Study on oxidative stress and inflammatory/antioxidant substance levels in autism spectrum disorder.

BACKGROUND: The etiology of autism spectrum disorder (ASD) includes oxidative stress and brain inflammation. We investigated the relationship among oxidative stress markers, in vivo inflammatory substances, and antioxidants that can be easily measured in the clinic and compared them between children with ASD and those with typical development (TD). METHODS: Sixty-one children with TD and 199 with untreated ASD were investigated. They were Japanese children aged 2-15 years and were divided into those aged <7 and ≥7 years. Serum levels of reactive oxygen metabolites (ROMs), high-sensitivity C-reactive protein (hsCRP), prolactin (PRL), albumin (Alb), total bilirubin (T-Bil), and uric acid (UA) were measured. These measurements were compared between TD and ASD, and the relationship between oxidative stress and relevant laboratory parameters was analyzed. RESULTS: The hsCRP and PRL levels were significantly higher in patients with ASD than in those with TD. Among those aged <7 years, hsCRP and PRL were significantly higher in those with ASD than in those with TD. Among those aged ≥7 years, ROMs, hsCRP, and PRL were significantly higher in those with ASD than in those with TD. In ASD, ROMs were significantly correlated with hsCRP, Alb, T-Bil, and PRL. In contrast, no significant correlations were found in the TD group except for the relationship between ROMs and hsCRP in those aged <7 years. CONCLUSION: The results suggest that serum levels of in vivo inflammatory substances, stress-related substances, and antioxidants are altered in ASD under oxidative stress.

[Developing a Biosafety Level 4 Laboratory user training program].

In recent years, numerous emerging and reemerging infectious diseases have occurred worldwide and have seriously threatened our society. As a countermeasure against the pathogens responsible for serious diseases (classified as class 4 pathogens), we are preparing for full operation of the first suit-type biosafety level 4 (BSL-4) facility available for basic and applied research at Nagasaki University. For the safe operation of these facilities, experienced and qualified personnel with appropriate skills and knowledge of biorisk management must be certified. Developing an appropriate training system is a prerequisite for ensuring the safety of users involved in research in a BSL-4 laboratory. Here, we introduce an overview of the content of the training program that we are currently establishing for the BSL-4 facility at Nagasaki University. We are designing this program to follow national and international guidelines and regulations in part by referring to experiences and materials derived from multiple BSL-4 facilities in other countries. The established training program system, including the formulation processes, will serve as a reference and will provide practical materials for other research organizations to develop their own high-containment laboratory training programs.

Subcellular localization of nucleocapsid protein of SFTSV and its assembly into the ribonucleoprotein complex with L protein and viral RNA.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus that causes novel zoonotic diseases in Asian countries including China, Japan, South Korea, and Vietnam. In phleboviruses, viral proteins play a critical role in viral particle formation inside the host cells. Viral glycoproteins (GPs) and RNA-dependent RNA polymerase (RdRp) are colocalized in the Golgi apparatus and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The nucleocapsid (N) protein was widely expressed in the cytoplasm, even in cells coexpressing GP. However, the role of SFTSV N protein remains unclear. The subcellular localization of SFTSV structural proteins was investigated using a confocal microscope. Subsequently, minigenome and immunoprecipitation assays were carried out. The N protein interacts with viral RNA (vRNA) and further shows translational activity with RdRp which is L protein and localized in the ERGIC and Golgi apparatus when co-expressed with GP. On the other hand, mutant N protein did not interact with vRNA either localized in the ERGIC or Golgi apparatus. The interaction between the N protein of SFTSV and vRNA is important for the localization of viral proteins and viral assembly. This study provides useful insights into the life cycle of SFTSV, which will lead to the detection of antiviral targets.

Identification of Novel Rodent-Borne Orthohantaviruses in an Endemic Area of Chronic Kidney Disease of Unknown Etiology (CKDu) in Sri Lanka.

We reported the genetic evidence of circulating hantaviruses from small mammals captured in a chronic kidney disease of unknown etiology (CKDu) hotspot area of Sri Lanka. The high seroprevalence of anti-hantavirus antibodies against Thailand orthohantavirus (THAIV) has been reported among CKDu patients and rodents in Sri Lankan CKDu hotspots. We captured 116 small mammals from CKDu endemic regions in the Polonnaruwa District of Sri Lanka. Seven animals (five out of 11 Mus booduga and two out of 99 Rattus rattus) were PCR-positive for the hantavirus. A rat-borne sequence was grouped with a THAIV-like Anjozorobe virus. In contrast, Mus-borne sequences belonged to the THAIV lineage, suggesting a novel orthohantavirus species according to the phylogenetic analyses and whole-genome comparisons. Our genetic evidence indicates the presence of two THAIV-related viruses circulating in this CKDu endemic area, suggesting a basis for further investigations to identify the infectious virus in patients with CKDu and the CKDu induction mechanism of these viruses.

The NF-κB inhibitor, SC75741, is a novel antiviral against emerging tick-borne bandaviruses.

Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV) cause viral hemorrhagic fever-like illnesses in humans due to an aberrant host inflammatory response, which contributes to pathogenesis. Here, we established two separate minigenome (MG) systems based on the M-segment of SFTSV and HRTV. Following characterization of both systems for SFTSV and HRTV, we used them as a platform to screen potential compounds that inhibit viral RNA synthesis. We demonstrated that the NF-κB inhibitor, SC75741, reduces viral RNA synthesis of SFTSV and HRTV using our MG platform and validated these results using infectious SFTSV and HRTV. These results may lead to the use of MG systems as potential screening systems for the identification of antiviral compounds and yield novel insights into host-factors that could play role in bandavirus transcription and replication.

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.

The Polarity of an Amino Acid at Position 1891 of Severe Fever with Thrombocytopenia Syndrome Virus L Protein Is Critical for the Polymerase Activity.

Severe fever with thrombocytopenia syndrome virus subclone B7 shows strong plaque formation and cytopathic effect induction compared with other subclones and the parental strain YG1. Compared to YG1 and the other subclones, only B7 possesses a single substitution in the L protein at the amino acid position 1891, in which N is changed to K (N1891K). In this study, we evaluate the effects of this mutation on L protein activity via a cell-based minigenome assay. Substitutions of N with basic amino acids (K or R) enhanced polymerase activity, while substitutions with an acidic amino acid (E) decreased this activity. Mutation to other neutral amino acids showed no significant effect on activity. These results suggest that the characteristic of the amino acid at position 1891 of the L protein are critical for its function, especially with respect to the charge status. Our data indicate that this C-terminal domain of the L protein may be crucial to its functions in genome transcription and viral replication.

Assessment of oxidative stress in autism spectrum disorder using reactive oxygen metabolites and biological antioxidant potential.

There are several studies on oxidative stress of Autism Spectrum Disorder (ASD), but in these cases there is no study to measure oxidative stress and antioxidant capacity at the same time or studies considering childhood development. Therefore, this study comprehensively assessed the level of oxidative stress in ASD children by simultaneously measuring reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP). The subjects were Japanese, 77 typical development (TD) children, 98 ASD children, samples were plasma. The subjects were divided into age groups: toddlers/preschool age (2-6 years) and school age (7-15 years), to compare the relationships among the d-ROMs levels and BAP/d-ROMs ratios. Furthermore, the correlations between the Parent-interview ASD Rating Scales (PARS) scores and the measured values were analyzed. The levels of d-ROMs were significantly higher in the ASD (7-15 years) than in TD (7-15 years). The PARS scores were significantly higher in the ASD and were significantly correlated with d-ROMs levels. These results suggested that d-ROMs and BAP/d-ROMs ratios could be objective, measured indicators that could be used in clinical practice to assess stress in ASD children.

Evaluation of the efficacy of drug treatment based on measurement of the oxidative stress, using reactive oxygen metabolites and biological antioxidant potential, in children with autism spectrum disorder and attention deficit hyperactivity disorder.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder, mainly characterized by impairment of social communication and restricted interests. ASD is frequently accompanied by attention deficit hyperactivity disorder (ADHD), which is characterized by carelessness, hyperactivity and impulsivity (ASD/ADHD). It has been suggested that ASD and ADHD are associated with oxidative stress, that is, that patients with ASD/ADHD are in a state of increased oxidative stress. There are currenr tly no objective or biological test criteria for evaluating the efficacy of drug therapy in these patients. The purpose of this study was to evaluate whether oxidative stress markers [serum reactive oxygen metabolites (d-ROMs) levels and biological antioxidant potential (BAP)] can be used as objective indicators for evaluating the efficacy of drug treatment in ASD/ADHD patients. METHODS: The subjects of this study subjects were 50 Japanese patients with ASD/ADHD aged 4 to 14 years old. Serum samples were obtained from the patients to measure the serum levels of d-ROMs and the serum BAP. The study subjects were divided into two age groups: preschool children (4 to 6 years old) and school-age children (7 to 14 years old), and the serum levels of d-ROMs, serum BAP, serum BAP/d-ROMs ratio (hereinafter, the prefix serum will be dropped), and scores on the Parent-interview ASD Rating Scales-Text Revision (PARS-TR) and ADHD Rating Scale (ADHD-RS) were determined before and after drug therapy and compared between the two groups. In addition, changes in the d-ROMs, BAP and BAP/d-ROMs ratio and changes in the scores on the PARS-TR and ADHD-RS after treatment were also analyzed. RESULTS: Significant decrease of the d-ROMs, BAP, and scores on the PARS-TR and ADHD-RS, with a significant increase of the BAP/d-ROMs ratio, was observed after treatment. In addition, a significant correlation was observed between the changes in the d-ROMs and changes in the scores on the PARS-TR and ADHD-RS after treatment in the school-age ASD/ADHD children. CONCLUSION: Our results suggest the possibility that the serum level of d-ROMs may be useful as an objective assessment marker to supplement the subjective assessment of the effects of drug treatment in school-age children with ASD/ADHD.

Reply to Comments by Yih et al. (Exposure to Hantavirus is a Risk Factor Associated with Kidney Diseases in Sri Lanka: A Cross-Sectional Study).

Dear Drs. W. Katherine Yih, Martin Kulldorff, Jessica H. Leibler, David J. Friedman, and Daniel R. Brooks [...].

Exposure to Hantavirus is a Risk Factor Associated with Kidney Diseases in Sri Lanka: A Cross Sectional Study.

Chronic kidney disease of unknown etiology (CKDu) imposes a substantial burden on public health in Sri Lankan agricultural communities. High seroprevalences of hantavirus have been reported in CKDu patients in several locations of Sri Lanka. We carried out a cross-sectional study followed by an unmatched case-control comparison in two geographically distinct areas of Sri Lanka, Girandurukotte (CKDu endemic) and Kandy (CKDu non-endemic) to determine whether exposure to hantaviruses is a potential risk factor in patients with kidney disease. An indirect immunofluorescent antibody assay using two antigens, Thailand orthohantavirus-infected and recombinant N protein-expressing Vero E6 cells, were used for serodiagnosis. Participants' demographic and other socio-economic data were collected through a structured questionnaire. Fifty kidney disease patients and 270 controls from Kandy and 104 kidney disease patients and 242 controls from Girandurukotte were examined. Seropositivities were 50% and 17.4% in kidney patients and controls, respectively, in Girandurukotte, and they were 18% and 7% in Kandy. The odds of exposure to hantaviruses were higher for kidney disease patients than for controls in both Girandurukotte (OR:3.66, 95% CI:2.01 to 6.64) and Kandy (OR:2.64, 95% CI:1.07 to 6.54) in binary logistic regression models. According to statistical analysis, individuals exposed to hantaviruses had a higher risk of developing renal impairment. Therefore, hantavirus infection might be an important risk factor for development of kidney disease in Sri Lanka.

Serological Evidence of Thailand Orthohantavirus or Antigenically Related Virus Infection Among Rodents in a Chronic Kidney Disease of Unknown Etiology Endemic Area, Girandurukotte, Sri Lanka.

We have reported high seroprevalence to Thailand orthohantavirus (THAIV) or THAIV-related orthohantavirus (TRHV) among patients with chronic kidney disease of unknown etiology in Girandurukotte, Sri Lanka. THAIV or TRHV infection is considered to be transmitted by rodent hosts in this area, but its reservoir rodents have not yet been identified. Hence, 116 rodents were captured, and seroprevalences were examined by indirect immunofluorescent antibody assay (immunofluorescence assay [IFA]) using antigens of THAIV strain Thai749-infected Vero E6 cells and recombinant nucleocapsid protein of THAIV expressed in Vero E6 cell. Molecular biological species identification of rodents was carried out by sequencing rag1, irbp, and mitochondrial cytb genes. The majority (112/116) of the captured rodents were lineage Ib of black rats (Rattus rattus). Among them, 19.6% (22/112) of the rats possessed antibodies against THAIV. Also, a lesser bandicoot rat (Bandicota bengalensis), which belongs to the Sri Lankan endemic genetic lineage, was seropositive (1/1). Two Mus booduga and one Murinae sp. were seronegative. Rodent sera showed less cross-reactivities to antigens of Vero E6 cells infected with Hantaan orthohantavirus (HTNV), Seoul orthohantavirus (SEOV), and Puumala orthohantavirus (PUUV) in IFA. These results suggest that the hantavirus present in rodents in Sri Lanka is related to THAIV or TRHV rather than to SEOV, HTNV, or PUUV. However, it might be serologically distinct from the prototype THAIV strain, Thai749, used in this study. This study revealed that black rats and lesser bandicoot rats belonging to Sri Lankan endemic lineages are possible reservoirs for THAIV or TRHV in Girandurukotte. Further multiple geographical studies are needed to confirm the THAIV or TRHV reservoir status of black and lesser bandicoot rats in Sri Lanka.

Evaluation of oxidative stress and antioxidant capacity in healthy children.

BACKGROUND: Measurement of the levels of the derivatives of reactive oxygen metabolites (d-ROMs) and of the biological antioxidant potential (BAP) enables simultaneous assessment of oxidation degree and antioxidant capacity, using the same sample and testing equipment. At present, reference values of healthy adults are clarified, but the reference value of healthy children is unknown. This study was undertaken to clarify the age-related changes and the reference values of d-ROMs and BAP in healthy children. METHODS: The study population consisted of 77 children, ranging in age from 2 to 15 years, in normal mental and physical health as examined by a pediatrician, and seven healthy adult volunteers. Serum samples were obtained from the subjects for assay. Using these samples, d-ROMs and BAP values were measured, and the relationship with age was analyzed. RESULTS: The d-ROMs level decreased as the age increased, while the BAP showed no correlation with the age. The d-ROMs level was significantly higher in 2-6 years group than in 7-11 years group, 12-15 years group, or healthy adults group. The BAP/d-ROMs ratio, an index of antioxidant capacity, increased significantly with higher age. CONCLUSION: This study was carried out for the first time in healthy children in oxidative stress assessment using d-ROMs and BAP. In the infancy 2-6 years, the d-ROMs value was significantly higher and the BAP/d-ROMs ratio was significantly lower. From this, it was suggested that age should be considered when performing oxidative stress assessment using d-ROMs and BAP in children.

Involvement of CD8+ T cells in the development of renal hemorrhage in a mouse model of hemorrhagic fever with renal syndrome.

Hemorrhagic fever with renal syndrome (HFRS) is caused by hantavirus infection. Although host immunity is thought to be involved in the pathogenesis of HFRS, the mechanism remains to be elucidated. A mouse model of HFRS, which showed renal hemorrhage similar to that seen in patients, has been developed previously. In this study, we aimed to clarify whether CD4+ and CD8+ T cells are involved in the development of renal hemorrhage in the mouse model. At 2 days before virus inoculation, CD4+ or CD8+ T cells in 6-week-old BALB/c mice were depleted by administration of antibodies. The CD4+ T cell-depleted mice developed signs of disease such as transient weight loss, ruffled fur and renal hemorrhage as in non-depleted mice. In contrast, the CD8+ T cell-depleted mice showed no signs of disease. After determination of CTL epitopes on the viral glycoprotein in BALB/c mice, the quantity of virus-specific CTLs was analyzed using an MHC tetramer. The quantity of virus-specific CTLs markedly increased in spleens and kidneys of virus-infected mice. However, the quantity in high-pathogenic clone-infected mice was comparable to that in low-pathogenic clone-infected mice. We previously reported that the high-pathogenic clone propagated more efficiently than the low-pathogenic clone in kidneys of mice during the course of infection. Therefore, there is a possibility that the balance between quantities of the target and effector is important for disease outcome. In conclusion, this study showed that CD8+ T cells are involved in the development of renal hemorrhage in a mouse model of HFRS.

Targeting of severe fever with thrombocytopenia syndrome virus structural proteins to the ERGIC (endoplasmic reticulum Golgi intermediate compartment) and Golgi complex.

Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) is a newly emerged phlebovirus identified in China, Japan, and South Korea. Phlebovirus glycoproteins (GP) play a key role in targeting viral structural components to the budding compartments in the ER-Golgi intermediate compartment (ERGIC) and Golgi complex. However, the role of SFTSV GP in targeting structural proteins to the ERGIC and Golgi complex remains unresolved. In this study, we show that SFTSV GP plays a significant role in targeting RNA-dependent RNA polymerase (L) and nucleocapsid protein (NP) to the budding sites. Confocal microscopy was used to investigate the subcellular localization of SFTSV structural proteins. In SFTSV-infected cells, GP and L localized to the ER, ERGIC and Golgi complex, whereas NP localized to the ERGIC and Golgi complex. In addition, GP colocalized with L and NP in infected cells. In cells singly transfected with GP, L or NP, GP localized to the same subcellular compartments as in infected cells. However, L or NP alone did not localize to the ER, ERGIC, or Golgi complex. Cotransfection experiments showed that GP altered the localization of L to the ERGIC and Golgi complex but not that of NP. Interestingly, plasmid-expressed NP fused with a hemagglutinin tag localized to the ERGIC and Golgi complex when expressed in SFTSV-infected cells and colocalised with GP, suggesting that GP plays a role in the subcellular localization of L and NP in infected cells. Thus, the SFTSV structural components start to assemble at the ERGIC to Golgi complex. GP is required for transporting L and NP to the ERGIC and Golgi complex. In addition, targeting of NP requires interaction with other factors besides GP.

Pathogenic analysis of the pandemic 2009 H1N1 influenza A viruses in ferrets.

The pandemic 2009 H1N1 influenza A virus emerged in humans and caused the first influenza pandemic of the 21st century. Mexican isolates, A/Mexico/4108/2009 (H1N1) (Mex4108) and A/Mexico/InDRE4478/2009 (H1N1) (Mex4487) derived from a mild case and from a cluster of severe cases, showed heterogeneity in virulence in a cynomolgus macaque model. To compare the more pathogenic differences, we generated recombinant viruses and compared their virulence in ferrets. Ferrets infected with recombinant Mex4487 displayed a slightly higher rate of viral replication and severe pneumonia in the early stage of infection. In contrast, prolonged lower virus shedding of recombinant Mex4108 than that of recombinant Mex4487 was detected in throat swabs. Thus, Mex4487 induces severe pneumonia in infected individuals, whereas Mex4108 might have wide-spreading potential with mild disease.

Evidence of infection with Leptospira interrogans and spotted fever group rickettsiae among rodents in an urban area of Osaka City, Japan.

We examined 33 rodents captured in an urban area of Osaka City, Japan for IgG antibodies against Seoul virus, severe fever with thrombocytopenia syndrome virus, hepatitis E virus, Leptospira interrogans, Yersinia pestis, spotted fever, typhus and scrub typhus group rickettsiae. We found that 3 (9.1%) and 1 (3.0%) of the 33 rodents had antibodies against L. interrogans and spotted fever group rickettsiae, respectively. DNAs of leptospires were detected from 2 of the 3 seropositive rodents, but DNA of rickettsia was not detected. Phylogenetic analysis and multiple locus sequence typing revealed that the 2 leptospires were L. interrogans belonging to a novel sequence type. There is a potential risk for acquiring rodent-borne zoonotic pathogens even in cities in developed countries.

The amino acid at position 624 in the glycoprotein of SFTSV (severe fever with thrombocytopenia virus) plays a critical role in low-pH-dependent cell fusion activity.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel phlebovirus responsible for causing an emerging zoonotic disease. We previously established subclones from SFTSV strain YG1 based on differences in low-pH-dependent cell fusion activities and found two amino acid substitutions, Y328H and R624W, in the envelope glycoprotein (GP) of high fusion subclones. In this study, we show that transiently expressed GP with the R624W mutation, but not the Y328H mutation, induced cell fusion under acidic conditions. GP possessing either tryptophan, serine, glycine or aspartic acid at position 624 induced cell fusion, whereas GP possessing basic amino acids such as arginine or lysine did not induce cell fusion. These results indicated that the amino acid at position 624 has an important role for inducing low-pH-dependent cell fusion.

Genetic Predisposition To Acquire a Polybasic Cleavage Site for Highly Pathogenic Avian Influenza Virus Hemagglutinin.

Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes.IMPORTANCE Influenza A viruses are divided into subtypes based on the antigenicity of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase. Of the 16 HA subtypes (H1 to -16) maintained in waterfowl reservoirs of influenza A viruses, H5 and H7 viruses often become highly pathogenic through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been known since the 1980s, the genetic basis for nucleotide insertions has remained unclear. This study shows the potential role of the viral RNA secondary structure for nucleotide insertions and demonstrates a key mechanism explaining why the acquisition of the polybasic HA cleavage site is restricted to particular HA subtypes in nature. Our findings will contribute to better understanding of the ecology of influenza A viruses and will also be useful for the development of genetically modified vaccines against H5 and H7 influenza A viruses with increased stability.

Appearance of renal hemorrhage in adult mice after inoculation of patient-derived hantavirus.

BACKGROUND: Hemorrhagic fever with renal syndrome (HFRS) caused by hantavirus infection is characterized by fever, renal dysfunction and hemorrhage. An animal model mimicking symptoms of HFRS remains to be established. In this study, we evaluated the pathogenicity of an HFRS patient-derived Hantaan virus (HTNV) in adult mice. METHODS: Five clones of HTNV strain KHF 83-61 BL (KHFV) that was derived from blood of an HFRS patient were obtained by plaque cloning. The pathogenicity of the virus clones was evaluated by using 6-week-old female BALB/c mice. Sequence analysis of the viral genome was performed by conventional methods. RESULTS: All of the mice intravenously inoculated with KHFV clone (cl)-1, -2, -3 and -5 showed signs of disease such as transient body weight loss, ruffled fur, reduced activity and remarkably prominent hemorrhage in the renal medulla at 6 to 9 days post-inoculation (dpi) and then recovered. In contrast, mice intravenously inoculated with KHFV cl-4 did not show any signs of disease. We selected KHFV cl-5 and cl-4 as representative of high-pathogenic and low-pathogenic clones, respectively. Quantities of viral RNA in kidneys of KHFV cl-5-infected mice were larger than those in KHFV cl-4-infected mice at any time point examined (3, 6, 9 and 12 dpi). The quantities of viral RNA of KHFV cl-5 and cl-4 peaked at 3 dpi, which was before the onset of disease. Sequence analysis revealed that the amino acid at position 417 in the glycoprotein Gn was the sole difference in viral proteins between KHFV cl-5 and cl-4. The result suggests that amino acid at position 417 in Gn is related to the difference in pathogenicity between KHFV cl-5 and cl-4. When the inoculum of KHFV cl-5 was pretreated with a neutralizing antibody against HTNV strain 76-118, which belongs to the same serotype as KHFV clones, mice did not show any signs of disease, confirming that the disease was caused by KHFV infection. CONCLUSION: We found that an HFRS patient-derived HTNV caused renal hemorrhage in adult mice. We anticipate that this infection model will be a valuable tool for understanding the pathogenesis of HFRS.