Hantaviruses: molecular biology, evolution and pathogenesis

Hantaviruses are tri-segmented negative sense single stranded RNA viruses that belong to the family Bunyaviridae. In nature, hantaviruses are exclusively maintained in the populations of their specific rodent hosts. In their natural host species, hantaviruses usually develop a persistent infection with prolonged virus shedding in excreta. Humans become infected by inhaling virus contaminated aerosol. Unlike asymptomatic infection in rodents, hantaviruses cause two acute febrile diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The mortality rate varies from 0.1% to 40% depending on the virus involved. Hantaviruses are distributed world wide, with over 150,000 HFRS and HPS cases being registered annually. In this review we summarize current knowledge on hantavirus molecular biology, epidemiology, genetic diversity and co-evolution with rodent hosts. In addition, special attention was given in this review to describing clinical manifestation of HFRS and HPS, and advances in our current understanding of the host immune response, treatment, and prevention.     

Development of small-molecule inhibitors against hantaviruses

Hantavirus (HV), a pathogen of animal infectious diseases that poses a threat to humans, has attracted extensive attention. Clinically, HV can cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), between which HFRS is mostly in Eurasia, and HPS is mostly in the Americas. This paper reviews the research progress of small-molecule inhibitors of HV.     

Andes virus infection of lymphatic endothelial cells causes giant cell and enhanced permeability responses that are rapamycin and vascular endothelial growth factor C sensitive

Hantaviruses primarily infect endothelial cells (ECs) and nonlytically cause vascular changes that result in hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Acute pulmonary edema during HPS may be caused by capillary leakage and failure of lymphatic vessels to clear fluids. Uniquely regulated lymphatic ECs (LECs) control fluid clearance, although roles for lymphatics in hantavirus disease remain undetermined. Here we report that hantaviruses productively infect LECs and that LEC infection by HPS causing Andes virus (ANDV) and HFRS causing Hantaan virus (HTNV) are inhibited by α(v)β(3) integrin antibodies. Although α(v)β(3) integrins regulate permeabilizing responses directed by vascular endothelial growth factor receptor 2 (VEGFR2), we found that only ANDV-infected LECs were hyperpermeabilized by the addition of VEGF-A. However, VEGF-C activation of LEC-specific VEGFR3 receptors blocked ANDV- and VEGF-A-induced LEC permeability. In addition, ～75% of ANDV-infected LECs became viable mononuclear giant cells, >4 times larger than normal, in response to VEGF-A. Giant cells are associated with constitutive mammalian target of rapamycin (mTOR) activation, and we found that both giant LECs and LEC permeability were sensitive to rapamycin, an mTOR inhibitor, and VEGF-C addition. These findings indicate that ANDV uniquely alters VEGFR2-mTOR signaling responses of LECs, resulting in giant cell and LEC permeability responses. This suggests that ANDV infection alters normal LEC and lymphatic vessel functions which may contribute to edematous fluid accumulation during HPS. Moreover, the ability of VEGF-C and rapamycin to normalize LEC responses suggests a potential therapeutic approach for reducing pulmonary edema and the severity of HPS following ANDV infection.     

Second external quality assurance study for the serological diagnosis of hantaviruses in Europe

Hantaviruses are endemic throughout the world and hosted by rodents and insectivores. Two human zoonoses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), are caused by hantaviruses and case fatality rates have reached 12% for HFRS and 50% for HPS in some outbreaks. Symptomatic hantavirus infections in Europe are summarised as HFRS mainly due to Puumala, Dobrava-Belgrade and Saaremaa virus. While HFRS has an overall low incidence in Europe, the number of cases varies from 100 per year in all Eastern and Southern Europe up to 1,000 per year only in Finland. To assess the quality of hantavirus diagnostics, the European Network for the Diagnostics of "Imported" Viral Diseases (ENIVD) organised a first external quality assurance (EQA) in 2002. The purpose of this second EQA study is to collect updated information on the efficiency and accurateness of hantavirus serological methods applied by expert laboratories. A serum panel of 14 samples was sent to 28 participants in Europe of which 27 sent results. Performance in hantavirus diagnosis varied not only on the method used but also on the laboratories and the subclass of antibodies tested. Commercial and in-house assays performed almost equally. Enzyme immunoassays were mainly used but did not show the best performances while immunoblot assays were the less employed and showed overall better performances. IgM antibodies were not detected in 61% of the positive IgM samples and IgM detection was not performed by 7% of the laboratories indicating a risk of overlooking acute infections in patients. Uneven performances using the same method is indicating that there is still a need for improving testing conditions and standardizing protocols.     

汉坦病毒糖蛋白的结构及功能研究进展

汉坦病毒感染人类可导致肾综合征出血热和汉坦病毒肺综合征,其包膜糖蛋白(GP)是重要的结构蛋白,本文就汉坦病毒糖蛋白的结构和功能的研究进展作一综述。     

NK Cell Activation in Human Hantavirus Infection Explained by Virus-Induced IL-15/IL15Rα Expression

Clinical infection with hantaviruses cause two severe acute diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). These diseases are characterized by strong immune activation, increased vascular permeability, and up to 50% case-fatality rates. One prominent feature observed in clinical hantavirus infection is rapid expansion of natural killer (NK) cells in peripheral blood of affected individuals. We here describe an unusually high state of activation of such expanding NK cells in the acute phase of clinical Puumala hantavirus infection. Expanding NK cells expressed markedly increased levels of activating NK cell receptors and cytotoxic effector molecules. In search for possible mechanisms behind this NK cell activation, we observed virus-induced IL-15 and IL-15Rα on infected endothelial and epithelial cells. Hantavirus-infected cells were shown to strongly activate NK cells in a cell-cell contact-dependent way, and this response was blocked with anti-IL-15 antibodies. Surprisingly, the strength of the IL-15-dependent NK cell response was such that it led to killing of uninfected endothelial cells despite expression of normal levels of HLA class I. In contrast, hantavirus-infected cells were resistant to NK cell lysis, due to a combination of virus-induced increase in HLA class I expression levels and hantavirus-mediated inhibition of apoptosis induction. In summary, we here describe a possible mechanism explaining the massive NK cell activation and proliferation observed in HFRS patients caused by Puumala hantavirus infection. The results add further insights into mechanisms behind the immunopathogenesis of hantavirus infections in humans and identify new possible targets for intervention.     

Lambda interferon (IFN-lambda) in serum is decreased in hantavirus-infected patients, and in vitro-established infection is insensitive to treatment with all IFNs and inhibits IFN-gamma-induced nitric oxide production

Hantaviruses, causing hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), are known to be sensitive to nitric oxide (NO) and to pretreatment with type I and II interferons (alpha interferon [IFN-alpha]/IFN-beta and IFN-gamma, respectively). Elevated serum levels of NO and IFN-gamma have been observed in HFRS patients, but little is known regarding the systemic levels of other IFNs and the possible effects of hantaviruses on innate antiviral immune responses. In Puumala virus-infected HFRS patients (n = 18), we report that the levels of IFN-alpha and IFN-beta are similar, whereas the level of IFN-lambda (type III IFN) is significantly decreased, during acute (day of hospitalization) compared to the convalescent phase. The possible antiviral effects of IFN-lambda on the prototypic hantavirus Hantaan virus (HTNV) replication was then investigated. Pretreatment of A549 cells with IFN-lambda alone inhibited HTNV replication, and IFN-lambda combined with IFN-gamma induced additive antiviral effects. We then studied the effect of postinfection treatment with IFNs. Interestingly, an already-established HTNV infection was insensitive to subsequent IFN-alpha, -beta, -gamma, and -lambda stimulation, and HTNV-infected cells produced less NO compared to noninfected cells when stimulated with IFN-gamma and IL-1beta. Furthermore, less phosphorylated STAT1 after IFN treatment was observed in the nuclei of infected cells than in those of noninfected cells. The results suggest that hantavirus can interfere with the activation of antiviral innate immune responses in patients and inhibit the antiviral effects of all IFNs. We believe that future studies addressing the mechanisms by which hantaviruses interfere with the activation and shaping of immune responses may bring more knowledge regarding HFRS and HCPS pathogenesis.     

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.     

Hantavirus-infection Confers Resistance to Cytotoxic Lymphocyte-Mediated Apoptosis

Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardio-pulmonary syndrome (HCPS; also called hantavirus pulmonary syndrome (HPS)), both human diseases with high case-fatality rates. Endothelial cells are the main targets for hantaviruses. An intriguing observation in patients with HFRS and HCPS is that on one hand the virus infection leads to strong activation of CD8 T cells and NK cells, on the other hand no obvious destruction of infected endothelial cells is observed. Here, we provide an explanation for this dichotomy by showing that hantavirus-infected endothelial cells are protected from cytotoxic lymphocyte-mediated induction of apoptosis. When dissecting potential mechanisms behind this phenomenon, we discovered that the hantavirus nucleocapsid protein inhibits the enzymatic activity of both granzyme B and caspase 3. This provides a tentative explanation for the hantavirus-mediated block of cytotoxic granule-mediated apoptosis-induction, and hence the protection of infected cells from cytotoxic lymphocytes. These findings may explain why infected endothelial cells in hantavirus-infected patients are not destroyed by the strong cytotoxic lymphocyte response.     

Degrons at the C terminus of the pathogenic but not the nonpathogenic hantavirus G1 tail direct proteasomal degradation

Pathogenic hantaviruses cause two human diseases: hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). The hantavirus G1 protein contains a long, 142-amino-acid cytoplasmic tail, which in NY-1 virus (NY-1V) is ubiquitinated and proteasomally degraded (E. Geimonen, I. Fernandez, I. N. Gavrilovskaya, and E. R. Mackow, J. Virol. 77: 10760-10768, 2003). Here we report that the G1 cytoplasmic tails of pathogenic Andes (HPS) and Hantaan (HFRS) viruses are also degraded by the proteasome and that, in contrast, the G1 tail of nonpathogenic Prospect Hill virus (PHV) is stable and not proteasomally degraded. We determined that the signals which direct NY-1V G1 tail degradation are present in a hydrophobic region within the C-terminal 30 residues of the protein. In contrast to that of PHV, the NY-1V hydrophobic domain directs the proteasomal degradation of green fluorescent protein and constitutes an autonomous degradation signal, or "degron," within the NY-1V G1 tail. Replacing 4 noncontiguous residues of the NY-1V G1 tail with residues present in the stable PHV G1 tail resulted in a NY-1V G1 tail that was not degraded by the proteasome. In contrast, changing a different but overlapping set of 4 PHV residues to corresponding NY-1V residues directed proteasomal degradation of the PHV G1 tail. The G1 tails of pathogenic, but not nonpathogenic, hantaviruses contain intervening hydrophilic residues within the C-terminal hydrophobic domain, and amino acid substitutions that alter the stability or degradation of NY-1V or PHV G1 tails result from removing or adding intervening hydrophilic residues. Our results identify residues that selectively direct the proteasomal degradation of pathogenic hantavirus G1 tails. Although a role for the proteasomal degradation of the G1 tail in HPS or HFRS is unclear, these findings link G1 tail degradation to viral pathogenesis and suggest that degrons within hantavirus G1 tails are potential virulence determinants.     

Role of mixed Th1 and Th2 serum cytokines on pathogenesis and prognosis of hantavirus pulmonary syndrome

The hantavirus pulmonary syndrome (HPS) is an emerging syndrome in the Americas. The disease results from intense immune activation and changes in vascular permeability. The aim of this study was to determine the profile of serum cytokines in HPS patients looking for correlation with the clinical parameters, severity and outcome of illness. Studying 21 HPS patients, we found that IL-6 may have an important role in the pathogenesis of HPS, being associated with fatal outcome. Our results also support a mixed Th1/Th2 immune response during the course of HPS and that the magnitude of Th1 response effector cytokines is correlated to HPS severity. The decreased levels of TGF-beta observed in HPS patients suggest that immunoregulatory activity could be damaged in these patients.     

Andes Hantavirus-Infection of a 3D Human Lung Tissue Model Reveals a Late Peak in Progeny Virus Production Followed by Increased Levels of Proinflammatory Cytokines and VEGF-A

Andes virus (ANDV) causes hantavirus pulmonary syndrome (HPS), a severe acute disease with a 40% case fatality rate. Humans are infected via inhalation, and the lungs are severely affected during HPS, but little is known regarding the effects of ANDV-infection of the lung. Using a 3-dimensional air-exposed organotypic human lung tissue model, we analyzed progeny virus production and cytokine-responses after ANDV-infection. After a 7–10 day period of low progeny virus production, a sudden peak in progeny virus levels was observed during approximately one week. This peak in ANDV-production coincided in time with activation of innate immune responses, as shown by induction of type I and III interferons and ISG56. After the peak in ANDV production a low, but stable, level of ANDV progeny was observed until 39 days after infection. Compared to uninfected models, ANDV caused long-term elevated levels of eotaxin-1, IL-6, IL-8, IP-10, and VEGF-A that peaked 20–25 days after infection, i.e., after the observed peak in progeny virus production. Notably, eotaxin-1 was only detected in supernatants from infected models. In conclusion, these findings suggest that ANDV replication in lung tissue elicits a late proinflammatory immune response with possible long-term effects on the local lung cytokine milieu. The change from an innate to a proinflammatory response might be important for the transition from initial asymptomatic infection to severe clinical disease, HPS.     

Hantavirus pulmonary syndrome-associated hantaviruses contain conserved and functional ITAM signaling elements

Hantaviruses infect human endothelial and immune cells, causing two human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). We have identified key signaling elements termed immunoreceptor tyrosine-based activation motifs (ITAMs) within the G1 cytoplasmic tail of all HPS-causing hantaviruses. ITAMs direct receptor signaling within immune and endothelial cells and the presence of ITAMs in all HPS-causing hantaviruses provides a means for altering normal cellular responses which maintain vascular integrity. The NY-1 G1 ITAM was shown to coprecipitate a complex of phosphoproteins from cells, and the G1 ITAM is a substrate for the Src family kinase Fyn. The hantavirus ITAM coprecipitated Lyn, Syk, and ZAP-70 kinases from T or B cells, while mutagenesis of the ITAM abolished these interactions. In addition, G1 ITAM tyrosines directed intracellular interactions with Syk by mammalian two-hybrid analysis. These findings demonstrate that G1 ITAMs bind key cellular kinases that regulate immune and endothelial cell functions. There is currently no means for establishing the role of the G1 ITAM in hantavirus pathogenesis. However, the conservation of G1 ITAMs in all HPS-causing hantaviruses and the role of these signaling elements in immune and endothelial cells suggest that functional G1 ITAMs are likely to dysregulate normal immune and endothelial cell responses and contribute to hantavirus pathogenesis.     

Hantavirus pulmonary syndrome in Uberaba,Minas Gerais,Brazil

This report describes the epidemiological and clinical-evolutive characteristics of eight patients with hantavirus pulmonary syndrome (HPS) in Uberaba, Minas Gerais, Brazil. A positive history of contact with rodents was present in 100% of the cases. The time between the onset of symptoms and hospital care was, on average, 3.6 days. All patients showed clinical and laboratory findings suggestive of HPS. Elevated urea and creatinine levels were observed in 6 (75%) cases, PO2 was < 60 mmHg in 100% of the cases, and a chest X-ray demonstrated a bilateral interstitial-alveolar infiltrate. The diagnosis was confirmed by the detection of IgM antibodies against Sin Nombre virus by ELISA. Three patients died as a direct consequence of HPS.     

In vitro and in vivo activity of ribavirin against Andes virus infection

Pathogenic hantaviruses are a closely related group of rodent-borne viruses which are responsible for two distinct diseases in humans, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (HPS, otherwise known as hantavirus cardiopulmonary syndrome, HCPS). The antiviral effect of ribavirin against Old World hantaviruses, most notably Hantaan virus, is well documented; however, only a few studies have addressed its inhibitory effect on New World hantaviruses. In the present study, we demonstrate that ribavirin is highly active against Andes virus (ANDV), an important etiological agent of HPS, both in vitro and in vivo using a lethal hamster model of HPS. Treatment of ANDV infected Vero E6 cells with ribavirin resulted in dose-dependent reductions in viral RNA and protein as well as virus yields with a half maximal inhibitory concentration between 5 and 12.5 μg ml(-1). In hamsters, treatment with as little as 5 mg kg(-1) day(-1) was 100% effective at preventing lethal HPS disease when therapy was administered by intraperitoneal injection from day 1 through day 10 post-infection. Significant reductions were observed in ANDV RNA and antigen positive cells in lung and liver tissues. Ribavirin remained completely protective when administered by intraperitoneal injections up to three days post-infection. In addition, we show that daily oral ribavirin therapy initiated 1 day post-infection and continuing for ten days is also protective against lethal ANDV disease, even at doses of 5 mg kg(-1) day(-1). Our results suggest ribavirin treatment is beneficial for postexposure prophylaxis against HPS-causing hantaviruses and should be considered in scenarios where exposure to the virus is probable. The similarities between the results obtained in this study and those from previous clinical evaluations of ribavirin against HPS, further validate the hamster model of lethal HPS and demonstrate its usefulness in screening antiviral agents against this disease.     

A Lethal Disease Model for Hantavirus Pulmonary Syndrome in Immunosuppressed Syrian Hamsters Infected with Sin Nombre Virus

Sin Nombre virus (SNV) is a rodent-borne hantavirus that causes hantavirus pulmonary syndrome (HPS) predominantly in North America. SNV infection of immunocompetent hamsters results in an asymptomatic infection; the only lethal disease model for a pathogenic hantavirus is Andes virus (ANDV) infection of Syrian hamsters. Efforts to create a lethal SNV disease model in hamsters by repeatedly passaging virus through the hamster have demonstrated increased dissemination of the virus but no signs of disease. In this study, we demonstrate that immunosuppression of hamsters through the administration of a combination of dexamethasone and cyclophosphamide, followed by infection with SNV, results in a vascular leak syndrome that accurately mimics both HPS disease in humans and ANDV infection of hamsters. Immunosuppressed hamsters infected with SNV have a mean number of days to death of 13 and display clinical signs associated with HPS, including pulmonary edema. Viral antigen was widely detectable throughout the pulmonary endothelium. Histologic analysis of lung sections showed marked inflammation and edema within the alveolar septa of SNV-infected hamsters, results which are similar to what is exhibited by hamsters infected with ANDV. Importantly, SNV-specific neutralizing polyclonal antibody administered 5 days after SNV infection conferred significant protection against disease. This experiment not only demonstrated that the disease was caused by SNV, it also demonstrated the utility of this animal model for testing candidate medical countermeasures. This is the first report of lethal disease caused by SNV in an adult small-animal model.     

Pathogenic hantaviruses elicit different immunoreactions in THP-1 cells and primary monocytes and induce differentiation of human monocytes to dendritic-like cells

Hantaviruses cause two important human illnesses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Both syndromes are believed to be immune-mediated diseases. Monocytes/macrophages are thought to be the main target cells for hantaviruses and important sources of and targets for cytokines/chemokines secretion. THP-1 cells have been used extensively as models for primary monocytes in biocompatibility research. The aim of our study was to determine if hantaviruses induce the same immunoreactions in THP-1 cells and primary monocytes/ macrophages and might therefore be suitable for immune studies of hantaviral infections. For that purpose we compared various cytokines/chemokines and their receptors in THP-1 cell line and primary monocytes/macrophages. Infected primary monocytes/macrophages induced mostly beta-chemokines and their receptors. In contrast, THP-1 cells, expressed receptors for CXC chemokines. Surprisingly, infected macrophages underwent morphological changes toward dendritic-like cells and increased expression of co-stimulatory molecules: CD40, CD80, CD83 and CD86. Our data indicate that THP-1 cells are not ideal for in vitro research of the immunopathogenesis of hantaviruses in humans. Further, our studies revealed potential roles for cytokines/chemokines in HFRS/HPS immunopathogenesis and point to intriguing possibilities for the possible differentiation of infected macrophages to dendritic-like cells.     

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.     

Misdiagnosis of scrub typhus as hemorrhagic fever with renal syndrome and potential co-infection of both diseases in patients in Shandong Province,China, 2013–2014

BackgroundScrub typhus, caused by Orientia tsutsugamushi, an obligate intracellular gram-negative bacterium, along with hemorrhagic fever with renal syndrome (HFRS), caused by hantaviruses, are natural-focus infectious diseases prevalent in Shandong Province, China. Both diseases have similar clinical manifestations in certain disease stages and similar epidemic seasons, which has caused difficulties for physicians in distinguishing them. The aim of this study was to investigate whether misdiagnosis of scrub typhus as HFRS occurred in patients in Shandong Province.MethodsSerum samples (N = 112) of clinically suspected HFRS patients from 2013 to 2014 in Shandong Province were analyzed with enzyme-linked immunosorbent assay (ELISA) for antibodies to both hantavirus and Orientia tsutsugamushi.ResultsELISA showed that 56.3% (63/112) and 8.0% (9/112) of clinically suspected HFRS patients were IgM antibody positive to hantavirus and O. tsutsugamushi, respectively. Among the hantavirus IgM antibody positive patients, 7.9% (5/63) were also IgM antibody positive to O. tsutsugamushi. Among the hantavirus IgM antibody negative sera, 8.2% (4/49) of sera were positive to O. tsutsugamushi.ConclusionsWe concluded that some scrub typhus patients were misdiagnosed as HFRS and co-infection of scrub typhus and HFRS might exist in China. Due to the different treatments for scrub typhus and HFRS, physicians should carefully differentiate between scrub typhus and HFRS and consider administering anti-rickettsia antibiotics if treatment for HFRS alone does not work.