of the paired samples of 110 Indigenous people who participated in both stages of the study enabled identification of four individuals who had seroconverted during the study period. Identifying the circulation of orthohantaviruses in the Utiariti Indigenous land highlights a serious public health problem in viral expansion and highlights the need to implement preventive measures appropriate to the sociocultural reality of these communities.

Prevalence of Orthohantavirus-Reactive Antibodies in Humans and Peri-Domestic Rodents in Northern Ethiopia.

In 2012, Tigray orthohantavirus was discovered in Ethiopia, but its seasonal infection in small mammals, and whether it poses a risk to humans was unknown. The occurrence of small mammals, rodents and shrews, in human inhabitations in northern Ethiopia is affected by season and presence of stone bunds. We sampled small mammals in two seasons from low- and high-density stone bund fields adjacent to houses and community-protected semi-natural habitats in Atsbi and Hagere Selam, where Tigray orthohantavirus was first discovered. We collected blood samples from both small mammals and residents using filter paper. The presence of orthohantavirus-reactive antibodies in blood was then analyzed using immunofluorescence assay (human samples) and enzyme linked immunosorbent assays (small mammal samples) with Puumala orthohantavirus as antigen. Viral RNA was detected by RT-PCR using small mammal blood samples. Total orthohantavirus prevalence (antibodies or virus RNA) in the small mammals was 3.37%. The positive animals were three Stenocephalemys albipes rats (prevalence in this species = 13.04%). The low prevalence made it impossible to determine whether season and stone bunds were associated with orthohantavirus prevalence in the small mammals. In humans, we report the first detection of orthohantavirus-reactive IgG antibodies in Ethiopia (seroprevalence = 5.26%). S. albipes lives in close proximity to humans, likely increasing the risk of zoonotic transmission.

Structural Basis for a Neutralizing Antibody Response Elicited by a Recombinant Hantaan Virus Gn Immunogen.

Hantaviruses are a group of emerging pathogens capable of causing severe disease upon zoonotic transmission to humans. The mature hantavirus surface presents higher-order tetrameric assemblies of two glycoproteins, Gn and Gc, which are responsible for negotiating host cell entry and constitute key therapeutic targets. Here, we demonstrate that recombinantly derived Gn from Hantaan virus (HTNV) elicits a neutralizing antibody response (serum dilution that inhibits 50% infection [ID50], 1:200 to 1:850) in an animal model. Using antigen-specific B cell sorting, we isolated monoclonal antibodies (mAbs) exhibiting neutralizing and non-neutralizing activity, termed mAb HTN-Gn1 and mAb nnHTN-Gn2, respectively. Crystallographic analysis reveals that these mAbs target spatially distinct epitopes at disparate sites of the N-terminal region of the HTNV Gn ectodomain. Epitope mapping onto a model of the higher order (Gn-Gc)4 spike supports the immune accessibility of the mAb HTN-Gn1 epitope, a hypothesis confirmed by electron cryo-tomography of the antibody with virus-like particles. These data define natively exposed regions of the hantaviral Gn that can be targeted in immunogen design. IMPORTANCE The spillover of pathogenic hantaviruses from rodent reservoirs into the human population poses a continued threat to human health. Here, we show that a recombinant form of the Hantaan virus (HTNV) surface-displayed glycoprotein, Gn, elicits a neutralizing antibody response in rabbits. We isolated a neutralizing (HTN-Gn1) and a non-neutralizing (nnHTN-Gn2) monoclonal antibody and provide the first molecular-level insights into how the Gn glycoprotein may be targeted by the antibody-mediated immune response. These findings may guide rational vaccine design approaches focused on targeting the hantavirus glycoprotein envelope.

Cytokine Profiles and Antibody Response Associated to Choclo Orthohantavirus Infection.

Background: New World Hantaviruses (NWHs) are the etiological agent underlying hantavirus cardiopulmonary syndrome (HCPS), a severe respiratory disease with high mortality rates in humans. In Panama, infections with Choclo Orthohantavirus (CHOV) cause a much milder illness characterized by higher seroprevalence and lower mortality rates. To date, the cytokine profiles and antibody responses associated with this milder form of HCPS have not been defined. Therefore, in this study, we examined immune serological profiles associated with CHOV infections. Methods: For this retrospective study, sera from fifteen individuals with acute CHOV-induced HCPS, were analyzed alongside sera from fifteen convalescent phase individuals and thirty-three asymptomatic, CHOV-seropositive individuals. Cytokine profiles were analyzed by multiplex immunoassay. Antibody subclasses, binding, and neutralization against CHOV-glycoprotein (CHOV-GP) were evaluated by ELISA, and flow cytometry. Results: High titers of IFNγ, IL-4, IL-8, and IL-10 serum cytokines were found in the acute individuals. Elevated IL-4 serum levels were found in convalescent and asymptomatic seropositive individuals. High titers of IgG1 subclass were observed across the three cohorts analyzed. Neutralizing antibody response against CHOV-GP was detectable in few acute individuals but was strong in both convalescent and asymptomatic seropositive individuals. Conclusion: A Th1/Th2 cytokine signature is characteristic during acute mild HCPS caused by CHOV infection. High expression of Th2 and IL-8 cytokines are correlated with clinical parameters in acute mild HCPS. In addition, a strong IL-4 signature is associated with different cohorts, including asymptomatic individuals. Furthermore, asymptomatic individuals presented high titers of neutralizing antibodies.

MAIT cell activation is associated with disease severity markers in acute hantavirus infection.

Hantaviruses are zoonotic RNA viruses that cause severe acute disease in humans. Infected individuals have strong inflammatory responses that likely cause immunopathology. Here, we studied the response of mucosal-associated invariant T (MAIT) cells in peripheral blood of individuals with hemorrhagic fever with renal syndrome (HFRS) caused by Puumala orthohantavirus, a hantavirus endemic in Europe. We show that MAIT cell levels decrease in the blood during HFRS and that residual MAIT cells are highly activated. This activation correlates with HFRS severity markers. In vitro activation of MAIT cells by hantavirus-exposed antigen-presenting cells is dependent on type I interferons (IFNs) and independent of interleukin-18 (IL-18). These findings highlight the role of type I IFNs in virus-driven MAIT cell activation and suggest a potential role of MAIT cells in the disease pathogenesis of viral infections.

Immune response during hantavirus diseases: implications for immunotherapies and vaccine design.

Orthohantaviruses, previously named hantaviruses, cause two emerging zoonotic diseases: haemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. Overall, over 200 000 cases are registered every year worldwide, with a fatality rate ranging between 0·1% and 15% for HFRS and between 20% and 40% for HCPS. No specific treatment or vaccines have been approved by the U.S. Food and Drug Administration (FDA) to treat or prevent hantavirus-caused syndromes. Currently, little is known about the mechanisms at the basis of hantavirus-induced disease. However, it has been hypothesized that an excessive inflammatory response plays an essential role in the course of the disease. Furthermore, the contributions of the cellular immune response to either viral clearance or pathology have not been fully elucidated. This article discusses recent findings relative to the immune responses elicited to hantaviruses in subjects suffering HFRS or HCPS, highlighting the similarities and differences between these two clinical diseases. Also, we summarize the most recent data about the cellular immune response that could be important for designing new vaccines to prevent this global public health problem.

Serological methods for detection of infection with shrew-borne hantaviruses: Thottapalayam, Seewis, Altai, and Asama viruses.

The infectivity of shrew-borne hantaviruses to humans is still unclear because of the lack of a serodiagnosis method for these viruses. In this study, we prepared recombinant nucleocapsid (rN) proteins of Seewis orthohantavirus, Altai orthohantavirus (ALTV), Thottapalayam thottimvirus (TPMV), and Asama orthohantavirus. Using monospecific rabbit sera, no antigenic cross-reactivity was observed. In a serosurvey of 104 samples from renal patients and 271 samples from heathy controls from Sri Lanka, one patient serum and two healthy control sera reacted with rN proteins of ALTV and TPMV, respectively. The novel assays should be applied to investigate potential infectivity of shrew-borne hantaviruses to humans.

Humoral Immunity to Hantavirus Infection.

Hantaviruses are zoonotic pathogens found in parts of Europe, Asia, South America, and North America, which can cause renal and respiratory failure with fatality rates up to 40%. There are currently no FDA-approved vaccines or therapeutics for hantavirus-related diseases; however, it is evident that a robust neutralizing antibody response is critical for protection from severe disease. Although virologists first described this family of viruses in the 1950s, there is limited information on the neutralizing epitopes that exist on the hantavirus antigenic glycoproteins, Gn and Gc, and sites important for the design of effective therapeutics and vaccines. We provide a thorough summary of the hantavirus field from an immunological perspective. In particular, we discuss our current structural knowledge of antigenic proteins Gn and Gc, identification of B cell neutralizing epitopes, previously isolated monoclonal antibodies and their cross-reactivity between different hantavirus strains, and current developments toward vaccines and therapeutics. We conclude with some outstanding questions in the field and emphasize the need for additional studies of the human antibody response to hantavirus infection.IMPORTANCE Hantaviruses are pathogens that sometimes pass from animals to humans, and they are found in parts of Europe, Asia, and North and South America. When human infection occurs, these viruses can cause kidney or lung failure, and as many as 40% of infected people die. Currently, there are no vaccines or therapeutics for hantavirus-related diseases available. A first step in developing prevention measures is determining what type of immune response is protective. Increasingly it has become clear that the induction of a type of response called a neutralizing antibody response is critical for protection from severe disease. Although virologists first described this family of viruses in the 1950s, there is limited information on what features on the surface of hantaviruses are recognized by the immune system. Here, we review the current state of knowledge of this information, which is critical for the design of effective therapeutics and vaccines.

RIG-I-like receptor activation drives type I IFN and antiviral signaling to limit Hantaan orthohantavirus replication.

Pathogenic hantaviruses, genus Orthohantaviridae, are maintained in rodent reservoirs with zoonotic transmission to humans occurring through inhalation of rodent excreta. Hantavirus disease in humans is characterized by localized vascular leakage and elevated levels of circulating proinflammatory cytokines. Despite the constant potential for deadly zoonotic transmission to humans, specific virus-host interactions of hantaviruses that lead to innate immune activation, and how these processes impart disease, remain unclear. In this study, we examined the mechanisms of viral recognition and innate immune activation of Hantaan orthohantavirus (HTNV) infection. We identified the RIG-I-like receptor (RLR) pathway as essential for innate immune activation, interferon (IFN) production, and interferon stimulated gene (ISG) expression in response to HTNV infection in human endothelial cells, and in murine cells representative of a non-reservoir host. Our results demonstrate that innate immune activation and signaling through the RLR pathway depends on viral replication wherein the host response can significantly restrict replication in target cells in a manner dependent on the type 1 interferon receptor (IFNAR). Importantly, following HTNV infection of a non-reservoir host murine model, IFNAR-deficient mice had higher viral loads, increased persistence, and greater viral dissemination to lung, spleen, and kidney compared to wild-type animals. Surprisingly, this response was MAVS independent in vivo. Innate immune profiling in these tissues demonstrates that HTNV infection triggers expression of IFN-regulated cytokines early during infection. We conclude that the RLR pathway is essential for recognition of HTNV infection to direct innate immune activation and control of viral replication in vitro, and that additional virus sensing and innate immune response pathways of IFN and cytokine regulation contribute to control of HTNV in vivo. These results reveal a critical role for innate immune regulation in driving divergent outcomes of HTNV infection, and serve to inform studies to identify therapeutic targets to alleviate human hantavirus disease.

Neutralizing Monoclonal Antibodies against the Gn and the Gc of the Andes Virus Glycoprotein Spike Complex Protect from Virus Challenge in a Preclinical Hamster Model.

Hantaviruses are the etiological agent of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The latter is associated with case fatality rates ranging from 30% to 50%. HCPS cases are rare, with approximately 300 recorded annually in the Americas. Recently, an HCPS outbreak of unprecedented size has been occurring in and around Epuyén, in the southwestern Argentinian state of Chubut. Since November of 2018, at least 29 cases have been laboratory confirmed, and human-to-human transmission is suspected. Despite posing a significant threat to public health, no treatment or vaccine is available for hantaviral disease. Here, we describe an effort to identify, characterize, and develop neutralizing and protective antibodies against the glycoprotein complex (Gn and Gc) of Andes virus (ANDV), the causative agent of the Epuyén outbreak. Using murine hybridoma technology, we generated 19 distinct monoclonal antibodies (MAbs) against ANDV GnGc. When tested for neutralization against a recombinant vesicular stomatitis virus expressing the Andes glycoprotein (GP) (VSV-ANDV), 12 MAbs showed potent neutralization and 8 showed activity in an antibody-dependent cellular cytotoxicity reporter assay. Escape mutant analysis revealed that neutralizing MAbs targeted both the Gn and the Gc. Four MAbs that bound different epitopes were selected for preclinical studies and were found to be 100% protective against lethality in a Syrian hamster model of ANDV infection. These data suggest the existence of a wide array of neutralizing antibody epitopes on hantavirus GnGc with unique properties and mechanisms of action.IMPORTANCE Infections with New World hantaviruses are associated with high case fatality rates, and no specific vaccine or treatment options exist. Furthermore, the biology of the hantaviral GnGc complex, its antigenicity, and its fusion machinery are poorly understood. Protective monoclonal antibodies against GnGc have the potential to be developed into therapeutics against hantaviral disease and are also great tools to elucidate the biology of the glycoprotein complex.

Meeting report: Eleventh International Conference on Hantaviruses.

The 2019 11th International Conference on Hantaviruses (ICH 2019) was organized by the International Society for Hantaviruses (ISH), and held on September 1-4, 2019, at the Irish College, in Leuven, Belgium. These ICHs have been held every three years since 1989. ICH 2019 was attended by 158 participants from 33 countries. The current report summarizes research presented on all aspects of hantavirology: ecology; pathogenesis and immune responses; virus phylogeny, replication and morphogenesis; epidemiology; vaccines, therapeutics and prevention; and clinical aspects and diagnosis.

Analytical Performance of the RIDASCREEN® Hantavirus Puumala IgG/IgM ELISA Assay.

The National Reference Center for Hantavirus in Belgium is currently using the Hantavirus IgM/IgG ELISA Progen kit (Heidelberg, Germany) for the detection of the most prevalent Hantavirus in Western Europe, Puumala virus (PUUV). Two commercially available PUUV kits were compared: Progen and RIDASCREEN® Hantavirus Puumala IgM/IgG ELISA assay (Darmstadt, Germany). METHODS: The sensitivity was evaluated with a panel of 68 samples from patients with an acute infection (n = 44) or a past infection (n = 24). Specificity was evaluated with a panel of 62 samples from patients with potentially false borderline results (n = 7) (no seroconversion), seronegative samples (n = 25) and potentially cross reacting samples (n = 30). Discordances were resolved by immunoblot. Substantial agreement was calculated using Cohen kappa coefficient. RESULTS: The RIDASCREEN® kit showed a higher specificity (IgM: 94.3%; IgG: 94.4%) than the Progen kit (IgM: 77.0% IgG: 93.0%). The sensitivity for IgM ELISA was 100% for both assays. IgG sensitivity was, respectively, 98.3% and 100% for Progen and RIDASCREEN®. A Cohen kappa coefficient of 0.76 and 0.90 was found between Puumala IgM and IgG, respectively. CONCLUSIONS: This study showed a higher specificity for the RIDASCREEN® kit than the Progen kit, while the sensitivity was as good as for the Progen kit.

Investigation of Orthohantavirus Seroprevalence in Northern Rural Areas of Denizli Province, Turkey.

Orthohantaviruses infect humans via inhalation of the viral particles in the excreta of infected rodents or direct contact with infected rodents. The infections caused by Puumala orthohantavirus (PUUV) and Dobrava-Belgrade orthohantavirus (DOBV) have been reported in Turkey. Serum samples of 346 healthy volunteers who are in the high-risk group of Orthohantavirus infections among the residents of Çal, Baklan, Çivril, and Bekilli counties, located in the northeast part of Denizli province, were used in this study. The samples were screened and confirmed using commercial ELISA and immunoblot tests, which detect IgG antibodies against DOBV, PUUV, and Hantaan orthohantavirus. IgG antibodies against PUUV were detected in the samples of 2 volunteers (2/346, 0.6%). One was a veterinarian and the other a farmer and they live in the Baklan and Çal counties, respectively. Both of them have a high probability of exposure to the virus, based on their occupation and living conditions. However, no symptoms were found in the clinical findings of both cases. This study is the first publication of reported PUUV seropositivities from the southwestern part of Turkey.

Suggestive Serological Evidence of Infection with Shrew-Borne Imjin Virus (Hantaviridae) in Humans.

The pathogenicity of the shrew-borne Imjin virus (MJNV) is unknown. The objective of our study was to find serological evidence of MJNV infection in humans. Partial MJNV nucleocapsid protein (NP) was cloned and expressed as an antigen for double-antigen sandwich ELISA, IgM capture ELISA, and dot blot to detect MJNV specific antibodies in hemorrhagic fever with renal syndrome (HFRS) patients' and healthy persons' sera from endemic areas in China. The purified recombinant NP reacted with neither the 90 healthy individuals' sera from non-endemic areas of MJNV nor the 100 antisera to HFRS-causing virus, indicating that the MJNV NP had no cross-reaction with normal human sera and HFRS-causing viral antibodies. As determined by screening ELISA and dot blot analysis, IgG antibodies against MJNV NP were detected in sera from two of 385 healthy individuals from MJNV-endemic areas, suggesting infection with MJNV or MJNV-like thottimvirus. Based on the suggestive evidence, healthcare workers should be alert to febrile diseases occurring among individuals with exposure to shrew-infested habitats.